CN103756973A - Indirect ELISA (enzyme linked immunosorbent assay) detection kit for grass carp reoviruses - Google Patents

Abstract

The invention discloses a monoclonal antibody resistant to grass carp IgM, a hybridoma cell strain and an application thereof. The monoclonal antibody resistant to grass carp IgM is generated by the secretion of the hybridoma cell strain S467 with a collection number of CCTCC NO: C2013193; an indirect ELISA detection kit for grass carp reoviruses is created by virtue of the monoclonal antibody, and the kit comprises the enzyme marker of the monoclonal antibody resistant to grass carp IgM, and an ELISA plate coated with an antigen. The indirect ELISA detection kit for grass carp type-II reoviruses, created by the monoclonal antibody disclosed by the invention can be used for qualitatively or quantitatively detecting the specific antibody of the grass carp type-II reoviruses, and can be used for detecting a plurality of serum samples of grass carp and achieving the purpose of early diagnosis simultaneously, thus having high clinic use and promotion values for diagnosis on the hemorragic disease of grass carp, epidemiological investigation and pathogen monitoring, and providing a technical guarantee on reduction for great economic losses brought by the hemorragic disease of grass carp, during an aquaculture process.

Description

A kind of indirect ELISA testing kit of GCRV

Technical field

The invention belongs to biological technical field, be specifically related to monoclonal antibody, hybridoma cell strain and the GCRV indirect ELISA testing kit of a kind of anti-grass carp IgM.

Background technology

GCRV (Grass carp reovirus, GCRV) is subordinate to Aquareovirus, is Reoviridae one newcomer, is the first strain fishes virus that China's Mainland separates.This virus mainly causes that the grass carp kind in Asian countries's freshwater aquicultures such as China, Vietnam, Burma, in the fingerling stage, hemorrhagic disease occurs, mortality ratio is generally 30%-50%, reach as high as 60%-80%, and can also infect black carp, pseudorasbora parva, Bu Shi meal bar fish etc., and can make these several fish generation hemorrhagic disease symptoms and death, this viral prevalence is wide, harm is large, mortality ratio is high, morbidity season is long, has had a strong impact on numerous culturists' enthusiasm and the sound development of China's freshwater aquaculture industry.GCRV tool double capsid, virus particle mean diameter is 60 nm-70nm, icosahedron symmetry, without cyst membrane, genome is comprised of 11 segmented double-stranded RNAs.Oneself is through having reported more than 20 strain isolated at present, comprise GCRV854, GCRV861, GCRV873, GCRV875, GCRV876, GCRV991, H962, ZV-8802, GCHV-854, GCRV HZ08, JX09-01, GCRV-104, GD10 etc., different isolates differs greatly at aspects such as genome sequence, genome banding pattern, cytopathy, virulencies to grass carp.GCRV does not also carry out phylogenetic systematics to it so far in serology or genotype.According to existing strain isolated sequence information, carry out that nucleotide sequence and aminoacid sequence are compared and constructing system phylogenetic analysis, institute's toxic strain can be divided into 3 large classes generally, be divided into 3 genotype, be GCRV I type (representative strains is GCRV-873 and GCRV-JX09-01), GCRV II type (representative strains is GCRV-HZ08 and GCRV-GD10) and GCRV III type (GCRV-104, GCRV-HB1007).Current, in the epidemic strain that all parts of the country are separated to, three class hypotypes all have report, and the independent infection having, also has polyinfection.By our 2009 to 2013 these hemorrhagic disease of grass carp of 4 years monitoring and epidemiological survey and analysis, show, causing at present popular and the main of outburst of hemorrhagic disease of grass carp is GCRV II type.The specific diagnosis of the hemorrhagic disease of grass carp therefore, GCRV II C-type virus C being caused is significant.

The detection method of GCRV has multiple, each tool advantage and defect, virus separation, electron microscopic observation, nucleic acid belt type analysis are still the method that GCRV generally adopts that detects so far, but these method methods waste time and energy, operate more complicated, can not carry out quick diagnosis to virus, be unfavorable for the epidemiology survey work of hemorrhagic disease of grass carp.Totivirus diagnosis relates to virus culture, purifying and the process such as concentrated, not only complex manufacturing process, and consuming time, cost is higher, but also potential loose poison is dangerous, is therefore unfavorable for promoting the use of.Current, for the detection of GCRV, the most conventional method is the various molecular biology for detections of PCR-based amplification technique, comprises conventional RT-PCR and quantitative fluorescent PCR etc., has higher susceptibility and specificity.But these molecular biology methods are all to detect viral nucleic acid whether to exist, and method is single, often causes false positive and false negative result, is difficult to this disease to accomplish to make a definite diagnosis.Therefore, be badly in need of other method and make up the deficiency of molecular biology for detection, as various immunologys (serology) detection method.

Up to now, for the control of hemorrhagic disease of grass carp, also there is no special effective medicine and method, the most effectively way remains vaccinate, carries out immunoprophylaxis.At present, for the vaccine of hemorrhagic disease of grass carp, be no matter popular local method vaccine on the cell weak-toxic vaccine of rules license or market, even some cell deactivation epidemic diseases, the overwhelming majority is all the vaccine for GCRV II type.And evaluation to these immune effect of vaccine, just according to the mortality ratio of immune grass carp, come according to a preliminary estimate, once the outburst of other epidemic disease causes immune grass carp death, just cannot understand fully it is because vaccine effect is bad, hemorrhagic disease of grass carp still break out cause and death, or due to the death that the outburst of other epidemic disease causes, more cannot evaluate immune effect of vaccine.Therefore, be also badly in need of wanting a kind of immunological method that detects GCRV specific antibody.

Summary of the invention

Our nearest research is found: the proteins encoded (SEQ ID NO:2) of GCRV-HZ08 strain S10 gene (SEQ ID NO:1) is structural protein, and than other structural protein induction of immunity animal of GCRV, produce the more specific antibody of high-titer, there is better immunogenicity; Can specific recognition GCRV II type strain for the antibody of S10 proteins encoded, and the type strain is had to very strong neutralising capacity; Therefore, can utilize S10 gene coded protein to catch the specific antibody in body, thereby realize the detection of specific antibody.

Therefore, one object of the present invention is to provide a strain of hybridoma strain S467, on December 10th, 2013, be preserved in Chinese Typical Representative culture collection center (CHINA CENTER FOR TYPE CULTURE COLLECTION), address: Wuhan, Wuhan University, deposit number is CCTCC NO:C2013193.

It is the monoclonal antibody of the anti-GCRV II type of the hybridoma cell strain secretion generation of CCTCC NO:C2013193 by above-mentioned deposit number that another object of the present invention is to provide a kind of.

Another object of the present invention is to provide a kind of GCRV indirect ELISA testing kit.

The technical solution used in the present invention is:

Hybridoma cell strain S467, is preserved in Chinese Typical Representative culture collection center on December 10th, 2013, and deposit number is CCTCC NO:C2013193.

A monoclonal antibody of anti-grass carp IgM, its hybridoma cell strain or secretion of its passage cell strain that is CCTCC NO:C2013193 by deposit number produces.

The application in the anti-GCRV II type of preparation and/or hemorrhagic disease of grass carp detection reagent or diagnostic reagent of above-mentioned hybridoma cell strain or monoclonal antibody.

A kind of GCRV indirect ELISA testing kit, described test kit comprises the enzyme labelling thing of the monoclonal antibody of anti-grass carp IgM, is coated with the enzyme plate of antigen.

Hybridoma cell strain or the secretion of its passage cell strain that the monoclonal antibody of described anti-grass carp IgM is CCTCC NO:C2013193 by deposit number produce.

Described antigen is the proteins encoded of GCRV II type S10 gene, and its sequence is as shown in SEQ ID NO:2.

In described test kit, also comprise standard serum, TMB nitrite ion, stop buffer and washings.

A kind of GCRV indirect ELISA detection method, comprises the steps:

(1) application of sample: sample to be checked is added in the coated plate reacting hole of enzyme mark of the proteins encoded (SEQ ID NO:2) that is coated with GCRV II type S10 gene;

(2) incubation: with the rearmounted 37 ℃ of incubations of shrouding film shrouding;

(3) washing: take shrouding film off, with PBST washings detersive enzyme target;

(4) add two to resist: in reacting hole, add ELIAS secondary antibody, described ELIAS secondary antibody is the horseradish peroxidase-labeled thing of the monoclonal antibody of anti-grass carp IgM;

(5) incubation: operation is with (2);

(6) washing: operation copper (3);

(7) colour developing: add TMB developer in reacting hole, 37 ℃ of lucifuge colour developings;

(8) mensuration and result judgement.

The invention has the beneficial effects as follows:

1, hybridoma cell strain S467 of the present invention can also continue to produce anti-grass carp Immunoglobulin IgM monoclonal antibody by infinite multiplication, and this monoclonal antibody specificity is strong, the height of tiring.

2, the antibody of the envelope antigen S10 proteins encoded in the present invention can specific recognition GCRV II type strain, can use it to catch the specific antibody in body, thereby realize qualitative detection and the quantitative analysis of specific antibody.

3, GCRV II type ELISA detection kit of the present invention can be to the qualitative or detection by quantitative of carrying out of GCRV II type specificity antibody, while can be detected many parts of serum samples of grass carp and reach the object of early diagnosis, and diagnosis, epidemiology survey and cause of disease monitoring to hemorrhagic disease of grass carp have higher clinical use and promotional value; And can be used for the detection of antibody after vaccine immunity injection, carry out the evaluation of immune effect of vaccine, for the prevention and control of hemorrhagic disease of grass carp and immunologic surveillance etc. provide the technique means of science.By the application of this test kit, will provide technical guarantee for China controls and finally eliminate hemorrhagic disease of grass carp, thereby reduce the tremendous economic loss bringing because of hemorrhagic disease of grass carp in aquaculture process.

Accompanying drawing explanation

Fig. 1 is the SDS-PAGE glue detection figure (1 is Marker, and 2 is anti-grass carp IgM monoclonal antibody) of anti-grass carp IgM monoclonal antibody;

Fig. 2 is the SDS-PAGE glue detection figure (M is Marker, and 1 is the S10 albumen after purifying, and 2 was that Ni post stream is worn liquid, and 3 is bacterial protein) of GCRV-HZ08 strain S10 gene coded protein.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.

The molecular biology experiment technology adopting in following examples comprises that pcr amplification, plasmid transform, DNA fragmentation connects, enzyme is cut, gel electrophoresis etc., if no special instructions, conventionally according to ordinary method, operate, specifically can be referring to < < molecular cloning experiment guide > > (third edition) (Sambrook J, Russell DW, Janssen K, the yellow training hall of Argentine J. waits to be translated, 2002, Beijing: Science Press), or the condition of advising according to manufacturer.

Embodiment

preparation, purifying and the mark of the anti-grass carp IgM of embodiment 1 monoclonal antibody

1. antigen preparation

Blood of Ctenopharyngodon is taked in 1.1 dockings, is placed in Glass tubing, under room temperature, solidifies, and places 2h, then spend the night in 4 ℃ of placements at 37 ℃, and after serum is fully separated out, the centrifugal 1min of 1000g/min, gets its supernatant.

1.2 pillar preparations: 6ml Protein A Agarose is dissolved with the molten peptide damping fluid of 1.5ml (0.1M NaHCO3,0.5M NaCl, pH8.3), pack centrifuge tube into, 4 ℃ of centrifugal 1min of 600g/min, abandon supernatant.Every pipe adds 1mL to contain the PBS damping fluid (pH7.2) of Protein A, puts upside down and mixes 30min.Then sucking-off upper prop, crosses post with the molten peptide damping fluid of 5 times of column volumes.With sealing damping fluid, (0.1M Tris-HCl, after pH8.0 sealing 2h, by washing lotion, ((0.1M N Tris-HCl, 0.5M NaCl, pH8.0) washes post 3 times.Then by pillar, by PBS balance to ultraviolet registration, be 0.

1.3 loadings: measure 30ml serum loading, regulating ultraviolet registration is 0, regulates flow velocity (1mL/min) loading of constant flow pump.Treat that registration rises to more than 50, collect effluent liquid.After complete on serum, with PBS, wash.After ultraviolet registration is less than 50, do not regather effluent liquid.When registration is 0, allow PBS flow to glue face tangent.Add elution buffer to soak wash-out after 1min, collect the elutriant that registration is greater than 0.With PBS balance to registration be 0.Then repeat loading wash-out.Next day, the centrifugal precipitation of going, surveys concentration, and-20 ℃ save backup.

The detection of IgM after 1.4 purifying: by the purification effect of IgM in SDS-PAGE electrophoresis detection serum, sample mixes rear with equal-volume sample buffer, 100 ℃ of boiling water water-bath 3-5min, loading after centrifugal.Resolving gel concentration is 12%, and electrophoretic voltage is 100V, and electrophoresis time is 1h, uses coomassie brilliant blue staining after electrophoresis.

Detected result shows, through the sample of Protein A affinity chromatography column purification, carries out SDS-PAGE detection, obtains two bands that size is about 75kD and 25kD, close with light chain molecular weight with the grass carp IgM heavy chain of expection.

2. animal immune

The immunity Balb/C mouse in 6 weeks age take the grass carp IgM of above-mentioned purifying as specific antigens, gets 5 female BALB/c mouse subcutaneous injection in 6 weeks age antigens (20 μ g/ only), immunity and the emulsification of equivalent Freund's complete adjuvant for the first time, 0.2 mL/; After two weeks, carry out immunity for the second time, antigen and the emulsification of equivalent Freund's incomplete adjuvant, subcutaneous injection, 0.2 mL/ is only; After two weeks, carry out immunity for the third time, immunization method and dosage are with immunity for the second time.After immune one week for the third time, mouse orbit blood sampling is surveyed it and is tired, and after immune two weeks for the third time, selects to tire the highest mouse, with the antigen booster immunization that does not add adjuvant, gets mouse boosting cell and SP2/0 cytogamy after 3 d.

3. cytogamy

3.1 myeloma cells' preparation

Select growth conditions good SP2/0 cell, while covering the bottle end 80%, abandon supernatant, after incomplete substratum washing once, cell is blown down gently with the incomplete substratum of 10 mL.

The preparation of 3.2 splenic lymphocyte

Get after booster immunization the mouse of 3 days, extract eyeball and take a blood sample as positive serum; Neck dislocation is lethal by mouse, with 75% alcohol disinfecting body surface 10 min, puts into immediately on the dissection plate of super clean bench, and belly upward, fix by four limbs; Spleen is taken out in the aseptic abdominal cavity of opening, and with basic medium washing, and carefully removes the reticular tissue adhering to around; Subsequently spleen is transferred in another plate that fills DMEM.With grinding rod extruding, splenocyte is fully discharged, make splenocyte suspension.

The preparation of 3.3 feeder cell

Get a ripe healthy ICR mouse, pluck eyeball and take a blood sample as negative serum, neck dislocation is put to death; Body surface sterilization and fixing after, expose peritonaeum, cotton ball soaked in alcohol sterilization peritonaeum; With l0 mL syringe, No. 12 syringe needles, draw 10 mL HAT substratum to abdominal cavity, the right hand is fixed syringe, and left hand is held cotton ball soaked in alcohol and is massaged gently belly, draws back intraperitoneal liquid, injects oneself ready container.

3.4 merge

The myeloma cell of above-mentioned preparation and splenocyte are mixed in the fusion pipe with cover of 50 mL, and centrifugal 10 min of 1000 rpm, abandon supernatant.Fusion pipe is placed in to palm, and friction bottom, fully mixes two kinds of cells gently; In 37 ° of C water-baths, in 45 s, slowly add the PEG-1500 of 1 mL preheating in fusion pipe, limit edged shakes up gently; The fast incomplete DMEM substratum that drips 37 ° of C preheatings of 30 mL after first slow in 90 s immediately, makes PEG dilution and ineffective, 37 ° of standing 10 min of C, centrifugal 10 min of 1000 rpm; Abandon supernatant, with the 60 mL HAT substratum sedimentation cell that suspends gently, then add appropriate peritoneal macrophage; Be sub-packed in 96 porocyte culture plates, then culture plate put in 37 ° of C, 6% CO2 incubator and cultivated; After 5 d with the fresh HAT substratum half substratum that swaps out; After 10 d with the HT of the preheating HAT that swaps out; Observe the growing state of hybridoma, until its cells and supernatant flavescence or clone, be distributed to 1/10 when above of hole floorage, draw appropriate cell conditioned medium and carry out ELISA detection, twice positive positive colony hole that is made as of detected result.

The cloning of 3.5 hybridomas and hybridoma cell strain are set up

Adopt limiting dilution assay to carry out cloning to the cell strain of double test positive.Positive cell counting, get 100 cells and put into the nutrient solution of 5 mL containing feeder cell, i.e. 20 cell/mL, 100 μ L/ holes drip in 96 porocyte culture plates, be 1, every hole cell: again by remaining cell suspension doubling dilution, cell count is 10/mL, and 100 μ L/holes drip in 96 porocyte culture plates, i.e. 0.5, every hole cell.During 8-9 d, naked eyes visible cell clone, screens by the indirect ELISA method of screening positive hybridoma cell the hole that occurs individual cells clone again.Carry out continuously more than three times subclone, be judged to positive cell strain enlarged culturing, obtain hybridoma cell strain, on December 10th, 2013, be preserved in Chinese Typical Representative culture collection center (CHINA CENTER FOR TYPE CULTURE COLLECTION), address: Wuhan, Wuhan University, deposit number is CCTCC NO:C2013193.Positive hybridoma cell strain is put into liquid nitrogen cryopreservation.

3.6 ascites preparation and antibody purifications

Get 4 BALB/C mice, every injection 0.5ml paraffin oil, after 7 days, get hybridoma (the higher while cell state of tiring is good) and be resuspended in serum free medium, by 1 × 106 cell/0.5ml/, only measure injection paraffin mouse, after the about 7-14d of injection cell, collect ascites.First use Binding Buffer balance protein G affinity column steady to baseline; By ascites sample upper prop, collect stream and wear liquid; Stream is worn to liquid upper prop again, continue balance steady to baseline; Add Eluting Buffer wash-out, with 0.01M, after the PBS dialysis that pH is 7.2, collect elution peak, make the antibody after purifying be kept at 0.01M, in the environment of pH7.2 PBS; By the purity of antibody after SDS-PAGE glue detection purifying.Result shows, by the antibody of protein G affinity column purifying, its purity is shown in Fig. 1 higher than 95%().

4. the preparation of horseradish peroxidase labeling antibody

The sodium periodate method of sampling improvement is resisted grass carp IgM monoclonal antibody and is carried out horseradish peroxidase (HRP) mark, and its concrete steps are as follows:

(1) get 5mg HRP and be dissolved in 0.5mL distilled water, add 1%2,4 and dinitrofluorobenzene (DNFB) ethanol solution 0.1mL, the lower gentle agitation effect 1h of room temperature (20 ℃ ±).

(2) add 1mL 0.06mol/L NaIO4, under room temperature, lucifuge is gently stirred 30min, and solution is yellow-green colour.

(3) add 1mL0.16mol/L ethylene glycol, under room temperature (20 ℃ ±), gently stir effect 1 h, stop oxidizing reaction.

(4) add 5mg antibody (anti-IgM monoclonal antibody), pack dialysis tubing into, set to 0 .05mol/L, in pH9.6 carbonate buffer solution (distilled water adds to 1 000 mL for anhydrous sodium carbonate 1.5g, sodium bicarbonate 2.93g) 1 000 mL, 4 ℃ of dialysed overnight, change 3 times.

(5) take out liquid (approximately 3 mL) in dialysis tubing, add 4 ℃ of 2h of 5m,g/m,LNa,HB4 0.2 mL or spend the night.

(6) add 50% saturated ammonium sulphate (NH4) 2SO4 solution (ammonium sulfate 850g, distilled water adds to 1 000 mL, adjusts pH7.2 with 30% ammoniacal liquor), 6 mL precipitation binding substancess, put after 4 ℃ of 30min, the centrifugal 30min of 3 000r/min, abandons supernatant, gets precipitation.

(7) throw out is dissolved in to PBS(0.02M pH7.4) in, pack dialysis tubing into, and with this damping fluid dialysis equilibrium 6-12h, change liquid 3 times.

(8) in binding substances, add BSA to protein concentration be 10mg/ mL, or add equivalent 60% glycerine, after mensuration work is tired, packing in a small amount, cryopreservation is standby.

great expression and the purifying of embodiment 2 GCRV II type S10 gene coded proteins

Utilize Qiagen RNA extract test kit in infecting the CIK cell of GCRV HZ08 strain virus (the chamber preservation of Zhujiang River aquatic products institute's fish disease) extraction RNA, viral RNA reverse transcription is become to cDNA with TAKARA reverse transcription test kit.Utilize upstream primer: 5'-ATA gGA TCCaTG GCG GGT GTG TCT CT CAA CA-3'(SEQ ID NO:3); Downstream primer: 5'-GCT aAG CTTcAG CAT CTG CGC AAA TAT ACG TC-3'(SEQ ID NO:4), in upstream and downstream primer sequence, insert respectively BanH I and Hand III restriction enzyme site (with the gene of underscore), take the cDNA of above-mentioned preparation as masterplate, S10 gene is carried out to pcr amplification.

PCR product detects through 1% agarose gel electrophoresis, finds to occur at about 110bp place an expection specific amplification band of the same size, is S10 gene, and as shown in SEQ ID NO:1, its coded protein sequence is as shown in SEQ ID NO:2.With glue, reclaim test kit and reclaim purifying object band, through sequence verification, conform to expection.

Utilize ordinary method that the S10 gene order of GCRV HZ08 strain (SEQ ID NO:1) is inserted in prokaryotic expression carrier pET-32a, recombinant plasmid transformed is arrived to e. coli bl21 (DE3), picking LB/Amp plate screening obtains positive single bacterium colony to 1mL LB/Amp culture medium culturing 10 ~ 12h, then be inoculated into 1L LB/Amp substratum, 37 ℃, 200 rpm/min constant-temperature tables are cultivated, abduction delivering under the expression condition of optimizing.The bacterium liquid of abduction delivering, at the centrifugal 5min of 10000 rpm/min, is collected to thalline, use the resuspended centrifuge washing of 400ml PBS 2 times, then add 100 ml PBS resuspended, on ice, ultrasonic disruption to solution is clarified.By centrifugal broken liquid collection inclusion body (this albumen solubility verifies as inclusion body protein), then with reference to Ni-gel-purified test kit (inclusion body protein purifying) specification sheets purifying target protein.With 50ml Binding Buffer, dissolve inclusion body and spend the night, the centrifugal 20min of lysate 10000 rpm/min is collected to supernatant, then the membrane filtration of 0.45 μ m, by 10 times of column volumes of the Ni column flow rate that in supernatant load, balance is good/hour, collect stream and wear liquid; Wash away not in conjunction with albumen and foreign protein with the Binding Buffer of 15 times of column volumes, then use 20mlElution Buffer wash-out target protein, collect elution peak (whole purge process is carried out in 4 ℃ of refrigerators).Pack the albumen under wash-out on Ni post into dialysis tubing, put into successively containing 6M, 4M, 2M, 1M, 0.5M, dialyses in the PBS solution of 0M urea, each concentration dialysis 12h(all processes carries out in 4 ℃ of refrigerators).After having dialysed, add PEG-6000,4 ℃ of refrigerators are standing concentrated, are distributed into 1mL/ pipe after concentrated 50%, by running SDS-PAGE glue, analyze its purification effect (see figure 2), with after ultraviolet light absorption Spectrophotometry for Determination concentration-20 ℃ save backup.

the foundation of embodiment 3 GCRV indirect ELISA detection methods

1. antigen and primary antibodie working concentration determines

By the proteins encoded of the restructuring S10 gene of purifying, with carbonate buffer solution (pH9.6) dilution of 50mM, be 20 μ g/mL, 15 μ g/mL, 10 μ g/mL, 8 μ g/mL, 6 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL concentration gradient, coated 96 hole enzyme plates, every hole adds 100uL, 4 ℃ of coated spending the night of wet box.Every hole adds the skimming milk of 200 μ L 5% to seal, and in 37 ℃ of incubators, hatches 1 h, with PBST washing 3 times, and each 3-5 min.To resist GCRV positive serum, grass carp negative serum to carry out respectively 1:25,1:50,1:100,1:200,1:400 and 1:800 and doubly dilute, every hole adds 100 uL to carry out the test of ELISA square formation, with PBST washing 3 times, and each 3-5 min.By the mouse-anti grass carp IgM antibody 1:5000 of the HRP mark of above-mentioned preparation, every hole 100uL, hatches 1h in 37 ℃ of incubators, with PBST washing 3 times, and each 3-5 min.Add 50 μ L chromogenic substrates (TMB), 37 ℃ of lucifuges, 10 min that develop the color.With 50 μ L 2mol/L H ₂sO ₄termination reaction.Measure each hole OD ₄₅₀nm value, defined antigen and primary antibodie working concentration.

Square formation titration results shows, when antigen coated concentration is that 8 μ g/mL, serum to be checked are that P/N value is maximum, positive serum OD with 1:50 dilution _450nmvalue is higher than 1.0, negative serum OD _450nmvalue is less than 0.2.Therefore, the suitableeest coated concentration of defined antigen is 8 μ g/mL, and serum optimal dilution is 1:50.

2. best confining liquid and off-period determines

Selecting respectively 1% skim-milk, 2.5% skim-milk, 5% skim-milk, 10% skim-milk and 1% BSA is that confining liquid carries out blocking test.Select best confining liquid to seal respectively 30min, 60min, 90min, 120min in 37 ℃ of incubators, determine best off-period.

Result shows, the highest as the P/N value of confining liquid with 5% skim-milk, and positive serum OD _450nmvalue is greater than 1.0, negative serum OD _450nmvalue is less than 0.2, and sealing effect is best.While sealing 60min under 37 ℃ of conditions, P/N value is the highest.

3. two anti-working concentrations determines

The anti-grass carp IgM monoclonal antibody of horseradish peroxidase (HRP) mark that embodiment 1 is prepared is as ELIAS secondary antibody.

ELIAS secondary antibody is pressed to 1:1000,1:2500,1:5000,1:10000 dilution, and antigen and serum are pressed optimum dilution degree dilution.Result shows, when ELIAS secondary antibody working concentration is done 1:2500 dilution, P/N maximum, therefore, determines that two anti-optimum dilution degrees are 1:2500.

4. ELISA criterion determines

By above-mentioned definite ELISA method, to 30 parts, through virus neutralization tests, turn out to be negative serum and check, according to OD _450nmmean value (X) and the standard deviation (SD) of value are set up criterion.Threshold value is decided to be X+3XSD.According to 30 parts of negative serum OD _450nmmean value (X=0.203) and standard variance (SD=0.012), determine that its threshold value is 0.213+0.052 × 3=0.239, definition sample OD _450nmbe judged to be the positive at>=0.239 o'clock.

5. the foundation of GCRV indirect ELISA detection method

(1) preparation of samples: take Blood of Ctenopharyngodon with disposable syringe, be placed in Glass tubing, solidify under room temperature, at 37 ℃, place 2h, then spend the night in 4 ℃ of placements, after serum is fully separated out, the centrifugal 1min of 1000g/min, gets its supernatant, as detecting sample.

(2) application of sample: detection sample PBST(pH7.4 prepared by above-mentioned steps (1)) by 1:50, dilute, the sample having diluted is added in the enzyme plate being coated with by the amount in 100 uL/ holes.

(3) incubation: with hatching 1h in the rearmounted 37 ℃ of incubators of shrouding film shrouding.

(4) washing: by sample sucking-off from each hole, with PBST washing 3 times, each 3-5 min.

(5) dilution of ELIAS secondary antibody: by PBST(pH7.4 for ELIAS secondary antibody (the anti-grass carp IgM monoclonal antibody of horseradish peroxidase (HRP) mark)) do 1:2500 dilution.

(6) add two to resist: the ELIAS secondary antibody of having diluted is added in the enzyme plate having washed in above-mentioned steps (4) by the amount in 100 uL/ holes.

(7) incubation: same step (3);

(8) washing: same step (4);

(9) colour developing: add freshly prepared substrate solution (TMB:H2O2=1:1) 50 μ L/ holes, with the rearmounted 37 ℃ of lucifuges colour developing of shrouding film shrouding 10min.

(10) stop: add the H2SO4 stop buffer of 50 μ L/ hole 2M, termination reaction.

(11) measure: adopt the microplate reader of wavelength 450nm to measure OD value.

(12) result judgement: OD _450nmbe judged to be the positive at>=0.239 o'clock.

6. the foundation of GCRV indirect ELISA testing kit:

Detection kit of the present invention comprises following reagent: horseradish peroxidase (HRP) marker of the monoclonal antibody of anti-grass carp IgM, is coated with enzyme plate, positive serum (GCRV II type positive serum), negative serum (GCRV negative serum), TMB nitrite ion, stop buffer and the PBST washings of the proteins encoded of GCRV II type S10 gene.

embodiment 4 specific detection

The ELISA method of setting up according to claim 3, take GCRV negative serum and GCRV II type positive serum as contrast, with indirect ELISA detection GCRV I type positive serum, GCRV III type positive serum, Aeromonas hydrophila positive serum, the Pseudomonas fluorescens positive serum set up, analyze the specificity of the indirect ELISA method of setting up.

Detected result shows, GCRV II type positive serum OD _450nmvalue is greater than 1.0, GCRV negative serum, GCRV I type positive serum, GCRV III type positive serum, Aeromonas hydrophila positive serum, Pseudomonas fluorescens positive serum OD _450nmvalue is all less than 0.239.Show that the method has good specificity.

embodiment 5 repeated experiments

The ELISA method of setting up by embodiment 3 detects 4 parts of GCRV positive serums in same plate, and every part of serum sample repeats 6 holes, calculates with a serum sample OD _450nmthe variation coefficient (CV) of value, to detect the repeatability of sample in inspection panel.The restructuring S10 proteins encoded coated elisa plate of using under the same conditions 4 batches of expression and purifications, detects 4 parts of GCRV positive serums and 1 part of negative serum, and every part of parallel 3 holes of doing of serum, calculate its CV, as CV is less than 10%, meet the repeated requirement of detection.

Result shows, the replica test variation coefficient in plate is at 2.1%-6.7%, and between plate, the replica test variation coefficient, between 4.86%-7.98%, is all less than 10%, illustrates that this ELISA method has good repeatability.

embodiment 6 clinical samples detect

Applying this test sets up indirect ELISA detection method the Grass Carp Serum sample of the doubtful ill hemorrhagic disease of grass carp that picks up from Jiangxi Province, cities of Guangdong area is detected, in 60 parts of bovine serum samples, the GCRV II type specificity antibody of 49 parts of serum is positive as a result, and seroprevalence is 81.7%.

Above embodiment is only for introducing preferred case of the present invention, and to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention, all should be regarded as a part of the present invention.

<110> China's Pearl River Fishery Research Institute of Aquatic Science Research Institute

The indirect ELISA testing kit of a <120> GCRV

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1038

<212> DNA

<213> GCRV HZ08

<400> 1

atggcgggtg tgtctctcaa catcaatcgc aacatctcaa actcggcatc aacgatcttt 60

ctcgaagata ttccattact gtcatgttca gtgcggtgtg aacccggtaa aggacgcgaa 120

ttaccaaaat ttaacatgag ctgccctgct attaacgcga tgggtcgatg tcttaatcca 180

atgaagttca ttgctgagca ctgggtcccc aacagtccaa gtcgtaaacc atccagacag 240

cattggcgta atgttttgaa tggactggaa ttcagtaatg gtcgtggatt cgatgtgctg 300

agtttctcac cagcgggcat ggctgttctt cgtgacatcc tgacagaaga tagcgtgaac 360

tattgctttg acgagagtaa cacttgcagt ttgttcacct tgctgtacac tctgtgttgc 420

gatgccgcag gcgtagaacc gatggacttg gattcacgtc agacggacgc cagtgcccgg 480

atggtgagct accaagatcg cgctatcgtg ctgacctcta atgaggcagg agatagaatt 540

gagccgtgga atgttgagct cgacaaggag tttggaaatc cagatctgct cagccgtctg 600

aacatctcat atggcgtgca acgatatggc gactccaaag ccagcacaga cactctgacc 660

ttggctgatg ccccagagag gtccaagcct gctctgatta ctgtgcaacc cttgttggtg 720

gctatgtgca tcaaacaatc tttggatggc ttgctggctt tatctgattt gcgcctgaga 780

ttcgatcagt atcctggata cgcaaatgct ctcatgaatg ctatggccat gtacgcttgc 840

ctagatcgtg acttgatgcg ttttctgctc cgcttagaaa tgactcacgc gagcacggtg 900

tctgaagtgg ctgagtgctg gaggaactct cgcaactctc gcgatgcaac aggttgtcat 960

attgtcccac gtcaaggttt gctcatcatc gtttccggag atgtcgaggt aagacgtata 1020

tttgcgcaga tgctgtag 1038

<210> 2

<211> 345

<212> PRT

<213> GCRV HZ08

<400> 2

Met Ala Gly Val Ser Leu Asn Ile Asn Arg Asn Ile Ser Asn Ser Ala

1 5 10 15

Ser Thr Ile Phe Leu Glu Asp Ile Pro Leu Leu Ser Cys Ser Val Arg

20 25 30

Cys Glu Pro Gly Lys Gly Arg Glu Leu Pro Lys Phe Asn Met Ser Cys

35 40 45

Pro Ala Ile Asn Ala Met Gly Arg Cys Leu Asn Pro Met Lys Phe Ile

50 55 60

Ala Glu His Trp Val Pro Asn Ser Pro Ser Arg Lys Pro Ser Arg Gln

65 70 75 80

His Trp Arg Asn Val Leu Asn Gly Leu Glu Phe Ser Asn Gly Arg Gly

85 90 95

Phe Asp Val Leu Ser Phe Ser Pro Ala Gly Met Ala Val Leu Arg Asp

100 105 110

Ile Leu Thr Glu Asp Ser Val Asn Tyr Cys Phe Asp Glu Ser Asn Thr

115 120 125

Cys Ser Leu Phe Thr Leu Leu Tyr Thr Leu Cys Cys Asp Ala Ala Gly

130 135 140

Val Glu Pro Met Asp Leu Asp Ser Arg Gln Thr Asp Ala Ser Ala Arg

145 150 155 160

Met Val Ser Tyr Gln Asp Arg Ala Ile Val Leu Thr Ser Asn Glu Ala

165 170 175

Gly Asp Arg Ile Glu Pro Trp Asn Val Glu Leu Asp Lys Glu Phe Gly

180 185 190

Asn Pro Asp Leu Leu Ser Arg Leu Asn Ile Ser Tyr Gly Val Gln Arg

195 200 205

Tyr Gly Asp Ser Lys Ala Ser Thr Asp Thr Leu Thr Leu Ala Asp Ala

210 215 220

Pro Glu Arg Ser Lys Pro Ala Leu Ile Thr Val Gln Pro Leu Leu Val

225 230 235 240

Ala Met Cys Ile Lys Gln Ser Leu Asp Gly Leu Leu Ala Leu Ser Asp

245 250 255

Leu Arg Leu Arg Phe Asp Gln Tyr Pro Gly Tyr Ala Asn Ala Leu Met

260 265 270

Asn Ala Met Ala Met Tyr Ala Cys Leu Asp Arg Asp Leu Met Arg Phe

275 280 285

Leu Leu Arg Leu Glu Met Thr His Ala Ser Thr Val Ser Glu Val Ala

290 295 300

Glu Cys Trp Arg Asn Ser Arg Asn Ser Arg Asp Ala Thr Gly Cys His

305 310 315 320

Ile Val Pro Arg Gln Gly Leu Leu Ile Ile Val Ser Gly Asp Val Glu

325 330 335

Val Arg Arg Ile Phe Ala Gln Met Leu

340 345

<210> 3

<211> 31

<212> DNA

<213> artificial sequence

<400> 3

ataggatcca tggcgggtgt gtctctcaac a 31

<210> 4

<211> 32

<212> DNA

<213> artificial sequence

<400> 4

gctaagcttc agcatctgcg caaatatacg tc 32

Claims (8)

1. hybridoma cell strain S467, is preserved in Chinese Typical Representative culture collection center on December 10th, 2013, and deposit number is CCTCC NO:C2013193.

2. a monoclonal antibody of anti-grass carp IgM, its hybridoma cell strain or secretion of its passage cell strain that is CCTCC NO:C2013193 by deposit number produces.

3. hybridoma cell strain claimed in claim 1 or monoclonal antibody claimed in claim 2 application in the anti-GCRV II type of preparation and/or hemorrhagic disease of grass carp detection reagent or diagnostic reagent.

4. a GCRV indirect ELISA testing kit, described test kit comprises the enzyme labelling thing of the monoclonal antibody of anti-grass carp IgM, is coated with the enzyme plate of antigen.

5. indirect ELISA testing kit according to claim 4, is characterized in that, the monoclonal antibody of described anti-grass carp IgM is monoclonal antibody claimed in claim 2.

6. indirect ELISA testing kit according to claim 4, is characterized in that, described antigen is the proteins encoded of GCRV II type S10 gene, and its sequence is as shown in SEQ ID NO:2.

7. indirect ELISA testing kit according to claim 4, is characterized in that, also comprises standard serum, TMB nitrite ion, stop buffer and washings in described test kit.

8. a GCRV indirect ELISA detection method, comprises the steps:

(1) application of sample: sample to be checked is added in the coated plate reacting hole of enzyme mark of the proteins encoded (SEQ ID NO:2) that is coated with GCRV II type S10 gene;

(2) incubation: with the rearmounted 37 ℃ of incubations of shrouding film shrouding;

(3) washing: take shrouding film off, with PBST washings detersive enzyme target;

(4) add two anti-: in reacting hole, add ELIAS secondary antibody, described ELIAS secondary antibody is the horseradish peroxidase-labeled thing of the monoclonal antibody of anti-grass carp IgM described in claim 2;

(5) incubation: operation is with (2);

(6) washing: operation is with (3);

(7) colour developing: add TMB developer in reacting hole, 37 ℃ of lucifuge colour developings;

(8) mensuration and result judgement.

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