CN110456047B

CN110456047B - Infectious bursal disease virus antibody competition ELISA detection method and kit

Info

Publication number: CN110456047B
Application number: CN201910846665.7A
Authority: CN
Inventors: 欧阳伟; 王永山; 王晓丽; 夏兴霞; 钱晶; 诸玉梅; 王晶宇; 马孙婷
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2019-09-09
Filing date: 2019-09-09
Publication date: 2023-05-02
Anticipated expiration: 2039-09-09
Also published as: CN110456047A

Abstract

The invention relates to an infectious bursal disease virus antibody competition ELISA detection method and a kit, and belongs to the technical field of biology. The IBDV VP2 gene is optimally designed, a prokaryotic expression plasmid pET28+VP2 is constructed, escherichia coli is transformed, and the molecular weight of recombinant VP2 protein is about 50kDa. IBDV VP2 mAb was screened using purified rVP2 protein as screening antigen. An IBDV antibody competition ELISA detection method (rVP 2-cELISA) was established using rVP2 protein as coating antigen and one of the HRP-labeled VP2 monoclonal antibodies as competition antibody. Compared with the existing commercial IBDV antibody detection kit, the infectious bursal disease virus antibody competition ELISA detection method has the advantages of shorter time consumption, better specificity, wider detection sample range, lower cost, more convenient production and the like, and has huge production and application values.

Description

Infectious bursal disease virus antibody competition ELISA detection method and kit

1. Technical field

The invention relates to an infectious bursal disease virus antibody competition ELISA detection method and a kit, and belongs to the technical field of biology.

2. Background art

Infectious Bursal Disease (IBD) is an infectious disease caused by Infectious Bursal Disease Virus (IBDV) and characterized by the invasion of young poultry bursa of Fabricius, which is one of the major infectious diseases causing serious economic losses in the poultry industry in China and the world. The disease not only causes death of the diseased animal, but also causes immunosuppression of the body, resulting in reduced immune defenses and failure of vaccination against multiple vaccines. Vaccination of susceptible chicken flocks is the most effective method for preventing this disease, but due to the differences in virulence and antigenicity of the widely circulating IBDV strains, the economic losses due to IBD immune failure or immunosuppression are enormous each year. In view of this, further analysis of the molecular epidemiology of current IBDV, development of an economical and rapid detection method remains an important topic to be solved in current IBD prevention and control efforts.

The main methods for detecting chicken IBDV antibodies are: neutralization test, agar amplification, indirect Immunofluorescence (IFA) and indirect ELISA (the existing commercial kit coating antigen is whole virus). Although the neutralization test is a gold standard for evaluating chicken antibodies, the neutralization test is complicated in operation, takes a long time, requires cell culture and is not suitable for common farms. Although the agar-agar experiment is not complicated in operation, the agar-agar experiment is not sensitive enough and takes a long time, and the aims of rapidness and sensitivity cannot be achieved. IFA is also cumbersome to handle, requires cell culture, and requires expensive fluorescence microscopy for observation. ELISA has the characteristics of specificity, simplicity and rapidness. Therefore, indirect EILSA is currently used to evaluate IBDV antibody levels in chicken flocks. However, the existing commercial IBDV antibody ELISA detection kits all adopt indirect ELISA, two steps are needed for detecting samples, the time is approximately 2 hours, and the cost is high. In this regard, competition ELISA was used for detection of IBDV antibodies in this study. The method can detect the serum sample in one step, takes 30 minutes, and is more rapid and efficient compared with indirect ELISA.

3. Summary of the invention

Technical problem

Infectious Bursal Disease (IBD) is an acute, febrile, highly contagious infectious disease of chickens and turkeys caused by Infectious Bursal Disease Virus (IBDV), and is one of the major infectious diseases causing serious economic losses in the poultry industry in China and the world.

Indirect EILSA is currently used to evaluate IBDV antibody levels in chicken flocks. However, the existing commercial IBDV antibody indirect ELISA detection kit is expensive and is not beneficial to popularization and application; the coating antigen of the domestic IBDV antibody indirect ELISA detection kit is purified IBDV whole virus, and the culture of the IBDV whole virus requires cells or chickens, so that the culture cost is high, and the IBDV purification requires a relatively expensive ultracentrifuge, so that the time is long. In contrast, the IBDV antibody competition ELISA detection kit developed in the research adopts prokaryotic expression recombinant VP2 protein (rVP 2) as a coating antigen, adopts HRP-marked VP2 monoclonal antibody as a competition antibody, and can detect the result of a sample within 30 min. Compared with the existing IBDV antibody indirect ELISA detection kit at home and abroad, the kit has 4 advantages: the method has the advantages of shorter time consumption, better specificity, wider range of detection samples, lower cost, more convenient production and the like, and has huge production and application values.

Technical proposal

An infectious bursal disease virus antibody competition ELISA detection method is characterized in that the method uses a preservation number of CCTCC NO: monoclonal antibody hybridoma cell strain 1H6 monoclonal antibody against the infectious bursal disease virus VP2 protein of C201966 is a competing antibody.

rVP2 recombinant VP2 protein is used as a coating antigen, and the preparation method comprises the following steps: optimally designing IBDV VP2 gene according to the registered IBDV VP2 sequence EU417824.1 of GenBank and the preference of escherichia coli codons, removing ATG of the VP2 gene, synthesizing total genes, directionally cloning into a prokaryotic expression vector pET-28a (+) to construct prokaryotic expression plasmid pET28+VP2, and converting escherichia coli Rosetta (DE 3); following IPTG induction, bacteria were collected by centrifugation and rVP2 was purified according to HisTrap HP instructions.

Diluting purified rVP2 recombinant protein to 5 mug/L with carbonate buffer solution with pH of 9.6, adding the diluted rVP2 recombinant protein into an ELISA plate, coating the ELISA plate with 100 mug/L of each hole at 4 ℃ overnight, sealing the holes by using PBST sealing liquid containing 10% FBS at 37 ℃ for 2 hours, washing the PBST for 3 times each time for 3 minutes, and drying the ELISA plate by beating; adding 50 mu L/well 96 parts of clinical sample diluted by 1:500, simultaneously adding 50 mu L/well of HRP-VP2 monoclonal antibody, diluting by 1:20000, washing by 1h and PBST for 5 times at 37 ℃ for 3min each time, and drying by beating; adding 100 mu L/hole of TMB color reagent and developing color in a cassette; 2M H ₂ And (5) stopping SO4, measuring an A450 value, and judging a result. Determination criteria: pi=100—sample OD value/negative average OD value x100, positive when PI > 50; and when PI is less than or equal to 50, judging as negative.

The infectious bursal disease virus antibody competition ELISA detection method can be directly applied. The competition ELISA detection method of the infectious bursal disease virus antibody can also be used for constructing a kit.

Advantageous effects

The infectious bursal disease virus antibody competition ELISA detection method established by the invention is compared with the existing commercial IBDV antibody detection kit: the method has the advantages of shorter time consumption, better specificity, wider range of detection samples, lower cost, more convenient production and the like, and has huge production and application values.

The invention has the following characteristics and advantages:

the invention adopts prokaryotic expression recombinant VP2 protein (rVP 2) as a coating antigen, adopts HRP-marked VP2 monoclonal antibody as a competitive antibody, and can detect the result of a sample within 30 min. Compared with the existing IBDV antibody indirect ELISA detection kit at home and abroad, the kit has 4 advantages;

1. the specificity is better: the kit adopts HRP-marked VP2 monoclonal antibody as competitive antibody, while the existing indirect ELISA kit adopts IBDV polyclonal antibody, and the monoclonal antibody has better specificity than polyclonal antibody as is known.

2. The range of detection samples is wider: the kit adopts HRP-marked VP2 monoclonal antibody as a competitive antibody, can not be limited by the source of a detection sample, and can detect various animal sources or non-animal sources; the enzyme-labeled secondary antibody of the existing indirect ELISA kit is aimed at a chicken serum sample, so that the detection range of the existing indirect ELISA kit can only be the chicken serum sample.

3. Less time consuming: when the kit is used for detecting a sample, the sample to be detected and the HRP-marked VP2 monoclonal antibody are added simultaneously, and the detection time is about 30 min; the existing indirect ELISA kit needs 2 steps, and needs to react the serum to be detected with the coating antigen (about 1 h), then add the enzyme-labeled secondary antibody for reaction (about 1 h), and the total time is more than 2 h.

4. Lower cost and more convenient production: from the coated antigen, the IBDV antibody competition ELISA detection kit developed in the research adopts prokaryotic expressionThe recombinant VP2 protein (rVP 2) of (2) is used as a coating antigen, and a large amount of the coating antigen (rVP 2) required by people can be produced only by a common shaking table, while the coating antigen of the existing commercial indirect ELISA kit is IBDV, and cells or chickens are required for culturing the IBDV, so that the production cost is high and the production is inconvenient. In addition, the competitive antibody used in the kit is a VP2 monoclonal antibody, only ascites is prepared from hybridoma cells through mice, about 15ml of ascites can be prepared from 1 mouse, 4ml of HRP-marked VP2 monoclonal antibody can be obtained from 1ml of ascites, when a sample is detected, the HRP-marked VP2 monoclonal antibody is diluted by more than 20000, and in conclusion, the total amount of HRP-marked VP2 monoclonal antibody working solution can be obtained from the ascites prepared from 1 mouse: 15ml ascites×4 mHRP-labeled VP2 mab×20000=1.2×10 ⁶ The HRP-marked VP2 monoclonal antibody working solution required by the ml and 1 kit is about 100ml, so that the ascites prepared by 1 mouse can meet the requirements of more than 10000 kits, and the cost is far lower than the production cost of polyclonal antibodies.

4. Description of the drawings

FIG. 1 10% SDS-PAGE analysis of VP2 full length expression in E.coli Transsetta (DE 3)

FIG. 2Westernblot analysis of reactivity of HRP-VP2 mab with rVP2

Preservation of organisms

The cell line 1H6 was deposited with the China center for type culture Collection, address: the preservation number of the Chinese university of Wuhan is CCTCC NO: c201966, classification naming: a monoclonal antibody hybridoma cell strain 1H6 of the infectious bursal disease virus VP2 protein.

5. Detailed description of the preferred embodiments

1 main experimental materials

Infectious Bursal Disease Virus (IBDV) B87 medium virulent attenuated vaccine strain was purchased from Nanjing Tianbang biotechnology Co., ltd; e.coli Rosetta (DE) ₃ Competent cells were purchased from Shanghai Biotechnology Inc.; plasmid purification kits were purchased from Omega company; DNA gel recovery kit was purchased from full gold biotechnology Co., ltd; restriction enzymes were purchased from TaKaRa.

2 Experimental methods

2.1 Expression of IBDV VP2 Gene in E.coli

2.1.1 Optimization of codons of IBDV VP2 Gene

The VP2 gene is optimally designed according to the registered IBDV VP2 sequence (EU 417824.1) of GenBank and the preference of the escherichia coli codon, the ATG of the VP2 gene is removed, the His tag on pET28a (+) is fused, and fusion expression is carried out in escherichia coli.

2.1.2 Construction of recombinant expression plasmid for IBDV VP2 Gene

The optimally designed VP2 gene is synthesized through total gene synthesis, bamHI and XhoI enzyme cutting sites are added at the 5 'end and the 3' end of the VP2 gene, and the optimally designed VP2 gene is synthesized by a general biological System (Anhui) Limited company and cloned into a prokaryotic expression vector pET28a (+). Double digestion identification was performed with XhoI and MluI, and sequencing analysis was performed. The obtained recombinant plasmid was named pET28-VP2 (4-1350). Analysis by electrophoresis on an EB-containing 1% agarose gel revealed that 2 DNA fragments of about 4.0Kb and 2.5Kb in length, respectively, were consistent with the theoretical values of pET28a (+) and VP2 gene fragments.

2.1.3 Expression of IBDV VP2 Gene in E.coli

Transforming recombinant plasmid pET28-VP2 (4-1350) into E.coli Rosetta (DE) ₃ Competent cells were plated on 1.5% agar plates prepared with LB, cultured at 37℃for 14h, single colonies were picked and inoculated into LB, and cultured with shaking for 14h. The following day, LB medium (containing 100. Mu.g/ml of calicheamicin) was inoculated with 1% of each, followed by shaking culture, at A ₆₀₀ At a value of 0.8, IPTG was added at a final concentration of 1.0mM, and induction was performed for 4 hours, respectively. The induced bacterial culture was centrifuged at 5000g for 5min, and the expression of VP2 fragments in the cells was analyzed by 12% SDS-PAGE. Test synchronization set-up pET28a (+) empty plasmid transformed E.coli Rosetta (DE) ₃ Is used as a control. A protein band of one entry was found at a molecular weight of about 50kDa by 12% SDS-PAGE electrophoresis, whereas pET28a (+) transformed E.coli Rosetta (DE ₃ ) There is no 50 kDa-sized protein band.

2.1.4 Western blot analysis of expression level of IBDV VP2 protein

Centrifuging 5000g of recombinant bacteria with 3ml of induced expression for 10min, removing supernatant, and adding 160 μl deionized water for resuspensionThen 40. Mu.L of 5 XSDS-Loading Buffer was added, SDS-PAGE analysis of 12% separation gel and 5% concentration gel was performed according to the conventional method, transferred onto nitrocellulose membrane, after transfer, nitrocellulose membrane was blocked overnight with 5% skim milk powder at 4℃and then reacted with primary antibody (His monoclonal antibody or chicken anti-IBDV hyperimmune serum) and secondary antibody (HRP-labeled goat anti-mouse IgG antibody or HRP-labeled rabbit anti-chicken IgG antibody) respectively, and DAB substrate was developed to observe specific protein bands. Test synchronization establishment of empty plasmid pET28a (+) transformed E.coli Rosetta (DE ₃ ) Is used as a control. After electrophoresis, transfer and DAB development by 12% SDS-PAGE, the recombinant VP2 protein may have an entry protein band with His mab or chicken anti-IBDV hyperimmune serum at a molecular weight of about 50kDa, whereas pET28a (+) transformed E.coli Rosetta (DE ₃ ) There is no 50 kDa-sized protein band.

2.1.5 Purification of IBDV VP2 protein

200mL of the culture for inducing expression is taken, bacteria are collected by centrifugation, 30mL of a binding buffer solution is used for resuspension, 20 mu L of 100mmol/L PMSF is added, ultrasonic disruption is carried out under the ice bath condition until the cell suspension is clear, centrifugation is carried out for 30min at 4 ℃ and 10000g, cell fragments are removed, the supernatant is filtered by a filter membrane with a pore diameter of 0.45 mu m, and then the recombinant VP2 protein fragment is purified according to the HisTrap HP specification. The ultrasonic precipitation contains a large amount of recombinant VP2 protein, which indicates that the recombinant VP2 protein exists mainly in the form of inclusion bodies.

2.2 HRP-labeled IBDV VP2 gene monoclonal antibody

2.2.1 Preparation of IBDV VP2 Gene monoclonal antibody

Female BALB/c mice of 6-8 weeks old were immunized with purified IBDV, 1 time every 14d, and after the 4 th immunization, the immunized BALB/c mice spleen cells were aseptically taken and fused with SP2/0 cells in the logarithmic growth phase. The rVP2 protein is used as a coating antigen, and an indirect ELISA method is used for measuring culture supernatant of the fusion cells, so that 8 IBDV VP2 monoclonal antibodies are screened. Positive hybridoma cells were selected for cloning by limiting dilution. The cloned positive hybridoma cells are subjected to expansion culture, and are injected into BALB/c female mice with the age of 8-10 weeks in an intraperitoneal mode to prepare ascites.

2.2.2 Horseradish peroxidase (HRP) labeled IBDV VP2 mab

Take 0.5Equal amount of PBS was added to the ml ascites fluid for dilution, 3000r/min and 10min, and the supernatant was filtered through a 0.45 μm pore size filter, followed by purification of VP2 mab according to HiTrap ProteinAHP instructions. Labeling purified VP2 mAb with horseradish peroxidase (HRP) by sodium periodate method ^[1] 。

[1] Luo Jiali, shen Binggui, song Guangcheng, chen Caiyun, xue Haichou. Simple method of labelling antibodies with horseradish peroxidase (HRP) [ J ]. Proc. Biochemical and BioPhysics, 1981 (01): 1-8.

2.2.3 Westernblot analysis of reactivity of IBDV VP2 monoclonal antibodies with rVP2

Transformation of pET28-VP2 (4-1350) into E.coli Rosetta (DE) ₃ E.coli Rosetta (DE) transformed with the empty plasmid pET28a (+) ₃ ) Induced expression was performed, SDS-PAGE analysis was performed, transferred to nitrocellulose membrane, blocked at 5% nonfat milk powder at 4℃overnight, and then developed with HRP-VP2 monoclonal antibody as primary antibody, DAB substrate, and specific protein bands were observed.

After electrophoresis, transfer and DAB development by 10% SDS-PAGE, HRP-VP2 mab was associated with a single-entry protein band at a molecular weight of about 50kDa with the prokaryotic-expressed recombinant VP2 protein, whereas pET28a (+) transformed E.coli Rosetta (DE ₃ ) There is no protein band. The HRP-VP2 mab of the selected cell line 1H6 can have an entry protein band with the prokaryotic expressed recombinant VP2 protein at a molecular weight of about 50kDa.

2.3 Establishment of IBDV antibody competition ELISA detection method

Diluting purified rVP2 recombinant protein to 5 mug/L with carbonate buffer solution with pH of 9.6, adding into an ELISA plate, coating at 4 ℃ for overnight, sealing the holes with sealing solution (PBST containing 10% FBS) at 37 ℃ for 2h, washing with PBST for 3 times and 3min each time, and drying by beating; 50. Mu.L/well (96 parts) of clinical sample diluted 1:500 was added, and simultaneously 50. Mu.L/well of HRP-VP2 mab (HRP-VP 2 mab 1:20000 dilution) was added, washed 5 times with PBST at 37℃for 1h, 3min each time, and dried by flash; adding 100 mu L/hole of TMB color reagent and developing color in a cassette; 2M H ₂ SO ₄ Termination, test A ₄₅₀ And (5) value and judging result. Determination criteria: pi=100—sample OD value/negative average OD value x100, positive when PI > 50; and when PI is less than or equal to 50, judging as negative.

2.4 Preliminary application of IBDV antibody competition ELISA detection method

2.4.1 Competition ELISA

Diluting purified rVP2 recombinant protein to 5 mug/L with carbonate buffer solution with pH of 9.6, adding into an ELISA plate, coating at 4 ℃ for overnight, sealing the holes with sealing solution (PBST containing 10% FBS) at 37 ℃ for 2h, washing with PBST for 3 times and 3min each time, and drying by beating; 50. Mu.L/well (96 parts) of clinical sample diluted 1:500 was added, and simultaneously 50. Mu.L/well of HRP-VP2 mab (HRP-VP 2 mab 1:20000 dilution) was added, washed 5 times with PBST at 37℃for 1h, 3min each time, and dried by flash; adding 100 mu L/hole of TMB color reagent and developing color in a cassette; 2M H ₂ SO ₄ Termination, test A ₄₅₀ And (5) value and judging result. Meanwhile, an IDEXX IBDV antibody indirect ELISA kit is used for detecting clinical samples and is used as a commercial control group.

2.4.2 neutralization test

96 clinical samples are respectively diluted to 1:64 by 2 times of serial dilution from 1:2; 100. Mu.L of each of the cells was mixed with an equal volume of an adaptive virus dilution of IBDV B87 cells (virus content 200TCID50/0.1 mL), reacted at 37℃for 1h, inoculated in 96-well plates containing DF-1 monolayers, and 100. Mu.L/well (virus content 100TCID 50/well) was repeated 4 times per serum to be tested; at the same time, virus control, positive serum control, negative serum control and cell control are established. Culturing at 37deg.C with 5% CO2, observing pathological changes every day, recording, calculating virus neutralizing antibody titer of each serum sample according to Reed-Muench method, and judging positive according to national standard GB/T19167-2003, wherein neutralizing titer >1:32, and negative otherwise.

IBDV antibody Competition ELISA, IDEXX-ELISA (IBDV) and neutralization assay established with recombinant VP2 protein 96 clinical samples were tested simultaneously, as a result: compared with a neutralization test, the detection method of the I BDV antibody cELISA established by the recombinant VP2 protein and the IDEXX-ELISA (IBDV) have the relative sensitivities of 94.12 percent and 100 percent respectively; the relative specificities were 95.16% and 25.8%, respectively; the compliance rates were 94.79% and 52.08%, respectively. Specific results are as follows (tables 1 and 2).

TABLE 1 comparison of rVP2-cELISA with neutralization assay

Relative sensitivity: 32/34×100% =94.12%;

relative specificity: 59/62×100% =95.16%;

coincidence rate: (32+59)/96×100% =94.79%

TABLE 2 comparison of IDEXX-ELISA with neutralization assay

Relative sensitivity: 34/34×100% = 100%;

relative specificity: 16/62×100% = 25.8%;

coincidence rate: (34+16)/96×100% =52.08%.

Claims

1. An infectious bursal disease virus antibody competition ELISA detection kit is characterized in that the kit adopts a CCTCC NO: the monoclonal antibody secreted by hybridoma cell line 1H6 of C201966, which secretes the monoclonal antibody against infectious bursal disease virus VP2 protein, is a competing antibody.

2. The infectious bursal disease virus antibody competition ELISA detection kit as claimed in claim 1, wherein rVP2 recombinant VP2 protein is used as a coating antigen, and the preparation method comprises the following steps: optimally designing IBDV VP2 genes, optimally designing VP2 genes according to the registered IBDV VP2 sequence EU417824.1 of GenBank and the preference of escherichia coli codons, removing ATG of the VP2 genes, synthesizing the complete genes, directionally cloning the complete genes into a prokaryotic expression vector pET-28a (+) to construct prokaryotic expression plasmid pET28+VP2, and converting escherichia coli Rosetta (DE 3); following IPTG induction, bacteria were collected by centrifugation and rVP2 was purified according to HisTrap HP instructions.

3. The infectious bursal disease virus antibody of claim 2 competing ELISA detection kit, its characterized in that: diluting purified rVP2 recombinant protein to 5 mu g/L with carbonate buffer solution with pH of 9.6, adding the diluted rVP2 recombinant protein into an ELISA plate, coating the ELISA plate at a temperature of 100 mu L per hole and a temperature of 4 ℃ overnight, sealing the hole by using PBST sealing liquid containing 10% FBS, sealing the hole at a temperature of 37 ℃, washing the PBST for 3 times at a time of 2 hours, and drying the ELISA plate by beating; adding 96 parts of 50 [ mu ] L/hole of a clinical sample diluted by 1:500, simultaneously adding 50 [ mu ] L/hole of HRP-VP2 monoclonal antibody, diluting by 1:20000, washing for 5 times at 37 ℃ for 1h by PBST, and drying for 3min each time; adding TMB color reagent 100 mu L/hole, and developing color by a cassette; 2M H ₂ SO ₄ And (3) ending, measuring an A450 value, judging a result and judging a standard: pi=100—sample OD value/negative average OD value x100, positive when PI > 50; and when PI is less than or equal to 50, judging as negative.