CN110346554A

CN110346554A - A kind of double-antibody method enzyme immunochromatographytest test kit and preparation method and application

Info

Publication number: CN110346554A
Application number: CN201810283311.1A
Authority: CN
Inventors: 田克恭; 王莹
Original assignee: Luoyang Zhongke Gene Detection And Diagnosis Center Co Ltd; Luoyang Pu Laikewantai Bioisystech Co Ltd
Current assignee: Luoyang Zhongke Gene Detection And Diagnosis Center Co Ltd; Luoyang Pu Laikewantai Bioisystech Co Ltd
Priority date: 2018-04-02
Filing date: 2018-04-02
Publication date: 2019-10-18
Anticipated expiration: 2038-04-02
Also published as: CN110346554B

Abstract

It include enzyme immunochromatographydetecting detecting test strip, sample treatment liquid, buffer and buffer supply unit the present invention relates to a kind of kit, buffer includes any one of phosphate buffer, high molecular weight protein, Tween-20 and preservative and carbamide peroxide, hydrogen peroxide urea, hydrogen peroxide；Enzyme immunochromatographydetecting detecting test strip includes substrate pad, is adsorbed with the zymolyte made of drying after substrate pad diluted thereon, substrate pad dilution includes dehydrated alcohol, pH7.40.1M PBS solution and 0.5%~4%W/V PEG6000 or PEG4000；Enzyme immunochromatographydetecting detecting test strip includes enzyme mark pad, being adsorbed with the drying after enzyme labelled antibody diluted thereon, into enzyme labelled antibody, enzyme labelled antibody dilution includes any one of pH7.4 0.1M PBS solution and 0.5%V/V PEG4000, PEG6000 or PEG8000；Enzyme immunochromatographydetecting detecting test strip includes detection line and nature controlling line, and the sessile antibody through sessile antibody diluted is fixed in detection line, and the sessile antibody dilution includes pH7.4 0.1M PBS solution, 0.5%V/V trehalose, 0.1%V/V Tween-20.

Description

A kind of double-antibody method enzyme immunochromatographytest test kit and preparation method and application

Technical field

The present invention relates to a kind of double-antibody method enzyme immunochromatographytest test kits and the preparation method and application thereof, belong to life Analyte detection field.

Background technique

Currently, field of biological detection using double-antibody sandwich detection antigen it is relatively conventional, wherein have by enzyme label or The detection method that the mode chromatographed after enzyme label is established, such as (Zhang Lihuai etc., detects avian influenza virus antigen to Zhang Lihuai document Double-antibodies sandwich ELISA establishes Chinese Medicine biotechnology, 2009,4 (1): 62-64) in report it is anti-with monoclonal The Enzyme-linked Immunosorbent Assay method ELISA that the sandwich principle of body-polyclonal antibody is established detects avian influenza virus antigen, and such as China The enzyme chromatography test strip of patent CN104062430A report established with monoclonal antibody-monoclonal antibody sandwich principle Detect influenza virus.Wherein, most commonly seen with the sandwich detection antigen of monoclonal antibody-monoclonal antibody (double monoclonal antibodies).

But such phenomenon is had found in the repetition test based on the prior art is groped: being related to double antibody (double lists It is anti-) the quick testing product of sandwich principle is after determining that 2 strain antibodies can be used for double-antibody sandwich detection antigen, collocation mould therewith Formula result of study show wherein 1 plant of monoclonal antibody be only used for label and another 1 plant of monoclonal antibody can only be used as detection line The antibody of upper immobilization that is, by the location swap of 2 plants of monoclonal antibodies in product, then can cause if the collocation mode of 2 strain antibodies is changed Non-specific responding reduces detection sensitivity.

In addition, sample treatment liquid, buffer ingredient are different in existing product, it is anti-to often lead to target in actually detected Original, which is not cracked sufficiently or reacted, to be not enough.And different kit sample treatment fluids come generally only for specific object to be checked Source sample is effective, to other animal epidemic antigens may not effectively (referring to the specification of commercial kit, indicate " cannot make more With the sample treatment liquid or Sample dilution of other kits "), this cause it is actually detected using other kinds of sample when it is sensitive It spends that low, sensibility is bad, or even non-specific responding occurs, influence to detect speed, can make that sb.'s illness took a turn for the worse or leads to virus when serious It propagates wantonly.

Therefore, lacking one kind in the prior art can be such that the labelled antibody of double antibodies sandwich detection antigen and sessile antibody exchanges Kit, also lack a kind of reagent that is able to solve and can sufficiently handle to guarantee detection effect the sample of separate sources Box.

Summary of the invention

To solve the deficiencies in the prior art, it is an object of the present invention to provide a kind of inspections of double-antibody method enzyme immunochromatography Test agent box, the kit include that enzyme immunochromatographydetecting detecting test strip, sample treatment liquid, buffer and buffer supply are single Member, which is characterized in that

The buffer includes phosphate buffer, high molecular weight protein, Tween-20 and preservative, and hydrogen peroxide Any one of urea, hydrogen peroxide urea, hydrogen peroxide；The preferably described phosphate buffer is pH7.4 0.1M PBS solution； The preferably described high molecular weight protein is one of BSA, OVA, fetal calf serum or a variety of；The preferably described preservative is that celebrating is big mould Any one of element, kanamycin sulfate, Sodium azide, thimerosal and its esters, Proclin300；The carbamide peroxide, mistake Aoxidize urea hydrogen, the concentration of hydrogen peroxide is 0.02%~0.2%W/V；The preferably described carbamide peroxide, hydrogen peroxide urea, The concentration of hydrogen peroxide is 0.05%~0.1%W/V；Further preferably comprising pH7.4 0.1M PBS solution, 0.1%~ 0.5%V/V Tween-20,0.02%V/V Proclin300,0.05%V/V carbamide peroxide and 0.5%~1%W/V Any BSA, OVA and fetal calf serum；

The enzyme immunochromatographydetecting detecting test strip includes substrate pad, is adsorbed with after substrate pad diluted thereon dry Made of zymolyte, the substrate pad dilution includes dehydrated alcohol, pH7.4 0.1M PBS solution and 0.5%~4%W/V PEG6000 or PEG4000；Preferably, substrate pad dilution includes 2%V/V dehydrated alcohol, 0.1%V/VPEG4000, pH7.4 0.1M PBS；

The enzyme immunochromatographydetecting detecting test strip includes enzyme mark pad, is adsorbed with does after enzyme labelled antibody diluted thereon It is dry to form enzyme labelled antibody, the enzyme labelled antibody dilution include pH7.40.1M PBS solution and 0.5%V/V PEG4000, Any one of PEG6000 or PEG8000；Preferably, the enzyme labelled antibody dilution includes 0.5%V/VPEG4000, pH7.4 0.1M PBS；

The enzyme immunochromatographydetecting detecting test strip includes detection line and nature controlling line, is fixed in the detection line through fixed anti- The sessile antibody of body diluted, the sessile antibody dilution include pH7.4 0.1M PBS solution, 0.5%V/V seaweed Sugar, 0.1%V/V Tween-20.

Enzyme immunochromatographytest test kit of the invention, it is anti-by the buffer of specific composition, substrate pad dilution, enzyme mark Body dilution, sessile antibody dilution solve non-interchangeable between two kinds of antibody in former double-antibody method enzyme immunochromatography Problem is also further improving detection sensitivity with clock synchronization using former monoclonal antibody, and anti-using the monoclonal exchanged Body can be preceding suitable or more excellent with exchange with clock synchronization detection sensitivity.

As one embodiment of the present invention, in enzyme immunochromatographytest test kit of the present invention, the reagent Box includes test strip, sample treatment liquid, buffer and buffer supply unit, wherein the test strip is from longitudinal direction On successively include substrate supply area, sample supply area, detection zone；The substrate supply area includes the substrate pad, is adsorbed thereon There is dry zymolyte, the preferably described zymolyte is tetramethyl benzidine；The sample supply area includes the enzyme mark pad, On be adsorbed with enzyme labelled antibody, the enzyme is preferably horseradish peroxidase, alkaline phosphatase or beta galactosidase, the enzyme bottom Object can generate chromogenic reaction with the enzyme on the enzyme labelled antibody；The detection zone includes the detection line and the nature controlling line, Immobilization has sheep anti mouse mostly anti-or sheep anti mouse secondary antibody on the nature controlling line；The buffer supply unit is used for the buffer It supplies to the substrate supply area of the test strip, and makes buffer and the sample treatment liquid with buffer diffusion, enzyme bottom Object and enzyme labelled antibody are migrated along the longitudinal direction of test strip to one end far from substrate supply area.

As one embodiment of the present invention, in enzyme immunochromatographytest test kit of the present invention, the enzyme mark The concentration that antibody is dissolved in enzyme labelled antibody dilution is 0.5~10 μ g/ml, the sessile antibody is dissolved in sessile antibody dilution The concentration that concentration in liquid is 0.5~1mg/ml, sheep anti mouse is mostly anti-or sheep anti mouse secondary antibody is dissolved in sessile antibody dilution is 1 ~3mg/ml, zymolyte are dissolved in final concentration of 0.5%~4%W/V of substrate dilution.

Enzyme labelled antibody of the invention, sessile antibody, zymolyte concentration take specific range, can step detection sensitivity It further increases.

As one embodiment of the present invention, in enzyme immunochromatographytest test kit of the present invention, the detection Line, nature controlling line line-to-line are away from >=5mm.

As one embodiment of the present invention, in enzyme immunochromatographytest test kit of the present invention, enzyme labelled antibody In monoclonal antibody and sessile antibody in monoclonal antibody be CCTCC No:C2014198 mouse hybridoma cell 3G12 point The pig circle for the porcine circovirus 2 type monoclonal antibody 3G12 and CCTCC No:C2014199 mouse hybridoma cell 2F8 secretion secreted Any combination of 2 type monoclonal antibody 2F8 of circovirus virus；Or for Porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB2 and Any combination of Porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB1, the monoclonal antibody PEDV-McAB2 heavy chain can Become area into SEQ ID No.1 shown in, light chain variable region be that the monoclonal antibody PEDV-McAB1 is weighed shown in SEQ ID No.2 Chain variable region is shown in SEQ ID No.3, light chain variable region is shown in SEQ ID No.4；It or is transmissible gastroenteritis of swine disease Any combination of malicious monoclonal antibody TGEV-3D2 and monoclonal antibody of transmissible gastro-enteritis virus TGEV-4B4, the Dan Ke Grand antibody TGEV-3D2 heavy chain variable region is shown in SEQ ID No.5, light chain variable region is the list shown in SEQ ID No.6 Clonal antibody TGEV-4B4 heavy chain variable region is shown in SEQ ID No.7, light chain variable region is shown in SEQ ID No.8；Or For anti-influenza type A virus nucleoprotein monoclonal antibody IV-McAB1 and anti-influenza type A virus nucleoprotein monoclonal antibody IV- Any combination of McAB2, the monoclonal antibody IV-McAB1 heavy chain variable region be SEQ ID No.9 shown in, light chain variable region For shown in SEQ ID No.10, the monoclonal antibody IV-McAB2 heavy chain variable region is shown in SEQ ID No.11, light chain can Become area as shown in SEQ ID No.12；Or secreted for 10B11 plants of mouse bone marrow cells hybridoma of CCTCC No:C201578 10H4 plants of mouse bone marrow cells hybridoma of CPV-10B11 and CCTCC No:C201579 of canine parvovirus monoclonal antibody secretion Any combination of canine parvovirus monoclonal antibody CPV-10H4；It or is CCTCC No:C2015201 mouse bone marrow cells hybridoma Canine distemper virus monoclonal antibody CDV-1G5 and CCTCC the No:C2015202 mouse bone marrow cells hybridoma of cell 1G5 secretion Any combination of the canine distemper virus monoclonal antibody CDV-6E11 of 6E11 plants of secretions；It or is hepatitis infectiosa canis virus monoclonal antibody Any combination of CAV-5G4 and hepatitis infectiosa canis virus monoclonal antibody CAV-1A1, the monoclonal antibody CAV-5G4 heavy chain variable region For shown in SEQ.ID No.13, light chain variable region be the monoclonal antibody CAV-1A1 weight chain variable shown in SEQ.ID No.14 Area is shown in SEQ.ID No.15, light chain variable region is shown in SEQ.ID No.16.

As one embodiment of the present invention, the enzyme labelled antibody is PCV2-3G12, and the sessile antibody is PCV2- 2F8；Or the enzyme labelled antibody is PCV2-2F8, the sessile antibody is PCV2-3G12.

As one embodiment of the present invention, the enzyme labelled antibody is PEDV-McAB2, and the sessile antibody is PEDV- McAB1；Or the enzyme labelled antibody is PEDV-McAB1, the sessile antibody is PEDV-McAB2.

As one embodiment of the present invention, the enzyme labelled antibody is TGEV-3D2, and the sessile antibody is TGEV- 4B4；Or the enzyme labelled antibody is TGEV-4B4, the sessile antibody is TGEV-3D2.

As one embodiment of the present invention, the enzyme labelled antibody is IV-McAB1, and the sessile antibody is IV- McAB2；Or the enzyme labelled antibody is IV-McAB2, the sessile antibody is IV-McAB1.

As one embodiment of the present invention, the enzyme labelled antibody is CPV-10B11, and the sessile antibody is CPV- 10H4；Or the enzyme labelled antibody is CPV-10H4, the sessile antibody is CPV-10B11.

As one embodiment of the present invention, the enzyme labelled antibody is CDV-1G5, and the sessile antibody is CDV- 6E11；Or the enzyme labelled antibody is CDV-6E11, the sessile antibody is CDV-1G5.

As one embodiment of the present invention, the enzyme labelled antibody is CAV-5G4, and the sessile antibody is CAV-1A1； Or enzyme labelled antibody is CAV-1A1, the sessile antibody is CAV-5G4.

As one embodiment of the present invention, in enzyme immunochromatographytest test kit of the present invention, the sample Treatment fluid includes pH7.4 0.1M PBS solution, CHAPS, saponin(e and preservative；Preferably, the sample treatment liquid includes PH7.4 0.1M PBS solution, CHAPS, saponin(e and Proclin300；It is highly preferred that the sample treatment liquid includes pH7.4 0.1M PBS solution, 0.5%~2.0%W/V CHAPS, 0.5%~2.0%W/V saponin(e and 0.02%V/V Proclin300； Most preferably, the sample treatment liquid include pH7.4 0.1M PBS solution, 0.5%W/V CHAPS, 1%W/V saponin(e, 0.02%V/V Proclin300.

By the present invention in that the detection to a variety of detection target samples is realized with the sample treatment liquid of specific composition, gram The defect of one or more of detection target samples can only be detected by having taken a kind of enzyme immunochromatographytest test kit in the prior art.

As one embodiment of the present invention, in enzyme immunochromatographytest test kit of the present invention, solid sample The meltage for being dissolved in the sample treatment liquid is 0.08~0.5g/500-1500 μ l, and fluid sample is dissolved at the sample The meltage for managing liquid is 500~1500 μ l/500-1500 μ l；Through sample treatment liquid dissolution sample detection dosage be 50~ 150μl。

As one embodiment of the present invention, in enzyme immunochromatographytest test kit of the present invention, the sample Selected from tissue, serum, anus secretion, excrement, pharynx nasal discharge, eye nasal discharge and viral cultures.

Enzyme immunochromatographytest test kit of the invention can detect various samples.

Another object of the present invention is to provide a kind of preparation method of mentioned reagent box, comprising:

Step (1) prepares buffer；

Step (2) is diluted substrate using substrate pad dilution, and is adsorbed in adsorbate up to substrate pad, after dry It is placed in test strip；

Step (3) labeled monoclonal antibody is enzyme labelled antibody, is diluted with enzyme labelled antibody dilution, and be adsorbed in absorption Object is placed in test strip up to enzyme mark pad, drying；

The cured antibody diluent of step (4) sessile antibody is diluted and is fixed in test strip as detection line, sheep Anti- mouse is mostly anti-or sheep anti mouse monoclonal antibody is fixed on nature controlling line, dry；

Step (5) prepares sample treatment liquid；And

Step (6) supplies the enzyme immunochromatographydetecting detecting test strip, the sample treatment liquid, the buffer and buffer Unit is answered to be assembled into kit.

Kit prepared by the present invention solves 2 strain antibodies in the existing product using the preparation of double-antibody sandwich principle The technical problem of location swap effectively reduces the risk that nonspecific reaction occurs, improves detection sensitivity.

Another object of the present invention be to provide based on non-diagnostic mesh come using mentioned reagent box come viral in test sample Method, the detection method include:

Step (1) pre-processes sample to be tested using the sample treatment liquid；

Step (2) will be added in the test strip through the pretreated sample of step (1)；

Step (3) supplies the buffer to the test strip by the buffer supply unit, and stands anti- It answers 30 minutes；And

The test strip is observed after step (4) reaction, if nature controlling line is without band, no matter whether detection line has band It is invalid that this detection is all determined as；If nature controlling line has band, it is the positive that detection line, which has band, and detection line is yin without band Property.

Another object of the present invention is to provide a kind of sample treatment liquid, and the sample treatment liquid includes pH7.4 0.1M PBS Solution, CHAPS, saponin(e and preservative；Preferably, the sample treatment liquid includes pH7.4 0.1M PBS solution, CHAPS, soap Glycosides and Proclin300；It is highly preferred that the sample treatment liquid includes pH7.4 0.1M PBS solution, 0.5%~2.0%W/V CHAPS, 0.5%~2.0%W/V saponin(e and 0.02%V/V Proclin300；Most preferably, the sample treatment liquid includes PH7.4 0.1M PBS solution, 0.5%W/V CHAPS, 1%W/V saponin(e, 0.02%V/V Proclin300.

Sample treatment liquid of the invention can be used cooperatively from different commercial kits.When sample treatment liquid of the invention When being combined with commercial kit, the detection sensitivity of the more former commercial kit of detection sensitivity is more preferable；And it can be right Various samples are detected, and one or more of inspections can only be detected by overcoming a kind of enzyme immunochromatographytest test kit in the prior art Survey the defect of target sample.

As one embodiment of the present invention, sample treatment liquid of the invention sample detected be selected from tissue, serum, Anus secretion, excrement, pharynx nasal discharge, eye nasal discharge and viral cultures.

Specific embodiment

Hereinafter, some illustrative embodiments of the invention are described in detail so that the present invention is easier to by this field skill Art personnel are understood.

For the sake of simple and clear, some embodiments partially enumerated in " summary of the invention " of the invention are " specific at this Embodiment " partially repeats no more.

" enzyme chromatography test strip ", " enzyme immunochromatographydetecting detecting test strip " or " test strip " can in the present invention It is used interchangeably.

" enzyme labelled antibody " can be used interchangeably with " labelled antibody " in the present invention, be marked with enzyme based on monoclonal antibody. Term " enzyme " includes but is not limited to horseradish peroxidase, alkaline phosphatase, beta galactosidase, glucose oxidase, calf Intestinal alkaline phosphatase.Wherein, substrate used in horseradish peroxidase HRP is o-phenylenediamine (OPD), tetramethyl benzidine (TMB), aminosalicylic acid, adjacent biphenyl methylamine or 2,2'- connect amido -2 (3- ethyl-and thiazole sulfonate moiety -6) ammonium salt, preferably Tetramethyl benzidine (TMB)；Substrate used in alkaline phosphatase is p- nitrophenyl phosphate (p-NPP) or naphthols-AS-Mx Phosphate+diazonium salt；Substrate used in glucose oxidase is that ABTS+HRP+ glucose or glucose+methylthio phenol hold in the mouth or the eyes+thiazole It is blue；Substrate used in beta galactosidase is 4-methyl umbelliferone-β-D galactoside (4MUG) or nitrophenols galactoside (ONPG)。

" sessile antibody " also known as captures antibody in the present invention, is fixed on test strip detection line (abbreviation T line), is used for The compound that the antigen and labelled antibody for capturing chromatography are formed.

Term " phosphate buffer " refers to containing phosphoric acid or its salt and the solution for being brought to desired pH, is biochemistry The most widely used a kind of buffer in research.Generally, phosphate buffer is (including but unlimited from phosphoric acid or phosphate In sodium and sylvite) preparation.Some phosphate, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, phosphoric acid hydrogen has been known in the art Disodium and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate.Known phosphate be in the form of the hydrate of salt existing for.Due to slow The second level dissociation of fliud flushing, the pH value range of buffering is very wide, for example, about pH4 to the range of about pH10, preferably from about pH5 to pH9 Range, more have choosing about pH7 to the range of about pH8, most preferably from about pH7.4.It is further preferred that the phosphate buffer is The phosphate buffer of sodium chloride-containing and potassium chloride." phosphate buffer " is used interchangeably with PBS in the present invention.

Term " high molecular weight protein " includes but is not limited to bovine serum albumin(BSA) BSA, oralbumin OVA, fetal calf serum.

Term " preservative " include but is not limited to gentamicin, kanamycin sulfate, Sodium azide, thimerosal and its esters, Proclin300。

Heretofore described " adsorbate " is by that can discharge adsorbed target with adsorbed target object and in buffer The material of object is constituted.Adsorbate does not change the chemical property for the object to be adsorbed.For example, in some embodiments of the present invention Middle adsorbate can be with immunoabsorbent substrate pads dilution and substrate pad is made, and substrate pad then can discharge bottom under the action of buffer Object.The example of adsorbate includes but is not limited to glass fibre membrane, nitrocellulose filter and polyester film.

Term " glass fibre membrane " abbreviation glass fibre, including glass fibre element film, glass fiber filter and glass fibre Filter paper.

Term " saponin(e " is also known as saponin, alkali soap body, saponin, saponin(e, saponin or saponarin, be aglycon be triterpene or A kind of glucosides of spirostane class compound, mode classification is a variety of, can be divided into monodesmosidic saponin, bisdesmoside, three sugar chain soaps Glycosides, and acid saponin(e, neutral saponin(e can be divided into, it can also be divided into steroid saponin, triterpenoid saponin.The saponin(e include steroid saponin, Triterpenoid saponin, preferably digitonin.Term " digitonin " be also known as digitonin, digitonin, water caltrop take off soap Glycosides, digitophyllin.

Term " CHAPS " is a kind of detergent or detergent, also known as 3- [(3- cholesterol aminopropyl) dimethylamino] -1- Propane sulfonic acid, 3- ((3- bile aminopropyl) dimethyl amine) -1- propane sulfonic acid, in 3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid Salt, 3- [(3- cholesterol aminopropyl) dimethylamino] -1- propane sulfonic acid (CHAPS) and 3- [3- (gallbladder amido propyl) diformazan ammonia Base] propane sulfonic acid inner salt.

Term " nature controlling line " is also known as control line.

The present invention provides a kind of enzyme immunochromatographytest test kit, the kit includes enzyme immunity chromatography detection test paper Item, sample treatment liquid, buffer and buffer supply unit, which is characterized in that

The buffer includes phosphate buffer, high molecular weight protein, Tween-20 and preservative, and hydrogen peroxide Any one of urea, hydrogen peroxide urea, hydrogen peroxide；

The enzyme immunochromatographydetecting detecting test strip includes substrate pad, is adsorbed with after substrate pad diluted thereon dry Made of zymolyte, the substrate pad dilution includes dehydrated alcohol, pH7.4 0.1M PBS solution and 0.5%~4%W/V PEG6000 or PEG4000；

The enzyme immunochromatographydetecting detecting test strip includes enzyme mark pad, is adsorbed with does after enzyme labelled antibody diluted thereon It is dry to form enzyme labelled antibody, the enzyme labelled antibody dilution include pH7.40.1M PBS solution and 0.5%V/V PEG4000, Any one of PEG6000 or PEG8000；

As an embodiment of kit of the present invention, the kit includes test strip, sample treatment liquid, delays Fliud flushing and buffer supply unit, wherein

The test strip successively includes substrate supply area, sample supply area, detection zone from longitudinal direction；The substrate supplies Answering area includes the substrate pad, is adsorbed with dry zymolyte thereon；The sample supply area includes the enzyme mark pad, is inhaled thereon With enzyme labelled antibody, the zymolyte can generate chromogenic reaction with the enzyme on the enzyme labelled antibody；The detection zone includes described Detection line and the nature controlling line, immobilization has sheep anti mouse mostly anti-or sheep anti mouse secondary antibody on the nature controlling line；

The buffer supply unit is used to supply the buffer to the substrate supply area of the test strip, and So that buffer and with buffer diffusion sample treatment liquid, zymolyte and enzyme labelled antibody along the longitudinal direction of test strip Xiang Yuan One end migration from substrate supply area.

Can be realized the buffer supply unit of above-mentioned function is known in the prior art.Such as the buffering Liquid supply unit can be the structure as described in Chinese patent CN104062430A, and buffer is stored in substrate buffer slot, Buffer is supplied to detection and in the expansion fluid cushion immersion substrate buffer slot for connecting the substrate pad with test strip The substrate supply area of test strips, and by the way that water absorption pad is arranged after the detection zone of test strip, utilize the capillary of water absorption pad Guan Li migrates buffer along the longitudinal direction of test strip to detection zone.Alternatively, can be put by will test test strips Be placed in substrate supply area it is higher and on the lower bearing slope of detection zone, using the gravity of buffer itself make supplied to substrate supply The buffer in area is answered to migrate to detection zone.

The present invention is with substrate tetramethyl used in horseradish peroxidase HRP labelled antibody and horseradish peroxidase HRP It will be described in detail in example 2 for base benzidine (TMB).Further, horseradish peroxidase HRP institute of the present invention The hydrogen acceptor needed is with hydrogen peroxide H₂O₂Solid or liquid as main component, including but not limited to carbamide peroxide, peroxide Change urea hydrogen, hydrogen peroxide etc..

Porcine monoclonal antibody according to the present invention specifically includes that

(1) porcine circovirus 2 type monoclonal antibody 3G12 (abbreviation PCV2-3G12) (is protected by mouse hybridoma cell 3G12 Hiding number is CCTCC No:C2014198) secretion acquisition, porcine circovirus 2 type monoclonal antibody 2F8 (abbreviation PCV2-2F8) is by small Mouse hybridoma 2F8 (deposit number is CCTCC No:C2014199) secretion obtains.2 plants of mouse hybridoma cells are preserved in China typical culture collection center, preservation address are Wuhan, China Wuhan University, and the deposit date is on November 3rd, 2014. Referring to Chinese patent CN105717293A.

(2) Porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB2, Porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB1, wherein the monoclonal antibody PEDV-McAB2 heavy chain variable region be SEQ ID No.1 shown in, light chain variable Area be SEQ ID No.2 shown in, the monoclonal antibody PEDV-McAB1 heavy chain variable region be SEQ ID No.3 shown in, light chain Variable region is shown in SEQ ID No.4, referring to Chinese patent CN105461805A.

(3) monoclonal antibody of transmissible gastro-enteritis virus TGEV-3D2, the amino acid sequence of heavy chain variable region are SEQ ID No.5, light chain variable region amino acid sequence be SEQ ID No.6；Monoclonal antibody of transmissible gastro-enteritis virus TGEV- 4B4, the amino acid sequence of heavy chain variable region is SEQ ID No.7, the amino acid sequence of light chain variable region is SEQ ID No.8。

Birds monoclonal antibody according to the present invention specifically includes that

(4) anti-influenza type A virus nucleoprotein monoclonal antibody IV-McAB1, anti-influenza type A virus nucleoprotein monoclonal are anti- Body IV-McAB2, wherein the monoclonal antibody IV-McAB1 heavy chain variable region be SEQ ID No.9 shown in, light chain variable region For shown in SEQ ID No.10, the monoclonal antibody IV-McAB2 heavy chain variable region is shown in SEQ ID No.11, light chain can Become area into shown in SEQ ID No.12, referring to Chinese patent CN104062430A.

Canine monoclonal antibody according to the present invention specifically includes that

(5) canine parvovirus monoclonal antibody 10B11 (abbreviation CPV-10B11) is by mouse bone marrow cells hybridoma 10B11 Strain (deposit number be CCTCC No:C201578) secretion obtains, canine parvovirus monoclonal antibody 10H4 (abbreviation CPV-10H4) by 10H4 plants of mouse bone marrow cells hybridoma (deposit number is CCTCC No:C201579) secretion obtains.2 plants of mouse hybridoma cells It is preserved in China typical culture collection center, preservation address is Wuhan, China Wuhan University, and the preservation time is 2015 May 20 days.Referring to Chinese patent CN104928258A.

(6) canine distemper virus monoclonal antibody 1G5 (abbreviation CDV-1G5) is by mouse bone marrow cells hybridoma 1G5 (deposit number It secretes and obtains for CCTCC No:C2015201), canine distemper virus monoclonal antibody 6E11 (abbreviation CDV-6E11) is by mouse bone marrow cells 6E11 plants of hybridoma (deposit number is CCTCC No:C2015202) secretion obtains.2 strain of hybridoma are preserved in China Type Tissue Collection, preservation address are Wuhan, China Wuhan University, and the preservation time is on November 25th, 2015.Referring to Chinese patent CN105695420A.

(7) hepatitis infectiosa canis virus monoclonal antibody CAV-5G4, heavy chain variable amino acid sequence are shown in SEQ.ID No.13 Amino acid sequence, chain variable region amino acid sequence be SEQ.ID No.14 shown in amino acid sequence；With hepatitis infectiosa canis virus list Clonal antibody CAV-1A1, heavy chain variable amino acid sequence are amino acid sequence shown in SEQ.ID No.15, and light chain can Change region amino acid sequence is amino acid sequence shown in SEQ.ID No.16.

The present invention also provides a kind of buffers for enzyme immunochromatographytest test kit, and it includes phosphate-buffereds Liquid, high molecular weight protein, Tween-20 and preservative.

The present invention also provides a kind of sample treatment liquids for enzyme immunochromatographytest test kit, and it includes pH7.4 0.1M PBS solution, CHAPS, saponin(e and preservative.

Sample treatment liquid prepared by the present invention, can be used for virus liquid, excrement, serum, microorganism swab (nose swab containing eye, Nasopharyngeal swabs, nephrodinic swab), the dissolution of tissue sample after grinding, exempt to chromatograph with the enzyme for the sandwich principle building of double monoclonal antibodies Sample after detecting the accurate detection processing of class test strips.

The present invention also provides the substrate pad dilutions that a kind of use is prepared in enzyme immunochromatographytest test kit, comprising anhydrous Ethyl alcohol, PEG4000 and pH7.4 0.1M PBS solution, or include dehydrated alcohol, PEG6000 and pH7.4 0.1M PBS solution.

The present invention also provides a kind of enzyme labelled antibody dilutions for being used to prepare enzyme immunochromatographytest test kit, include PEG4000, pH7.4 0.1M PBS solution, or comprising PEG6000, pH7.4 0.1M PBS solution, or comprising PEG8000, PH7.4 0.1M PBS solution.

The present invention, which has been gone back, provides a kind of curing antibody dilution for being used to prepare enzyme immunochromatographytest test kit, includes PH7.4 0.1M PBS solution, trehalose and Tween-20.

The present invention also provides the preparation methods of the kit, comprising:

Step (1) prepares buffer；

Step (5) prepares sample treatment liquid；And

A kind of embodiment of preparation method as kit of the invention, the preparation method include:

Step (1) prepares buffer；

Step (2) is diluted substrate using substrate pad dilution, and is adsorbed in adsorbate up to substrate pad, after dry It is placed in the substrate supply area of test strip；

Step (3) labeled monoclonal antibody is enzyme labelled antibody, is diluted with enzyme labelled antibody dilution, and be adsorbed in absorption Object is placed in the sample supply area of test strip up to enzyme mark pad, drying；

Step (4) sessile antibody is through sessile antibody diluted and sheep anti mouse is mostly anti-or sheep anti mouse monoclonal antibody is through phosphoric acid Salt buffer or curing antibody diluted, are fixed in the detection zone of test strip respectively as detection line, nature controlling line, It is dry；

Step (5) prepares sample treatment liquid；And

Step (6) assembles kit, so that the test strip successively includes substrate supply area, sample confession from longitudinal direction Area, detection zone are answered, and buffer supply unit is supplied the buffer to the substrate confession of the test strip Area is answered, migrates buffer to one end far from substrate supply area along the longitudinal direction of test strip.

Step (1) prepares buffer；

Substrate is diluted to 0.5%~4%W/V using substrate pad dilution by step (2), and is adsorbed in adsorbate i.e. Substrate pad is obtained, drying is placed in the substrate supply area of test strip；

Step (3) labeled monoclonal antibody is enzyme labelled antibody, is diluted to 0.5~10 μ g/ with enzyme labelled antibody dilution Ml, and adsorbate is adsorbed in up to enzyme mark pad, drying is placed in the sample supply area of test strip；

Step (4) sessile antibody is through sessile antibody diluted to 0.5~1mg/ml and sheep anti mouse resists more or goat-anti Mouse monoclonal antibody is and solid respectively as detection line, nature controlling line through phosphate buffer or curing antibody diluted to 1~3mg/ml Due in the detection zone of test strip, dry；

Step (5) prepares sample treatment liquid；And

A kind of embodiment of preparation method as kit of the invention, in the preparation method, the detection Spacing >=5mm of line and the nature controlling line.

A kind of embodiment of preparation method as kit of the invention, in the preparation method, the buffer Comprising phosphate buffer, high molecular weight protein, Tween-20 and preservative, the preferably described phosphate buffer is pH7.4 0.1M PBS solution, the preferably described high molecular weight protein are one of BSA, OVA, fetal calf serum or a variety of, preferably described Preservative be any one of gentamicin, kanamycin sulfate, Sodium azide, thimerosal and its esters, Proclin300, it is more excellent Selection of land also includes any one of carbamide peroxide, hydrogen peroxide urea, hydrogen peroxide, further preferably includes pH7.4 0.1M PBS solution, 0.5%~1%W/V BSA, 0.1%~0.5%V/V Tween-20,0.02%V/V Proclin300 and peroxide Change hydrogen urea, most preferably comprises carbamide peroxide, 0.1M PBS of pH7.4,1%W/VBSA, 0.1%V/VTween-20,0.02% V/VProclin300；The sample treatment liquid includes pH7.40.1M PBS solution, CHAPS, saponin(e and preservative, is preferably comprised PH7.4 0.1M PBS solution, CHAPS, saponin(e and Proclin300 more preferably include pH7.4 0.1M PBS solution, 0.5% ~2.0%W/V CHAPS, 0.5%~2.0%W/V saponin(e and 0.02%V/V Proclin300, most preferably comprise pH7.4 0.1M PBS solution, 0.5%W/V CHAPS, 1%W/V saponin(e and 0.02%V/V Proclin300；The substrate pad dilution Comprising dehydrated alcohol, PEG4000 and pH7.4 0.1M PBS solution, or include dehydrated alcohol, PEG6000 and pH7.4 0.1M PBS solution preferably comprises 2%V/V dehydrated alcohol, 0.1%V/V PEG4000 and pH7.4 0.1M PBS solution, or includes 2% V/V dehydrated alcohol, 0.1%V/V PEG6000 and pH7.4 0.1M PBS solution；The enzyme labelled antibody dilution includes PEG4000, pH7.4 0.1M PBS solution, or comprising PEG6000, pH7.4 0.1M PBS solution, or comprising PEG8000, PH7.4 0.1M PBS solution preferably includes 0.5%V/V PEG4000, pH7.4 0.1M PBS solution, or includes 0.5% V/V PEG6000, pH7.4 0.1M PBS solution, or include 0.5%V/V PEG8000, pH7.4 0.1M PBS solution；And The curing antibody dilution includes pH7.4 0.1M PBS solution, trehalose and Tween-20, preferably comprises pH7.4 0.1M PBS solution, 0.5%V/V trehalose, 0.1%V/V Tween-20.

A kind of embodiment of preparation method as kit of the invention, buffer described in the step (1) are PH7.4 0.1M PBS solution, 0.5%~1%W/V BSA, 0.1%~0.5%V/V Tween-20,0.02%V/V Proclin300；

Substrate pad dilution described in the step (2) is 2%V/V dehydrated alcohol, 0.1%V/V PEG4000, pH7.4 0.1M PBS solution or 2%V/V dehydrated alcohol, 0.1%V/V PEG6000, pH7.4 0.1M PBS solution；

Enzyme labelled antibody dilution described in the step (3) be 0.5%V/V PEG4000, pH7.40.1M PBS solution, Or 0.5%V/V PEG6000, pH7.4 0.1M PBS solution or 0.5%V/V PEG8000, pH7.4 0.1M PBS solution；

Curing antibody dilution described in the step (4) be pH7.4 0.1M PBS solution, 0.5%V/V trehalose, 0.1%V/V Tween-20；

Sample treatment liquid described in the step (5) is pH7.4 0.1M PBS solution, 0.5%W/V CHAPS, 1%W/V Saponin(e, 0.02%V/V Proclin300.

As one embodiment of detection method, the detection method includes:

Step (1) pre-processes sample to be tested using the sample treatment liquid, is dissolved in solid sample at the sample The meltage for managing liquid is 0.08~0.5g/500-1500 μ l, and the meltage that fluid sample is dissolved in the sample treatment liquid is 500 ~1500 μ l/500-1500 μ l；

Step (2) supplies the sample that the test strip is added through step (1) pretreated sample of 50~150 μ l Area；

The detection zone of the test strip is observed after step (4) reaction, if nature controlling line is without band, no matter detection line is It is no to there is this detection of band to be all determined as in vain；If nature controlling line has band, it is the positive that detection line, which has band, and detection line is without item Band is feminine gender.

It is qualitative fixed that the animal epidemic antigen detection of the non-diagnostic purpose is carried out including epidemiological analysis, in vitro tissue Amount detection, Epitope Identification research.

However, kit of the invention, which is not precluded, in this can be used in the viral diagnosis activity of other purposes.

Pass through body detoxification approach after the toxin expelling approach of institute's sample to be tested is infected primarily directed to target antigen in the present invention It is metabolized, as pig circular ring virus mainly passes through tissue, blood toxin expelling, Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine Virus is mainly by anus swab, excrement toxin expelling, and A type influenza mainly passes through pharynx nose swab toxin expelling, and canine parvovirus is mainly anus Swab, excrement toxin expelling, canine distemper virus mainly pass through a nose swab, anus swab, excrement toxin expelling, and hepatitis infectiosa canis virus mainly passes through The toxin expellings such as eye nose swab, anus swab, excrement, blood.

As a kind of embodiment of detection method of the invention, the sample to be tested includes but is not limited to pig, fowl, dog institute The epidemic disease antigen of generation.That is, the application of kit of the present invention includes but is not limited to pig, fowl, epidemic disease antigen caused by dog.

As a kind of embodiment of detection method of the invention, the sample to be tested original includes porcine circovirus 2 type, pig Epidemic diarrhea virus, transmissible gastro-enteritis virus, influenza A, canine parvovirus, canine distemper virus, hepatitis infectiosa canis virus.

Buffer and sample treatment liquid of the invention can make when being applied in combination with double antibody (especially double monoclonal antibodies) sandwich original The 2 plants of monoclonal antibody positions realization exchange for managing fixation used in the existing product of preparation, label, completes different collocation moulds The product of the preparation of 2 plants of monoclonal antibodies of formula does not cause non-specific responding after test sample and properly increases sensitivity.

The present invention is further described below with reference to certain specific embodiments, the advantages and features of the present invention will be with retouching It states apparent.Examples are merely exemplary for these, not implements or use the unique forms of the specific embodiment of the invention, because It is not intended to limit the scope of the present invention in any way for this.Multiple specific embodiments are covered in embodiments described below Feature and for construction and operate these specific embodiments method and step and its sequence.Those skilled in the art should manage Solution, without departing from the spirit and scope of the invention can details to technical solution of the present invention and form modify or Replacement, but these modifications and replacement are fallen within the protection scope of the present invention.

It is as follows to prepare example for used PBS buffer solution in the embodiment of the present invention: sodium chloride 8g, potassium chloride 0.2g, phosphoric acid hydrogen Disodium 1.44g, potassium dihydrogen phosphate 0.24g adjust pH7.4, constant volume 1L, including but not limited to this formula；Chemical reagent is analysis It is pure, it is purchased from Chinese medicines group.

Experimental method of the present invention, if being conventional method without specified otherwise；The biomaterial, if without spy Different explanation, commercially obtains.

Monoclonal antibody situation analysis used in 1 existing product of embodiment

Based on double-antibody sandwich principle, the technology of the present invention team and other R&D teams have developed a series of products, briefly It is as follows:

Porcine circovirus 2 type monoclonal antibody 2F8 (abbreviation PCV2-2F8), 3G12 in Chinese patent CN105717293A When (abbreviation PCV2-3G12) matches antibody activity detection, it is PCV2-2F8, labelled antibody PCV2- that discovery, which only has coated antibody, The enzyme immunochromatographydetecting detecting test strip prepared when 3G12 can use, if being PCV2- by 2 strain antibody transpositions, that is, coated antibody There are non-specific respondings for maximum probability when 3G12, labelled antibody PCV2-2F8.

Porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB1, PEDV- in Chinese patent CN105461805A McAB2, the colloid prepared when it is PEDV-McAB1 that discovery, which only has sessile antibody, when matched pair study, labelled antibody is PEDV-McAB2 Golden test strip or enzyme immunochromatographydetecting detecting test strip can just be applied, and detect if 2 transpositions are prepared into product, As a result maximum probability is false positive.

Influenza A monoclonal antibody 1 (abbreviation IV-McAB1), 2 (abbreviation IV- in Chinese patent CN104062430A McAB2), the enzyme layer analysis prepared when it is IV-McAB2 that discovery, which only has sessile antibody, when matched pair study, labelled antibody is IV-McAB1 Method test strip just can be used, and detect if 2 transpositions are prepared into product, and as a result maximum probability occurs non-specific Property reaction.

Canine parvovirus monoclonal antibody 10B11,10H4 in Chinese patent CN104928258A, discovery is only when matched pair study The enzyme chromatography test strip prepared when having sessile antibody is 10B11, labelled antibody is 10H4 just can be used, if mutual by 2 Change place is prepared into product and is detected, and as a result false negative occurs in maximum probability.

Canine distemper virus monoclonal antibody 6E11,1G5 in Chinese patent CN105695420A, discovery only has when matched pair study The enzyme immunochromatographydetecting detecting test strip or colloidal gold colloidal gold detection test paper strip prepared when sessile antibody is 6E11, labelled antibody is 1G5 is It can be used, detected if 2 transpositions are prepared into product, as a result false negative occurs in maximum probability.

It is detected in Chinese patent CN101149377A and only has sessile antibody anti-for 9D47A1, label in 2 strain antibodies of aspergillus When body is 7E11A1 the product for preparing can generate strongest signal, avoid it is non-specific.

Based on this, the present inventor has carried out a series of researchs to solve to be unable to transposition with 2 strain antibody of clock synchronization, or exchanges Sensitivity decrease is caused behind position, is generated non-specific responding, is caused the technical problems such as false positive false negative.

When in view of test strips clinical application required detection target mainly include a nose swab, anus swab, excrement, blood with And treated tissue etc., wherein impurities are more in excrement, thus the present invention first just detect excrement dog canine parvovirus It is explained in detail for enzyme immunochromatographydetecting detecting test strip.

The preparation and evaluation of 2 canine parvovirus toxenzyme immunochromatographytest test kit of embodiment

Preferably to evaluate canine parvovirus toxenzyme immunochromatographytest test kit, the present invention is first 10H4 with sessile antibody, Labelled antibody carries out test strips for 10B11 and prepares (2.1 part of corresponding embodiment), and by AV298 plants of canine parvovirus CVCC (purchased from Chinese veterinary microorganism culture presevation administrative center), S2 plants of canine parvovirus epidemic strain CPV 2a type and S0425 plants, dog S0304 plants of parvovirus epidemic strain CPV 2b type is (referring to Preparation and application of two monoclonal antibodies against canine parvovirus vaccine and field strains, Journal of Vaccines and Immunology, 2017,3 (1): 001-004) and 20 parts of positive excrement, 20 parts of yin Property excrement (being respectively positive, negative through canine parvovirus PCR qualification result) be used as sample disc, test strips are divided comprehensively Analysis.

The preparation of 2.1 buffers

Each constituent content of buffer is shown in Table 1, wherein meaning surplus by the component (i.e. if not indicating concrete content PBS or citric acid -0.2M disodium hydrogen phosphate) composition.Sampling carries out appearance detection, steriling test (it is attached to press existing Chinese veterinary pharmacopoeia Record carries out) and storage life (2~30 DEG C of preservations) research, it the results are shown in Table 2.

Component contained by the different buffers of table 1 summarizes

1~A16 of buffer solution A, substrate pad (preparing by embodiment 2.2.a, substrate pad dilution is B3), enzyme mark pad (are pressed Embodiment 2.2.b preparation, enzyme labelled antibody dilution is C3), detection zone (is prepared, curing antibody dilution is by embodiment 2.2.c D4), be sequentially prepared by embodiment 2.4 it is corresponding at kit a1~a16, and by embodiment 2.5 by sample disc sample embodiment The sample treatment liquid F4 dilution of 2.3 preparations is detected, and as a result (being shown in Table 2) shows: buffer appearance is clarified, steriling test is equal Qualification, storage life except A1 be 12 months it is outer remaining be 15 months, when buffer for the test strips that are prepared when A6 in the detection not The sensitivity for false positive results and detection CPV strain especially epidemic strain occur is higher.

Testing result summarizes after the different buffer testing results of table 2 and reagent preparation box

Note: positive excrement, negative excrement result indicate that test strips testing result/PCR detects positive or negative total sample number.

To evaluate whether the Contents of Main Components of buffer solution A 6 influences testing result, spy's setting range (be shown in Table 1 number A6~ A13 it) is further debugged, as a result (being shown in Table 2) shows: when buffer is that (corresponding buffer solution is 0.5% to A6~A9 ~1%W/VBSA, 0.1%~0.5%V/VTween-20) when prepare test strips detection sensitivity height (10^3.2~ 10^3.5TCID₅₀/ ml), detection clinical sample is consistent with PCR testing result；The test strips prepared when buffer is A10~A13 Detection sensitivity decline (> 10^3.5TCID₅₀/ ml), it is higher when detecting clinical sample non-specific responding probability occur.

For the final concentration of carbamide peroxide in evaluation buffer, spy successively adjusts the concentration of carbamide peroxide in buffer solution A 6 It is whole be 0.01%, 0.02%, 0.2%, 0.3%W/V to prepare test strips, as a result: when being 0.01%W/V by carbamide peroxide There are negative findings for having maximum probability when detecting positive excrement in the test strips of preparation, when carbamide peroxide is 0.3%W/V The test strips of preparation have maximum probability when detecting negative excrement and positive findings occur, that is, there are problems that false positive false negative；And The detection sensitivity and A6 result of the test strips prepared when carbamide peroxide is 0.02%, 0.2%W/V are suitable, and detection is positive Excrement, negative excrement result are correct, so the final concentration of carbamide peroxide in buffer is set as 0.02%~0.2%W/ V。

It substitutes BSA to evaluate other high molecular weight proteins such as OVA and is debugged and (be shown in Table A14 in 1), testing result (is shown in Table 2) Consistent with the buffer assembling testing result of test strips containing BSA, show: other macromoleculars also can be used as buffer composition and can Reach the preferable effect of detection.

In addition, being debugged and (being shown in Table A15~A16 in 1) hydrogen peroxide urea, hydrogen peroxide substitution carbamide peroxide, detect As a result it is consistent with the buffer assembling testing result of test strips containing carbamide peroxide (to be shown in Table 2), shows: hydrogen peroxide urea, Hydrogen peroxide also can be used as buffer composition and can reach the preferable effect of detection.

The preparation of 2.2 test strips

2.2.a the preparation of the substrate pad of substrate zone

Substrate tetramethyl benzidine (TMB) substrate pad dilution (being shown in Table 3) is diluted, keeps TMB final concentration of 0.5%~4%W/V is adsorbed in the glass fibre membrane as adsorbate, uses after dry.

3 substrate pad dilution component of table

Number	Substrate pad dilution component
		B1	2%V/V dehydrated alcohol
B2	The 0.1M PBS of 2%V/V dehydrated alcohol, 0.1%V/VPEG2000, pH7.4
		B3	The 0.1M PBS of 2%V/V dehydrated alcohol, 0.1%V/VPEG4000, pH7.4
B4	The 0.1M PBS of 2%V/V dehydrated alcohol, 0.1%V/VPEG6000, pH7.4
		B5	The 0.1M PBS of 2%V/V dehydrated alcohol, 0.1%V/VPEG8000, pH7.4

By buffer solution A 6 (preparing by embodiment 2.1), (final concentration of 1%) of TMB, enzyme mark pad (press embodiment to substrate pad 2.2.b it prepares, enzyme labelled antibody dilution (is pressed for C3), detection zone

Embodiment 2.2.c preparation, curing antibody dilution are D4), it is sequentially prepared kit b1~b5 by embodiment 2.4, And detect the sample treatment liquid F4 dilution that sample disc sample is prepared with embodiment 2.3 by embodiment 2.5, as a result (it is shown in Table 4) it shows: to the sensitive of CPV strain especially epidemic strain when the test strips detection prepared when substrate pad dilution is B3 or B4 It spends higher by (10^3.2~10^3.5TCID₅₀/ ml), detection clinical sample is consistent with PCR testing result；When substrate pad dilution be B1, The detection sensitivity of the test strips prepared when B2, B5 declines (> 10^3.5TCID₅₀/ ml), detection clinical sample occurs non-specific anti- It answers.

Testing result summarizes after the different substrate pad dilution reagent preparation boxes of table 4

TMB is diluted to final concentration of by the final concentration of TMB when adsorbing for evaluation substrate pad, spy with substrate pad dilution 0.4%, 0.5%, 4%, 5%W/V is to prepare test strips, and as a result: the test strips prepared when being 0.4%W/V by TMB are used to examine There is negative, the result when test strips prepared when TMB is 5%W/V detect negative excrement in result maximum probability when surveying positive excrement There is the positive in maximum probability, that is, there are problems that false positive false negative；And the test strips prepared when TMB is 0.5%, 4%W/V Detection sensitivity is suitable with B3 result, and it is correct to detect positive excrement, negative excrement result, so the final concentration of TMB is set For 0.5%~4%W/V.

2.2.b in sample supply area enzyme mark pad preparation

2.2.b.1 the preparation of enzyme labelled antibody

The label of horseradish peroxidase HRP is carried out using improvement Over-voltage protection.Weigh 20mg horseradish peroxidase HRP is dissolved in 1ml ultrapure water, and the NaIO of 1ml Fresh is added₄Solution (30mg NaIO₄It is dissolved in 1ml ultrapure water), it mixes, 2 ~8 DEG C are protected from light effect 30 minutes；40 μ l ethylene glycol are added in the above solution, 2~8 DEG C are protected from light effect 30 minutes；It is passed through according to 1mg Octanoic acid-the monoclonal antibody of saturated ammonium sulphate method after purification adds the ratio of the 100 above-mentioned mixed liquors of μ l, after the two is mixed, It is added in bag filter, mixes, dialysed 6 hours with coating buffer (pH9.6,0.05mol/L carbonate buffer solution).Entire behaviour Work need to be carried out in the dark；Mixed liquor after dialysis is transferred in 1.5ml EP pipe, the NaBH of 10 μ l Fresh is added₄Solution (20mg NaBH₄It is dissolved in 1ml ultrapure water), room temperature acts on 2 hours, mixes every 30 minutes primary；Isometric saturation sulphur is added Sour ammonium, after mixing, 4 DEG C are acted on 15 minutes.12000r/min is centrifuged 10 minutes, abandons supernatant.Will precipitating with the bodies such as antibody purification The mixed liquor (V: V=1: 1) of long-pending PBS and glycerol blows outstanding.

2.2.b.2 the identification of enzyme labelled antibody

Ocular estimate: it is rufous liquid at room temperature, has no that floccule precipitates.

Quality evaluation: with UV spectrophotometer measuring enzyme labelled antibody in the light absorption value A of 403nm and 280nm, " label is pressed Rate=A_403nm/A_280nm" calculate mark rate；Monoclonal antibody amount after enzyme labelled antibody, i.e. IgG amount, by " IgG amount (mg/ml)= (A_280nm-A_403nm× 0.3) × 0.62 × extension rate " is calculated.It the results are shown in Table 5:

The quality evaluation result of 5 enzyme labelled antibody of table summarizes

Labelled antibody title	IgG amount (mg/ml)	Mark rate
			CPV-10B11	1.0	60%
CPV-10H4	1.5	58%

2.2.b.3 the preparation of enzyme mark pad

By the different enzyme labelled antibody dilution (being shown in Table 6) of enzyme labelled antibody be diluted to monoclonal antibody final concentration of 0.5~ It is adsorbed on glass fibre membrane after 10 μ g/ml, is used after dry.

6 enzyme labelled antibody dilution component of table summarizes

Number	Enzyme labelled antibody dilution component
		C1	The 0.1M PBS of pH7.4
C2	The 0.1M PBS of 0.5%V/VPEG2000, pH7.4
		C3	The 0.1M PBS of 0.5%V/VPEG4000, pH7.4
C4	The 0.1M PBS of 0.5%V/VPEG6000, pH7.4
		C5	The 0.1M PBS of 0.5%V/VPEG8000, pH7.4

Buffer solution A 6 (preparing by embodiment 2.1), substrate pad (are prepared, substrate pad dilution is by embodiment 2.2.a B3), enzyme mark pad (enzyme labelled antibody content is 2 μ g/ml), detection zone (are prepared, curing antibody dilution is by embodiment 2.2.c D4), it is sequentially prepared kit c1~c5 by embodiment 2.4, and prepares sample disc sample embodiment 2.3 by embodiment 2.5 Sample treatment liquid F4 dilution detected, as a result (being shown in Table 7) show: when enzyme labelled antibody dilution be C3, C4 or C5 when prepare Test strips it is higher to the detection sensitivity of CPV strain by (10^3.0~10^3.5TCID₅₀/ ml), detection clinical sample and PCR detection are tied Fruit is consistent；The detection sensitivity of the test strips prepared when substrate pad dilution is C1, C2 declines (> 10^4.0TCID₅₀/ ml), inspection It surveys clinical sample and non-specific responding occurs.

Testing result summarizes after the different enzyme labelled antibody dilution reagent preparation boxes of table 7

The use concentration of enzyme labelled antibody content, spy use enzyme labelled antibody dilution by enzyme labelled antibody when preparing for evaluation enzyme mark pad Being diluted to working concentration is 0.4,0.5,10,12 μ g/ml to prepare test strips, as a result: when enzyme labelled antibody working concentration is 0.4 μ The test strips prepared when g/ml decline 10~100 times to the sensitivity of detection CPV epidemic strain, when enzyme labelled antibody working concentration is 12 When μ g/ml, negative excrement result maximum probability is detected with the test strips of preparation and the positive occurs, that is, there are false positive results；And work as enzyme The detection sensitivity and C3 result for the test strips that labeling antibody working concentration is prepared when being 0.5,10 μ g/ml are suitable, detect positive excrement Just, negative excrement result is correct, so enzyme labelled antibody working concentration is set as 0.5~10 μ g/ml.

2.2.c the preparation of detection zone

Sessile antibody sessile antibody (being shown in Table 8) diluted is to 0.5~1mg/ml, and nature controlling line is with antibody pH7.4 0.1M PBS buffer solution or curing antibody dilution D4 are diluted to 1mg/ml, are adsorbed in one end of nitrocellulose filter respectively, and two Line spacing >=5mm uses after dry.

8 curing antibody dilution component of table summarizes

Buffer solution A 6 (preparing by embodiment 2.1), substrate pad (are prepared, substrate pad dilution is by embodiment 2.2.a B3), enzyme mark pad (preparing by embodiment 2.2.b, enzyme labelled antibody dilution is C3), nitrocellulose filter are (fixed anti-in fixing line Bulk concentration is 0.8mg/ml), it is sequentially prepared kit d1~d6 by embodiment 2.4, and sample disc sample is used by embodiment 2.5 Sample treatment liquid F4 dilution prepared by embodiment 2.3 is detected, and as a result (being shown in Table 9) shows: when preparing detection line, solidification is anti- The test strips that body dilution is prepared when being D4 are higher by (10 to the sensitivity of detection CPV strain^3.0~10^3.5TCID₅₀/ ml), detection Clinical sample is consistent with PCR testing result；The test strips prepared when curing antibody dilution is D1, D2, D3, D5 or D6 are being examined Sensitivity declines (> 10 when sample^4.0TCID₅₀/ ml), and there is non-specific responding in clinical sample.

Testing result summarizes after the different curing antibody dilution reagent preparation boxes of table 9

Sessile antibody, is diluted to by sessile antibody concentration when to evaluate nitrocellulose film preparation with curing antibody dilution Working concentration be 0.4,0.5,1.0,1.2mg/ml to prepare test strips, as a result: when sessile antibody working concentration be 0.4mg/ml When the test strips that prepare 3~100 times are declined to the sensitivity of detection CPV epidemic strain, when sessile antibody working concentration is 1.2mg/ The test strips prepared when ml result maximum probability when detecting negative excrement is the positive, that is, there are false positive results；And when fixed anti- The detection sensitivity and D4 result for the test strips that bulk concentration is prepared when being 0.5,1.0mg/ml are suitable, detect positive excrement, feminine gender Excrement result is correct, so sessile antibody working concentration is set as 0.5~1.0mg/ml.

The preparation of 2.3 sample treatment liquids

It prepares following sample treatment liquid (being shown in Table 10), sampling carries out appearance detection, steriling test (presses existing Chinese veterinary pharmacopoeia Annex carries out) and storage life research, it the results are shown in Table 11.

Component contained by 10 sample treatment liquid of table summarizes

Buffer solution A 6 (preparing by embodiment 2.1), substrate pad (are prepared, substrate pad dilution is by embodiment 2.2.a B3), enzyme mark pad (by embodiment 2.2.b prepare, enzyme labelled antibody dilution be C3), detection zone (by embodiment 2.2.c prepare, Gu Change antibody diluent is D4), it is sequentially prepared kit f1~f6 by embodiment 2.4, and sample disc sample is used by embodiment 2.5 Sample treatment liquid F1~F6 dilution prepared by embodiment 2.3 is detected, and as a result (being shown in Table 11) shows: when sample treatment liquid is F4 When, the sensitivity for detecting CPV strain is higher by (10^3.2~10^3.5TCID₅₀/ ml), detection clinical sample is consistent with PCR testing result； When sample treatment liquid is F1, F2, F3, F5, F6, the sensitivity of test sample declines (> 10^4.0TCID₅₀/ ml), and clinical sample There is non-specific responding in product.

Testing result summarizes after the different sample treatment liquid reagent preparation boxes of table 11

For the content of CHAPS in evaluation sample treatment liquid, other compositions are remained unchanged, CHAPS content is adjusted to 0.4%, 0.5%, 2.0%, 2.5%W/V with reagent preparation box and is detected, as a result: as CHAPS contained in sample treatment liquid The sensitivity of the kit detection CPV prepared when being 0.4% declines 10~100 times；When CHAPS contained in sample treatment liquid is It is the positive that the positive excrement of kit detection prepared when 2.5%, which is negative, negative excrement, and there are serious non-specific；And work as sample The kit detection sensitivity and F4 result prepared when CHAPS content is 0.5%, 2.0% in product treatment fluid is suitable, and detection is positive Excrement, negative excrement result are correct, so CHAPS content in sample treatment liquid is set as 0.5%~2.0%W/V.

For the content of saponin(e in evaluation sample treatment liquid, other compositions are remained unchanged, saponin content is adjusted to 0.4%, 0.5%, 2.0%, 3.0%W/V with reagent preparation box and is detected, as a result: when saponin(e contained in sample treatment liquid is 0.4% When the sensitivity of kit detection CPV for preparing decline 10 times and detect negative excrement as the positive；When contained in sample treatment liquid It is the positive that the positive excrement of kit detection prepared when CHAPS is 3.0%, which is negative, negative excrement, and there are serious non-specific； And the kit detection sensitivity and F4 result prepared when saponin content is 0.5%, 2.0% in sample treatment liquid is suitable, inspection It is correct to survey positive excrement, negative excrement result, so saponin content in sample treatment liquid is set as 0.5%~2.0%W/ V。

In addition, for evaluation debugging after buffer solution A 6, substrate pad dilution B3 or B4, enzyme labelled antibody dilution C3 or C4 or Whether C5, sessile antibody influence the testing result of test strips with each main component in solution of dilution D4, its transposition is carried out Evaluation.As the result is shown: detecting negative excrement after each main component in solution transposition and non-specific responding occur, and detect CPV The sensitivity of epidemic strain declines 10~1000 times.Thus, it is only fixed in kit or specific position uses corresponding buffer Kit sensibility and specificity when detecting is just able to achieve with dilution.In other words, different from the position of 2 plants of monoclonal antibodies It sets (basically position embodies reaction sequence), the position of buffer and dilution cannot exchange.

The preparation method of 2.4 kits

Step (1) prepares buffer；

Substrate is diluted to 0.5%~4%W/V with dilution using substrate pad by step (2), and is adsorbed in adsorbate Up to substrate pad, drying is placed in the substrate supply area of test strip；

Step (4) sessile antibody is through sessile antibody diluted to 0.5~1mg/ml and sheep anti mouse resists more or goat-anti Mouse monoclonal antibody is and solid respectively as detection line, nature controlling line through phosphate buffer or curing antibody diluted to 1~3mg/ml Due in the detection zone of test strip, make line-to-line away from >=5mm, it is dry；

Step (5) prepares sample treatment liquid, 1ml/ pipe；And

Step (6) assembles kit, will test test strips and is connected to buffer supply unit, and step (1) is prepared Buffer injects buffer supply unit, and buffer supply unit is enable to supply the buffer to the test strip Substrate supply area；Direction of the test strip certainly gradually far from buffer supply unit includes substrate supply area (cellulose nitrate Plain film left end, including substrate pad, are adsorbed with dry zymolyte thereon), sample supply area (among nitrocellulose filter, including Enzyme mark pad is adsorbed with enzyme labelled antibody thereon, and the zymolyte can generate chromogenic reaction with the enzyme on the enzyme labelled antibody, and in nitre To the distal migration away from the buffer supply unit on sour tunica fibrosa), detection zone (nitrocellulose filter right end, including detection Line, nature controlling line, immobilization has sessile antibody in the detection line, and immobilization has sheep anti mouse mostly anti-or sheep anti mouse on the nature controlling line Secondary antibody), wherein substrate pad, whole section of enzyme mark pad are successively adhered to nitrocellulose filter from left to right；Step (5) are prepared into tubulature Sample treatment liquid is fitted into kit.

The detection method of 2.5 kits

The detection method of kit includes:

Step (1) acquires sample to be tested, solid sample and/or fluid sample according to the toxin expelling approach of animal epidemic antigen It is dissolved in sample treatment liquid.

By treated, sample is added dropwise in the sample supply area that the test strip is added step (2)；

S2 plants of CPV epidemic strain of selection, positive excrement debug sample detection dosage, with kit (buffer solution A 6, bottom The combination of enzyme labelled antibody dilution C3, curing antibody dilution D4 and sample treatment liquid F4 on object pad dilution B3, enzyme mark pad) It is detected, as a result (is shown in Table 12): under conditions of sample detection dosage is 100 μ l, when solid sample is dissolved in 500-1500 μ Testing result is negative (there are false negatives) when the amount of l sample treatment liquid is 0.05g, goes out when the meltage of solid sample is 0.6g Existing sample treatment liquid is unable to complete chromatography process and causes result invalid, only the detection knot when solid sample is 0.08~0.5g Fruit is effectively and accurate；Similarly under conditions of sample detection dosage is 100 μ l, when fluid sample is dissolved in 500-1500 μ l sample Testing result is negative (there are false negatives) when the amount of product treatment fluid is 300 μ l or 2000 μ l, only molten when fluid sample Result is just correct when solution amount is 500~1500 μ l.The testing result when the detection dosage of the sample through sample treatment liquid is 25 μ l It is terminated after chromatography process carries out half due to causes testing result invalid, when sample detection dosage is 200 μ l due to reaction Excessively lead to false negative result, only testing result is just effectively and accurate when sample detection dosage is 50~150 μ l.

12 sample detection dosage of table and the debugging result that dosage is added dropwise

According to the above debugging result, the present invention only (is resisted buffer solution A 6, substrate pad dilution B3, enzyme mark with this condition The combination of body dilution C3, curing antibody dilution D4 and sample treatment liquid F4, solid sample are dissolved in sample treatment liquid Meltage is 0.08~0.5g/500-1500 μ l, and the meltage that fluid sample is dissolved in sample treatment liquid is 500~1500 μ l/ 500-1500 μ l is 50~150 μ l through the dissolved sample detection dosage of sample treatment liquid) for the similar kit for preparing and It is evaluated is described in detail with application.

The kit evaluation of 2.6 2 plants of monoclonal antibody difference collocation mode preparations

2.6.a the kit remolding sensitivity of different collocation modes preparations compared with

By 2 plants of monoclonal antibody transpositions (being shown in Table 13) in test strips, and evaluated with sample disc, while using quotient Pin Hua South Korea pacifies prompt canine parvovirus colloidal gold colloidal gold detection test paper strip and carries out parallel test, as a result (is shown in Table 13): according to the prior art With the sample in CPV kit 3 (referring to Chinese patent CN104928258A) test sample disk of 2 plants of monoclonal antibody pairing modes preparation Sensitivity is 10^3.2~10^3.5TCID₅₀/ml；The constant 2 test sample disk of CPV kit in the monoclonal antibody position obtained according to the present invention In sensitivity be 10^2.0~10^2.5TCID₅₀/ ml, than CPV kit 1 and commercially available common colloidal gold product high sensitivity 10~ 50 times；Existing 2 plants of monoclonal antibody pairing modes are changed, i.e., behind exchange monoclonal antibody position, the CPV kit 1 that obtains according to the present invention Detection sensitivity is suitable or more excellent with CPV kit 3, and detection is positive, negative sample result is accurate, does not occur non-specificity Reaction.

Testing result summarizes after the different collocation mode reagent preparation boxes of table 13

2.6.b the kit specific detection of different collocation mode preparations

Detect specific sample using CPV kit 1,2,3 respectively, including canine distemper virus liquid, canine parainfluenza virus liquid, 1 type virus liquid of hepatitis infectiosa canis virus, 2 type virus liquid of hepatitis infectiosa canis virus, rabies venom, Healthy Dogs excrement, canine distemper virus positive dog Excrement, dog rotavirus positive dog excrement, canine coronavirus cell toxicant sample, result is feminine gender, is shown specific good.

2.6.c the kit repetitive research of different collocation mode preparations

CPV kit 1,2 is carried out between criticizing with sample, specific sample in sample disc respectively, batch interior repetitive research, knot Fruit: CPV kit 1,2 batch between, batch in repeatability it is good.

2.6.d the kit storage life research of different collocation mode preparations

Respectively by CPV kit 1,2 in 2~8 DEG C of placements progress sensitivity, specific detection in 6,9,12,15 months, knot Fruit: testing result coincidence rate is 100%, shows that kit 1,2 can save even 15 months 12 months at 2~8 DEG C.

2.6.e the clinical application of the kit of different collocation mode preparations

It will be accredited as 200 parts positive, negative 200 parts of clinical samples through PCR to be detected with CPV kit 1,2 respectively, tie Fruit (is shown in Table 14): it is that detect total coincidence rate be 97% to 94%, CPV kit 2 that CPV kit 1, which detects total coincidence rate, can replace behaviour When making trouble and PCR detection method that is cumbersome, needing special technical staff, special instrument and equipment to be just able to achieve carries out clinic Fast and accurately identify.

The different kit detection clinical sample results of table 14 summarize

Kit	The positive/identification total positives	Feminine gender/identification is always negative	Total coincidence rate
				CPV kit 1	176/200	224/200	94%
CPV kit 2	187/200	213/200	97%

The preparation of other enzyme immunochromatographytest test kits of embodiment 3

The animals such as the other antigens of pig, fowl, cat, dog can be met further to evaluate each condition that embodiment 2 is optimized Application in disease diagnosis detection, it is special by porcine circovirus 2 type, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, A Type influenza virus, canine distemper virus, hepatitis infectiosa canis virus etc. are debugged one by one and are evaluated, as a result: (1) sensitivity, former collocation mode The kit sensitivity of preparation is superior to commercially available or existing (10~50 times high), the examination of the collocation mode preparation after transposition The sensitivity of agent box is suitable or more excellent with before the debugging of original collocation mode；(2) specific, well, characteristic appearance is sent out nothing but It is raw；(3) repeated, well；(4) storage life is all satisfied requirement；(5) detection application effect is preferable, can quickly, on the spot, it is quasi- Really detect.It is below that representative is briefly illustrated with regard to porcine circovirus 2 type, influenza A.

3.1 porcine circovirus 2 type enzymes exempt from chromatography detecting test paper strip

3.1.a the kit remolding sensitivity of different collocation modes preparations compared with

By 2 plants of monoclonal antibody transpositions (being shown in Table 15) in test strips, and with PCV2 different subtype strain and PCR Positive clinical serum, the negative serum of identification are evaluated, and as a result (are shown in Table 15, assaypositive tissue, negative tissue, positive serum, yin Property serum identified through PCR): the PCV2 kit 3 that is prepared according to the prior art with 2 plants of monoclonal antibody pairing modes (referring to it is Chinese specially Sharp CN105717293A) the sample sensitivity in test sample disk is 10^4.0~10^4.2TCID₅₀/ml；It obtains according to the present invention Sensitivity in 2 test sample disk of PCV2 kit is 10^3.8~10^4.0TCID₅₀/ ml, than 1 spirit of PCV2 kit of the prior art Sensitivity is 10~15 times high；Will monoclonal antibody pairing mode change after the PCV2 kit 1 that obtains according to the present invention, detection sensitivity with PCV2 kit 3 is suitable or more excellent, and detection is positive, negative sample result is accurate, does not occur nonspecific reaction.

Testing result summarizes after the different collocation mode reagent preparation boxes of table 15

3.1.b the kit specific detection of different collocation mode preparations

PCV2 kit 1,2 is detected to specific sample respectively, including 1 type virus liquid of pig circular ring virus, pig breed and exhale Inhale syndrome virus virus liquid, high-pathogenicity porcine reproductive and respiration syndrome disease virus liquid, pig parvoviral liquid, porcine pseudorabies disease Venom, hog cholera venom, Porcine Epidemic Diarrhea virus liquid, result are feminine gender, are shown specific good.

3.1.c the kit repetitive research of different collocation mode preparations

PCV2 kit 1,2 is carried out between criticizing with sample, specific sample in sample disc respectively, batch interior repetitive research, As a result: PCV2 kit 1,2 batch between, batch in repeatability it is good.

3.1.d the kit storage life research of different collocation mode preparations

Respectively by PCV2 kit 1,2 in 2~8 DEG C of placements progress sensitivity, specific detection in 6,9,12,15 months, knot Fruit: testing result coincidence rate is 100%, shows that kit 1,2 can save even 15 months 12 months at 2~8 DEG C.

3.1.e the clinical application of the kit of different collocation mode preparations

250 parts positive, negative 250 parts of clinical samples will be accredited as (after serum, grinding through Virus Isolation and PCR Tissue sample) detected respectively with PCV2 kit 1,2, as a result (be shown in Table 16): PCV2 kit 1 detects total coincidence rate and is It is 94% that 91%, PCV2 kit 2, which detects total coincidence rate, is superior to PCV2 kit 3 (original collocation mode, the prior art) As a result, can replace operating virus purification that is time-consuming and cumbersome, needing special technical staff, special instrument and equipment to be just able to achieve Identification or PCR detection method carry out clinical fast and accurately identification.

The different kit detection clinical sample results of table 16 summarize

Kit	The positive/identification total positives	Feminine gender/identification is always negative	Total coincidence rate
				PCV2 kit 1	205/250	295/250	91%
PCV2 kit 2	220/250	280/250	94%
				PCV2 kit 3	200/250	300/250	90%

3.2A type influenza endonuclease exempts from chromatography detecting test paper strip

3.2.a the kit remolding sensitivity of different collocation modes preparations compared with

By 2 plants of monoclonal antibody transpositions (being shown in Table 17) in test strips, and with influenza different subtype strain and move The sample disc of forward and backward microorganism swab (the i.e. negative swab, positive swab) composition collected of object challenge test is evaluated, and is tied Fruit (is shown in Table 17~18): with original 2 plants of monoclonal antibody pairing modes according to the IV kit 3 of prior art preparation (referring to Chinese patent CN104062430A) sensitivity of test sample disk sample is 0.01~0.1HA or 10^4.5~10^5.5EID₅₀/100μl；With original The sensitivity in 2 test sample disk of IV kit that 2 plants of monoclonal antibody pairing modes obtain according to the present invention be 0.005~01HA or 10^3.5~10^4.0EID₅₀/ 100 μ l, than 10~50 times of high sensitivity before optimization；Original 2 plants of monoclonal antibody pairing modes are changed, i.e., After transposition, the detection sensitivity of the IV kit 1 obtained according to the present invention is suitable or more excellent with IV kit 3, and detects The positive, negative sample result are accurate, do not occur nonspecific reaction.

The different collocation mode reagent preparation boxes of table 17

Table 18 is summarized with different collocation mode reagent preparation box testing results

3.1.b the kit specific detection of different collocation mode preparations

IV kit 1,2 is detected to specific sample, pharynx, cloacal swab sample including negative SPF chicken, health respectively The pharynx of chicken, cloacal swab sample, the Nasopharyngeal swabs sample of health pig, the Nasopharyngeal swabs sample of Healthy Dogs, result is feminine gender, Show specific good.

3.1.c the kit repetitive research of different collocation mode preparations

IV kit 1,2 is carried out between criticizing with sample, specific sample in sample disc respectively, batch interior repetitive research, knot Fruit: IV kit 1,2 batch between, batch in repeatability it is good.

3.1.d the kit storage life research of different collocation mode preparations

Respectively by IV kit 1,2 in 2~8 DEG C of placements progress sensitivity, specific detection in 6,9,12,15 months, as a result: Testing result coincidence rate is 100%, shows that kit 1,2 can save even 15 months 12 months at 2~8 DEG C.

200 parts positive, negative 200 parts of clinics will be accredited as through SPF egg inoculation virus isolation procedure and RT-PCR method Sample is detected with IV kit 1,2,3 respectively, as a result (is shown in Table 19): IV kit 1 detects total coincidence rate as 94%, IV examination It is 96% that agent box 2, which detects total coincidence rate, is superior to the coincidence rate of IV kit 3 (original collocation mode, the prior art) detection 92%, it can replace operating SPF chicken embryo that is time-consuming and cumbersome, needing special technical staff, special instrument and equipment to be just able to achieve Virus inoculation separation identification or PCR detection method carry out clinical fast and accurately identification.

The different kit detection clinical sample results of table 19 summarize

Kit	The positive/identification total positives	Feminine gender/identification is always negative	Total coincidence rate
				IV kit 1	176/200	224/200	94%
IV kit 2	185/200	215/200	96%
				IV kit 3	168/200	232/200	92%

The other enzyme labelled antibodies of embodiment 4 prepare enzyme and exempt to chromatograph detection kit and its application

4.1 other enzyme labelled antibodies

Alkali phosphatase enzyme mark antibody: using glutaraldehyde two-step method labeled monoclonal antibody, dissolves alkali with 2% glutaraldehyde solution Acid phosphatase to its final concentration of 20mg/ml, in biochemical cultivation case 25 DEG C stand overnight after go to bag filter, in 2~8 DEG C PBS solution dialysed overnight, calculate hydroformylation concentration after be adjusted to 2mg/ml, with 1mg monoclonal antibody mix after go to bag filter, use It is coated with buffer (pH9.6,0.05mol/L carbonate buffer solution change liquid 3 times) dialysed overnight, PBS solution is changed to later and (changes Liquid 3 times) continue dialysed overnight, take out dialysate, add freeze after isometric glycerol it is spare.The identification of alkali phosphatase enzyme mark antibody Identical as HRP enzyme labelled antibody, reference implementation example 2 carries out, and the results are shown in Table 20.Beta galactosidase labelled antibody: glutaraldehyde method is used 5mg monoclonal antibody and 10mg beta galactosidase are dissolved in 2ml 0.1mol/L kaliumphosphate buffer by labeled monoclonal antibody (pH6.8) it mixes, in 2~8 DEG C of dialysed overnights, dilutes glutaraldehyde to 1% with 0.1mol/L kaliumphosphate buffer (pH6.8), to The glutaraldehyde that 100 μ l diluted is added in dialysis mixed liquor, 2mol/L glycine liquid is added after being stirred at room temperature three hours makes its end Concentration is placed at room temperature for 2 hours after being 0.1mol/L, by mixed liquor in 2~8 DEG C with PBS dialysed overnight, 10000 revs/min 4 DEG C from The heart 30 divides, and takes supernatant, add freeze after isometric glycerol it is spare.The identification of beta galactosidase labelled antibody and HRP enzyme labelled antibody Identical, reference implementation example 2 carries out, and the results are shown in Table 20.

The quality evaluation result of the other enzyme labelled antibodies of table 20 summarizes

4.2 other enzyme labelled antibodies prepare enzyme and exempt to chromatograph the assembling and its application of detection kit

Alkali phosphatase enzyme mark antibody, the beta galactosidase labelled antibody H1~H4 prepared with embodiment 4.1 is pressed respectively Canine parvovirus toxenzyme is prepared according to embodiment 2 to exempt to chromatograph detection kit h1~h4, and is detected with the sample in sample disc, is tied Fruit (being shown in Table 21) shows: the enzyme of other enzyme labelled antibody preparations, which exempts from chromatography detection kit, can also realize effective detection, and kit In after two strain antibody transpositions testing result it is suitable.

Testing result summarizes after the other enzyme labelled antibody reagent preparation boxes of table 21

In conclusion combine kit of the present invention buffer, substrate pad dilution and by enzyme labelled antibody dilution, consolidate When determining the test strips of antibody diluent preparation, it can make to exempt from used in chromatography detecting test paper strip with the enzyme of the sandwich principle preparation of double monoclonal antibodies The anti-position of 2 plants of monoclonals of the fixation, label arrived, which is realized, to be exchanged, to realize with 2 plants of monoclonal antibodies of different collocation modes Prepare detection kit, and with not only not causing non-specific responding that can also improve spirit after such kit test sample Sensitivity.

SEQUENCE LISTING

<110>Luoyang Pu Laikewantai Bioisystech Co., Ltd

Genetic test diagnostic center Co., Ltd, section in Luoyang

<120>a kind of double-antibody method enzyme immunochromatographytest test kit and preparation method and application

<160> 16

<170> PatentIn version 3.3

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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Thr Lys Pro Gly Ala

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<212> PRT

<213>hybridoma (hybridoma)

<400> 16

Asp Thr Val Met Thr Gln Ser Gln Lys Phe Ile Ser Thr Ser Ile Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Thr Ala Ser Gln Asn Val Gly Thr Phe

20 25 30

Val Val Trp Tyr Gln Arg Lys Ser Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Lys Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asp Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

Claims

1. a kind of double-antibody method enzyme immunochromatographytest test kit, the kit include enzyme immunochromatographydetecting detecting test strip, Sample treatment liquid, buffer and buffer supply unit, which is characterized in that

The buffer includes phosphate buffer, high molecular weight protein, Tween-20 and preservative, and carbamide peroxide, mistake Aoxidize any one of urea hydrogen, hydrogen peroxide；The preferably described phosphate buffer is pH7.4 0.1M PBS solution；Preferably The high molecular weight protein is one of BSA, OVA, fetal calf serum or a variety of；The preferably described preservative is gentamicin, sulphur Any one of sour kanamycins, Sodium azide, thimerosal and its esters, Proclin300；The carbamide peroxide, hydrogen peroxide Urea, hydrogen peroxide concentration be 0.02%~0.2%W/V；The preferably described carbamide peroxide, hydrogen peroxide urea, hydrogen peroxide Concentration be 0.05%~0.1%W/V；It further preferably include pH7.4 0.1M PBS solution, 0.1%~0.5%V/V Tween-20,0.02%V/V Proclin300,0.05%V/V carbamide peroxide and 0.5%~1%W/V it is any BSA, OVA and fetal calf serum；

The enzyme immunochromatographydetecting detecting test strip includes substrate pad, is adsorbed with the drying after substrate pad diluted thereon and forms Zymolyte, the substrate pad dilution includes dehydrated alcohol, pH7.4 0.1M PBS solution and 0.5%~4%W/V PEG6000 or PEG4000；Preferably, substrate pad dilution includes 2%V/V dehydrated alcohol, 0.1%V/VPEG4000, pH7.4 0.1M PBS；

The enzyme immunochromatographydetecting detecting test strip includes enzyme mark pad, be adsorbed with after enzyme labelled antibody diluted thereon it is dry and At enzyme labelled antibody, the enzyme labelled antibody dilution include pH7.40.1M PBS solution and 0.5%V/V PEG4000, Any one of PEG6000 or PEG8000；Preferably, the enzyme labelled antibody dilution includes 0.5%V/VPEG4000, pH7.4 0.1M PBS；

The enzyme immunochromatographydetecting detecting test strip includes detection line and nature controlling line, is fixed in the detection line dilute through sessile antibody Release the diluted sessile antibody of liquid, the sessile antibody dilution include pH7.4 0.1M PBS solution, 0.5%V/V trehalose, 0.1%V/V Tween-20.

2. enzyme immunochromatographytest test kit according to claim 1, which is characterized in that the kit includes detection examination Paper slip, sample treatment liquid, buffer and buffer supply unit, wherein

The test strip successively includes substrate supply area, sample supply area, detection zone from longitudinal direction；The substrate supply area Including the substrate pad, it is adsorbed with dry zymolyte thereon, the preferably described zymolyte is tetramethyl benzidine；The sample Supply area includes the enzyme mark pad, is adsorbed with enzyme labelled antibody thereon, and the enzyme is preferably horseradish peroxidase, alkaline phosphatase Or beta galactosidase, the zymolyte can generate chromogenic reaction with the enzyme on the enzyme labelled antibody；The detection zone includes institute Detection line and the nature controlling line are stated, immobilization has sheep anti mouse mostly anti-or sheep anti mouse secondary antibody on the nature controlling line；

The buffer supply unit makes for supplying the buffer to the substrate supply area of the test strip Buffer and the sample treatment liquid, zymolyte and the enzyme labelled antibody that spread with buffer are along the longitudinal direction of test strip and to the bottom of far from One end of object supply area migrates.

3. enzyme immunochromatographytest test kit according to claim 2, which is characterized in that the enzyme labelled antibody is dissolved in institute The concentration in enzyme labelled antibody dilution is stated to be dissolved in the sessile antibody dilution for 0.5~10 μ g/ml, the sessile antibody Concentration be 0.5~1mg/ml, sheep anti mouse is mostly anti-or sheep anti mouse secondary antibody is dissolved in phosphate buffer or the sessile antibody is dilute Release final concentration of 0.5%~4%W/V that the concentration in liquid is 1~3mg/ml, zymolyte is dissolved in substrate pad dilution.

4. enzyme immunochromatographytest test kit according to claim 2, which is characterized in that the detection line and the Quality Control Line line-to-line is away from >=5mm.

5. kit according to claim 1, which is characterized in that in the monoclonal antibody and sessile antibody in enzyme labelled antibody Monoclonal antibody

For CCTCC No:C2014198 mouse hybridoma cell 3G12 secretion porcine circovirus 2 type monoclonal antibody 3G12 and Any combination of the porcine circovirus 2 type monoclonal antibody 2F8 of CCTCC No:C2014199 mouse hybridoma cell 2F8 secretion； Or

For Porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB2 and Porcine epidemic diarrhea virus monoclonal antibody PEDV- Any combination of McAB1, the monoclonal antibody PEDV-McAB2 heavy chain variable region be SEQ ID No.1 shown in, light chain variable Area be SEQ ID No.2 shown in, the monoclonal antibody PEDV-McAB1 heavy chain variable region be SEQ ID No.3 shown in, light chain Variable region is shown in SEQ ID No.4；Or

For monoclonal antibody of transmissible gastro-enteritis virus TGEV-3D2 and monoclonal antibody of transmissible gastro-enteritis virus TGEV- Any combination of 4B4, the monoclonal antibody TGEV-3D2 heavy chain variable region is shown in SEQ ID No.5, light chain variable region is Shown in SEQ ID No.6, the monoclonal antibody TGEV-4B4 heavy chain variable region be SEQ ID No.7 shown in, light chain variable region For shown in SEQ ID No.8；Or

For anti-influenza type A virus nucleoprotein monoclonal antibody IV-McAB1 and anti-influenza type A virus nucleoprotein monoclonal antibody IV- Any combination of McAB2, the monoclonal antibody IV-McAB1 heavy chain variable region be SEQ ID No.9 shown in, light chain variable region For shown in SEQ ID No.10, the monoclonal antibody IV-McAB2 heavy chain variable region is shown in SEQ ID No.11, light chain can Become area as shown in SEQ ID No.12；Or

The canine parvovirus monoclonal antibody CPV- secreted for 10B11 plants of mouse bone marrow cells hybridoma of CCTCC No:C201578 The canine parvovirus monoclonal antibody CPV- of 10H4 plants of mouse bone marrow cells hybridoma of 10B11 and CCTCC No:C201579 secretions Any combination of 10H4；Or

For the canine distemper virus monoclonal antibody CDV-1G5 of CCTCC No:C2015201 mouse bone marrow cells hybridoma 1G5 secretion The canine distemper virus monoclonal antibody CDV-6E11 secreted with 6E11 plants of mouse bone marrow cells hybridoma of CCTCC No:C2015202 Any combination；Or

For any combination of hepatitis infectiosa canis virus monoclonal antibody CAV-5G4 and hepatitis infectiosa canis virus monoclonal antibody CAV-1A1, the Dan Ke Grand antibody CAV-5G4 heavy chain variable region is shown in SEQ.ID No.13, light chain variable region is the list shown in SEQ.ID No.14 Clonal antibody CAV-1A1 heavy chain variable region is shown in SEQ.ID No.15, light chain variable region is shown in SEQ.ID No.16.

6. enzyme immunochromatographytest test kit according to claim 1, which is characterized in that the sample treatment liquid includes PH7.4 0.1M PBS solution, CHAPS, saponin(e and preservative；Preferably, the sample treatment liquid includes pH7.4 0.1M PBS Solution, CHAPS, saponin(e and Proclin300；It is highly preferred that the sample treatment liquid include pH7.4 0.1M PBS solution, 0.5%~2.0%W/V CHAPS, 0.5%~2.0%W/V saponin(e and 0.02%V/V Proclin300；Most preferably, described Sample treatment liquid includes pH7.4 0.1M PBS solution, 0.5%W/V CHAPS, 1%W/V saponin(e, 0.02%V/V Proclin300。

7. enzyme immunochromatographytest test kit according to claim 6, which is characterized in that solid sample is dissolved in the sample The meltage of product treatment fluid is 0.08~0.5g/500-1500 μ l, and fluid sample is dissolved in the meltage of the sample treatment liquid For 500~1500 μ l/500-1500 μ l；It is 50~150 μ l through the dissolved sample detection dosage of sample treatment liquid；

The sample is selected from tissue, serum, anus secretion, excrement, pharynx nasal discharge, eye nasal discharge and viral cultures.

8. a kind of sample treatment liquid for any one of the claim 1-7 kit, which is characterized in that the sample treatment Liquid includes pH7.4 0.1M PBS solution, CHAPS, saponin(e and preservative；Preferably, the sample treatment liquid includes pH7.4 0.1M PBS solution, CHAPS, saponin(e and Proclin300；It is highly preferred that the sample treatment liquid includes pH7.40.1M PBS Solution, 0.5%~2.0%W/V CHAPS, 0.5%~2.0%W/V saponin(e and 0.02%V/V Proclin300；Most preferably Ground, the sample treatment liquid include pH7.4 0.1M PBS solution, 0.5%W/V CHAPS, 1%W/V saponin(e, 0.02%V/V Proclin300。

9. sample treatment liquid according to claim 8, which is characterized in that the sample of the sample treatment liquid detection is selected from group It knits, serum, anus secretion, excrement, swallow nasal discharge, eye nasal discharge and viral cultures.

10. a kind of method for preparing any one of claim 1-8 kit characterized by comprising

Step (1) prepares buffer；

Step (2) is diluted substrate using substrate pad dilution, and is adsorbed in adsorbate up to substrate pad, and drying is placed on In test strip；

Step (3) labeled monoclonal antibody is enzyme labelled antibody, is diluted with enzyme labelled antibody dilution, and be adsorbed in adsorbate i.e. Enzyme mark pad is obtained, drying is placed in test strip；

The cured antibody diluent of step (4) sessile antibody is diluted and is fixed in test strip as detection line, sheep anti mouse More anti-or sheep anti mouse monoclonal antibodies are fixed on nature controlling line, dry；

Step (5) prepares sample treatment liquid；And

Step (6) supplies the enzyme immunochromatographydetecting detecting test strip, the sample treatment liquid, the buffer and buffer single Member is assembled into kit.