CN112180089A

CN112180089A - Bovine herpesvirus type I antibody blocking ELISA detection method

Info

Publication number: CN112180089A
Application number: CN202010993414.4A
Authority: CN
Inventors: 宋佰芬; 翟璐; 宋丽; 马金柱; 佟春玉; 于永忠; 崔玉东
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2021-01-05

Abstract

The invention discloses a bovine herpes virus I-type antibody blocking ELISA detection method, belongs to a serological detection method for antibody detection, and is mainly used for detecting bovine herpes virus I. The detection method is that the antigen coated by the ELISA plate in the ELISA detection method is herpes virus I type gD recombinant protein and horseradish peroxidase-labeled monoclonal antibody 2B 4. The detection method has strong specificity and high sensitivity, and can directly detect the bovine herpes virus I type infected person in the latent period.

Description

Bovine herpesvirus type I antibody blocking ELISA detection method

Technical Field

The invention relates to an ELISA detection method.

Background

Infectious Bovine rhinotracheitis, also known as necrotic rhinitis and red nose disease, is a contact infectious disease of cattle caused by Bovine herpes virus type I (Bovine herpesvirus 1, BoHV-1), and the establishment of a Bovine herpes virus type I detection method is helpful for taking prevention and control measures for the early spread of viruses. At present, the separation and identification of bovine herpes virus type I virus, electron microscopy, histopathology, polymerase chain reaction, serology and other common detection methods have various advantages and disadvantages, wherein the former three methods cannot carry out rapid high-flux detection, and the polymerase chain reaction cannot detect virus antigens in a latent period.

Disclosure of Invention

The invention provides a bovine herpes virus type I antibody blocking ELISA detection method for solving the problems that the conventional bovine herpes virus type I detection method cannot carry out rapid high-flux detection or cannot detect virus particles with low emission in a latent period.

The detection method of bovine herpes virus type I antibody blocking ELISA is carried out according to the following steps:

firstly, an antigen recombinant gD protein coated by an enzyme label plate coats a reaction plate, and BSA is used for sealing;

secondly, after the serum to be detected and the negative and positive control serum are processed, a reaction plate is added for blocking;

thirdly, the monoclonal antibody 2B4 marked by horseradish peroxidase is combined with unblocked antigen gD protein for color development;

fourthly, stopping the reaction, measuring OD450nm, and calculating the inhibition rate; the detection is completed.

The antigen recombinant gD protein in the first step is prepared by the following method:

firstly, extracting bovine herpes virus type I genome DNA;

secondly, recovering and purifying the amplified PCR product, wherein the primer sequences are as follows:

gD-T1：5’-GGAATTCATGCAAGGGCCGACATTGG-3’

gD-T2：5’-CCCTCGAGCAGCGCGCTGTAGTTGACGTT-3’

thirdly, connecting the PCR product with a T vector, transferring the connecting product into competent cells, carrying out enzyme digestion identification, screening positive clones, and carrying out sequence determination;

fourthly, carrying out double enzyme digestion on plasmids of the positive clone and pGEX-6P-1 by Eco RI-HF and Xho I, constructing a pGEX-6P-1-gD expression vector, transferring the vector into a competent cell, culturing, selecting the positive clone, and carrying out double enzyme digestion identification;

and fifthly, inducing and expressing the gD recombinant protein in the culture solution of the positive clone by IPTG.

The invention has the following advantages:

1. the horseradish peroxidase-labeled monoclonal antibody 2B4 used in the invention can identify bovine herpes virus type I and locate the gD envelope protein thereof, has higher specificity, can directly detect the result by using the method of the invention, does not need to add secondary antibody, and has the characteristics of simplifying operation steps, saving time and saving cost.

2. The 2B4 antibody is modified by horseradish peroxidase, can be used as a direct detection antibody of bovine herpes virus type I, and is convenient.

3. The replication of virus particles in the latent period is reduced, the discharge amount is low, but the host generates a certain amount of antibody after the virus infects the host for the first time, and then the virus enters the latent period, but the antibody level is maintained for a longer time and is relatively constant, so that the low amount of antibody generated by the virus in the latent period can be detected by using the detection method provided by the invention.

The detection method is very suitable for measuring the concentration of the small molecule antibody in a complex sample, and has high specificity, repeatability and coincidence rate.

Drawings

FIG. 1 is a graph showing the effect of a BSA blocking solution in a first embodiment;

FIG. 2 is a graph showing the results of mAb 2B4 in the first embodiment;

FIG. 3 is a PCR map of the gD recombinant protein gene;

FIG. 4 shows the purification of gD recombinant protein gene.

Detailed Description

The first embodiment is as follows: the detection method of bovine herpes virus type I antibody blocking ELISA according to the present embodiment is performed by the following steps:

In the first step of the present embodiment, the preparation method of the BSA blocking solution is as follows:

firstly, preparing PBS with pH 7.4: weighing 8g NaCl, 0.2g KCl and 1.42g Na₂HPO₄·12H₂O and 0.27g KH₂PO₄Add 800mL ddH₂Stirring until the O is completely dissolved, adjusting the pH to 7.4, fixing the volume to 1L, sterilizing at 121 ℃ for 20min under high pressure, and storing at room temperature;

secondly, 5g of Bovine Serum Albumin (BSA) is weighed and added into 100mL of PBS in the first step, namely BSA blocking solution (5% BSA).

FIG. 1 is a graph showing the effect of BSA blocking solutions, and as shown in FIG. 1, FIG. 1 shows the blocking effect of different blocking solutions, and it can be seen from FIG. 1 that the blocking rate of 5% BSA is the highest.

The fourth termination reaction in the present embodiment is 2M H₂SO₄(ii) a Inputting all the inhibition rates into MedCalc software to draw an ROC curve, obtaining a critical value of 47.7 percent according to the ROC curve to serve as a diagnostic standard, and obtaining the blocking rate>47.7% of the total percentRate of failure<47.7% was judged negative, and 0.471 to 0.527 was the suspicious interval. Wherein, the formula of the serum inhibition rate is as follows:

the serum inhibition ratio (%) - (negative control OD450 nm-test serum OD450 nm)/negative control OD450nm × 100%.

The preparation method of the monoclonal antibody 2B4 of the embodiment is as follows:

firstly, aiming at BoHV-1gD mAbs (monoclonal antibody 2B4), gD recombinant protein with the concentration of 0.2mg/mL and Freund's adjuvant are adopted according to the proportion of 1: emulsifying at a volume ratio of 1, injecting BALB/c mice subcutaneously at multiple points at a dose of 500 mu L/mouse, boosting the immunization once every two weeks, collecting tail vein serum of the mice 14 days after the third immunization, determining the optimal working concentration of the coated gD protein and the positive serum of the mice by a square matrix titration method, and simultaneously determining the titer of the serum of the mice after the third immunization; BALB/c mice were boosted 3 days prior to fusion with 50. mu.L dose of gD protein.

Taking the whole spleen of the immunized BALB/c mouse, grinding to obtain whole spleen cells, and mixing the whole spleen cells with mouse myeloma cells sp2/0 in a logarithmic growth phase according to the ratio of 1:1, uniformly mixing and fusing, adding a 96-pore plate, sequentially screening for one week through an HAT incomplete culture medium and an HT incomplete culture medium, observing the liquid condition of each pore to timely replenish liquid to form a single-strain cell strain, and then detecting whether the cell supernatant has antibody titer and performing 4-round screening through an indirect ELISA (enzyme-linked immunosorbent assay) method to finally obtain 3 monoclonal antibodies; 1E7, 2B4, 3H 4;

and thirdly, the antibodies 2B4 are labeled by HRP and selected as detection antibodies through 1 affinity detection and blocking rate.

FIG. 2 is a graph showing the results of mAb 2B 4; as shown in FIG. 2, A represents the monoclonal antibody after screening, B represents the hybridoma cell line during screening, and it is understood that we have successfully screened 3 monoclonal antibodies.

Wherein, monoclonal antibody 2B4(BoHV-1gD mAbs) is prepared by mixing gD recombinant protein with concentration of 0.2mg/mL and Freund's adjuvant according to the ratio of 1: after emulsification according to the volume ratio of 1, injecting BALB/c mice subcutaneously in a multi-point way at the dose of 500 mu L/mouse, boosting the immunization once every two weeks, collecting tail vein serum of the mice 14 days after the third immunization, and determining the optimal working concentration of the coated gD protein and the positive serum of the mice by a square matrix titration method, wherein the result is shown in table 1, the table 1 shows the optimal dilution ELISA detection result of the antigen and the serum, and the optimal dilution multiple of the antigen is 1:800 and the optimal dilution multiple of the antibody is 1: 800. And simultaneously determining the serum titer of the mice after three times of immunization, wherein the result is shown in table 2, the table 2 shows the antibody titer of the serum of the three-immune mice, and the antibody titer reaches over 1: 64000. BALB/c mice were boosted 3 days prior to fusion with 50. mu.L dose of gD protein.

TABLE 1 optimal dilution ELISA test results for antigen and serum

Dilution factor of antibody	1:200	1:400	1:800	1:1600	1:3200	1:6400	1:12800
								1:200(+)	3.154	3.081	2.727	2.099	1.628	1.145	1.086
1:200(-)	0.150	0.143	0.112	0.107	0.136	0.118	0.116
								1:400(+)	3.017	2.855	2.681	2.190	1.769	1.28	1.045
1:400(-)	0.148	0.147	0.13	0.134	0.134	0.145	0.147
								1:800(+)	2.441	2.800	2.507	1.958	1.180	0.774	0.546
1:800(-)	0.099	0.119	0.091	0.092	0.092	0.099	0.095
								1:1600(+)	2.981	2.940	2.489	1.853	1.127	0.699	0.419
1:1600(-)	0.123	0.122	0.122	0.121	0.120	0.121	0.126
								1:3200(+)	2.568	2.670	2.229	1.589	1.010	0.605	0.390
1:3200(-)	0.111	0.097	0.091	0.090	0.096	0.091	0.098
								1:6400(+)	2.475	2.377	1.725	1.198	0.764	0.465	0.302
1:6400(-)	0.114	0.111	0.115	0.121	0.126	0.126	0.134
								1:12800(+)	2.082	1.892	1.277	0.874	0.584	0.332	0.227
1:12800(-)	0.088	0.096	0.090	0.092	0.104	0.096	0.098

Note: (+) positive serum, (-) negative serum;

TABLE 2BALB/c mice third immunotiter test results

Taking the whole spleen of the immunized BALB/c mouse, grinding to obtain whole spleen cells, and mixing the whole spleen cells with mouse myeloma cells sp2/0 in a logarithmic growth phase according to the ratio of 1:1, uniformly mixing and fusing, adding a 96-well plate, sequentially carrying out one-week screening by using an HAT incomplete culture medium and an HT incomplete culture medium, observing the liquid condition of each well to timely replenish liquid to form a single-strain cell strain, then carrying out 4-round screening by using an indirect ELISA (enzyme-Linked immuno sorbent assay) method for detecting whether the cell supernatant has antibody titer and subcloning, and finally obtaining 3 monoclonal antibodies, wherein the antibody titer is shown in table 3, the table 3 shows the antibody titer of hybridoma cell culture supernatant and ascites, the 1E7 supernatant titer in the three monoclonal antibodies is 1:1600, and the ascites titer is 1: 16000; 2B4 the titer of the supernatant is 1:800 and the titer of the ascites is 1: 16000; the titer of the 3H4 supernatant was 1:800, and the titer of ascites was 1: 16000. The antibody 2B4 was selected as the detection antibody by HRP labeling and 1 affinity detection and blocking rate, as shown in Table 4, the antibody titers and blocking rates of the three monoclonal antibodies labeled with horseradish peroxidase are shown in Table 4, and it is known from the Table that the titers of the three monoclonal antibodies are consistent and are 1:800, but the blocking effect of 2B4 is the highest, so the subsequent experiments all use the 2B4 monoclonal antibody labeled with horseradish peroxidase.

TABLE 3 antibody titers of hybridoma cell culture supernatants and ascites

TABLE 4 antibody titer and serum blockade rate

The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the antigen recombinant gD protein of the first step is prepared according to the following method:

firstly, extracting bovine herpes virus type I genome DNA;

gD-T1：5’-GGAATTCATGCAAGGGCCGACATTGG-3’

gD-T2：5’-CCCTCGAGCAGCGCGCTGTAGTTGACGTT-3’

and fifthly, inducing and expressing the gD recombinant protein in the culture solution of the positive clone by IPTG. Other steps and parameters are the same as those in the first embodiment.

In the second step of this embodiment, a pair of primers is designed based on the nucleotide sequence of BoHV-1US6 published by GenBank, and the target product is 1251 kb. Respectively as follows: 30s at 98 ℃; PCR products were obtained at 98 ℃ for 10s, 63 ℃ for 30s, and 72 ℃ for 45s, setting 25 cycles.

In the embodiment, the obtained PCR gene fragment is inserted into a pGEX-6P-1 vector by means of connection transformation and is transferred into BL21(DE3) competent cells to prepare pGEX-6P-gD recombinant bacteria. Inducing the bacterial liquid in the logarithmic growth phase by IPTG, and purifying the gD recombinant protein by the methods of ultrasonic crushing of the bacterial liquid and affinity chromatography after inducing for 4h at 30 ℃, wherein the specific results are shown in figures 3 and 4, and

lanes

1 and 2 in figure 3 respectively show the negative result and the positive result of the PCR of the BoHV-1gD protein gene; 3. lane 4 shows the single cleavage of the recombinant gD protein gene; lane 5 shows the double cleavage of the recombinant gD protein gene. From the figure, it is known that the gD protein gene was successfully inserted into the pGEX-6p-1 plasmid.

In FIG. 4, lane 4 shows the recombinant gD protein after induction of expression, and lane 5 shows the gD protein after purification. From the figure, it is known that the gD protein gene can be successfully expressed in Escherichia coli BL21 after being inserted into pGEX-6p-1 plasmid, and purified by ultrasonication and affinity chromatography.

The third concrete implementation mode: the second embodiment is different from the first embodiment in that: the PCR amplification conditions in the second step are as follows: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 63 ℃ for 30s, extension at 72 ℃ for 45s, setting 25 cycles, and final extension at 72 ℃ for l0 min. Other steps and parameters are the same as those in the second embodiment.

In the present embodiment, the temperatures and times for pre-denaturation, annealing and extension are set by the specification of PCR enzyme with high GC content of Beijing Sizhengbai Biotech Co.

The fourth concrete implementation mode: the detection method of bovine herpes virus type I antibody blocking ELISA is carried out according to the following steps:

firstly, an antigen recombinant gD protein coated by an enzyme label plate coats a reaction plate, namely, the purified gD protein is diluted by 1:800 times of CBS buffer solution with pH 9.6 and then coated overnight at 4 ℃, the concentration of the coating solution is 100 mu L/hole, the coating solution is discarded the next day, and the plate is washed by PBST for 4 times and 5 min/time; blocking BSA, blocking 5% BSA at 100. mu.L/well at 37 ℃ for 1h, discarding the blocking solution, washing the plate with PBST for 4 times, 5 min/time;

treating the serum to be detected and the negative and positive control serum, namely treating the treated serum with PBS and the serum to be detected and the negative and positive control serum by using a ratio of 1: diluting at a ratio of 1, incubating at 100 μ L/well for 1.5h at 37 deg.C, discarding serum, washing the plate with PBST for 4 times, 5 min/time; adding a reaction plate for blocking;

thirdly, the enzyme-labeled antibody 2B4 is combined with unblocked antigen gD protein and is developed, namely, the antibody 2B4 is modified by horse radish peroxidase, the horse radish enzyme-labeled mAb is diluted by 1:8000 times by PBS, 100 mu L/hole is incubated for 1h at 37 ℃, the mAb is discarded, and the plate is washed by PBST for 4 times and 5 min/time;

fourthly, use 2M H₂SO₄The reaction is stopped, 50 mu L/hole is formed, and then the reading value of OD450nm is measured on a microplate reader; the detection is completed.

Example 1 detection of bovine herpes Virus type I antibodies Using the methods of the invention

The specific test method comprises the following steps:

1. according to the detection result of the IDEXX gE import kit, 18 serum samples are selected for batch repeat, the coated antigen and the labeled detection antibody prepared in the same batch are used for independently detecting the samples for 3 times by the established blocking ELISA method (adopting the fourth specific implementation mode) under the same condition, the blocking rate is calculated by statistical values, the variation coefficient is not more than 10 percent as shown in the table 5, the batch repeat test is shown, and the batch repeat test is good from the table.

2. And repeating the batches, applying the coated antigens and the marked detection antibodies prepared in different batches, independently detecting 16 serum samples for 2 times by using the established blocking ELISA method (adopting the fourth specific implementation mode) under the same condition, counting the detected values, wherein the variation coefficient is not more than 10% as shown in table 6, the table 6 shows batch repeat tests, and the batch repeatability is good.

3. BVDV with higher homology with BoHV-1 is selected as a control, and the detection is carried out by using the established blocking ELISA detection method (adopting the fourth embodiment), as shown in Table 7, the blocking rate of BVDV is lower, and as shown in Table 7, specificity detection is shown, and the method disclosed by the invention is good in specificity.

TABLE 5 in-batch repeatability test

TABLE 6 repeatability tests between batches

TABLE 7 specificity test

Test 2 comparative test

Detecting 146 parts of sample by adopting an established blocking ELISA detection method and a commercial kit, wherein the result shows that the positive serum detected by the commercial kit is 73 parts, and the negative serum is 73 parts; 66 parts of positive serum and 80 parts of negative serum are detected by blocking ELISA. As shown in Table 8, the established blocking ELISA detection method was found to have a percent compliance of 95.2% when compared to the commercially available kits imported from abroad. Although the detection results are basically consistent, the invention can be realized by only one-time detection, and the detection cost is greatly reduced by about 70%.

TABLE 8 recombination Rate measurements

Claims

1. a bovine herpes virus type I antibody blocking ELISA detection method, it is characterized in that the detection method that bovine herpes virus type I antibody blocking ELISA is carried out according to the following steps:

1. The antigen recombinant gD protein coated by the ELISA plate is coated with the reaction plate, and BSA is blocked;

2. After the serum to be tested and the negative and positive control serum are processed, add the reaction plate for blocking effect;

3. Horseradish peroxidase-labeled mAb 2B4 binds to the unblocked antigen gD protein, and develops color;

4. Terminate the reaction, measure OD450 nm, and calculate the inhibition rate; that is, the detection is completed.

2. a kind of bovine herpes virus type I antibody blocking ELISA detection method according to claim 1 is characterized in that the antigenic recombinant gD protein of step 1 is prepared according to the following method:

1. Extracting bovine herpesvirus type I genomic DNA;

2. Recovery and purification of amplified PCR products, primer sequences are as follows:

gD-T1: 5'-G GAATTC ATGCAAGGGCCGACATTGG-3'

gD-T2: 5'-CC CTCGAG CAGCGCGCTGTAGTTGACGTT-3'

3. The PCR product is ligated with the T vector, and the ligated product is transferred into competent cells, identified by enzyme digestion, screened for positive clones, and sequenced;

4. The plasmids of Eco RI-HF and Xho I double-enzyme-digested positive clones and pGEX-6P-1 were constructed to construct pGEX-6P-1-gD expression vector, which was transferred into competent cells. After culture, positive clones were picked and double-enzyme digested identification;

5. The culture medium of positive clones was induced to express gD recombinant protein with IPTG.

3. a bovine herpes virus type I antibody blocking ELISA detection method according to claim 2, is characterized in that in step 2, PCR amplification conditions are: 98 ℃ of pre-denaturation 30s; 98 ℃ of denaturation 10s, 63 ℃ of annealing 30s , extended at 72 °C for 45 s, set 25 cycles, and finally extended at 72 °C for 10 min.