CN102680682A - Kit for detecting antibody of reticuloendotheliosis virus (REV) through enzyme-linked immunosorbent assay (ELISA) - Google Patents

Abstract

The invention relates to the field of animal virology and in particular provides a kit for detecting an antibody of a reticuloendotheliosis virus (REV) through enzyme-linked immunosorbent assay (ELISA). The kit mainly comprises an REV envelope protein pre-coated ELISA plate, blocking solution, sample diluent, an enzyme conjugate, washing concentrate, enzyme substrate solution, stop solution, REV positive anti-chicken serum and SPF chicken serum, wherein the envelope protein is the env protein subjected to prokaryotic expression and purification. By adopting the kit, the specificity reaches 100% and the sensitivity is 1:700. The kit is used for detecting the level of the REV antibody of chicken to exactly know the REV infection conditions of the chicken.

Description

The one syndrome virus ELISA of stud bird reticuloendotheliosis antibody assay kit

Technical field

The present invention relates to the animal virology field a kind of kit is provided, stud bird reticuloendotheliosis syndrome virus (REV) ELISA antibody assay kit specifically is provided.

Background technology

Disease (the Reticuloendotlleliosis of fowl reticuloendotheliosis; RE) be the caused one group of pathology syndrome of reticuloendotheliosis's syndrome virus (REV) that belongs to by the fowl retroviruse, comprise the chronic tumour of reticuloendothellium cytoma, runting syndrome, lympha tumour and its hetero-organization etc.After fowl Marek's disease and avian leukosis, RE is the third infectiousness neoplastic disease of bird of having confirmed so far.REV strain the earliest is to suffer from the lymphadenomatous turkey spleen of internal organ from an example of the U.S. in 1958 being separated to, through a strain new virus that obtains continuous go down to posterity for 300 times of turkey and chicken.

The natural reservoir (of bird flu viruses) that REV infects has turkey, chicken, duck, goose etc., can cause the immunosupress of being infected fowl, disturbs other immunological effect, causes immuning failure, and causes scabies secondary infection easily, and the diagnosis of eqpidemic disease and prevention and control difficulty are strengthened.Existing nowadays domesticly also do not have effective vaccine and medicine to prevent and treat, and chicken crowd REV antibody horizontal is detected, and can definitely understand chicken crowd antibody horizontal, and then understand infection state, in time eliminates ill chicken, purification chicken crowd.The harm that receives the REV infection along with China's poultry husbandry is increasingly serious, and the clinical demand of development REV Serum Antibody Detection kit is more urgent.The terminal epi-position of the gp90C of the env gene code of REV standard strain SNV is positioned at the surface of infection cell, contains order and comformational epitope, is the immundominance albumen of virus, can excite infection host to produce neutralizing antibody; Therefore the env albumen of REV is considered to a kind of good selection of the REV of being applied to diagnosis.

At present, the REV virus detection techniques that adopts both at home and abroad mainly contains: viral separation and Culture, indirect immunofluorescence, immunohistochemistry, PCR (PCR) etc.Though above-mentioned technology and method can detect REV virus or antibody horizontal, also obtained certain effect in practice.But all have the experimental technique complicated operation, length consuming time needs specific professional skill and instrument and equipment etc., and shortcomings such as finished product costliness should not be used for Clinical detection.Domestic related patent U.S. Patent No. of also not seeing the REV antibody assay kit so far, though external specific detection REV antibody assay kit commercialization, since expensive, be difficult to use on a large scale.Compare with existing external commercial kit, the specificity of this kit is stronger, susceptibility is higher.

Summary of the invention

Many weak points that inventor of the present invention exists to existing REV virus detection techniques; One stud bird reticuloendotheliosis syndrome virus (REV) ELISA antibody assay kit is provided; This kit consists predominantly of: the ELISA Plate, confining liquid, sample diluting liquid, enzyme conjugates, concentrated cleaning solution, enzyme substrate solution, stop buffer, REV virus-positive chicken antiserum and the SPF chicken serum that encapsulate fowl reticuloendotheliosis syndrome virus envelope protein in advance; Wherein said envelope protein is through the env albumen behind prokaryotic expression and the purifying, adopts this kit specificity to reach 100%; Sensitivity is 1: 700.This kit is used for chicken crowd REV antibody horizontal is detected, and definitely understands chicken crowd REV infection state.

Concrete technical scheme of the present invention is:

The kit that is provided consists predominantly of: encapsulate ELISA Plate, confining liquid, sample diluting liquid, enzyme conjugates, concentrated cleaning solution, enzyme substrate solution, stop buffer, REV virus-positive chicken antiserum and the SPF chicken serum of fowl reticuloendotheliosis syndrome virus envelope protein in advance, wherein said envelope protein is through the env albumen behind prokaryotic expression and the purifying; And compare with conventional kit, the stop buffer of using among the present invention is the H of 3moL/L ₂SO ₄Solution, main cause are that the use of this stop buffer can make the result become yellow by blueness, are easier to the observation of people's naked eyes.

Wherein REV virus-positive chicken antiserum in the kit and SPF chicken serum are respectively as positive control and negative control, and it all adopts prior art under the raising condition of strictness, to obtain;

Wherein, described through the env albumen behind prokaryotic expression and the purifying, obtain through following method:

The DFI cell DNA of the REV standard strain SNV strain that directly (Chinese Academy of Sciences microorganism fungus kind storehouse) buied from market has been infected in extraction, according to REV-env gene order design specific primers: upstream primer 5 '-CCGGAATTCGCCATCCTACAGAAAACAAA-3 ', its gene order is shown in Seq ID No:1; Downstream primer 5 '-CGACTCGAGAACAATATGAGCCCAAACA-3 '; Its gene order and has been added EcoR I and Xhol I restriction enzyme site respectively in primer shown in Seq ID No:2, go out the env gene with pcr amplification; Expression vector pET-28a plasmid is gone in the env gene clone; Obtained recombinant vector pET-28a-env recombinant plasmid, used 0.2～0.3mmoL IPTG abduction delivering to go out to have the fusion His-env of His label, its molecular weight is about 45KD; Be 46.3KD more specifically; This albumen exists with the form of inclusion body, with the urea dissolving inclusion body of 8mol, gradient urea recombinant protein.

In other components in the kit:

Encapsulate the carbonate buffer solution that damping fluid is 0.05M pH9.6, contain 1.59gNa in the 1L solution ₂CO ₃, 2.93g NaHCO ₃, package amount is every hole 0.2～0.3 μ g; Confining liquid is that mass concentration is 2%～3% skim milk powder aqueous solution, and be 1h off-period; Sample diluting liquid is to contain 0.01mol/L that volumetric concentration is 0.5 ‰ Tween-20 and the phosphate buffer (PBS) of pH 7.2, and the diluted sample multiple is 600～700 times, and incubation time is 2h; Enzyme conjugates is horseradish peroxidase (HRP)-goat anti chicken IgG monoclonal antibody enzyme conjugates, and its extension rate is 5000 times, and incubation time is 2h; Concentrated cleaning solution is to contain 0.1mol/L that volumetric concentration is 5 ‰ Tween-20 and the phosphate buffer (PBS) of pH 7.2; Enzyme substrate solution is 3,3 ,-5,5, and-tetramethyl biphenyl amine aqueous solution (TMB), incubation time are 20-30min; Stop buffer is 3mol/L H ₂SO ₄Solution, positive control, negative control are placed in the box.

Various reaction conditionss in the said components are that inventor of the present invention is through after the test of long duration; The optimum reaction condition that provides; Judgment basis is: the data to each reaction are handled; Calculate the P/N value of reaction, maximum with the P/N value, and the P value is near 1 judgment basis as the indirect ELISA optimum reaction condition.

P/N value calculating method: P/N value=positive control hole OD ₄₅₀Nm average/negative control hole OD ₄₅₀The nm average.

The various parameters that obtain through said method are The optimum reaction conditions in this kit testing process, can reduce the error that possibly occur in the course of reaction to greatest extent, more accurately the truth of reaction detection sample; The confining liquid of using in the detection is cheap and easy to get, and incubation conditions is 37 ℃ of commonly used incubators of laboratory, and what each above-mentioned condition was detection normally provides advantage.

And the method for application of the above-mentioned syndrome virus ELISA of the fowl reticuloendotheliosis antibody assay kit of the present invention, step is following: each component room temperature (18-25 ℃) of rising again in the kit, and put upside down to shake and mix.

1) taking-up encapsulates plate, the record sample position;

2) every empty 300 μ L confining liquids, 37 ℃ of incubation 1h of adding;

3) be total to 3-5 time with the cleansing solution washing micropore after about 350 μ L dilution, clap liquid in the hole dried fully after the washing;

4) add positive control serum that 100 μ L need not dilute to A1 and B1 hole, the negative control sera that 100 μ L need not dilute is to C1 and D1 hole;

5) add the good sample of 100 μ L dilution to corresponding hole, all samples both can carry out diplopore detection also can carrying out single hole and detect; 37 ℃ of incubation 2h;

6) repeating step 3;

7) every hole adds 100 μ L enzymes mark goat-anti chicken antibody (HRPO), 37 ℃ of incubation 2h;

8) repeating step 3;

9) every hole adds 100 μ LTMB substrate solutions, 37 ℃ of incubator colour developing 20-30min;

10) every hole adds 100 μ L stop buffer cessation reactions;

11) measure its OD450nm light absorption value line item of going forward side by side.

The aforesaid operations step is simple, good reliability, and accuracy all is greatly improved with quick degree.

In sum; The invention provides stud bird reticuloendotheliosis syndrome virus (REV) ELISA antibody assay kit; This kit consists predominantly of: the ELISA Plate, confining liquid, sample diluting liquid, enzyme conjugates, concentrated cleaning solution, enzyme substrate solution, stop buffer, REV virus-positive chicken antiserum and the SPF chicken serum that encapsulate fowl reticuloendotheliosis syndrome virus envelope protein in advance; Wherein said envelope protein is through the env albumen behind prokaryotic expression and the purifying, adopts this kit specificity to reach 100%; Sensitivity is 1: 700.This kit is used for chicken crowd REV antibody horizontal is detected, and definitely understands chicken crowd REV infection state.Be greatly improved than existing REV virus detection techniques accuracy and quick degree

Description of drawings

Fig. 1 analyzes the existence form electrophoresis synoptic diagram of inducing different time env albumen for SDS-PAGE;

M is 170KD albumen Marker among the figure; 1 for inducing preceding inclusion body; 2 for inducing inclusion body behind the 1h;

3 for inducing inclusion body behind the 2h; 4 for inducing inclusion body behind the 3h; 5 for inducing supernatant behind the 3h;

Fig. 2 is that Western blot method detects the protein-specific electrophoretogram;

M is Protein Marker (KD) among the figure; 1 is env albumen;

Fig. 3 is kit optimization step figure of the present invention.

Embodiment

Except that special indicating, other all adopt existing routine techniques among the following embodiment

Embodiment 1

1.REV the clone of env gene and order-checking

The DFI cell DNA of the REV standard strain SNV strain that directly (Chinese Academy of Sciences microorganism fungus kind storehouse) buied from market has been infected in extraction, according to one section sequence at REV standard strain SNV strain provirus genomic DNA env gene two ends as primer.Upstream primer is 5 '-CCGGAATTCGCCATCCTACAGAAAACAAA-3 '; Its gene order is shown in Seq ID No:1; Downstream primer is 5 '-CGACTCGAGAACAATATGAGCCCAAACA-3 '; Its gene order and has been added EcoR I and Xhol I restriction enzyme site respectively in primer shown in Seq ID No:2.The PCR reaction conditions is: 95 ℃ of 5min fully get into circulating system: 95 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, totally 30 circulations after the sex change; 72 ℃ are extended 10min then.Use DNA purification kit purified pcr product.

PET-28a carrier and PCR purified product are used restriction enzyme EcoR I, Xhol I double digestion 6h respectively, reclaim the kit explanation according to glue and reclaim big fragment respectively, connect down in 16 ℃ and spend the night.Linked system is: pET-28a carrier 1 μ L, and distilled water 3 μ L, the DNA purpose fragment 4 μ L of purifying, T4 ligase 1 μ L, 10 * buffer, 1 μ L, cumulative volume are 10 μ L.

Converted product is transformed into the competent cell BL21 that the CaCl2 method prepares by conventional method, and enzyme cutting method is identified positive colony, and positive colony is sent to the order-checking of order-checking company.

2.His-env the expression of albumen and purifying

The above-mentioned BL21 bacterium that contains recombinant plasmid is inoculated in the LB solid medium that contains card sodium mycin, cultivated picking list colony inoculation in the LB fluid nutrient medium that contains Kan (final concentration is 30 μ g/ml) for 37 ℃, 37 ℃ of vibrations (200rpm) are cultured to OD ₆₀₀=0.6, adding IPTG is 0.2～0.3mmol/L to final concentration, and 37 ℃ are continued to cultivate 3h, establishes the recombinant plasmid transformed BL21 bacterium do not induced simultaneously as contrast.

Analyze demonstration through SDS-PAGE, destination protein His-env is with the inclusion body formal representation, and it is following to detect step:

Collect the engineering bacteria bacterium liquid of cultivating, 5000rpm, centrifugal 5min abandons supernatant; Take by weighing wet bacterium that 30g collects in the 500ml beaker, add the resuspended liquid of 300ml, add rotor and on magnetic stirring apparatus, stir 20min, thalline is uniformly dispersed; Above-mentioned beaker is placed ice bath, with carrying out ultrasonic bacteria breaking appearance carrying out ultrasonic bacteria breaking, 120W, work 4s, 4s intermittently, about ultrasonic 1h, with rifle head pressure-vaccum bacterium liquid, when treating that thalline no longer sticks together, carrying out ultrasonic bacteria breaking end for the first time; Carrying out ultrasonic bacteria breaking liquid is changed in the 500ml centrifuge tube, and high speed refrigerated centrifuges is put in trim, 12000rpm, 4 ℃, centrifugal 20min; Get deposition, add the resuspended liquid of 200ml, it is ultrasonic, centrifugal then to carry out second time with said method, approximately after ultrasonic 2～4 times, goes deposition, uses the SDS-PAGE electrophoresis detection after handling;

The purification step of His-env albumen is following:

With the cleansing solution I resuspended deposition of above-mentioned deposition with 9 times of volumes, leave standstill 5min after, 12000rpm, 15mi abandons supernatant.With the resuspended deposition of the cleaning solution II of 9 times of volumes, leave standstill 5min after, 12000rpm, 15min abandons supernatant, keeps the thickness supernatant.With the resuspended deposition of the cleaning solution II I of 9 times of volumes, leave standstill 5min after, 12000rpm, 15min abandons supernatant, keeps the thickness supernatant.Add the 4ml lysate by 100ml bacterium liquid, concuss 1h.12000rpm, centrifugal 15min.Get supernatant, be the inclusion body of dissolving.

The said reagent compound method of inclusion body purification:

(1) the resuspended liquid of bacterium (250ml): PBS (NaCl 2g, KCl 0.05g, Na2HPO40.36g KH2PO40.06g), 1mm/LEDTA.

(2) solution I (250ml): 500mmol/L Tris-Cl (PH=8.0), 100mmol/L NaCl, 100mmol/L EDTA

(3) solution II (250ml): solution I, 2M urea

(4) solution III (250ml): solution I, 4M urea

(5) solubilization of inclusion bodies liquid (250ml): solution I, 8M urea

The inclusion body of above-mentioned dissolving is packed in the bag filter, use 20mmol/L Tris-Cl (PH=8.0) successively, 4M urea dialysis 2h; 20mmol/L Tris-Cl (PH=8.0), 2M urea dialysis 2h, 20mmol/L Tris-Cl (PH=8.0); 1M urea dialysis 1h, 20mmol/L Tris-Cl (PH=8.0), 0.5M urea dialysis 2h; 20mmol/L Tris-Cl (PH=8.0); 20mmol/L Tris-Cl (PH=8.0) 6h that dialyses, 12000rpm is centrifugal, and 2min discards deposition, and the supernatant that obtains is the His-env fusion accomplished of purifying very.Use the albumen after the SDS-PAGE method detects purifying.

The albumen that obtains is measured protein content through the Bradford method, and it is subsequent use to be diluted to 0.5mg/ml packing-20 ℃ preservation.

The SDS-PAGE operation steps is following:

1) with the glass plate wash clean, fix with clip, the vertical placement disposed 12% separation gel, is injected into fast between two glass plates, and glue top adds 1mL distilled water seals, and keeps glue smooth.Treat that separation gel solidifies back (about 40min), remove the liquid on separation gel surface, inhale with filter paper and go residuary water.Inject concentrated glue, insert comb fast, and note preventing the generation of bubble.Wait for that it solidifies.

2) get the last appearance of His-env protein 10 0 μ l equivalent 2 * SDS buffer behind the purifying, boil 5-10min.

3) add 1 * Tris-glycine buffer toward electrophoresis tank, wait to concentrate gelling back (about 30min) admittedly, carefully pull out comb, wash well with damping fluid, with appearance on the microsyringe, 10 μ l/ holes correctly connect the electrophoresis tank both positive and negative polarity, opening power.Earlier with 80V voltage electrophoresis, treat that sample concentration becomes a line and gets into separation gel after, improve voltage to 150V, stop electrophoresis during the bottom of electrophoresis to bromophenol blue arrival glue.

4) dyeing: take off gel, use distilled water flushing, put into to examine and dye solution, more than the dyeing 4h.

5) decolouring: gel is put into destainer, places on the shaking table and decolour, during change destainer more than 3 times, treat that the gel blue background is all sloughed to stop, gel is put into distilled water stops decolouring.The preservation of taking pictures.

The His-env fusion that uses above-mentioned purifying to obtain encapsulates elisa plate, uses EUSA to detect the BA of this albumen.The positive contrast of chicken serum of the REV antibody assay kit test positive of producing with IDEXX company, the negative contrast of healthy SPF chicken serum, HRP mark goat-anti chicken IgG ELIAS secondary antibody by specification uses.The result shows that the OD value ratio of positive control and negative control greater than 2, explains that this albumen has good BA and antigenicity.

The optimization step of embodiment 2. antibody assay kits:

It is following that conventional indirect ELISA detects step:

1) encapsulates: get purified recombinant albumen His-env, (contain 1.59gNa in the 1L solution with carbonate buffer solution ₂CO ₃, 2.93gNaHCO ₃) be diluted to optium concentration, encapsulate reaction plate, 100 μ l/ holes, 4 ℃ are spent the night.

2) washing: get rid of the coating buffer in the ELISA Plate, fill it up with each hole with PBST (volumetric concentration be the 0.01mol/L of 0.5 ‰ Tween-20 and the phosphate buffer of pH 7.2), room temperature leaves standstill 3min, discards PBST, wash 3 times, and 3min/ time, bat is dried.

3) sealing: each hole adds the 5wt% skimmed milk WS, and 350 μ l/ holes are put 37 ℃ of incubators and hatched 2h, washs 3 times, 3min/ time, claps and does.

4) application of sample: serum to be checked added after by gradient dilution encapsulate plate, the contrast of yin and yang attribute serum is set up in 100 μ l/ holes simultaneously.Put 37 ℃ of incubation 1h, wash 3 times, 3min/ time, clap and do.

5) with confining liquid HRP goat-anti chicken IgG ELIAS secondary antibody was diluted by 1: 3000,100 μ l/ holes are put 37 ℃ of incubators and are hatched 1h, wash 3 times, 3min/ time, clap and do.

6) colour developing: use TMB one-component chromogenic reagent box (TIANGEN Company products) to add by 100 μ l/ holes and encapsulate plate, lucifuge colour developing 20min adds stop buffer, 3M H under the room temperature ₂SO ₄Color development stopping.

7) survey its OD value, negative control OD with enzyme-linked immunosorbent assay instrument in each hole of wavelength 450nm detection ₄₅₀Value is N, positive control OD ₄₅₀Value is P.If P/N＞2.0 are judged to the positive.

Each parameter of above common process, but the inventor finds that through overtesting above-mentioned for purposes of the invention is not The optimum reaction conditions, the inventor optimizes for this reason, and concrete optimizing process is following.

The judgment basis of optimal conditions is: the data to each reaction are handled, and calculate the P/N value of reaction, and are maximum with the P/N value, and the P value is near 1 judgment basis as the indirect ELISA optimum reaction condition.

P/N value calculating method: P/N value=positive control hole OD ₄₅₀Nm average/negative control hole OD ₄₅₀The nm average.

1. antigen-antibody optimum diluting multiple

With antigen (REN-env albumen) and antibody (REV positive serum to be checked) difference doubling dilution, orthogonal experiment carries out ELISA and detects.The result shows that the best package amount of antigen Hu is every hole 0.2～0.3 μ g, and serum optimum diluting multiple to be checked is 600～700 times.

2. the selection of best coating buffer

Detect as coating buffer with following six kinds of solution respectively:

CB:50mmol/L PH 9.6 carbonate buffer solution TB:20mmol/LPH 8.5Tris-Hcl

PBS:PH 7.4 PBST:PH 7.4 W: distilled water S: physiological saline

The result shows that best antigen coated liquid is CB:50mmol/L PH 9.6 carbonate buffer solutions; PBST and distilled water are not useable for encapsulating.

3. the selection of best confining liquid concentration

Skimmed milk testing laboratory is the most frequently used, that be easy to get most is mixed with 0.25%-7%9 different gradient concentration, detects respectively.

The result shows the skimmed milk WS that best confining liquid concentration is 2～3wt%.

4. the selection of best off-period

Make the off-period of 6 gradients of 0.5h-3h of the skimmed milk WS of 2～3wt% respectively and detect, the result shows that be 1h best off-period.

5. the selection of antiserum the best use of time

After antiserum and control serum diluted corresponding multiple, every hole added 100 μ L, is divided into four groups, hatches different time for every group, and the result shows that the serum the best use of time to be checked is 2h.

6. the selection of two anti-optimum dilution degrees

Two anti-are Beijing Bo Aosen company goat-anti chicken two anti-(HRP mark), and this two is anti-ly made six gradient dilutions of 1000-6000 respectively, detect respectively, and the result shows that two anti-optimum diluting multiples are 5000 times.

7. the selection of two anti-action times

Above two anti-dilutions are provided with six groups for 5000 times between 0.5h-3h, act on elisa plate respectively and detect, the result shows that two anti-the best use of times were 2h.

8. the selection of best developing time

In the 15min-45min interval, be divided into six groups with TMB one-component substrate colour developing liquid and detect respectively, testing result shows that best developing time is 20～30min.

9. the calculating of critical value

Randomly draw 40 parts of SPF chicken serums, detect, calculate the average OD of these 40 parts of serum with the ELISA method of optimizing ₄₅₀The mean value of nm (x) and standard deviation SD, yin and yang attribute critical value=x+3SD.As the sample OD that detects ₄₅₀During nm value>=x+3SD, can on 99.9% level, be judged to be the positive.Critical value result of calculation is 0.340.

Embodiment 3. experimental results and analysis

The method of application of the syndrome virus ELISA of the fowl reticuloendotheliosis antibody assay kit that the present invention is above-mentioned, step is following: each component room temperature (18-25 ℃) of rising again in the kit, put upside down to shake and mix.

1) taking-up encapsulates plate, the record sample position;

2) every empty 300 μ L confining liquids, 37 ℃ of incubation 1h of adding;

3) be total to 3-5 time with the cleansing solution washing micropore after about 350 μ L dilution, clap liquid in the hole dried fully after the washing;

4) add positive control serum that 100 μ L need not dilute to A1 and B1 hole, the negative control sera that 100 μ L need not dilute is to C1 and D1 hole;

5) add the good sample of 100 μ L dilution to corresponding hole, all samples both can carry out diplopore detection also can carrying out single hole and detect;

37 ℃ of incubation 2h;

6) repeating step 3;

7) every hole adds 100 μ L enzymes mark goat-anti chicken antibody (HRPO), 37 ℃ of incubation 2h;

8) repeating step 3;

9) every hole adds 100 μ LTMB substrate solutions, 37 ℃ of incubator colour developing 20-30min;

10) every hole adds 100 μ L stop buffer cessation reactions;

11) measure its OD450nm light absorption value line item of going forward side by side;

Use above-mentioned method of application, the inventor has carried out following detection:

1. specific detection

ELISA operating process according to optimizing is measured ALV-J, MDV, NDV, H5N1, H9N1 virus-positive serum,, carries out specificity and tests as contrast with REV positive serum and negative serum, and testing result is (table 1) as follows

Table 1 specificity test findings

The result shows ALV-J, MDV, NDV, H5N1, H9N1 virus-positive serum OD450nm value all less than 0.340, and with the equal no cross reaction of REV positive serum, kit of the present invention has good specificity.

2. replica test

2.1 repeat to use env albumen to encapsulate 1 fast ELISA Plate in the plate with a collection of preparation, in same ELISA Plate, detect 4 parts of positive serum samples, every part of blood serum sample repeats 5 holes, calculates its mean value, standard deviation and the coefficient of variation (CV), and testing result is (table 2) as follows

Replica test result in table 2 plate

Coefficient of variation minimum value in the plate is 2.4%, and maximal value is 10.1%, less than 15%, explains with the degree of variation of a serum sample in same block of plate lessly, has better repeatability.

2.2 repeat to use env albumen to encapsulate 5 ELISA Plates between plate with a collection of preparation; In 5 ELISA Plates, detect 4 parts of different positive serum samples under the same terms respectively; Calculate mean value, standard deviation and the coefficient of variation (CV) of each part blood serum sample, testing result is (table 3) as follows

Replica test result between table 3 plate

Coefficient of variation minimum value is 6.9% between plate, and maximal value is 11.3%, all less than 15%, explains with the degree of variation of a serum sample between different plates lessly, has better repeatability.

In sum, kit of the present invention has better repeatability.

3. with the comparison of existing commercial kit

Use this kit and existing commercial kit to detect the clinical sample of three batches of separate sources respectively, testing result compared:

First: gathered 200 parts of chicken serums of the purple kite animal husbandry in Weifang on November 11st, 2011,20,000 egg kind chickens are cultured by this plant, and 110 ages in days when taking a blood sample detect ALV-J antibody positive rate 0.5%.Baby chick derives from the exit of valley, Beijing, visible spleen enlargement when cuing open inspection.

This kit of table 4 testing result

Table 5 commercial kit testing result

Second batch: picked up from bright the kind chicken in Yiyuan, Zibo on June 4th, 2011,80,000 of breed scales, principal item have sea blue brown, black-bone chicken and numb chicken.Black-bone chicken and numb chicken mixed breed, about 8,000,310 ages in days during blood sampling; Black black-bone chicken is introduced by certain black-bone chicken field, south; The fiber crops chicken is to cooperate with the Shandong Province institute of agricultural sciences, and oneself breeds gained; Cut open the visible big hepatomegaly spleen of inspection, visible vessels knurl under the duodenum intestinal mucosa is used various kinds of drug and vaccine and is not all had remarkable result, suspects to be the inhibitive ability of immunity disease.

This kit of table 6 testing result

Table 7 commercial kit testing result

The 3rd batch: picked up from Zhou Cheng, Jining on June 18th, 2011,10,000 commodity eggs of breed scale, baby chick are from Jining dealer, 180 ages in days when taking a blood sample; Couple of days before this batch chicken, the last consignment of chicken was once broken out hemangioma; Cuing open the visible liver of inspection has tangible tumor nodule, and PCR detection demonstration ALV-J and MDV are all positive.

This kit of table 8 testing result

Table 9 commercial kit testing result

The analysis of table 10 pair three batches of chicken testing results

Interpretation of result: during batch detection, the matching rate of this kit and commercial kit is more than 90%; In 186 samples, this kit detects number positive with commercial kit and is respectively 94 and 91, explains that kit susceptibility provided by the present invention is higher.

Claims (6)

1. the syndrome virus ELISA of a stud bird reticuloendotheliosis antibody assay kit; It is characterized in that: include in the kit: encapsulate the ELISA Plate of fowl reticuloendotheliosis syndrome virus envelope protein in advance, wherein said envelope protein is through the env albumen behind prokaryotic expression and the purifying.

2. kit according to claim 1 is characterized in that: also contain confining liquid, sample diluting liquid, enzyme conjugates, concentrated cleaning solution, enzyme substrate solution, stop buffer, positive control and negative control in the said kit.

3. according to claim 1 or described kit, it is characterized in that: described envelope protein is that its preparation method is through the env albumen behind prokaryotic expression and the purifying:

The DFI cell DNA of commercial REV standard strain SNV strain has been infected in extraction; According to REV-env gene order design specific primers: upstream primer 5 '-CCGGAATTCGCCATCCTACAGAAAACAAA-3 ', its gene order shown in Seq ID No:1, downstream primer 5 '-CGACTCGAGAACAATATGAGCCCAAACA-3 '; Its gene order is shown in Seq ID No:2; And in primer, added EcoR I and Xhol I restriction enzyme site respectively, and go out the env gene with pcr amplification, expression vector pET-28a plasmid is gone in the env gene clone; Obtained recombinant vector pET-28a-env recombinant plasmid; Use 0.2～0.3mmoLIPTG abduction delivering to go out to have the fusion His-env of His label, its molecular weight is 46.3KD, and this albumen exists with the form of inclusion body.

4. kit according to claim 2 is characterized in that: described encapsulating in its 1L solution of carbonate buffer solution that damping fluid is 0.05M pH9.6 of adopting when encapsulating in advance contains 1.59gNa ₂CO ₃, 2.93g NaHCO ₃, package amount is every hole 0.2～0.3 μ g;

Confining liquid is that mass concentration is 2%～3% skim milk powder aqueous solution, and be 1h off-period;

Sample diluting liquid is to contain 0.01mol/L that volumetric concentration is 0.5 ‰ Tween-20 and the phosphate buffer (PBS) of pH 7.2, and the diluted sample multiple is 600～700 times, and incubation time is 2h;

Enzyme conjugates is horseradish peroxidase-goat anti chicken IgG monoclonal antibody enzyme conjugates, and its extension rate is 5000 times, and incubation time is 2h;

Concentrated cleaning solution is to contain 0.1mol/L that volumetric concentration is 5 ‰ Tween-20 and the phosphate buffer (PBS) of pH 7.2;

Enzyme substrate solution is 3,3 ,-5,5, and-tetramethyl biphenyl amine aqueous solution, incubation time are 20-30min;

Stop buffer is 3mol/L H ₂SO ₄Solution.

5. kit according to claim 2 is characterized in that: described positive control is a REV virus-positive chicken antiserum; Negative control is the SPF chicken serum.

6. the method for application of the syndrome virus ELISA of a stud bird reticuloendotheliosis antibody assay kit as claimed in claim 1, step is following: each component room temperature (18-25 ℃) of rising again in the kit, put upside down to shake and mix:

1) taking-up encapsulates plate, the record sample position;

2) every empty 300 μ L confining liquids, 37 ℃ of incubation 1h of adding;

3) be total to 3-5 time with the cleansing solution washing micropore after about 350 μ L dilution, clap liquid in the hole dried fully after the washing;

4) add positive control serum that 100 μ L need not dilute to A1 and B1 hole, the negative control sera that 100 μ L need not dilute is to C1 and D1 hole;

5) add the good sample of 100 μ L dilution to corresponding hole, all samples both can carry out diplopore detection also can carrying out single hole and detect; 37 ℃ of incubation 2h;

6) repeating step 3;

7) every hole adds 100 μ L enzymes mark goat-anti chicken antibody (HRPO), 37 ℃ of incubation 2h;

8) repeating step 3;

9) every hole adds 100 μ LTMB substrate solutions, 37 ℃ of incubator colour developing 20-30min;

10) every hole adds 100 μ L stop buffer cessation reactions;

11) measure its OD450nm light absorption value line item of going forward side by side.

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