CN105445462B - Enzyme linked immunosorbent assay kit for specific quantitative detection of foot and mouth disease O type Guangxi strain antigen and application thereof - Google Patents

Abstract

The invention discloses an enzyme linked immunosorbent assay kit for specific quantitative detection of a foot and mouth disease O type Guangxi strain (O/GX/09-7 strain) antigen and an application thereof. The kit is a double-antibody sandwich-method enzyme-linked immune quantitative detection kit prepared from an O/GX/09-7 strain specific monoclonal antibody, and the content of the O/GX/09-7 strain antigen in a sample is quantitatively detected through detection of changes of signals generated by an enzyme catalyzed substrate. The detection using the kit does not generate cross interference with other current foot and mouth disease vaccine strains such as O/Mya98/XJ/2010 strains, O/Mya98/BY/2010, O/JMS/2000, OZK/93 strains, Asia1/JSL/06 and type A A/WH/09, and the method can simply, rapidly, sensitively and stably detect the content of the foot and mouth disease O type Guangxi strain antigen and specifically distinguish a foot and mouth disease O type Guangxi strain and other strains.

Description

A kind of specific quantification detects the ELISA reagent of the O-shaped Guangxi Strain antigen of foot and mouth disease Box and its application

Technical field

The invention belongs to enzyme linked immunosorbent detection analysis technical field, and in particular to a kind of quantitative determination of specificity foot and mouth disease O The enzyme linked immunological kit of type Guangxi Strain (O/GX/09-7 strains) antigen, it is adaptable to the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) Special, quick, the accurate detection and analysis of antigen.

Background technology

Foot and mouth disease viruses (Foot and Mouth Disease Virus, FMDV) belong to Picornaviridae, hoof-and-mouth disease Poison category, the virus have 7 serotypes, it is various between react without cross protection.Country's Major Epidemic is O-shaped, Type Asia 1 at present (such as JSL/06 strains) and A types (A/WH/09 strains).Wherein, it is O-shaped including multiple strains, it is Xinjiang Strain (O/Mya98/XJ/ respectively 2010 plants), Jiamusi strain (O/JMS/2000 strains), 98 plants of Burma (O/Mya98/BY/2010 strains), OZK strains (OZK/93 strains) etc.. Guangxi Strain (O/GX/09-7 strains) be also it is O-shaped in one kind, be widely current at home.Quickly, accurately, special detection foot and mouth disease O Type Guangxi Strain antigen (O/GX/09-7 strains) contributes to diagnosis and the production of vaccine quality control of disease.

At present, the method for determining foot-and-mouth disease antigen 146S mainly has density－gradient centrifuga－tion method.But, density－gradient centrifuga－tion method Complex operation, needs the time of 3 days, and every supercentrifuge can only detect 5 parts of samples every time, while serotype can not be carried out Differentiation.At present, domestic foot-and-mouth disease vaccine is mostly multivalence Seedling, it means that contain various strains or serotype in the vaccine, The FMD tye O swine vaccine of such as Jinyu Baoling Biology Drugs Co., Ltd's production contains 2 kinds of O-shaped antigens, is new respectively Boundary strain (O/Mya98/Xj/2010 strains) and Guangxi Strain (O/GX/09-7 strains).The accurate quantitative analysis of every kind of antigen are to ensure vaccine peace Entirely, an effective important means.Existing density－gradient centrifuga－tion method cannot be distinguished by different aftosa serum types, can not distinguish The different strains of same serotype.Accordingly it is desirable to people find a kind of more special, sensitive and reliable determination method energy It is enough effectively to distinguish different serotypes and strain.

Immunoassay (Immunoassay) come out in eighties of last century the seventies, are resisted by using the antigen of high specificity Precursor reactant grow up one has high sensitivity, high specific and quick analysis method.At present, main flow on market Immunoassay mainly include elisa (enzyme-linked immuno sorbent assay, ELISA) and chemiluminescence immune assay (chemiluminescent immunoassay, CLIA)) and colloid gold immune paper Chromatography etc..At present, these methods have been widely used in Clinical detection, including in the project such as infectious disease, tumor and hormone. The sensitivity of elisa technique can be usually reached the ng/mL even level of pg/mL, typically can complete right in 30 minutes to 2 hours The detection of nearly hundred parts of samples, and special instrument and equipment is not needed, the needs of foot and mouth disease 146S Detection of antigen can be met completely. At present, the accidental enzyme-linked immunologic detecting kit that can detect foot-and-mouth disease antigen on market, but mostly employ Anti-TNF-α System is into such as Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's exploitation, the O-shaped foot and mouth disease 146S antigen quantitative ELISA of production Detection kit, the test kit have declared patent (patent No. CN103076451A), and patent declares that this test kit employs O Two kinds of polyclonal antibodies of rabbit anti-serum and guinea pig antiserum of type foot and mouth disease are made, and can distinguish O-shaped with A types, Type Asia 1 Open, but cannot be distinguished by O-shaped different strains, such as Schweineseuche O-shaped the going out of Jinyu Baoling Biology Drugs Co., Ltd's production Xinjiang Strain (O/Mya98/XJ/2010 strains) and Guangxi Strain (O/GX/09-7 strains) in live vaccine, thus just cannot be in the vaccine Every kind of foot and mouth disease 146S antigen carry out individually quantitative, its chief reason is that polyclonal antibody is directed to various antigen tables Position, and especially may there is identical epitope between the different strains of same serotype in different serotype.

1975, G.J.F. gram of molecular biologist is strangled and C. Millsteins are created on the basis of natural hybridization technology Monoclonal anti Antibody Production Techniques (hybridoma technology), can obtain the antibody for certain specific antigen epi-position by this technology, And prepare in a large number.Even to this day, the technology is highly developed, and is widely used in different fields.Due to difference The gene homology of the foot and mouth disease 146S antigen of serotype is higher, especially the homology between same serotype difference strain Very high, homology can may only have the difference of a few or tens aminoacid sequences more than 85%.Therefore, use Viral direct immunization prepares monoclonal antibody, and therefrom screening is obtained for particular serotype, especially same serotype is different The specific antibody of strain just becomes extremely difficult.Therefore, how quickly and accurately to find foot and mouth disease viruses different serotypes, Differentiated epitope (albumen or polypeptide) between different strains, and monoclonal antibody specific is prepared with this become exploitation Differentiation different serotypes, the foot and mouth disease 146S antigen ELISA test kit of the different strains for even distinguishing same serotype are primarily solved Problem certainly.

The content of the invention

First, the invention provides O-shaped Guangxi Strain (O/GX/09-7 strains) the antigen enzyme linked immunological of a species specific foot and mouth disease Quantitative determination reagent kit.

Test kit provided by the present invention, including one plant of specificity list with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) Clonal antibody is used as coated elisa plate made by capture antibody.

The test kit may also include the other one plant of specificity Dan Ke with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) Grand antibody is used as enzymic-labelled antibody made by detection antibody.

The detection antibody can make enzymic-labelled antibody with the enzyme labelling such as horseradish peroxidase or alkaline phosphatase, excellent Elect horseradish peroxidase as, horseradish peroxidase can pass through glutaraldehyde method or periodic acid method is crosslinked in detection antibody.

For convenience of result observation, the test kit also includes the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) positive quality control Product, concretely the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) 146S antigen purifications inactivation liquid, is diluted to sample diluting liquid The level of 8-64ng/mL, may be selected the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) the 146S antigen purifications of each vaccine factory production Inactivation liquid, the preferably O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) 146S of Jinyu Baoling Biology Drugs Co., Ltd's production Antigen purification inactivates liquid；The raw material of the positive quality control product is standard level, and purity is not less than 80%.

For convenience of use, the test kit may also include detection sample diluting liquid, cleaning mixture, assay chromogenic substrate solution, end Only liquid and description.

In mentioned reagent box, the formula of sample diluting liquid is：Na₂HPO₄·12H₂O 2.9g, NaH₂PO₄·2H₂O 0.296g, NaCl 8.5g, Proclin 3000.6mL, casein 10g, new-born calf serum 50mL are dissolved to distilled water 1000mL, adjustment pH value to 7.2-7.4.

The formula of cleaning mixture is：Na₂HPO₄·12H₂O 58g, NaH₂PO₄·2H₂O 5.92g, NaCl 170g, Tween- 201.0mL, Proclin 3000.6mL, is dissolved to 1000mL, adjustment pH value to 7.2-7.4 with distilled water；During using cleaning mixture 20 times are diluted with distilled water.

The formula of assay chromogenic substrate solution：

1st, the assay chromogenic substrate solution of horseradish peroxidase (HRP)

Assay chromogenic substrate solution includes A liquid and B liquid, wherein, the formula of chromogenic substrate A liquid is：Sodium acetate trihydrate 8.2g, ice vinegar Sour 1.2mL, concentration are 30% hydrogen peroxidase 10 .27mL, are dissolved to 1000mL, adjustment pH value to 5.0-5.6 with distilled water；

The formula of chromogenic substrate B liquid is：Citric acid 1.92g, sodium ethylene diamine tetracetate 0.372g, 10mol/L sodium hydroxide Solution 0.3mL, glycerol 50mL, tetramethyl biphenyl amine hydrochlorate 0.23g are dissolved to 1000mL with distilled water, and adjustment pH value is extremely 2.5-3.5。

A liquid is pressed into 1 with B liquid using front：1 equal proportion mixes.Need to be terminated with terminate liquid after the assay chromogenic substrate solution reaction, then Just can above machine testing.

Terminate liquid：Concentration is 98% sulphuric acid 27.8mL, is dissolved to 1000mL with distilled water.

2nd, the assay chromogenic substrate solution of alkali phosphatase (ALP)

Formula is：PNPP 1g, diethanolamine 100mL, anhydrous magnesium chloride 47.5mg, distilled water 900mL, pH9.8.

Terminate liquid is needed to terminate after the assay chromogenic substrate solution reaction, terminate liquid：53g sodium carbonate, distilled water 1000mL.

The special of the O-shaped Guangxi Strain of foot and mouth disease of the present invention (O/GX/09-7 strains) is prepared in accordance with conventional methods known in the art Property monoclonal antibody.Specifically, the monoclonal antibody specific of the O-shaped Guangxi Strain of foot and mouth disease of the present invention (O/GX/09-7 strains) Preparation method, it may include following steps：

1) specificity of O-shaped Guangxi Strain (O/GX/09-7 strains) the specific antigen fragment of foot and mouth disease is obtained by 2-DE electrophoresis Protein band, molecular weight about in 20-30KDa, preferably wherein 5 higher protein bands of abundance ratio be dried, grind after conduct Immunogen is used；

2) with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) specific antigen fragment as immunogen immune animal；Continuously Immunity 4 times, every minor tick 14 days, 3 times using multiple spot subcutaneous inoculation mode, the 4th takes the immunization wayses of lumbar injection, often Secondary 10 μ g/ animals；

3) splenocyte of separating immune animal, itself and myeloma cell fusion are hybridized with the screening of HAT selective mediums Oncocyte, is cloned with indirect ELISA method screening specific positive to hybridoma supernatant；

4) cultivate hybridoma；Separate and purification from the ascites fluid of cell culture fluid or inoculation hybridoma animal Go out the monoclonal antibody specific of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains).

In the preparation method of said monoclonal antibody, step 2) in for the immune animal for preparing monoclonal antibody can be The mammals such as Balb/c mices, LOU/c rats and rabbit, preferred Balb/c mices.

Step 3) in when the antibody level of serum of immunized animal is more than 1:When 50000, the splenocyte of separable animal is simultaneously Be prepared into single cell suspension, and under the induction of appropriate fusion agent (such as Polyethylene Glycol) with myeloma cell (preferably mice Myeloma cell SP2/0) merge to form hybridoma, then, cultivate thin to screen the hybridoma of fusion in HAT culture medium Born of the same parents, and can further use the specific antibody cell strain needed for the identification of the methods such as indirect ELISA method.

Step 4) in can be in external (such as in tissue culture flasks or porous fibre reactor) or internal (as mice abdomen Water) cultivate the selected secretion O-shaped Guangxi Strain (O/GX/09-7 strains) of foot and mouth disease monoclonal antibody specific hybridoma, from The specificity Dan Ke of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) is collected and is purified in cell culture fluid or mouse ascites liquid Grand antibody.

Present invention also offers a kind of with the O-shaped Guangxi Strain (O/GX/09-7 of above-mentioned ELISA kit quantitative determination foot and mouth disease Strain) antigen method, comprise the steps:

1) standard substance, positive quality control product and testing sample are prepared；

2) standard substance of positive quality control product, testing sample or concentration known, board-washing after reaction are added；

3) enzyme-added labeling antibody, board-washing after reaction；

4) add substrate colour developing；

5) terminating reaction；

6) OD values are determined；

7) analysis result, obtains the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) level.

Specifically, the detection method may include following steps:

1) with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) diluted sample that 146S antigens and test kit are provided after purification Liquid prepares standard substance, and standard concentration is：0th, 2,4,8,16,32,64ng/mL, measuring samples are equally diluted with sample diluting liquid Into 4-8 gradient；

2) lath required for taking out, remaining lath load in valve bag, are placed in 2-8 DEG C and save backup；

3) in hole 100 μ L standard substance series dilution point, positive quality control, measuring samples are added to be serially diluted a little respectively, gently The light vibration 10-20 seconds mix, and stick plate patch；

4) it is placed in 37 DEG C of incubators and incubates 60 minutes, deducts liquid in hole, and washed with cleaning mixture (with 20 times of front dilution) 5-7 time, pat dry on clean napkin or towel, remove the moisture of residual as far as possible；

5) per the enzyme marker that 100 μ L are added in hole, gently vibrating the 10-20 seconds mixes, sticks plate and pastes；

6) it is placed in 37 DEG C of incubators and incubates 30 minutes, deducts liquid in hole, and washed 5-7 time with cleaning mixture, clean Pat dry on napkin or towel, remove the moisture of residual as far as possible；

7) add per hole assay chromogenic substrate solution (assay chromogenic substrate solution (A liquid, B liquid each 1 drop) of horseradish peroxidase (HRP) or The assay chromogenic substrate solution (2 drop) of alkali phosphatase (ALP)), the 10-20 seconds are gently vibrated, 37 DEG C of lucifuges is placed in and is reacted 15 minutes, per hole 1 is added to drip terminate liquid (terminate liquid (1 drop) of the terminate liquid or alkali phosphatase (ALP) of horseradish peroxidase (HRP)), gently Vibration is mixed several times, makes solution be changed into yellow liquid completely, in 30 minutes, measures absorbance (OD) (horseradish peroxidase (HRP) with 630nm as reference wavelength, measure 450nm place absorbance, alkali phosphatase (ALP) measurement 405nm extinction Degree)；

8) analysis result, calculates the concentration of O-shaped Guangxi Strain (O/GX/09-7 strains) antigen of foot and mouth disease.

In above-mentioned detection method, step 1) in testing sample selection can be it is various, it is such as O-shaped for foot and mouth disease wide Western strain (O/GX/09-7 strains) the infected, can be from the biopsy article of the infected, tissue slice etc., alternatively vaccine production process In culture fluid, inactivation liquid, refined solution, finished product vaccine after concentrated solution and breakdown of emulsion etc..

Step 8) in interpretation of result method can be：Respectively standard concentration and correspondence OD absorbances are taken the logarithm, Standard curve is set up on double logarithmic chart, with standard concentration as abscissa, that OD absorbances draw standard for vertical coordinate is bent Line, finds the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) antigen in sample on standard curve with each specimen absorbance to be measured Concentration, multiple dilution factors calculate meansigma methodss.

The invention provides the enzyme linked immunological of the O-shaped Guangxi Strain (O/GX/09-7 strains) of quantitative determination of specificity foot and mouth disease Detection kit.The test kit is using double made by the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) monoclonal antibody specific Antibody sandwich enzyme-linked immune quantitative detection reagent box, by detecting the signal intensity of substrate for enzymatic activity generation come detection by quantitative mark The content of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) antigen in this, and with other strains of foot and mouth disease, such as Xinjiang Strain (O/ Mya98/XJ/2010 strains), 98 plants of Burma (O/Mya98/BY/2010), Jiamusi strain (O/JMS/2000), OZK strain (OZK/93 Strain), the strain no cross reaction such as Type Asia 1 (JSL/06) and A types (A/WH/09), there is no cross interference.

The more existing correlation technique of the present invention has advantages below：

1) present invention makes double using the monoclonal antibody of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) high degree of specificity Antibody sandwich ELISA detection kit, with tetramethyl benzidine or pNPP as substrate, detects corresponding signal intensity, by double Mathematical logarithmic model calculates the concentration of testing sample, and its sensitivity reaches the level of 1.2ng/mL, significantly larger than density gradient from Can at most detect in the heart and animal experiment method, and 2 hours that the detection speed of 90 parts of samples substantially reduces detection week Phase (every supercentrifuge of sucrose density gradient centrifugation 5 parts of samples of detection in usual 3 days) and instrument and equipment that need not be expensive, The quality control of control and Guangxi Strain (O/GX/09-7 strains) production of vaccine for foot and mouth disease disease provides more effectively side Method.

2) in this test kit, coated elisa plate is the list with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) high degree of specificity Clonal antibody is made, and cannot be only used for the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) and Type Asia 1, the differentiation of A types, it is also possible to For the differentiation of Guangxi Strain (O/GX/09-7 strains) and O-shaped other strains, such as Xinjiang Strain (O/Mya98/XJ/2010 strains), Burma 98 plants (O/Mya98/BY/2010), Jiamusi strain (O/JMS/2000), OZK strains (OZK/93 strains), are particularly suitable for similar Jin Yubao The independent quantitative inspection of Guangxi Strain (O/GX/09-7 strains) in the FMD tye O swine vaccine of clever biologics company limited production Survey, this is cann't be solved using the test kit of polyclonal antibody development.

3) compared with the test kit developed using polyclonal antibody, as this test kit employs monoclonal antibody completely, It is therefore, it is possible in a large number, stably prepare, reproducible.The polyclonal antibody of different batches can be led due to the difference of host individual Cause to have differences on affinity and specificity per the polyclonal antibody criticized.

In sum, instant invention overcomes the conventional defect of detection method, that is, substantially increase detection method sensitivity, In turn simplify operating procedure and save the operating time, be that one kind easy, quick, sensitive, stably can detect that foot and mouth disease is O-shaped Guangxi Strain (O/GX/09-7 strains) antigenic content and the O-shaped Guangxi Strain (O/GX/09-7 strains) of special differentiation foot and mouth disease and other strains (O/Mya98/XJ/2010 strains, O/Mya98/BY/2010, O/JMS/2000, OZK/93 strain, Type Asia 1 JSL/06 and A type A/ WH/09 etc.) test kit, with easy, quick, accuracy height, sensitivity height, specificity height, good stability, to instrument and equipment The advantages of requiring low and with low cost, can be applicable to the monitoring and detection of disease or epidemic situation, it can also be used to which foot-and-mouth disease vaccine is each Quality control of production link etc..

The present invention is described in further details with reference to specific embodiment.

Description of the drawings

Marks of the Fig. 1 for the enzyme-linked immune detection method of the O-shaped Guangxi Strain of quantitative determination of specificity foot and mouth disease (O/GX/09-7 strains) The linear graph of directrix curve.

Specific embodiment

The concept of protein group (Proteome) is proposed by Marc Wilkins at first, is referred to by a genome (Genome) Or cell, an all proteins (protein) for tissue expression.Two dimensional gel electrophoresis (2-DE) are at present using the most The method of extensive research proteomics.2-DE technologies utilize isoelectric point of protein and molecular weight difference by a genome Interior various protein make a distinction, and the electrophoresis pattern for comparing different serotypes or different strains by software can hold very much Change places and find their discrepant albumen.Based on the comparative study to a large amount of biotechnology, inventor confirms that 2-DE technologies can be Discrepant epitope is searched out between each serotype of foot and mouth disease viruses 146S antigens or each strain reliable technology is provided Means.

Therefore, the present invention select first using 2-DE technologies search out the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) 146S resist Former discrepant albumen, and the antibody of the O-shaped Guangxi Strain specificity of foot and mouth disease is prepared using classical hybridoma technology, and then develop One kind is capable of the ELISA detection kit of the O-shaped Guangxi Strain of specific detection foot and mouth disease (O/GX/09-7 strains).The test kit by In the higher monoclonal antibody of specificity is employed, can not only be by foot and mouth disease O-shaped Guangxi Strain (O/GX/09-7 strains) and Asia 1 Type (JSL/06), A types (A/WH/09) are distinguished, more can be by O-shaped Guangxi Strain (O/GX/09-7 strains) and other O-shaped strain area Separate, such as Xinjiang Strain (O/Mya98/XJ/2010 strains), 98 plants of Burma (O/Mya98/BY/2010), Jiamusi strain (O/JMS/ 2000), OZK strains (OZK/93 strains).This is just O-shaped Guangxi Strain (O/GX/09-7 in foot and mouth disease unit price or multivalence combined vaccines Strain) independent quantitative providing may.

In following embodiments, method therefor is conventional method if no special instructions, and concrete steps can be found in： 《Molecular Cloning:A Laboratory Manual》(Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor)。

The percent concentration is mass/mass (W/W, unit g/100g) percent concentration, matter if no special instructions Amount/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percent concentration.

The acquirement approach of the various biomaterials described in embodiment is only to provide a kind of approach of experiment acquisition to reach To specifically disclosed purpose, the restriction to biological material source of the present invention should not be become.In fact, used biomaterial Source be it is extensive, it is any keep on the right side of the law the biomaterial that can obtain with moral ethics can according to embodiment in carry Show that replacement is used.

Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and specific Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.

Embodiment 1, the enzyme linked immunological quantitative determination reagent kit for preparing the O-shaped Guangxi Strain (O/GX/09-7 strains) of foot and mouth disease

First, prepare the monoclonal antibody specific of the O-shaped Guangxi Strain (O/GX/09-7 strains) of foot and mouth disease

The monoclonal antibody specific of the O-shaped Guangxi Strain (O/GX/09-7 strains) of foot and mouth disease is prepared, concrete grammar includes following step Suddenly：

1st, prepare O-shaped Guangxi Strain (O/GX/09-7 strains) specific antigen of foot and mouth disease

Take the O-shaped Guangxi Strain of the foot and mouth disease after purified concentration (O/GX/09-7 strains) purifying antigen and (clever biologics are protected by Jin Yu The commodity that company limited provides), concentration is more than 100 μ g/mL, carries out 2-DE electrophoresis, and method is as follows：

First to isoelectrofocusing, concrete operation step：1) the aquation sample-loading buffer of -20 DEG C of freezen protectives is taken from refrigerator (5 μ l/mL of Urea 480mg/mL, CHAPS 20mg/mL, IPG Buffer, DTT 2.8mg/mL, trace bromophenol blue), puts room Temperature dissolving.2) the prefabricated adhesive tape of IPG of -20 DEG C of freezen protectives is taken from refrigerator, is placed 10 minutes in room temperature.3) along focus on disk or The edge of aquation disk bracket groove right linear addition sample to a left side.At groove two ends, each 1cm or so should not be loaded, middle sample liquid one Surely to link up.4) after all of protein example all has been added to focus in disk or aquation disk, with tweezers removal gently Protective layer in prefabricated IPG adhesive tape.5) distinguish the both positive and negative polarity of adhesive tape, lightly by IPG adhesive tape glue be placed face down on focusing disk or In aquation disk on sample solution so that the positive pole of adhesive tape is corresponding to the positive pole for focusing on disk.Guarantee that adhesive tape is contacted with electrode seal.6) 2-3mL mineral oil is covered in every adhesive tape, the evaporation of liquid during isoelectrofocusing is prevented.Mineral oil need to be slowly added, Along adhesive tape, mineral oil is made drop by drop slowly to be added in plastic support film.7) to good positive and negative electrode, close the lid, the electricity such as setting Focalizer, program are shown in Table 1.

The selection (18cmIPG adhesive tape) of the isoelectrofocusing parameter of 1 preparative 2-DE of table

Balance：By the IPG adhesive tape after isoelectrofocusing be put into 20mL balance liquids (1.5M Tris-HCl 1.34mL, Urea14.14g, Glycerol 12mL, SDS 0.8g, trace bromophenol blue) in, 15 minutes × 2 are vibrated on shaking table.Wherein, During once balance, 100mg DTT are added in 20mL balance liquids；And when balancing for the second time, 0.9g is added in 20mL balance liquids Iodoacetamide.

Second to SDS-PAGE, concrete operation step：1) prepare 10% acrylamide gel.2) by the IPG after balance The unnecessary balance liquid of adhesive tape filter paper absorption (adhesive tape is erected on filter paper, in order to avoid loss albumen or damage gel surface), then One end that adhesive tape is clamped with tweezers makes glue surface soak end completely in 1 × electrophoretic buffer (Tris-Glycine-SDS, Tris 6.04g, glycine 28.8g, SDS 2g are dissolved in 2000mL water) in.3) IPG adhesive tape is moved to the top of polyacrylamide gel, With the syringe needle of tweezers, spatula or tack, lightly adhesive tape is pushed down on, be allowed to be connect with polyacrylamide gel glue surface completely Touch.It is careful not to any bubble is produced below adhesive tape.When adhesive tape is pushed away with tweezers, spatula or tack syringe needle, it should be noted that be The support membrane at the gel back side is promoted, glue surface should not be encountered.4) it is last, fixed with 0.8% agarose closing, place 5min, make Low melting-point agarose sealing liquid thoroughly solidifies.After low melting-point agarose sealing liquid solidifies completely.Gel is transferred to into electrophoresis tank In.5) after electrophoresis tank adds electrophoretic buffer, switch on power, the low current or low-voltage when initial treats sample complete IPG adhesive tape is walked out, after concentration is into a line, then high current, stop electricity by when bromophenol blue indicator reaches bottom margin Swimming.6) after electrophoresis terminates, layer glass is gently pried open, takes out gel, and corner cut and (wear glove, prevent from polluting glue with marking Face).7) dye.

Gel after dyeing is placed on GS-710 scanning densitometers, the PDQUEST 2D softwares of the image after scanning (business software) is analyzed, and the protein spot of selected section matching is compared.5 abundant foot and mouth disease of final choice are O-shaped Guangxi Strain (O/GX/09-7 strains) specific antigen band, molecular weight is about as immunogen after 20-30KDa, drying, grinding Use.

2nd, prepare the hybridoma of the monoclonal antibody specific of the O-shaped Guangxi Strain (O/GX/09-7 strains) of secretion foot and mouth disease And obtain antibody purification

Comprise the following steps：

1) with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) specific antigen of step 1 acquisition as immunogen immune Balb/C mices (are purchased from Military Medical Science Institute's Experimental Animal Center), altogether immunity 4 times, time interval 14 days, 3 times immunity side Formula is multiple spot subcutaneous inoculation, is lumbar injection immunity, every 10 μ g of injected in mice for the last time.

2), after immune 4 times, mouse tail hematometry potency is taken, after potency is more than 50,000, the splenocyte of separating immune animal, By itself and growth conditions good myeloma S/P20 cell fusion, hybridoma is obtained with the screening of HAT selective mediums.

3) it is with indirect ELISA method screening specific hybrid oncocyte clone, final to obtain 3 specific positive clones.

With indirect ELISA method screening specific cell clone, concrete operation step：Take Guangxi Strain (O/GX/09-7 strains), Xinjiang Strain (O/Mya98/XJ/2010 strains), 98 plants of Burma (O/Mya98/BY/2010), Jiamusi strain (O/JMS/2000), OZK The strain 146S antigen purifications such as strain (OZK/93), Type Asia 1 (JSL/06) and A types (A/WH/09) inactivation liquid, uses 10mM PBS pH7.2-7.4(Na₂HPO₄·12H₂O, 2.9g；NaH₂PO₄·2H₂O, 0.296g；NaCl, 8.5g；Proclin 300,0.6mL； Distilled water, is dissolved to 1000mL；Adjustment pH to 7.2-7.4) the coating working solution of 2.5 μ g/mL is diluted to respectively, it is loaded with multiple tracks Device is added separately in 96 hole COSTAR ELISA Plate, adds 110 μ l, every kind of virus antigen to be at least coated with 2 pieces of microwell plates, put per hole In 2-8 DEG C overnight (16-24 hours), take out microwell plate and discard liquid in hole, pat dry in absorbent paper.With multiple tracks sample injector to every 300 μ l of Block buffer are added in hole, and (1000mL confining liquids include NaH₂PO₄·2H₂O 0.296g, Na₂HPO₄·12H₂O 2.9g, casein 10g, Proclin 3000.6mL, distilled water are dissolved to 1000mL, and measure pH value is 7.2-7.4), room temperature is put Put 3 hours.Confining liquid is got rid of, is patted dry in absorbent paper.Room temperature removal moisture drying carried out envelope after 24 hours, put 2-8 DEG C of preservation standby With.

Take 100 μ L of cells and supernatant and add to and be coated with the ELISA Plate of virus antigen, 37 DEG C are reacted 30 minutes, with washing Liquid (formula：Na₂HPO₄·12H₂O 58g, NaH₂PO₄·2H₂O 5.92g, NaCl 170g, Tween-201.0mL, Proclin 3000.6mL, is dissolved to 1000mL, adjustment pH value to 7.2-7.4 with distilled water；20 are diluted with distilled water using during cleaning mixture Times.) board-washing 5 times, then per hole in add concentration to be 1：2000 rabbit anti-mouse igg-HRP labels (being purchased from KPL companies of the U.S.), Its diluent be enzymic-labelled antibody diluent (trishydroxymethylaminomethane 12.1g, Sodium Chloride 8.8g, BSA 10g, Proclin3001.0mL, Food Red 0.1g, is dissolved to 1000mL with distilled water, adjusts pH value to 7.5 with 6M hydrochloric acid.), 37 DEG C are anti- Answer 30 minutes, it is with cleaning mixture board-washing 5 times, after patting dry, each in every hole to add substrate A liquid (formula of chromogenic substrate A liquid is：Three Hydrated sodium acetate 8.2g, glacial acetic acid 1.2mL, the hydrogen peroxidase 10 .27mL that concentration is 30%, are dissolved to 1000mL with distilled water, adjust Whole pH value is to 5.0-5.6.) and B liquid (formula of chromogenic substrate B liquid is：Citric acid 1.92g, sodium ethylene diamine tetracetate 0.372g, 10mol/L sodium hydroxide solution 0.3mL, glycerol 50mL, tetramethyl biphenyl amine hydrochlorate 0.23g, are dissolved to distilled water 1000mL, adjustment pH value to 2.5-3.5.) each 1 drop, room temperature lucifuge reaction 15 minutes.Terminate liquid (1000mL is added in every hole In contain 98% sulphuric acid 27.8mL) 1 drop.Vibration 10 seconds.With 630nm as reference wavelength, the absorbance at 450nm is measured.With It is positive criterion that absorbance is more than 2.1 times of negative control (negative control is cell culture fluid).

First with all of culture supernatant of screening micropore plate of Guangxi Strain (O/GX/09-7 strains) virus 146S antigen coats, from 25 plants of positive colonies of middle acquisition, then verify the specificity of this 25 plants of positive colonies, finally with remaining viral coated microwell plate 3 plants of specific cell clones are obtained, they only have strong signal reaction with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains), and Not with Xinjiang Strain (O/Mya98/XJ/2010 strains), 98 plants of Burma (O/Mya98/BY/2010), Jiamusi strain (O/JMS/2000), The strains such as OZK strains (OZK/93), Type Asia 1 (JSL/06) and A types (A/WH/09) react.This three plants of positive colonies are compiled respectively Number be 7G3,2B5,6E1.

4) hybridoma is cultivated, is separated and purification from the ascites fluid of cell culture fluid or inoculation hybridoma animal Go out the monoclonal antibody specific of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains).

Prepared by ascites and the purification process of monoclonal antibody specific is：Take 7G3,2B5 and 6E1 these three foot and mouth disease O-shaped Guangxi Strain (O/GX/09-7 strains) specific positive is cloned, and cell is collected by centrifugation after cell grows to culture bottle 50-80%, is injected Balb/c mices that Jing norphytanes (be purchased from Sigma) are processed (7-10 days before injection cell, every injected in mice norphytane 0.5mL, Its purpose is to reduce the immunologic function of mice, improve antibody production), every mice about injects 10⁵-10⁶Individual cell.7-10 Ascites is collected after it.With the affine filler of Protein G (being purchased from GE companies, article No. is 17-0618-02) according to GE Products description Affinity purification method monoclonal antibody purification.The absorbance of 280nm is measured with ultraviolet spectrophotometer, is removed with the absorbance With empirical coefficient 1.48, concentration of the result for obtaining for monoclonal antibody, unit is mg/mL.The monoclonal antibody of 7G3 secretions is dense Spend for 2.16mg/mL, the MAb concentration of 2B5 secretions is 5.40mg/mL, the MAb concentration of 6E1 secretions is 1.52mg/mL.For ease of the comparative analysiss tested below, different antibody clonings use 10mM PBS pH7.2-7.4 (Na₂HPO₄·12H₂O 2.9g, NaH₂PO₄·2H₂O 0.296g, NaCl 8.5g, Proclin 3000.6mL, distilled water are fixed molten To 1000mL, pH to 7.2-7.4 is adjusted) it is diluted to 1.5mg/mL.

3rd, the pairing mode of antibody purification confirms

1) coating microwell plate is prepared with the monoclonal antibody specific of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains)

By the 10mM PBS pH7.2-7.4 (Na of monoclonal antibody specific after purification₂HPO₄·12H₂O 2.9g, NaH₂PO₄·2H₂O 0.296g, NaCl 8.5g, Proclin 3000.6mL, are dissolved to 1000mL with distilled water, and adjustment pH is extremely The coating working solution of 1-10 μ g/mL 7.2-7.4) is diluted to, is added separately in 96 hole COSTAR ELISA Plate with multiple tracks sample injector, 110 μ l, every kind of antibody are added at least to be coated with 2 pieces of microwell plates per hole.It is placed in 2-8 DEG C overnight (16-24 hours).Take out microwell plate to abandon Liquid in hole is removed, is patted dry in absorbent paper.300 μ l of Block buffer are added in every hole with multiple tracks sample injector, and (1000mL is closed Liquid includes NaH₂PO₄·2H₂O0.296g, Na₂HPO₄·12H₂O 2.9g, casein 10g, Proclin 3000.6mL, with double steamings Water is dissolved to 1000mL, and measure pH value is 7.2-7.4), room temperature is placed 3 hours.Confining liquid is got rid of, is patted dry in absorbent paper.Room Warm removal moisture drying carried out vacuum sealing bag after 24 hours, puts 2-8 DEG C and saves backup.

2) prepare the specific monoclonal of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) of horseradish peroxidase-labeled Antibody

By monoclonal antibody specific glutaraldehyde oxidizing process and the Radix Cochleariae officinalises of foot and mouth disease O-shaped Guangxi Strain (O/GX/09-7 strains) Peroxidase (HRP) is coupled, and is fully dialysed with PBS, plus equivalent high-quality glycerol, and less than -20 DEG C preserve.Concrete operation step It is as follows：

1. 5mg HRP are dissolved in the PBS (formula of 0.1mol/L pHs 6.8 of the 0.2mL containing 1.25% glutaraldehyde： KH₂PO₄6.805g, Na₂HPO₄·12H₂O 17.9g, NaCl 8.5g, constant volume adjust pH6.8 in 1000mL distilled waters) in, Put room temperature 18 hours, fully dialysis removes unnecessary glutaraldehyde；

2. plus normal saline is to 1mL, it is subsequently adding the spy of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) of 2.5mg purification 1mol/L carbonate buffer solution (the formula of specific monoclonal antibodies and 0.1mL pH9.6：Take 1M sodium carbonate 3mL and 1M bicarbonates Sodium 7mL mixes), mix and place 24 hours after 2-8 DEG C of refrigerator；

3. the lysine solution of 0.1mL 0.2mol/L is added (to claim lysine 29.2mg to be dissolved in 0.01M pH9.5 carbonic acid to delay Rush in liquid 1mL), room temperature is placed 2 hours；

4. pH7.20.15mol/L PBS (NaCl 8.5g, Na are used₂HPO₄·12H₂O 2.89g, KH₂PO₄0.2g, constant volume in 1000mL, adjusts pH7.2) fully dialyse, precipitation is removed by centrifugation, supernatant is enzyme conjugates.Use enzyme marker diluent (trishydroxymethylaminomethane 12.1g, Sodium Chloride 8.8g, BSA 10g, Proclin3001.0mL, Food Red 0.1g, use distilled water 1000mL is dissolved to, and pH value is adjusted to 7.5) dilution is the working solution of enzyme marker according to a certain percentage with 6M hydrochloric acid.

3) pair verification between three specific antibodies

(edited referring to Li Jinming using Checkerboard titration experiment《Clinical Enzyme immunoassay technology》) determine three plants of monoclonal antibodies Pairing mode.

2 parts of samples are selected, a copy of it concentration is 10ng/mL；Portion concentration is 2ng/mL in addition.The different antibody of combination To experiment, two-step method is taken：The first step adds sample, and 37 DEG C are reacted 60 minutes；After board-washing 5 times, enzyme marker working solution is added, 37 DEG C are reacted 30 minutes.37 DEG C of substrate is added to develop the color after board-washing 15 minutes, it is each to add the drop terminating reaction of terminate liquid 1.In microplate reader On, with 630nm as reference wavelength, measure the absorbance at 450nm.With the absorbance of 10ng/mL samples divided by 2ng/mL's Absorbance obtains coefficient b (see the table below).B values are bigger, show that the sensitivity for detecting is better.Therefore preferably 7G3 is anti-as capture Body, 2B5 are used as detection antibody；Next to that 2B5 is used as capture antibody, 6E1 is used as detection antibody.In the original of economy Then, 7G3 may be selected 2.5 μ g/mL as capture antibody.

2nd, coating microwell plate is prepared with the monoclonal antibody specific of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains)

By the 10mM PBS pH7.2-7.4 (Na of monoclonal antibody specific after purification₂HPO₄·12H₂O 2.9g, NaH₂PO₄·2H₂O 0.296g, NaCl 8.5g, Proclin 3000.6mL, distilled water are dissolved to 1000mL, and adjustment pH is extremely The coating working solution of 2.5 μ g/mL 7.2-7.4) is diluted to, is added separately in 96 hole COSTAR ELISA Plate with multiple tracks sample injector, 110 μ l are added per hole, in 2-8 DEG C overnight (16-24 hours).Take out microwell plate and discard liquid in hole, pat dry in absorbent paper.With Multiple tracks sample injector adds 300 μ l of Block buffer in every hole, and (1000mL confining liquids include NaH₂PO₄·2H₂O 0.296g, Na₂HPO₄·12H₂O 2.9g, casein 10g, Proclin 3000.6mL, distilled water are dissolved to 1000mL, determine pH value and are 7.2-7.4), room temperature is placed 3 hours.Confining liquid is got rid of, is patted dry in absorbent paper.Room temperature removal moisture drying carried out vacuum after 24 hours Envelope, posts label, puts 2-8 DEG C and saves backup.

3rd, prepare the determination of enzyme labelled antibody and enzyme labelled antibody concentration

1st, prepare the specific monoclonal of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) of horseradish peroxidase-labeled Antibody

By monoclonal antibody specific glutaraldehyde oxidizing process and the Radix Cochleariae officinalises of foot and mouth disease O-shaped Guangxi Strain (O/GX/09-7 strains) Peroxidase (HRP) is coupled, and is fully dialysed with PBS, plus equivalent high-quality glycerol, and less than -20 DEG C preserve.Concrete operations are walked It is rapid as follows：

1) 5mg HRP are dissolved in the PBS (formula of 0.1mol/L pHs 6.8 of the 0.2mL containing 1.25% glutaraldehyde： KH₂PO₄6.805g, Na₂HPO₄·12H₂O 17.9g, NaCl 8.5g, constant volume adjust flat H6.8 in 1000mL distilled waters) in, Put room temperature 18 hours, fully dialysis removes unnecessary glutaraldehyde；

2) add normal saline to 1mL, be subsequently adding the spy of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) of 2.5mg purification The 1mol/L carbonate buffer solutions of specific monoclonal antibodies and 0.1mL pH9.6 (take 1M sodium carbonate 3mL and 1M sodium bicarbonate 7mL Mixing), mix and place 24 hours after 2-8 DEG C of refrigerator；

3) lysine solution of 0.1mL 0.2mol/L is added (to claim lysine 29.2mg to be dissolved in 0.01M pH9.5 carbonic acid to delay Rush in liquid 1mL), room temperature is placed 2 hours；

4) with pH7.20.15mol/L PBS (NaCl 8.5g, Na₂HPO₄·12H₂O 2.89g, KH₂PO₄0.2g, constant volume in 1000mL, adjusts pH7.2) fully dialyse, precipitation is removed by centrifugation, supernatant is enzyme conjugates.

2nd, monoclonal antibody specific of the Euplotes woodruffi of improvement to the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) Labelling alkali phosphatase (ALP)

1) the monoclonal antibody specific 4.0mg of ALP 2.0mg and the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) is taken, point It is not dissolved in normal saline, mixes, final volume is controlled in 0.50mL；

2) glutaraldehyde of 0.50mL 1% is added, room temperature magnetic agitation after 15 minutes, react 4 hours by lucifuge；

3) the ethanolamine 0.10mL of 1.0mol/L, incubation at room temperature 2 hours are added；

4) add magnetic stirrer over night (16-24 hours) to dialyse with PBS under the conditions of 2-8 DEG C, change dialysis solution 3-4 time；

5) dialysis product is moved into into reagent bottle addition equal-volume glycerol, mixes, add 1%BSA, closed freezen protective is standby With.

3rd, prepare enzyme labelled antibody diluent

Formula：Trishydroxymethylaminomethane 12.1g, Sodium Chloride 8.8g, BSA 10g, Proclin3001.0mL, Food Red 0.1g, is dissolved to 1000mL with distilled water, adjusts pH value to 7.5 with 6M hydrochloric acid.

4th, (edited referring to Li Jinming using Checkerboard titration experiment《Clinical Enzyme immunoassay technology》) determine the work of enzyme labelled antibody Make concentration range.

2 parts of samples are selected, a copy of it concentration is 10ng/mL, and a concentration is 2ng/mL in addition.The different antibody of combination To experiment, two-step method is taken：The first step adds sample, and 37 DEG C are reacted 60 minutes；After board-washing five times, enzyme marker work is added Liquid, 37 DEG C are reacted 30 minutes.37 DEG C of substrate is added to develop the color after board-washing 15 minutes, it is each to add the drop terminating reaction of terminate liquid one.In enzyme On mark instrument, with 630nm as reference wavelength, the absorbance at 450nm is measured.With the absorbance of 10ng/mL samples divided by 2ng/ The absorbance of mL obtains coefficient b.Select the Guangxi Strain sample OD that coefficient b is larger, background OD values are less than 0.050 and 64ng/mL Dilution ratio of dilution ratio of the value more than 2.0 as enzyme marker working solution.

Enzyme marker working solution is prepared according to the dilution ratio, subpackage, every bottle of 10.5mL post label, be placed in 2-8 DEG C it is standby With.

3rd, prepare O-shaped Guangxi Strain (O/GX/09-7 strains) positive quality control product of foot and mouth disease

For convenience of result observation, positive quality control of the test kit also including the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) Product, the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) the 146S antigen purifications inactivation liquid that concretely foot-and-mouth disease vaccine factory produces, The purification of preferably Jinyu Baoling Biology Drugs Co., Ltd's production inactivates liquid.The raw material of positive quality control product be standard level, purity It is not less than 80%.With O-shaped Guangxi Strain (O/GX/09-7 strains) positive quality control product of sample diluting liquid dilution foot and mouth disease to 8-64ng/mL, Every bottle of 1.0mL is distributed into, label is posted, -20 DEG C is placed in and is saved backup.

4th, prepare sample diluting liquid

The formula of sample diluting liquid is：Na₂HPO₄·12H₂O 2.9g, NaH₂PO₄·2H₂O 0.296g, NaCl 8.5g, Proclin 3000.6mL, casein 10g, new-born calf serum 50mL are dissolved to 1000mL, adjustment pH value to 7.2- with distilled water 7.4.Every bottle of 25mL of subpackage, posts label, be placed in 2-8 DEG C it is standby.

5th, prepare cleaning mixture

The formula of cleaning mixture is：Na₂HPO₄·12H₂O 58g, NaH₂PO₄·2H₂O 5.92g, NaCl 170g, Tween- 201.0mL, Proclin 3000.6mL, is dissolved to 1000mL, adjustment pH value to 7.2-7.4 with distilled water；Every bottle of 25mL of subpackage, Label is posted, room temperature is placed in standby.

6th, prepare assay chromogenic substrate solution

1st, the assay chromogenic substrate solution of horseradish peroxidase (HRP)

Assay chromogenic substrate solution includes A liquid, B liquid and terminate liquid, wherein, the formula of chromogenic substrate A liquid is：Sodium acetate trihydrate 8.2g, glacial acetic acid 1.2mL, the hydrogen peroxidase 10 .27mL that concentration is 30%, are dissolved to 1000mL with distilled water, and adjustment pH value is extremely 5.0-5.6；

The formula of chromogenic substrate B liquid is：Citric acid 1.92g, sodium ethylene diamine tetracetate 0.372g, 10mol/L sodium hydroxide Solution 0.3mL, glycerol 50mL, tetramethyl biphenyl amine hydrochlorate 0.23g are dissolved to 1000mL with distilled water, and adjustment pH value is extremely 2.5-3.5。

A liquid is pressed into 1 with B liquid using front：1 ratio mixes.

Every bottle of 7mL of subpackage, posts label, be placed in 2-8 DEG C it is standby.Wherein A liquid White-opalescent plastic dropping bottle；B liquid is adopted With black or brown opaque plastics drop bottle；Terminate liquid adopts colourless transparent plastic drop bottle.(have purchased from the bright magnificent industry of Shenzhen gold Limit company).

2nd, the assay chromogenic substrate solution of alkali phosphatase (ALP)

Formula is：PNPP 1g, diethanolamine 100mL, anhydrous magnesium chloride 47.5mg, distilled water 900mL, pH9.8.

Every bottle of 12mL of subpackage, posts label, be placed in 2-8 DEG C it is standby.

7th, prepare terminate liquid

The termination formula of liquid of horseradish peroxidase：The sulphuric acid of 27.8mL 98% is added in per 1000 milliliters of distilled waters.

The termination formula of liquid of alkali phosphatase：The sodium carbonate of 53g is added in per 1000mL distilled waters.

8th, assemble test kit

By said components subpackage, 3 parts are randomly selected, after every methodology index assay approval, be assembled into foot and mouth disease O The enzyme linked immunological quantitative determination reagent kit (attached operation instructions) of type Guangxi Strain (O/GX/09-7 strains).

The user of the enzyme linked immunological quantitative determination reagent kit of the O-shaped Guangxi Strain of embodiment 2, foot and mouth disease (O/GX/09-7 strains) Method

Detection is carried out to sample with the test kit of the present invention to comprise the following steps：

1) it is dilute with the purification inactivation liquid of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) 146S and the sample of test kit offer Release liquid and prepare standard substance, standard concentration is：0、2、4、8、16、32、64ng/mL.Testing sample is equally dilute with sample diluting liquid Release 4-8 gradient.

2) lath required for taking out, remaining lath load in valve bag, are placed in 2-8 DEG C of preservation.

3) in hole 100 μ l standard substance series dilution point, positive quality control, specimen is added to be serially diluted a little respectively.Gently shake The dynamic 10-20 seconds mix, and stick plate patch.

4) it is placed in 37 DEG C of incubators and incubates 60 minutes.Liquid in hole is deducted, and 5-7 is washed with washing liquid (being diluted with first 20 times) It is secondary.Pat dry on clean napkin or towel, remove the moisture of residual as far as possible.

5) per the enzyme marker that 100 μ l are added in hole, gently vibrating the 10-20 seconds mixes, sticks plate and pastes.

6) it is placed in 37 DEG C of incubators and incubates 30 minutes.Deduct liquid in hole, and with wash liquid 5-7 time.In clean paper Pat dry on towel or towel, remove the moisture of residual as far as possible.

7) add per hole assay chromogenic substrate solution (assay chromogenic substrate solution (A liquid, B liquid each 1 drop) of horseradish peroxidase (HRP) or The assay chromogenic substrate solution (2 drop) of alkali phosphatase (ALP)), the 10-20 seconds are gently vibrated, 37 DEG C of lucifuges is placed in and is reacted 15 minutes, per hole 1 is added to drip terminate liquid (terminate liquid (1 drop) of the terminate liquid or alkali phosphatase (ALP) of horseradish peroxidase (HRP)), gently Vibration is mixed several times, makes solution be changed into yellow liquid completely, in 30 minutes, measures absorbance (OD) (horseradish peroxidase (HRP) with 630nm as reference wavelength, measure 450nm place absorbance, alkali phosphatase (ALP) measurement 405nm extinction Degree).

8) analysis result：Calculate the concentration of O-shaped Guangxi Strain (O/GX/09-7 strains) antigen of foot and mouth disease.

Step 8) in interpretation of result method can be：Respectively standard concentration and correspondence OD absorbances are taken the logarithm, Standard curve is set up on double logarithmic chart, with standard concentration as abscissa, it is bent that OD absorbances draw standard for vertical coordinate Line, as shown in figure 1, the O-shaped Guangxi Strain (O/GX/ of foot and mouth disease in sample is found on standard curve with each specimen absorbance to be measured 09-7 strains) antigen concentration.If multiple dilution factors, meansigma methodss are calculated.

9) print test results report.

Above detection process about needs to take 2 hours, can at most detect nearly 90 parts of samples in once testing.

Test example 1, Precision Experiment

First, precision in analyzing

The purification inactivation liquid of foot and mouth disease O-shaped Guangxi Strain (O/GX/09-7 strains) 146S (is protected into spirit by Jin Yu with sample diluting liquid Biologics company limited provides) prepare low concentration (concentration is 15ng/mL), high concentration (concentration is 50ng/mL) positive quality control Product, determine their absorbance in a collection of experiment with the detection method of the test kit and embodiment 2 of embodiment 1, each Quality Control is parallel to do 10 holes, calculates precision in the analysis of test kit of the present invention.

As a result it is as shown in table 2, it can be seen that the interior precision of test kit of the present invention：CV%<15.0%, show that test kit is examined Survey result reproducible, testing result is stable, reliable.

Precision Analyze result in table 2

2nd, precision between analyzing

Ibid, low, high concentration quality-control product is prepared with sample diluting liquid, their concentration is determined respectively at different experiments day Value, continuous 5 days, 2 repetitions every time, 10 results, calculate precision between their analysis altogether, analyze test kit of the present invention Precision between analysis.

As a result it is as shown in table 3, it can be seen that precision between the analysis of test kit of the present invention：CV%<20%, even if showing Different time detects that as a result also very well, testing result is stable, reliable for repeatability.

3 Precision Analyze results of table

Test example 2, sensitivity for analysis is tested

Negative control (sample diluting liquid i.e. in test kit) is taken, 10 multiple holes is set, is entered with reference to the detection method of embodiment 2 Row measurement, calculates the sensitivity that its meansigma methods is this test kit plus the corresponding concentration value of the OD values that twice standard deviation is asked, Respectively with three batch test kit detection sensitivities, using maximum therein as the sensitivity for analysis of this test kit.

As a result 3 measurings of Jing, show that the sensitivity of this test kit is 1.2ng/mL, show the test kit inspection of the present invention Survey sensitivity high, even if the testing sample concentration as little as level of 1.2ng/mL can also be detected.

Test example 3, accuracy is tested：

Sample-adding recovery experiment：Sample (the Jin Yubao that concentration known is 100ng/mL is separately added into in 1mL sample diluting liquids The O/GX/09-7 strains 146S inactivation refined solutions of clever biologics company limited production, its concentration is 103.96 μ g/mL, uses reagent Sample diluting liquid in box is diluted to 100ng/mL) 50 μ l, 100 μ l, 200 μ l, 400 μ l, obtain product to be tested, then with a collection of Number test kit which part test kit is stored always to be detected to the O/GX/09-7 strain 146S antigen concentrations in product to be tested In 2-8 DEG C, also part kit has deposited 7 days in 37 DEG C.

As a result as shown in table 4, show that theoretical concentration value and the bias of measured concentration value are less than ± 10%, in various dose The equal coincidence detection of the response rate of the done sample of level requires (100 ± 10%).

4 test kit accuracy testing result of table

Test example 4, stability experiment

37 DEG C of the test kit of the present invention is placed 3,7,10 days, then detectable concentration is the positive quality control of 15,50ng/mL Product, 64ng/ in meter sensitivity (n=10), precision (n=10), the linearly dependent coefficient of standard curve and standard curve The ratio of the OD values of mL and the OD values of 0ng/mL.

As a result it is as shown in table 5, it can be seen that test kit is after 37 DEG C of stability assessments 10 days, although the coefficient of variation (CV%) It is increased slightly, but still is below 10%；The measured value of low value Quality Control and high level Quality Control is substantially unchanged；Sensitivity is also without substantially change Change.Show that the stability of test kit can meet needs, performance is basically unchanged within the effect duration of test kit.

The indices of the test kit after the placement 3 days, 7 days and 10 days of 537 DEG C of table

Test example 5, specificity and capacity of resisting disturbance experiment

By the foot and mouth disease of 64ng/mL O-shaped Guangxi Strain (O/GX/09-7 strains) antigen (Jinyu Baoling Biology Drugs Co., Ltd The concentration of production is the 146S purification inactivation liquid of 103.96 μ g/mL, is diluted with the sample diluting liquid in test kit) 8mL point Into 8 parts of each 1mL, the BHK host proteins (for the host cell proteins of O/GX/09-7 strains) of per part of each addition 1mL 5000ng/mL, Foot and mouth disease Xinjiang strain antigen 1 mL (O/Mya98/XJ/2010 strains) of 5000ng/mL, foot and mouth disease Jiamusi poison of 5000ng/mL Strain 1mL (O/JMS/2000), foot and mouth disease Burma strain antigen 1 mL (O/Mya98/BY/2010) of 5000ng/mL, 5000ng/mL Foot and mouth disease OZK strain antigen 1 mL (OZK/93 strains), foot and mouth disease Type Asia 1 strain 1mL (JSL/06) of 5000ng/mL, Foot-and-mouth disease a type strain 1mL (A/WH/09) and sample diluting liquid (doing negative control) 1mL of 5000ng/mL obtains testing sample, O/GX/09-7 strain 146S antigen concentrations in testing sample are detected simultaneously.

As a result it is as shown in table 6, it can be seen that compared with theoretical value, whether other O-shaped strains such as Xinjiang Strain, Burma 98 plants, Jiamusi strain, OZK strains, or Type Asia 1 and A types, do not affect the detection of Guangxi Strain (O/GX/09-7 strains) antigen, table The antibody specificity adopted by bright test kit is very high, can be completely used for typing (distinguish O-shaped with Type Asia 1 and A types), Can distinguish and be all O-shaped different strains (O/GX/09-7 strains are distinguished from other O-shaped strain).

6 test kit specific detection result of table (O/GX/09-7 strain 146S antigen concentration ng/mL)

Above result of the test shows the enzyme linked immunological quantitative determination of the O-shaped Guangxi Strain of foot and mouth disease of the present invention (O/GX/09-7 strains) The precision of test kit, accuracy, sensitivity, stability and specificity comply fully with requirement.

Embodiment 3, the enzyme linked immunological kit of the O-shaped Guangxi Strain (O/GX/09-7 strains) of use foot and mouth disease of the present invention are quantitatively surveyed Determine reagent detection sample

With the O-shaped Guangxi Strain (O/GX/09-7 strains) of using method detection foot and mouth disease of the test kit and embodiment 2 of embodiment 1 O/GX/09-7 strain 146S antigen concentrations (Quality Control purpose) in the sample and finished product vaccine of different production phases.These samples come From Jinyu Baoling Biology Drugs Co., Ltd.Wherein three finished product vaccines of serial number 5,6,7 are examined after breakdown of emulsion as follows Survey：The n-amyl alcohol of 20mL is added in 100mL vaccines, static 30 minutes after stirring 1 minute so as to which upper lower leaf, water intaking are mutually detected.

It is as a result as shown in table 7, it can be seen that：The test kit not only can detect culture fluid, inactivation liquid, refined solution and concentration Liquid, it is also possible to detect various finished product vaccines.That is, the links in inactivated foot-and-mouth disease vaccine production can be using this Test kit is detecting for quality control and monitoring.Wherein can detect containing O/GX/09-7 strains, does not contain O/GX/09- Even if 7 plants also can't check containing other O-shaped strains of foot and mouth disease.Show that the test kit not only can typing (the O-shaped and Asia of differentiation 1 type, A types), it is also possible to distinguish O-shaped different strains.Further disclose the specificity of the adopted antibody of this test kit and resist dry Disturb ability.

7 sample detection result of table

The comparison of embodiment 4, test kit of the present invention and its detection method and sucrose density gradient centrifugation method

To verify the actual property of application of test kit of the present invention, now contrasted with sucrose density gradient centrifugation method.

5 parts of Guangxi Strain (O/GX/09-7 strains) samples are detected with the test kit of the present invention, sample protects clever biological medicament by Jin Yu Product company limited provides, and Jing sucrose density gradient centrifugations are quantitative, and O/GX/09-7 strain 146S antigen concentration ranges are from 2.0- 104μg/mL。

As a result it is as shown in table 8, it can be seen that two methods coincidence rate is high, the fitted trend line in EXCEL, coefficient R² Reach 0.9835.

The comparative result that 8 distinct methods of table are detected to O/GX/09-7 strain 146S antigen concentrations

Sample number into spectrum	Test kit (μ g/mL) of the present invention	Sucrose density gradient centrifugation (μ g/mL)
			1	95.6	103.96
2	2.32	2.08
			3	25.4	29.2
4	70.7	65.3
			5	34.3	37.5

With reference to other embodiments of the invention, it can be deduced that such as draw a conclusion：

1st, test kit of the invention only needs to 2 hours and can at most detect 90 samples, and does not need expensive instrument Equipment, and sucrose density gradient centrifugation then needs 72 hours, and every supercentrifuge can only detect 5 parts of samples, it is such Detection speed seriously constrains the production of foot-and-mouth disease vaccine and quality control.

2nd, kit assay sensitivity of the invention can reach the level of 1.20ng/mL, the spirit of Sucrose density centrifugation method Sensitivity is about 100-200ng/mL (see patent《The ultraviolet quantitative method detection foot and mouth disease antigen 1 46S contents of sucrose density gradient Method》, number of patent application：200910092779.3).

3rd, test kit of the invention, reproducible (CV% ＜ 15%), testing result reliability.

Embodiment 5, detection by quantitative comparative experimentss

The Guangxi Strain (O/GX/09-7 strains) provided with Jinyu Baoling Biology Drugs Co., Ltd and Xinjiang Strain (O/Mya98/ XJ/2010) purification inactivation liquid, prepares three parts of samples, wherein：

A：Comprise only Guangxi Strain (O/GX/09-7 strains) antigen, it is known that antigen concentration is 1.2 μ g/mL；It is conventionally newborn Breakdown of emulsion (adds the n-amyl alcohol of 20mL, stirring in antigen after 100mL emulsifyings afterwards to change (emulsifying agent that 55mL is added in 45mL antigens) Static 30 minutes after 1 minute so as to upper lower leaf), water intaking is mutually detected.

B：Comprise only Xinjiang Strain (O/Mya98/XJ/2010) antigen, it is known that its antigen concentration is 1.2 μ g/mL；According to routine Breakdown of emulsion (adds positive the penta of 20mL in antigen after 100mL emulsifyings afterwards for method emulsifying (emulsifying agent of 55mL is added in 45mL antigens) Alcohol, static 30 minutes after stirring 1 minute so as to upper lower leaf), water intaking is mutually detected.

C：Containing two kinds of antigens of Guangxi Strain (O/GX/09-7 strains) and Xinjiang Strain (O/Mya98/XJ/2010), it is known that two kinds resist Original content is respectively 1.2 μ g/mL, and 146S antigens total concentration is 2.4 μ g/mL.Conventionally emulsifying (is added in 45mL antigens The emulsifying agent of 55mL) afterwards breakdown of emulsion (in antigen after 100mL emulsifyings add 20mL n-amyl alcohol, stirring 1 minute after static 30 points Clock so as to upper lower leaf), water intaking is mutually detected.

Resisted with the test kit of the present invention and the O-shaped 146S of foot and mouth disease of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's production Original amount ELISA detection kit detects 146S antigen concentrations according to respective description simultaneously, as a result as shown in table 9.

9 comparative result of table (146S antigen concentrations, μ g/mL)

Find from the above, the test kit of the present invention only detects Guangxi Strain (O/GX/09-7 strains), even if being wherein mixed with Xinjiang Strain (O/Mya98/XJ/2010) antigen nor affects on detection.Contrast agents box cannot be distinguished by two kinds of O-shaped antigens.If knot Another ELISA detection kit (the O-shaped immunity detection reagent of foot and mouth disease) of applicant's exploitation is closed, the test kit can be examined Xinjiang Strain (O/Mya98/XJ/2010) is surveyed, with Guangxi Strain (O/GX/09-7 strains) without intersecting.Spirit can be protected to golden space completely biological Two kinds of 146S antigens in the FMD tye O swine vaccine of medicine company limited production accomplish individually quantitative, quality control.It is right Total O-shaped antigen can only be detected according to test kit, it is impossible to which individually quantitative, this is unfavorable for the quality control in vaccine production process.

Claims (10)

1. a kind of enzyme linked immunological kit of the O-shaped Guangxi Strain of quantitative determination of specificity foot and mouth disease (O/GX/09-7 strains), including mouth hoof One plant of monoclonal antibody specific of the O-shaped Guangxi Strain of epidemic disease (O/GX/09-7 strains) is used as coated elisa plate made by capture antibody；Institute The monoclonal antibody specific for stating the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) is prepared by following steps：

1) specific proteinses of O-shaped Guangxi Strain (O/GX/09-7 strains) the specific antigen fragment of foot and mouth disease are obtained by 2-DE electrophoresis Band, molecular weight are made as immunogen after the higher protein bands drying of wherein five abundance ratios of selection, grinding in 20-30KDa With；

2) with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) specific antigen fragment as immunogen immune animal；Continuous immunity 4 times, per minor tick 14 days, 3 times using multiple spot subcutaneous inoculation mode, the 4th took the immunization wayses of lumbar injection, and every time 10 μ g/ animals；

3) splenocyte of separating immune animal, by itself and myeloma cell fusion, screens hybridoma with HAT selective mediums thin Born of the same parents, are cloned with indirect ELISA method screening specific positive to hybridoma supernatant；

4) cultivate hybridoma；Separate from the ascites fluid of cell culture fluid or inoculation hybridoma animal and purification outlet The monoclonal antibody specific of the O-shaped Guangxi Strain of fever aphthous (O/GX/09-7 strains).

2. test kit according to claim 1, it is characterised in that：The test kit is also included with the O-shaped Guangxi Strain of foot and mouth disease The other one plant of monoclonal antibody specific of (O/GX/09-7 strains) 146S antigens is used as enzyme labelling made by detection antibody Jing enzyme labelling Antibody；The marker enzyme is horseradish peroxidase or alkaline phosphatase, and horseradish peroxidase is by glutaraldehyde method or mistake Iodic acid method is crosslinked on antibody.

3. test kit according to claim 1, it is characterised in that：The test kit also includes the O-shaped Guangxi Strain (O/ of foot and mouth disease GX/09-7 strains) positive quality control product, select the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) the 146S antigens of each vaccine factory production Purification inactivates liquid；The raw material of the positive quality control product is standard level, and purity is not less than 80%, 8-64ng/mL is diluted to during use.

4. test kit according to claim 3, it is characterised in that：Positive quality control product protects the limited public affairs of clever biologics for golden space The O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) the 146S antigen purifications inactivation liquid of department's production.

5. the test kit according to claim 1 or 2 or 3 or 4, it is characterised in that：The test kit also includes detection Sample diluting liquid, cleaning mixture, assay chromogenic substrate solution, terminate liquid and description；

The formula of the sample diluting liquid is：Na₂HPO₄·12H₂O 2.9g, NaH₂PO₄·2H₂O 0.296g, NaCl 8.5g, 300 0.6mL of Proclin, casein 10g, new-born calf serum 50mL are dissolved to 1000mL with distilled water, and adjustment pH value is extremely 7.2-7.4；

The formula of the cleaning mixture is：Na₂HPO₄·12H₂O 58g, NaH₂PO₄·2H₂O 5.92g, NaCl 170g, Tween- 300 0.6mL of 20 1.0mL, Proclin, is dissolved to 1000mL, adjustment pH value to 7.2-7.4 with distilled water；Using cleaning mixture Front distilled water dilutes 20 times；

The formula of the assay chromogenic substrate solution is：

1) assay chromogenic substrate solution of horseradish peroxidase (HRP) includes A liquid and B liquid, wherein, the formula of chromogenic substrate A liquid is：Three Hydrated sodium acetate 8.2g, glacial acetic acid 1.2mL, the hydrogen peroxidase 10 .27mL that concentration is 30%, are dissolved to 1000mL with distilled water, adjust Whole pH value is to 5.0-5.6；

The formula of chromogenic substrate B liquid is：Citric acid 1.92g, sodium ethylene diamine tetracetate 0.372g, 10mol/L sodium hydroxide solutions 0.3mL, glycerol 50mL, tetramethyl biphenyl amine hydrochlorate 0.23g are dissolved to 1000mL, adjustment pH value to 2.5- with distilled water 3.5；

A liquid is pressed into 1 with B liquid using front：1 ratio mixes；

2) the chromogenic substrate formula of liquid of alkali phosphatase (ALP) is：PNPP 1g, diethanolamine 100mL, anhydrous magnesium chloride 47.5mg, distilled water 900mL, pH9.8；

The formula of the terminate liquid is：The terminate liquid of horseradish peroxidase is：It is 98% to contain concentration in 1000mL distilled waters Sulphuric acid 27.8mL；The terminate liquid of alkali phosphatase is：Sodium carbonate containing 53g in 1000mL distilled waters.

6. test kit according to claim 5, it is characterised in that：The step 2) in for preparing exempting from for monoclonal antibody Epidemic disease animal is including the mammal including Balb/C mices, Lou/C rats, rabbit；

The step 3) in when the antibody level of serum of immunized animal is more than 1:When 50000, the splenocyte of separating animal's is simultaneously made It is standby into single cell suspension, and merge to form hybridization with murine myeloma cell SP2/0 under the induction of fusion agent Polyethylene Glycol Tumor；

The step 4) in can be in external tissue culture flasks or porous fibre reactor or selected by mouse ascites culture in vivo The hybridoma of the monoclonal antibody specific of the O-shaped Guangxi Strain of secretion foot and mouth disease (O/GX/09-7 strains) selected, from cell culture fluid or The monoclonal antibody specific of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) is collected and is purified in mouse ascites liquid.

7. it is a kind of to use the arbitrary test kit of claim 1-6 and the O-shaped Guangxi Strain (O/GX/ of ELISA method quantitative determination foot and mouth disease 09-7 strains) antigen concentration method, for the quality control in vaccine production process, comprise the steps:

1) standard substance, positive quality control product and testing sample are prepared；

2) standard substance of positive quality control product, testing sample or concentration known, board-washing are added；

3) enzyme-added labeling antibody, board-washing；

4) add substrate colour developing；

5) terminating reaction；

6) OD values are determined；

7) analysis result, obtains the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) level.

8. method according to claim 7, it is characterised in that：Specifically include following steps：

1) matched somebody with somebody with the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) sample diluting liquid that 146S antigens and test kit are provided after purification Standard substance processed, standard concentration is：0th, 2,4,8,16,32,64ng/mL, measuring samples are equally diluted to 4-8 with sample diluting liquid Individual gradient；

2) lath required for taking out, remaining lath load in valve bag, are placed in 2-8 DEG C and save backup；

3) in hole add 100 μ L standard substance series dilution point, positive quality control, measuring samples to be serially diluted a little respectively, gently shake The dynamic 10-20 seconds mix, and stick plate patch；

4) be placed in 37 DEG C of incubators and incubate 60 minutes, deduct liquid in hole, and 5-7 is washed with the front cleaning mixture for diluting 20 times It is secondary, pat dry on clean napkin or towel, remove the moisture of residual as far as possible；

5) per the enzyme marker that 100 μ l are added in hole, gently vibrating the 10-20 seconds mixes, sticks plate and pastes；

6) it is placed in 37 DEG C of incubators and incubates 30 minutes, deducts liquid in hole, and washed 5-7 time with cleaning mixture, in clean napkin Or on towel, pat dry the moisture for removing residual；

7) A liquid, each 1 drop of B liquid or alkali in assay chromogenic substrate solution, the i.e. assay chromogenic substrate solution of horseradish peroxidase (HRP) is added per hole The assay chromogenic substrate solution 2 of acid phosphatase (ALP) is dripped, and gently vibrates the 10-20 seconds, is placed in 37 DEG C of lucifuges and reacts 15 minutes, and 1 is added per hole Drop terminate liquid, the i.e. terminate liquid 1 of the terminate liquid of horseradish peroxidase (HRP) or alkali phosphatase (ALP) are dripped, are gently vibrated several Secondary mixing, makes solution be changed into yellow liquid completely, in 30 minutes, measures absorbance (OD), horseradish peroxidase (HRP) With 630nm as reference wavelength, the absorbance at 450nm is measured, alkali phosphatase (ALP) measures the absorbance of 405nm；

8) analysis result, calculates the concentration of O-shaped Guangxi Strain (O/GX/09-7 strains) antigen of foot and mouth disease.

9. method according to claim 8, it is characterised in that：The step 1) in testing sample be vaccine production process in Culture fluid, inactivation liquid, refined solution, the finished product vaccine after concentrated solution and breakdown of emulsion.

10. detection method according to claim 8 or claim 9, it is characterised in that：The step 8) in interpretation of result method For：Respectively standard concentration and correspondence OD absorbances are taken the logarithm, standard curve is set up on double logarithmic chart, with standard Product concentration is abscissa, and OD absorbances draw standard curve for vertical coordinate, with each specimen absorbance to be measured in standard curve On find the concentration of the O-shaped Guangxi Strain of foot and mouth disease (O/GX/09-7 strains) antigen in sample, multiple dilution factors calculate meansigma methodss.