CN102495216A - Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus - Google Patents
Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus Download PDFInfo
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- CN102495216A CN102495216A CN2011104233897A CN201110423389A CN102495216A CN 102495216 A CN102495216 A CN 102495216A CN 2011104233897 A CN2011104233897 A CN 2011104233897A CN 201110423389 A CN201110423389 A CN 201110423389A CN 102495216 A CN102495216 A CN 102495216A
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Abstract
The invention relates to colloidal gold test paper for rapidly detecting an antibody of a porcine reproductive and respiratory syndrome virus (PRRSV). The test paper is characterized in that: the test paper is composed of a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad which are sequentially attached to a nonabsorbent supporting sheet, wherein the combination pad is covered by PRRSVNsp9 proteins marked by colloidal gold; and the nitrocellulose membrane is respectively covered by a detection line T line composed of staphylococcus proteins A (SPA) and a quality control line C line composed of PRRSVNsp9 monoclonal antibodies 2D6. According to the invention, by utilizing an immunological principle that antigens and antibodies can be specially combined, the antigen marked by the colloidal gold is combined with the corresponding antibody and the conjugate of the antigen and the antibody is combined with the SPA to form an antigen-antibody-SPA conjugate, so as to form a color reaction on the nitrocellulose membrane (NC membrane) to be observed by naked eyes. The test paper provided by the invention has the characteristics of simplicity, fastness, sensitivity, good specificity and the like. And the price is low, so that the test paper is applicable to basically or clinically detecting the antibody of the porcine reproductive and respiratory syndrome virus. The test paper has the obvious advantages of strong specificity, high flexibility, simplicity in operation and fastness in diagnosis, and can be used for rapidly diagnosing the porcine reproductive and respiratory syndrome virus.
Description
Technical field
The present invention relates to a kind of porcine reproductive and respiratory syndrome virus antibody colloidal gold fast diagnose test paper bar, belong to a kind of new animal diagnostic reagent, be mainly used in diagnosis porcine reproductive and respiratory syndrome virus infected animals; Be applied to animal and veterinary and learn the field.
Background technology
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome; PRRS) infect by porcine reproductive and respiratory syndrome virus (PRRSV) and cause, the breeding difficulty that principal character is that farrowing sow is miscarried, premature labor and stillborn foetus etc. are serious and the breathing problem of piglet and growing and fattening pigs.PRRS is a kind of new communicable disease of breaking out in the later stage eighties in last century; Ran initially in north America region in 1987; In subsequently several years, just promptly in Europe, many countries in U.S. continent are popular, spread some countries to the Asia subsequently again; Become worldwide being very popular, caused enormous economic loss for global pig industry.China breaks out this disease at the bottom of nineteen ninety-five, and bamboo telegraph becomes China large scale of pig farm field and causes one of main eqpidemic disease of breeding difficulty in all parts of the country.PRRS is persistent infection in recent years, and causes secondary infection to increase, serious threat the development of pig industry.
The sick technical method that detects main employing of porcine reproductive and respiratory syndrome virus has at present: ELISA, reverse transcription-polymerase chain reaction (RT-PCR) technology, immunofluorescence technique, fluorescence RT-PCR, ring mediated reverse transcription isothermal detect the isolation identification of (RT-LAMP), genetic chip and virus etc.Technology is ELISA and fluorescence RT-PCR more fast and accurately, but these 2 kinds of technology all need special instrument and equipment, and detection time all more than 2 hours, it is expensive to detect cost, is not easy in good time and quick diagnosis.These have limited the sick prevention and control of porcine reproductive and respiratory syndrome virus, therefore develop a kind of high specificity, highly sensitive, simple diagnostic method is extremely urgent.
Summary of the invention
Invention relates to a kind of preparation method of porcine reproductive and respiratory syndrome virus antibody colloidal gold antibody fast test strip; This method is to utilize gene engineering expression porcine reproductive and respiratory syndrome virus Nsp9 albumen; Utilize immunology antigen-antibody ability specificity combination principle; Colloid gold label antigen and antibodies, in fast detecting pig blood or the serum with the virus replication associated antibodies; This test paper slip can be widely used in the detection of clinical porcine reproductive and respiratory syndrome virus; With other viral no cross reactions, the coincidence rate that detects with ELISA, two kinds of methods of neutralization test is respectively 95.3% and 93.12%.Compare with ELISA, the recombinant antigen immune colloid gold has remarkable advantages: security is good, need not to cultivate virus itself, has avoided because of operating the virus diffusion that virus causes; Can prepare in enormous quantities, technology is simple, low production cost; The antigenic component stable uniform, easy and simple to handle laborsaving, without instrument, the testing result specificity is high, good reproducibility.Whole experiment only needs 15 minutes.Easy and simple to handle, quick, accurate, highly sensitive, directly perceived, result judges easily.
Technical scheme of the present invention is achieved in that a kind of porcine reproductive and respiratory syndrome virus antibody colloidal gold fast diagnose test paper bar, it is characterized in that: test strips is sticked on the support slice (5) that does not absorb water successively by sample pad (1), pad (2), nitrocellulose filter (3), absorption pad (4); Wherein be coated with the porcine reproductive and respiratory syndrome virus Nsp9 recombinant protein of colloid gold label on the pad (2); Encapsulate the detection line T line (6) of SPA formation and the nature controlling line C line (7) that anti-Nsp9 protein monoclonal antibody IgG constitutes on the nitrocellulose filter (3) respectively.
The pig breeding of the colloid gold label that encapsulates on the described pad (2) is following with the preparation method of respiratory virus Nsp9 albumen: (1) at first utilizes genetic engineering bacterium prokaryotic expression Nsp9 albumen; Prepared antigen is porcine reproductive and respiratory syndrome virus Nsp9 albumen; And be viral peculiar albumen, this has just determined the specificity of test strips;
(2) with the PRRSV Nsp9 protein Preparation of purifying gold mark antigen, transfer the pH value to 8.2 of collaurum with 0.1M sal tartari, press 100ug/milliliter collaurum adding Nsp9 albumen, magnetic stirring apparatus mixing 30 minutes, add BSA to final concentration be 1%, left standstill 1 hour.Centrifugal 30 minutes of 4 ℃ of 13000rpm abandon supernatant, and deposition is preserved the liquid washed twice with the mark washing, and with the resuspended deposition of washing preservation liquid of 1/10th initial collaurum volumes, 4 ℃ subsequent use.
(3) pad was soaked in confining liquid promptly among 2% BSA, 0.5% Tween-20,2.5% sucrose, 0.05% Sodium azide, the 0.01M PBS 30 minutes, 37 ℃ of oven dry; The gold mark antigen that will prepare then evenly is sprayed on the pad, spreads 20 square centimeters, freeze-drying, preservation for every milliliter.
The detection line T line (6) that is coated with SP (SPA) formation on the described nitrocellulose filter (3) is to select for use a kind of albumen (SPA) that can combine with IgG molecule Fc fragments specific to be sprayed on the detection line; With encapsulating damping fluid SPA is diluted to 50ug/ml; The adjustment machine is scribed ss the T line; Be detection line near pad, apart from the about 5mm of pad one end.
Described PRRSV Nsp9 monoclonal antibody 2D6 (IgG
1) the nature controlling line C line (7) that constitutes be with PRRSV Nsp9 albumen as antigen, immune mouse utilizes hybridoma technology to prepare anti-PRRSV Nsp9 monoclonal antibody; Obtain cell line 2D6; And the acquisition antibody purified, with encapsulating damping fluid monoclonal antibody being diluted to 50-100ug/ml, the adjustment machine is scribed ss the C line; Be control line near absorption pad, apart from the about 3mm of absorption pad end; Two linear distance 5-8mm on the nature controlling line on the nitrocellulose filter NC film, can mark antigen with gold and combine, and check the validity of test strips with this.
The detection method of described test strips is that anti-PRRSV Nsp9 antibody Nsp9 antigen first and colloid gold label combines in the sample serum, because capillarity, the reaction compound is along coated film swimming forward; If in the sample PRRSV antibody is arranged, run into the SPA that is coated on the nitrocellulose filter when arriving detection line, will form gold mark Ag-Ab-SPA compound; Thereby aggegation forms specific red precipitate line on detection line, does not have then can directly pass through detection line with the gold mark antigen of antibodies; Be enriched in and form the red precipitate line on the nature controlling line, promptly be judged to positive findings, if no PRRSV Nsp9 antibody in the sample; The reaction compound runs into SPA when arriving detection line just can not form compound, and the reaction compound is enriched on the nature controlling line through detection line; Form red precipitate, promptly be judged to negative findings.
Advantage of the present invention is
1, have biological safety, its employed gold mark antigen is the recombinant viral proteins that genetic engineering bacterium is expressed, and does not contain porcine reproductive and respiratory syndrome virus, does not therefore have the danger of the poison that looses.
2, high specificity, colloidal gold strip gold mark antigen can be specific and PRRSV Nsp9 antibodies, and the SPA on the detection line can combine with IgG molecule Fc fragments specific, is capture antigen, and the two has improved the susceptibility and the specificity of detection.
3, but its fast detecting is 10-15 minute; Do not need special instruments and equipment, can be used for execute-in-place; Simple to operate, a step accomplishes, and need not professional's operation; The detection cost is low; Convenient for storing.
Description of drawings
Fig. 1 is a PRRSV recombinant protein involved in the present invention.
Fig. 2 is the tactic pattern figure of test strips of the present invention.
Fig. 3 judges synoptic diagram for test strips result of the present invention.
Embodiment
Below in conjunction with instantiation the present invention is elaborated.Experimental technique among the following embodiment like no specified otherwise, is conventional method.
Embodiment 1Express porcine reproductive and respiratory syndrome virus Nsp9 protein Preparation
1, the amplification of Nsp9 gene 7817 – 8657nt: with reference to PRRSV BJ-4 sequence, design and synthesize 1 pair of primer, the part fragment (7817 – 8657nt) of amplification Nsp9 gene.
The upstream primer underscore is the HindIII sequence, and the downstream primer underscore is the XhoI sequence; The PCR reaction conditions does, 94 ℃ of warm starts 5 minutes, and 94 ℃ of sex change 60 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and last 72 ℃ were extended 10 minutes, and amplified production is 840bp, through 1% agarose electrophoresis; 1.2Nsp9 the clone of genetic fragment in pMD-18T: utilize glue to reclaim kit amplified production and reclaim; Utilize the T-A principle; Be connected with the pMD-18T carrier, transform DH5 α competence Escherichia coli, coat the LB sheet plate of ampicillin; The white single bacterium colony of picking typical case is cultivated after 12 hours, carries DNA.Identify called after pMD-Nsp9 through HindIII and XhoI double digestion; 1.3Nsp9 the clone of gene in pET-28a: prokaryotic expression carrier pET-28a is the efficient expression vector that the upper reaches have the His label, with HindIII and XhoI double digestion recombinant plasmid pMD-Nsp9, inserts fragment 840bp; Reclaim wherein 840bp,, judge connection with the Nsp9 gene that has identical cohesive end with HindIII and XhoI double digestion pET-28a; Transformed into escherichia coli BL21 competence bacteria; The screening of card Na penicillin resistance, a small amount of nutrient culture media incubated overnight of picking list bacterium colony is carried DNA for a short time; Identify recombinant plasmid through restriction enzyme, obtain recombinant clone called after pET-28a-Nsp9.
2, the abduction delivering of pET-28a-Nsp9: the single bacterium colony of fresh Escherichia coli (BL-21) 37 ℃ of shaken cultivation in the LB nutrient culture media that will contain recombinant plasmid are spent the night; Next day is by 1% volume ratio switching 5ml fresh culture; 37 ℃ are continued to cultivate; When bacterium liquid OD600 value reaches 0.6-0.8, add aseptic IPTG solution, making its final concentration is 1mmol/L.Induce to stop after 3-4 hour cultivating, get the centrifugal collection thalline of 1ml, PBS (pH7.4) washing with 50 μ l PBS suspension thalline, mixes with 10 μ l, 5 * SDS sample-loading buffer, boils sex change 5 minutes.Carry out SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the acrylamide concentration of separation gel is 12%, the Escherichia coli that only contain carrier is induced as negative control equally the dyeing of 0.25% Coomassie brilliant blue; After identifying, shake bacterium in a large number, the centrifugation thalline, ultrasonication is after electroelution obtains highly purified recombinant protein, and is subsequent use in-80 ℃ of preservations.
Embodiment 2The preparation of anti-PRRSV Nsp9 protein monoclonal antibody:
1, animal immune is selected the PRRSV Nsp9 albumen of 6-8 health Balb/C in age in week mouse with Freund's complete adjuvant emulsification purifying; Every mouse peritoneal is injected about 100 μ g; Behind the 14d with incomplete Freund emulsified protein lumbar injection 100 μ g; During last booster immunization, directly lumbar injection 100 μ g purified proteins merge preceding 3～4d tail vein injection, 50 μ g purified proteins.
2, the Fusion of Cells splenocyte and the SP2/0 that get immune mouse is mixed in the fusion pipe, with centrifugal 10 min of 300g, supernatant discarded; The vibration cell mixes two kinds of cells as far as possible, slowly drips the PEG-4000 solution of preheating then in the 60s; 1640 nutrient culture media that slowly add serum-free again stop merging; Again with centrifugal 10 min of 1 000 r/min, add the HAT nutrient culture media after the supernatant discarded after leaving standstill, make cell suspension and mixing; In 96 well culture plates, cultivate, 14d begins to change liquid and begins to detect screening
3, indirect ELISA is adopted in hybridoma screening and cloning clone's screening, with the PRRSV Nsp9 albumen of purifying as envelope antigen.Culture supernatant to obtaining positive hybridoma cell is screened; The positive hybridoma cell of ELISA screening is carried out cloning, with the positive hole that detects time cloning and subclone again, all positive up to all holes.Finally obtain hybridoma cell strain 2D6 through three cell clones, antibody subtype is IgG1.
4, purifying antibody is expelled to the cell line that obtains in the Balb/C mouse peritoneal, collects ascites after the week, the monoclonal antibody that obtains with IgG purifying pillar purifying.
Embodiment 3The preparation of gold mark antigen
1, the colloid gold particle preparation is mixed with 0.01% WS with chlorauride; After getting 100ml and boiling 2min; Add 1% trisodium citrate 2ml while stirring, boil become claret to solution colour after, continue to boil to suitable concentration OD535=0.9312; The cooling back adds DDW and returns to original volume, puts 4 ℃ of preservations;
2, with PRRSV Nsp9 albumen doubling dilution to be marked, get 100 μ l respectively and add in the 1ml collaurum, add 10%NaCl 100 μ L, 4 ℃ of static 1h behind the 10min.The highly diluted multiple of getting that the collaurum color do not change is as the criterion, and adds in the colloidal gold solution of pH 8.0 in the ratio of the minimum protein concentration that the records PRRSV Nsp9 albumen with purifying, and the limit edged stirs, and room temperature leaves standstill 30 min; Add 10% bovine serum albumin(BSA) (BSA), making its final concentration in solution is 1%, and room temperature leaves standstill 15 min; 4 ℃, centrifugal 30 min of 8 778 * g inhale and remove supernatant, are suspended into original volume with resuspended liquid I (containing BSA etc.), and 4 ℃, centrifugal 30 min of 8 778 * g remove supernatant, and deposition is hanged with resuspended liquid II (containing sucrose, BSA, Tween-20 etc.) 1/5 volume, and 4 ℃ of preservations are subsequent use.
Embodiment 4The preparation of immune serum
Immune serum is as anti-PRRSV Nsp9 hyper-immune serum when collecting monoclonal antibody and preparing, and is subsequent use in-80 ℃ of preservations.
Embodiment 5The acquisition of SPA
SP (SPA) is purchased the company in sigma, and SPA is made into 50ug/ml, preserves subsequent use.
Embodiment 6The development of test strips
1, the composition of test strips is sprayed on the colloid gold label albumen composition on the collaurum pad with Membrane jetter, SPA albumen and anti-PRRSV Nsp9 albumen monoclonal antibody 2D6 interval 4mm is sprayed on the nitrocellulose membrane NCM, respectively as detecting band and quality control band; The NCM that encapsulates is put into the PBS that contains 3%BSA, spend the night, seal all the other protein binding sites; Outwell confining liquid, use PBS, wash 2 times; Each 5min, 37 ℃ of dry 1h are subsequent use, and nitrocellulose membrane, collaurum pad, sample pad, adsorptive pads etc. once are bonded on the PVC plate; That is, spun glass is connected on the NCM edge and attached on the NCM; The cotton pulp pad is connected with NCM attached on the spun glass; The absorbent filter plate is connected the NCM lower end; Edge and attached on the NCM is cut into the test strips that 60mm is long, 4mm is wide with the PVC material that glues, and promptly processes the porcine reproductive and respiratory syndrome virus colloidal gold antibody fast test strip; Test strips is sealed in the aluminium foil bag 4 ℃ of preservations;
2, being coated with the detection line T line (6) that SPA constitutes on the described nitrocellulose filter (3) is the recombination staphylococcus aureus albumin A of selecting for use genetic engineering bacterium to express; It is sprayed on the detection line; With encapsulating damping fluid SPA is diluted to 20-50ug/ml; The adjustment machine is scribed ss the T line, is detection line near pad, apart from the about 5mm of pad end.
3, the nature controlling line C line (7) of described mouse anti PRRSV Nsp9 monoclonal antibody IgG formation is the antibody of secreting with the cell line to the single epi-position of PRRSV Nsp9 that hybridoma technology obtains; With encapsulating damping fluid it is diluted to 50-100ug/ml; The adjustment machine is scribed ss the C line; Be control line near absorption pad, apart from the about 3mm of absorption pad end; Two linear distance 5-8mm on the nature controlling line on the nitrocellulose filter NC film, can mark antigen with gold and combine, and check the validity of test strips with this.
4, the detection method of said this paper slip is the Nsp9 protein combination of anti-porcine reproductive and respiratory syndrome virus Nsp9 antibody elder generation and colloid gold label in the sample serum; Because capillarity, reaction compound are along coated film swimming forward, if in the sample corresponding antibodies is arranged; Run into the SPA that is coated on the nitrocellulose filter when arriving detection line; Will form gold mark antigen-Nsp9 antibody-SPA compound, thereby aggegation forms specific red precipitate line on detection line; Do not have then can directly pass through detection line, be enriched in and form the red precipitate line on the nature controlling line, promptly be judged to positive findings with the gold mark antigen of antibodies.If no porcine reproductive and respiratory syndrome virus Nsp9 antibody in the sample; Sample runs into SPA when arriving detection line just can not form gold mark antigen-Nsp9 antibody-SPA compound, can not form the red precipitate line, and only be enriched on the nature controlling line; Form red precipitate, promptly be judged to negative findings.
Embodiment 7The application of colloidal gold strip
1, the method for application of test strips of the present invention
(1) specimen preparation: clinical collection animal blood serum to be checked is used for detecting.
(2) detect: take out test strips, equilibrium at room temperature 20 minutes splashes into serum to be checked in the test strips detection window, takes out, and keeps flat, and leaves standstill result of determination 10-15 minute.
(3) result judges: macroscopic aubergine nature controlling line only appearred in paper slip at that time, macroscopic aubergine detection line do not occur, was judged to feminine gender, was designated as "-"; When macroscopic aubergine nature controlling line appears in test strips, macroscopic aubergine detection line appears simultaneously, be judged to the positive, be designated as "+"; The detection line color depth is directly proportional with antibody horizontal to be checked, and color explains that more deeply serum titer to be checked is high more, and nature controlling line does not have band and then is judged to the test strips inefficacy.
Embodiment 8Test strips susceptibility specific assay
1, the sensitivity tests of test strips
With the mice serum of PRRSV Nsp9 protein immunization as detecting positive serum; Be used for the test of test strips sensitivity Detection; The positive serum titre is diluted to 2560 times, detects with test strips, the result when dilutability be that test strips detects negative more than 160 times the time; Dilutability is a test strips test positive below 80 times the time, and the result sees table 1
2, the specificity of test strips test
Detecting PRRSV with test strip, to infect serum positive, and detect deactivation PRRSV immune serum, pig circular ring virus, pig parvoviral, that swine influenza virus infects the serum result is negative.The proof test strips is comparatively special.The result sees table 2.
3, the replica test of test strips
5 parts of detections that repeat in batch, the result is consistent.The test strips of 4 different batches detects, and the result is consistent.The repeatability that this test strips is described is better.
Claims (5)
1. porcine reproductive and respiratory syndrome viral disease antibody colloidal gold fast diagnose test paper bar, it is characterized in that: test strips is sticked on the support slice (5) that does not absorb water successively by sample pad (1), pad (2), nitrocellulose filter (3), absorption pad (4); Wherein be coated with the reorganization PRRSV Nsp9 albumen of colloid gold label on the pad (2); Be coated with detection line T line (6) and PRRSV Nsp9 protein monoclonal antibody 2D6 (IgG that SP (SPA) constitutes on the nitrocellulose filter (3) respectively
1) the nature controlling line C line (7) that constitutes.
2. a boar according to claim 1 breeding with the sick antibody colloidal gold fast diagnose test paper bar of breath syndrome virus, the PRRSV Nsp9 protein preparation method of the colloid gold label that it is characterized in that encapsulating on the described pad (2) is following:
(A) at first prepare PRRSV Nsp9 antigen, utilize gene clone technology from the PRRSV genome, to clone the Nsp9 genetic fragment, it is linked among the pET-28a; Constitute the pET-28a-Nsp9 carrier, carrier is transformed among the Escherichia coli BL-21, express Nsp9 albumen; And with its purifying;-80 ℃ of preservations are subsequent use, PRRSV Nsp9 albumen only with anti-Nsp9 antibodies, this has just determined the specificity of test strips;
(B) the PRRSV Nsp9 protein labeling collaurum of selecting for use purifying to send out, preparation gold mark antigen is with the pH value to 8.2 of 0.1M sal tartari accent collaurum; Press 100ug antigen/milliliter collaurum and add Nsp9 albumen, magnetic stirring apparatus mixing 30 minutes, add BSA to final concentration be 1%; Left standstill 1 hour, centrifugal 30 minutes of 4 ℃ of 13000rpm abandon supernatant; Deposition is preserved the resuspended deposition of liquid with preserving the liquid washed twice with the washing of 1/10th initial collaurum volumes, and 4 ℃ subsequent use;
(C) pad was soaked in confining liquid promptly among 2% BSA, 0.5% Tween-20,2.5% sucrose, 0.05% Sodium azide, the 0.01M PBS 30 minutes, 37 ℃ of oven dry; The golden labeling antibody that will prepare then evenly is sprayed on the pad, spreads 20 square centimeters, freeze-drying, preservation for every milliliter.
3. boar breeding according to claim 1 and breathing syndrome viral disease antibody colloidal gold fast diagnose test paper bar; It is characterized in that being coated with on the described nitrocellulose filter (3) the detection line T line (6) that SP (SPA) constitutes is to select for use a kind of albumen (SPA) that can combine with IgG molecule Fc fragments specific to be sprayed on the detection line; With encapsulating damping fluid SPA is diluted to 50ug/ml; The adjustment machine is scribed ss the T line; Be detection line near pad, apart from the about 5mm of pad one end.
4. a kind of porcine reproductive and respiratory syndrome viral disease antibody colloidal gold fast diagnose test paper bar according to claim 1 is characterized in that described PRRSV Nsp9 monoclonal antibody 2D6 (IgG
1) the nature controlling line C line (7) that constitutes be with PRRSV Nsp9 albumen as antigen, immune mouse utilizes hybridoma technology to prepare anti-PRRSV Nsp9 monoclonal antibody; Obtain cell line 2D6; And the acquisition antibody purified, with encapsulating damping fluid monoclonal antibody being diluted to 50-100ug/ml, the adjustment machine is scribed ss the C line; Be control line near absorption pad, apart from the about 3mm of absorption pad end; Two linear distance 5-8mm on the nature controlling line on the nitrocellulose filter NC film, can mark antigen with gold and combine, and check the validity of test strips with this.
5. boar breeding and the sick collaurum fast diagnose test paper bar of breathing syndrome virus antibody, the detection method that it is characterized in that described test strips are that anti-PRRSV Nsp9 antibody combines with the Nsp9 antigen of colloid gold label earlier in the sample serum, owing to capillarity; The reaction compound if in the sample PRRSV antibody is arranged, runs into the SPA that is coated on the nitrocellulose filter along coated film swimming forward when arriving detection line; Will form gold mark Ag-Ab-SPA compound, thereby aggegation forms specific red precipitate line on detection line; Do not have then can directly pass through detection line, be enriched in and form the red precipitate line on the nature controlling line, promptly be judged to positive findings with the gold mark antigen of antibodies; If no PRRSV Nsp9 antibody in the sample; The reaction compound runs into SPA when arriving detection line just can not form compound, and the reaction compound is enriched on the nature controlling line through detection line; Form red precipitate, promptly be judged to negative findings.
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| CN104710528A (en) * | 2015-03-13 | 2015-06-17 | 西北农林科技大学 | Specific binding PRRS (Porcine Reproductive and Respiratory Syndrome) virus non-structural protein Nsp9 nanobody and application thereof |
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