CN107365364A - A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen - Google Patents
A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen Download PDFInfo
- Publication number
- CN107365364A CN107365364A CN201610310993.1A CN201610310993A CN107365364A CN 107365364 A CN107365364 A CN 107365364A CN 201610310993 A CN201610310993 A CN 201610310993A CN 107365364 A CN107365364 A CN 107365364A
- Authority
- CN
- China
- Prior art keywords
- adenovirus
- antigen
- antibody
- detection
- penton
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of adenoviral gene quantity sheet to reach artificial antigen；The method for preparing the antigen, this method includes the adenovirus penton gene order of artificial synthesized optimization, build prokaryotic expression carrier, Bacillus coli expression adenovirus penton antigen, using dialysis, gradient dilution method and gel chromatography renaturation inclusion body, acquisition has three-dimensional structure and immunocompetent recombined adhenovirus penton antigen；A kind of quick determination method for detecting adenovirus antibody, this method include applying adenovirus penton antigen；A kind of quick detection kit for adenovirus antibody detection, the kit includes adenovirus penton antigen, including possesses hemofiltration film in detection means, can be used directly to whole blood test.The invention provides a kind of Adenovirus Antigen, the antigen has the specificity of height；The invention provides the method for preparing the antigen, the quick method for determining adenovirus antibody, and for quickly determining the kit of adenovirus antibody.
Description
Technical field
The invention belongs to field of clinical medical detection, is related to immuno-chromatographic assay technology, and in particular to one kind is used to diagnose
The Adenovirus Antigen of adenovirus infection, the method for preparing the antigen, with the quick detection reagent of antigen detection adenovirus antibody
Box.
Background technology
Adenovirus (Adenovirus, Adv) is one of main pathogen of viral pneumonia all over the world, children or youth
People infects adenovirus and only causes upper respiratory disease, but can cause serious acute adenovirus pneumonia in infant.Adenopathy
Toxicity pneumonia was reported that follow-up research finds cause infant's adenoviral pneumonia popular in 1958 by Chang etc. first
Pathogen is mainly adenovirus 3,7 types, and mild and big-age-child's prognosis are preferable, severe infection and adenoviral pneumonia prognosis mala
Case fatality rate it is higher.
Adenovirus is a kind of particle for not having tunicary a diameter of 70~90 nm, by 252 capsomeres in twenty face body arrangement structure
Into.A diameter of 7~9 nm of each capsomere.It is wire double chain DNA molecule in capsid, containing about 35 000 bp.Adenovirus can infect
The organ such as respiratory tract, intestines and stomach, urethra and bladder, eye, liver.Known adenovirus has 6 hypotypes（A~F）It is different with 55
Serotype（By viral gene component type）.Known about more than 20 kind serotype can infect the mankind.Adenovirus pneumonia is mainly in June -2
Year children, mostly 3 types and 7 type adenovirus infections.The incubation period of adenovirus pneumonia infection is 2-14 days, and IgM antibody is in morbidity one
All left and right occur, sustainable to exist about 2-3 months.Except according in addition to epidemiology and clinical manifestation, make a definite diagnosis need examined by experiment
It is disconnected, mainly include virus purification and identification, serological test etc..
The test in laboratory of cause pathogeny imcrobe infection disease mainly includes detection to the pathogen of infection site etc. and right
Two methods of antibody test in patients serum, body fluid.Adenovirus detection at present mainly has isolated culture, polymerase chain reaction
Should（PCR）Method, Immunofluorescent Antibody technology（IFA）And EUSA（ELISA）Etc. technology.Cultivation is adenovirus
The conventional method of diagnosis, but generally time-consuming for this method, it is desirable to strict transportation and complicated technology, it is not suitable for
Quick diagnosis.PCR（PCR）Although Deng nucleic acid detection technique with higher specificity and sensitiveness, it is accurate
True property is not high, easily pollutes.
The detection of antibody is one of more satisfactory method of detection adenovirus infection.Currently used antibody test technology
There is Immunofluorescent Antibody technology（IFA）, EUSA（ELISA）Etc. technology.Specific IgG and IgM measure
The stage of infection can accurately be confirmed, the preventing and treating for cause of disease is significant.Adenovirus antibody detection reagent quality depends on
The performance of antigen, because the program that antigen is prepared from native adenoviral extraction is cumbersome, cost is higher and limits throughput.Quantity sheet reaches
Antigen is the best-of-breed technology scheme for preparing Adenovirus Antigen.In the antigen of adenovirus, penton（Penton base）It is a kind of
Optimal candidate antigen.Penton is one of major structural protein of adenovirus, and its conservative is preferable, body can be stimulated to produce corresponding
Antibody, so as to neutralize virion.Penton is also that excitating organism produces the most important viral antigen of protection antibody, and this causes five
Adjacent body is the first choice for developing diagnostic kit candidate antigens.
The content of the invention
The first object of the present invention is to provide a kind of quantity sheet and reaches adenovirus penton specific antigen, and the antigen has height
The specificity and immunoreactivity of degree.The advantages that antigen has that immunogenicity is high, no idiosyncrasy, and epitope is concentrated.
The second object of the present invention is to provide a kind of method of preparation engineering expression adenovirus penton antigen, this method profit
Prokaryotic expression is carried out with synthetic gene carrier construction, prepares the high specific antigen of activity by renaturing inclusion bodies, prepared by this method
Adenovirus Antigen have specificity and immunoreactivity.
The third object of the present invention is to provide a kind of kit for determining Anti-adenovirus antibody, and the kit is quick " one
Walk detection method " kit, there is high sensitivity, high specificity, detection speed is fast, easy to operate, without Special Equipment, is applied to
A variety of occasions such as clinical detection, epidemiology survey and place quarantine.
The fourth object of the present invention is to provide a kind of adenovirus antibody whole blood device for fast detecting, and it is that one kind is equipped with hemofiltration
The adenovirus whole blood test device that can filter out red blood cell of film.
The main contents of the present invention are as follows.
（1）A kind of Adenovirus Antigen, it is a kind of antigen prepared using technique for gene engineering.
（2）A kind of method for preparing Adenovirus Antigen, include the adenovirus penton antigen gene of artificial synthesized optimization, structure
Build prokaryotic expression carrier, Bacillus coli expression adenovirus penton antigen albumen, using dialysis, gradient dilution method and gel layer
Analysis method renaturation inclusion body, acquisition have three-dimensional structure and immunocompetent recombined adhenovirus penton antigen.
（3）A kind of adenovirus antibody quick determination method, including application is above（1）In Adenovirus Antigen.
（4）It is above-mentioned（3）Middle adenovirus antibody quick determination method, including（1）In Adenovirus Antigen be fixed to a kind of nitre
Acid cellulose film, a kind of labelled antibody（Secondary antibody）Fixed on solid phase carrier, testing sample and labelled antibody（Secondary antibody）Contact, if
Adenovirus antibody in testing sample be present, then adenovirus antibody forms antibody-marker secondary antibody compound with mark secondary antibody, " compound
Thing " moves horizontally in nitrocellulose filter, runs into and produces reaction fixed to the Adenovirus Antigen on film, with markd " compound
Thing " is just specifically fixed on antigen, and the position of immobilized antigen just has chromogenic reaction；If gland-containing virus do not resist testing sample
Body, then " compound " can not be fixed to antigen position on, herein just there is no chromogenic reaction, can be fixed according to nitrocellulose filter
Whether antigenic site has chromogenic reaction, to judge testing result, i.e., whether has adenovirus antibody in testing sample.
（5）It is above-mentioned（4）Adenovirus antibody detection method, wherein testing sample are the whole blood of people, blood plasma or serum.
（6）It is above-mentioned（4）Or（5）The method of middle measure adenovirus antibody, labelled antibody therein（Secondary antibody）It is the anti-of mark
People IgM or anti-human IgG antibodies.
（7）It is above-mentioned（4）-（6）Middle labelled antibody is the antibody of colloid gold particle or colored latex particle marker.
（8）A kind of kit of quick detection adenovirus antibody, its antigen used is above-mentioned（1）In Adenovirus Antigen.
（9）It is above-mentioned（8）In be used for quick detection adenovirus antibody kit, labelled antibody therein is colloidal metal
Grain labelled antibody or colored latex particle marker antibody.
（10）It is above-mentioned（8）In be used for detect kit, hemofiltration film therein is to be fixed with anti-erythrocyte monoclonal antibody or more
Anti- non-woven fabrics.
The present invention will be described in detail below.
The Adenovirus Antigen of the present invention, mainly by arriving prokaryotic expression carrier by adenovirus penton is gene constructed
PET30a, pET30a- penton expression vector convert Escherichia coli, screen positive restructuring bacterium, IPTG induction recombinant bacterium tables
Up to adenovirus penton protein, obtained through bacteria lysis, solubilization of inclusion bodies and recombinant protein structure renaturation and the steps such as purifying.
The method that the present invention prepares Adenovirus Antigen will be described below.
Adenovirus penton gene order is obtained from GenBank, artificial synthesized adenovirus penton gene order is simultaneously carried out
Codon optimization.Target gene is connected after double digestion with the prokaryotic expression carrier through same double digestion, transformed competence colibacillus cell
（Escherichia coli）, screening positive restructuring bacterium.Positive colony bacterium extraction plasmid, double digestion and sequencing identification, it was demonstrated that the purpose of insertion
Gene is correct.Recombinant bacterium through IPTG induced expressions, centrifuge to obtain recombinant bacterium thalline, thalline is resuspended in ultrasonic degradation after buffer solution, from
The heart obtains the albumen precipitation of inclusion bodies, is dissolved in urea liquid after scrubbed.Albuminate need by structure renaturation with
Purifying could obtain the immune response originality of native protein and reach the application standard of adenovirus antibody detection.
For adenovirus penton recombinant protein renaturing inclusion bodies method, dialysis, gradient dilution method, gel layer can be applied
The method that analysis method etc., preferably dialysis and gradient dilution method are combined, because they are easily controllable, protein renaturation rate is high.
For the purification process of adenovirus penton after renaturing inclusion bodies, gel chromatography, affinity chromatography etc. can be applied, preferably
Gel chromatography, because it is easiest to control, high income.
The adenovirus penton antigen obtained by genetic recombination has higher specificity and sensitiveness.
For the method for quick measure antibody, what is enumerated here is preferable method, is such as exempted from using various labelled antibodies
Epidemic disease measurement in chromatography, using immunity percolation method of various labelled antibodies etc..
Immunochromatographic measurement method is explained in detail below.
Anti-adenovirus antibody immunochromatographic measurement method comprises the steps：Adenovirus penton antigen carries fixed to solid phase
On body 1, labelled antibody（Secondary antibody）Fixed in solid phase 2, testing sample and labelled antibody（Secondary antibody）Contact, if being deposited in testing sample
In adenovirus antibody, then adenovirus antibody reacts to form antibody-marker secondary antibody compound with secondary antibody, should " compound " in solid phase 1
On move horizontally, with producing reaction fixed to the Adenovirus Antigen on carrier 1, with markd " compound " just by specifically solid
Determine onto antigen, the position of immobilized antigen just has chromogenic reaction on solid phase carrier；If testing sample is free of adenovirus antibody,
" compound " can not be fixed on the position of antigen, just do not have chromogenic reaction herein, can be according to immobilized antigen on solid phase carrier
Whether position has chromogenic reaction, to judge testing result, i.e., whether has Anti-adenovirus antibody in testing sample.
For solid phase carrier 1, such as nitrocellulose filter can be applied（NC films）, pvdf membrane etc..It is preferred that nitrocellulose filter.
For solid phase carrier 2, any form of such as glass fibre element film, non-woven fabrics etc. can be applied.It is preferred that non-woven fabrics
Make conjugate release pad, because it is easiest to control.
For the antibody of mark, the antibody with different mark substance markers is referred here to, refers here to anti-human IgM antibodies
Or anti-human IgG antibodies, can suitably it be applied according to the species of detected antibody.
Beneficial effects of the present invention：
（1）The present invention includes the Optimal Expression of adenovirus penton antigen, has synthesized the penton gene order of optimization, this sequence
The expression efficiency of row is higher, and specificity is stronger.And the present invention has also explored the inclusion body antigen purification of complete set and answered
Property method, the antigen high income prepared, immunogenicity is high.
（2）The present invention has also prepared a kind of kit of one-step method detection adenovirus antibody.The kit detects adenopathy
The IgG or IgM antibody of poison, beneficial to confirmation infective stage, the treatment suggesting effect for adenovirus is obvious.
（3）The present invention can also directly detect whole blood sample, have hemofiltration film on detection card, can use a small amount of whole blood sample
Detection.
Therefore, the present invention can be by the infection of primary sample one-step method whole blood test Respiratory Tract Adenovirus, suitable for basic unit
Medical institutions and hospital outpatient.
Brief description of the drawings
Fig. 1, it is shown that the surface structure schematic diagram of invention reagent-detection plate
1- detection plates；2- wells；3- inspection windows.
Fig. 2, it is shown that the external structure schematic diagram of invention reagent-detector bar
4- is loaded end absorbent paper layer；5- gold labeling antibody layers；6- control lines；7- detection lines；8- detection layers；9- suction sides water accepting layer；
10- liner plates.
Embodiment
The recombined adhenovirus penton protein antigen of embodiment 1 recombination expression, structure renaturation and purifying.
Adenovirus penton gene reference GenBank sequence KP270914.1, choose gene and synthesized, carried out during synthesis
E. coli codon optimizes, expression vector pET30a, and restriction enzyme site during connection is EcoR I/Xho I, and target gene is common
1635bp（544aa）, it is transformed into BL21（DE3）, the common 592aa of recombinant protein of expression, the kDa of molecular weight 67.6, isoelectric point 5.4.
Recombinant protein is expressed with inclusion bodies.
The structure of recombinant expression carrier and identification：Respectively with EcoR I and Xho I to adenovirus penton target gene piece
Section and pET30a plasmids carry out double digestion, and after digestion products purifying recovery, 16 DEG C of connections overnight, then are transferred toE. coliDH5 α feel
By state cell, picking conversion bacterium colony extraction plasmid enters performing PCR, digestion and sequencing identification.Identify through PCR and double digestion, confirm
There is size about 1700bp genetic fragment insertion vector, it is consistent with expected results.Sequencing result shows to insert target gene fragment
For 1635bp, nucleotide sequence is identical with submitting the adenovirus penton gene order of synthesis, and the expression vector of structure opens
Reading frame is correct, can express recombinant protein.
The induced expression of recombinant protein：Take 50 μ L pET30a-Pb（penton base）BL21 (DE3) bacterium solution is added to
In LB culture mediums of the 5mL containing 100 μ g/mL kanamycins, 37 DEG C, 200 rpm overnight incubations, next day press 1:50 increase bacterium in containing card
In the LB culture mediums of that mycin, cultivate to light absorption value OD600For 0.6 or so when, add IPTG to concentration be 1mmol/L, induction table
Up to 4 hours, 5000rpm was centrifuged 30 minutes, collected thalline, was suspended with TE buffer solutions, was fully mixed, -80 DEG C of multigelations 3 times,
Carrying out ultrasonic bacteria breaking, 4 DEG C, 12000 rpm, 15 min of centrifugation, collects supernatant, precipitation is washed 2 times with 2 M urea, 4 DEG C, 12000 rpm
15 min are centrifuged, residue precipitation is dissolved with 8 M urea, 4 DEG C of preservations.Precipitation uses SDS-PAGE analyzing proteins expression quantity.
The structure renaturation of recombinant protein and purifying：10mmol/L is used under room temperature condition, pH7.2 PBS is to adenopathy
The lysate of malicious penton recombinant expression protein 8M urea carries out gradient dialysis, by urea concentration in solution be respectively 6mol/L,
4mol/L, 3 mol/L control dilution gradient, and the time of each gradient dilution is respectively 3-4h.Containing for dilution refolding will be completed
4 DEG C of placement 24h of lysate of 3mol/L urea.The solution 10000r/min of dilution refolding is centrifuged off deposit, supernatant
Purified by the gel chromatography partition method using S-300 as medium, so as to obtain recombined adhenovirus penton antigen.Gained weight
Group adenovirus penton antigen component carries out SDS-PAGE, to determine the molecular weight of protein and purity of protein.Electrophoresis detection knot
Fruit shows main protein band at 67.6 kDa, and purity of protein reaches more than 95%.
A kind of preparation of adenovirus antibody IgG/IgM gold labeled quick detection reagent boxes of embodiment 2.
Gene recombinant adenovirus penton makees detection antigen obtained by above-mentioned, and detection line, ginseng are coated with nitrocellulose filter
According to Fig. 2, adenovirus antibody gold labeled quick detection reagent is prepared, its constituent includes：The water suction of sample-adding end is provided with liner plate 10
Layer 4, detection layers 8 and water accepting layer 9, gold mark Anti-adenovirus antibody layer 5 is provided between detection layers and sample-adding end water accepting layer 4, is being examined
Survey on layer 8 and be coated with detection line 7 and nature controlling line 6.Wherein it is loaded end water accepting layer 4 and suction side water accepting layer 9 is made up of multi-layer filter paper：
Detection layers 8 are nitrocellulose filter；Gold labeling antibody layer is that glass fibre or non-woven fabrics leach colloidal gold labeled monoclonal antibody.
Detection reagent preparation procedure includes：Nature controlling line 6, the coating of detection line 7 of gold labeling antibody layer 5 and detection layers 8 are prepared,
Then gold label test strip and detection card 1 are combined on liner plate 10 again, pastes the water suction of sample-adding end respectively at the both ends of plastics lining board 10
Ply of paper 4 and suction side water accepting layer 9；Section pastes the cellulose layer 8 of coating detection line and nature controlling line wherein, in sample-adding end blotting paper
Layer 4 and the handing-over position of cellulose film layer 8, the glass fibre of folder patch layer containing gold labeling antibody 5,4/5 part of glass fibre is adding
Among sample end absorbent paper layer 4,1/5 part is in cellulose film layer 8.Then according to 4 mm wides, 7 centimetres of long size slittings.Join again
According to Fig. 2, detector bar is assembled in plastic casing and forms detection card 1, the sample-adding end blotting paper of the face test-strips of well 2 on lid
Layer 4, the detection layers 8 of the face cellulose membrane of peep hole 3.
The preparation method of above-mentioned gold labeling antibody layer comprises the steps：The preparation of I collaurum, take 100mg gold chlorides molten
In 1000mL tri-distilled waters, the citric acid three sodium solution that 15mL concentration is 1% is added, boils 15 minutes, can obtain a diameter of 15-
50 nanometers of colloid gold particle solution；II colloid gold label anti-human igg or IgM monoclonal antibody, 100ml colloidal gold solutions are taken,
It is 8.4 to adjust pH with 0.2M solution of potassium carbonate, adds 1mg anti-human igg/IgM monoclonal antibody, stirs 20 minutes, add 240mg
Bovine serum albumin(BSA)（BSA）, continue stirring 5 minutes, 4 DEG C of standing 2-4 hours；III is by above-mentioned colloidal gold solution through 2000 revs/min
Zhongli's heart 10-15 minutes, go to precipitate, obtain supernatant；IV is precipitated supernatant for 60 minutes through 10000 revs/min of centrifugations；
The Tris-Hcl buffer solutions that precipitation is dissolved in 4mL 0.02M pH7.4 by V obtain colloidal gold solution, contain 0.25% in the solution
BSA and 0.02% Sodium azide；Colloidal gold solution is immersed glass fibre or non-woven fabrics untill liquid starts to ooze out by VI, 37 DEG C
Dry 2 hours and form gold labeling antibody layer 5.
Described detection layers 8 are that the detection line 7 that recombined adhenovirus penton antigen is formed is provided with nitrocellulose filter
With the nature controlling line 6 formed by sheep or rabbit-anti mouse IgM/IgG antibody.Its preparation method is to take above-mentioned 1）Prepared recombined adhenovirus
Penton antigen, degree of thickening are 1.5mg/mL, add 2% methanol, spray detection line 7 in cellulose membrane stage casing with Membrane jetter；Take again
Sheep or rabbit-anti mouse IgM/IgG antibody degree of thickening are 1.5mg/mL, with Membrane jetter in cellulose membrane stage casing, at detection line 0.5cm,
Nature controlling line 6 is sprayed, spray film amount is set by 2 μ l/cm, 37 DEG C of dryings 2 hours, then containing with 0.01mL pH7.0 are placed in after spraying film
The PBS of 10% calf serum is closed 30 minutes at 37 DEG C, 0.01mL pH7.0 PBS rinsings, 45 DEG C of dryings.
The measure of the adenovirus antibody of embodiment 3.
Serum or the μ l drops of plasma sample 10 are taken in the sample well 2 of detection plate 1, then be added dropwise 100 μ l sample diluting liquids in
In sample well 2, testing result is observed in observation window 3, in observation result 20 minutes effectively.If resist in sample containing anti-adenovirus
Body, then see that two red lines occur in detection line and nature controlling line in observation window, testing result is judged to the positive；If in serum not
Containing Anti-adenovirus antibody, then a red line is seen in observation window Quality Control line position, testing result is determined as feminine gender；If observation
A red all be can't see in window, then testing result is invalid.
The detection of the whole blood sample of embodiment 4.
The adenovirus immunisations chromatography quick detection kit that the present invention realizes can be detected directly to anti-freezing whole blood sample, without
Blood plasma or serum separation, its principle realized are, increase by a layers of blood separating film in sample pad, are solidified with hemofiltration film anti-human blood red
Cell antibody（Monoclonal antibody or polyclonal antibody）, during detection, after anticoagulation whole blood enters hemofiltration film in blood red blood cell by antibody
It is attached on hemofiltration film, blood plasma penetrates into sample pad, completes detection.
Sensitivity and Specificity of the embodiment 5 to clinical sample.
Adenovirus antibody is prepared with the Adenovirus Antigen and detection method of the present invention（IgM）Gold mark detection reagent, detection 44
Part adenovirus antibody IgM positive clinicals blood serum sample and 86 parts of adenovirus antibody IgM feminine gender clinical serum samples are special to carry out
Property and sensitivity tests.Meanwhile in order to verify the effect of the present invention, the gold mark detection to being carried out with the Adenovirus Antigen of the present invention
Reagent and the kit of Ou Meng companies production carry out coincidence rate analysis, the results are shown in Table 1, table 2.
Table 1：The specificity and sensitivity Detection result of the Adenovirus Antigen of the present invention
| Positive sample number | Negative sample number | |
| It is positive | 42 | 2 |
| It is negative | 2 | 84 |
| It is total | 44 | 86 |
The result of table 1 shows that the sensitiveness of antigen of the present invention reaches 95.45%, and specificity reaches 97.67%, antigen of the invention
Further research and development adenovirus antibody detection kit can be used as.
Table 2：With the detection reagent that antigen of the present invention is carried out and Ou Meng companies（EUROIMMUN）The coincidence rate analysis of reagent
The result of table 2 shows that detection kit of the present invention and the positive coincidence rate of Ou Meng companies reagent are 93.18%, and feminine gender meets
Rate is 97.67%, and total coincidence rate is 96.15%, illustrates the testing result and Ou Meng companies adenovirus antibody of kit of the present invention
Detection kit has preferable coincidence rate.Therefore, the present invention has preferable clinical value.
It is pointed out that the foregoing is merely illustrative of the preferred embodiments of the present invention, the present invention is not intended to limit, it is all at this
All any modification, equivalent and improvement done within the spirit and principle of invention etc., should be included in the protection model of the present invention
In enclosing.
Claims (6)
1. a kind of Adenovirus Antigen, it is a kind of using technique for gene engineering expression, and the size that renaturing inclusion bodies obtain is 67.6
KDa recombinant antigen.
2. a kind of renaturation and purification process of adenovirus penton antigen, it is characterised in that：10mmol/L is used under room temperature condition,
PH7.2 PBS carries out gradient dialysis dilution to the lysate of adenovirus penton recombinant expression protein 8M urea, by molten
Urea concentration is respectively 6mol/L, 4mol/L, 3 mol/L control dilution gradient in liquid, and the time of each gradient dilution is respectively
3-4h, 4 DEG C of the lysate that will complete the urea containing 3mol/L of dilution refolding place 24h, the solution 10000r/min of dilution refolding
Deposit is centrifuged off, supernatant is purified by the gel chromatography partition method using S-300 as medium, so as to be recombinated
Adenovirus penton antigen, gained recombined adhenovirus penton antigen component carries out SDS-PAGE, to determine the molecule of protein
Amount and purity of protein, deposition condition, concentration gum concentration is 5%, and resolving gel concentration 12%, protein staining is coomassie brilliant blue staining
Method, coomassie brilliant blue R_250 dyeing liquor dyeing 3h, acetic acid-ethanol decolorization liquid decolourize untill background is colourless, and main albumen one is taken out of
At present 67.6 kDa, purity of protein reaches more than 95%.
It is fine that 3. the Adenovirus Antigen in a kind of adenovirus antibody quick determination method, including application claim 1 is fixed to nitric acid
Tie up plain film, colloidal metal particles or colored latex particle marker antibody（Secondary antibody）Contact, resists if adenovirus in testing sample be present
Body, then adenovirus antibody is with marking secondary antibody to react to form antibody-marker secondary antibody compound, and " compound " is in nitrocellulose film water
Translation is dynamic, runs into and produces reaction fixed to the Adenovirus Antigen on nitrocellulose filter, just special with markd " compound "
Strange land is fixed on antigen, and the position of immobilized antigen just has chromogenic reaction, " multiple if testing sample is free of adenovirus antibody
Compound " can not be fixed on the position of antigen, just do not have chromogenic reaction herein, can be according to immobilized antigen position on nitrocellulose filter
Whether have chromogenic reaction, come in judgement sample whether have adenovirus antibody if putting；Detection antibody used is IgM/IgG, and sampling is convenient.
4. adenovirus antibody IgM/IgG detection kits according to claim 1, it is characterised in that the test strips are placed in
In the neck in cassette bottom inside cartridge, by substrate, double faced adhesive tape, sample pad, colloidal gold pad, adhesive tape, reaction film and sample suction pad group
Into being adsorbed with the efficient mouse source anti-human igg monoclonal antibody of colloid gold label in colloidal gold pad, quantity sheet be coated with reaction film
The five adjacent proteantigen of adenovirus reached（It is shown as detection line T）With quality control antibody sheep or rabbit anti-mouse igg antibody（It is shown as matter
Control line C）.
5. a kind of kit of quick detection adenovirus antibody, its antigen used is the penton protein of optimization, to forgive the bodily form
Existing for formula, by the expressing protein purified and renaturation obtains, method therefor is claim 2 methods described.
6. a kind of adenovirus antibody quick detection kit, its sample pad is by by glass fibre or non-woven fabrics and hemofiltration sample pad group
Close and form, the hemofiltration sample pad by the glass fibre through anti-human erythrocyte monoclonal antibody or more antiantibody curing process or
Non-woven fabrics is formed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610310993.1A CN107365364A (en) | 2016-05-12 | 2016-05-12 | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610310993.1A CN107365364A (en) | 2016-05-12 | 2016-05-12 | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107365364A true CN107365364A (en) | 2017-11-21 |
Family
ID=60304750
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610310993.1A Pending CN107365364A (en) | 2016-05-12 | 2016-05-12 | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107365364A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586589A (en) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | A kind of preparation method and applications of the Swollenin albumen from Guizhou trichoderma |
| CN109182354A (en) * | 2018-09-27 | 2019-01-11 | 浙江万里学院 | A kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules and application thereof |
| WO2020067418A1 (en) * | 2018-09-28 | 2020-04-02 | 株式会社カネカ | Method for producing capsid protein of adeno-associated virus, and use thereof |
| CN111474347A (en) * | 2020-04-20 | 2020-07-31 | 青岛爱博检测科技有限公司 | Novel coronavirus detection kit, preparation method and detection method thereof |
| CN111704656A (en) * | 2020-07-03 | 2020-09-25 | 安阳工学院 | Duck adenovirus type I Penton protein and its preparation method and application |
| CN115362257A (en) * | 2020-04-16 | 2022-11-18 | 电化株式会社 | Adenovirus immunoassay method and immunoassay device |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030219899A1 (en) * | 2001-04-17 | 2003-11-27 | Nikolay Korokhov | Mosaic adenoviral vectors |
| CN102445548A (en) * | 2011-11-15 | 2012-05-09 | 广西壮族自治区兽医研究所 | Indirect ELISA detecting kit for FAVI antibody based on penton protein |
| CN105238766A (en) * | 2015-10-15 | 2016-01-13 | 东莞市第八人民医院 | Application and preparation method of candidate strain of human type-3 adenovirus expressed human type-14 adenovirus neutralization epitope vaccine |
| CN105259345A (en) * | 2015-11-19 | 2016-01-20 | 国家纳米科学中心 | Colloidal gold test strip and test strip card for detecting IgM antibody, and preparation and detection method |
-
2016
- 2016-05-12 CN CN201610310993.1A patent/CN107365364A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030219899A1 (en) * | 2001-04-17 | 2003-11-27 | Nikolay Korokhov | Mosaic adenoviral vectors |
| CN102445548A (en) * | 2011-11-15 | 2012-05-09 | 广西壮族自治区兽医研究所 | Indirect ELISA detecting kit for FAVI antibody based on penton protein |
| CN105238766A (en) * | 2015-10-15 | 2016-01-13 | 东莞市第八人民医院 | Application and preparation method of candidate strain of human type-3 adenovirus expressed human type-14 adenovirus neutralization epitope vaccine |
| CN105259345A (en) * | 2015-11-19 | 2016-01-20 | 国家纳米科学中心 | Colloidal gold test strip and test strip card for detecting IgM antibody, and preparation and detection method |
Non-Patent Citations (2)
| Title |
|---|
| 姜海英: "人腺病毒五邻体基因重组质粒的构建及鉴定", 《哈尔滨医科大学学报》 * |
| 罗思思: "Ⅰ群禽腺病毒五邻体蛋白间接ELISA检测方法的建立", 《中国家禽》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586589A (en) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | A kind of preparation method and applications of the Swollenin albumen from Guizhou trichoderma |
| CN109182354A (en) * | 2018-09-27 | 2019-01-11 | 浙江万里学院 | A kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules and application thereof |
| WO2020067418A1 (en) * | 2018-09-28 | 2020-04-02 | 株式会社カネカ | Method for producing capsid protein of adeno-associated virus, and use thereof |
| CN115362257A (en) * | 2020-04-16 | 2022-11-18 | 电化株式会社 | Adenovirus immunoassay method and immunoassay device |
| CN111474347A (en) * | 2020-04-20 | 2020-07-31 | 青岛爱博检测科技有限公司 | Novel coronavirus detection kit, preparation method and detection method thereof |
| CN111704656A (en) * | 2020-07-03 | 2020-09-25 | 安阳工学院 | Duck adenovirus type I Penton protein and its preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104198703B (en) | People's mycoplasma pneumoniae gold label silver stain immunochromatographytest test kit and its preparation method and application | |
| CN111220803B (en) | Novel coronavirus antibody detection reagent, preparation method thereof and novel coronavirus antibody detection card | |
| CN107365364A (en) | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen | |
| CN110658339B (en) | Test paper and kit for detecting African swine fever virus and preparation method thereof | |
| CN104407137B (en) | A kind of CSFV velogen strain and low virulent strain differentiate Test paper | |
| CN211148669U (en) | S-antigen-based novel coronavirus 2019-nCoV antibody rapid detection kit | |
| CN105859843A (en) | Preparation method of respiratory syncytial virus antigen and rapid respiratory syncytial virus antibody detection kit prepared from antigen | |
| CN109187967B (en) | A dual rapid detection card for detecting and distinguishing O-type and A-type foot-and-mouth disease virus and preparation method thereof | |
| CN105319359B (en) | Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof | |
| CN113671178A (en) | African swine fever virus antibody detection test paper established based on capsid protein p72 and preparation method thereof | |
| CN106188248A (en) | A kind of Epstein-Barr virus antigen preparation procedure and utilize the quick detection kit of detection Epstein-Barr virus antibody prepared by this antigen | |
| CN105753981B (en) | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody | |
| CN102393460A (en) | Rapid detection device for helicobacter pylori | |
| CN112946294A (en) | Novel coronavirus 2019-nCoV antibody detection test strip and preparation method and application thereof | |
| CN102980997B (en) | EB virus capsid antigen IgM antibody colloidal gold method detection reagent and preparation method thereof | |
| CN117448279A (en) | Hybridoma cell strain secreting monoclonal antibody against human parainfluenza virus 3 type N protein, monoclonal antibody, application and kit | |
| CN117448280A (en) | Hybridoma cell strain secreting monoclonal antibody against human metapneumovirus N protein, monoclonal antibody, application and kit | |
| CN105585633B (en) | Antibody against human Haemophilus influenzae P6 protein and immunochromatographic kit using the antibody | |
| CN202453359U (en) | Dot gold immunofiltration kit of echinococcus multillocularis circulating antigen | |
| CN105753982B (en) | The immune chromatography reagent kit of anti-human streptococcus pneumonia fam1 family PspA protein antibodies and the application antibody | |
| CN115932255A (en) | Fluorescent microsphere immunochromatography detection kit for goat pox virus antibody and preparation method thereof | |
| CN108761091A (en) | Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof | |
| CN209280731U (en) | A kind of quickly detection viral infection of measles kit | |
| CN102305861B (en) | Reagent strip for carrying out joint detection on Toxoplasma gondii IgM (immunoglobulin M) and IgG (immunoglobulin G) antibodies and preparation method thereof | |
| CN105859885B (en) | Anti-human mycoplasma pneumoniae P30 protein antibody and immunochromatography kit applying same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171121 |