CN111751553B

CN111751553B - Peste des petits ruminants virus H protein antibody iELISA detection method and application

Info

Publication number: CN111751553B
Application number: CN202010563205.6A
Authority: CN
Inventors: 窦永喜; 钱榜; 李彦敏; 朱学亮; 张志东; 张学燕
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2022-01-25
Anticipated expiration: 2040-06-19
Also published as: CN111751553A

Abstract

The invention selects 4 antigen epitope peptides with better reactogenicity to be connected in series and then synthesized as coating antigen based on the B cell epitope of the screened Peste Des Petits Ruminants Virus (PPRV) H protein, establishes an iELISA method aiming at the PPRV H protein antibody, and the optimal coating amount of the multi-epitope antigen peptide is 1.5 multiplied by 10^‑6ug/hole, the critical dilution concentration of the negative and positive serums to be detected is 1:100, and the optimal dilution concentration of the enzyme-labeled secondary antibody is 1: 80000; the coating solution is carbonate buffer solution, the serum and the secondary antibody diluent are PBST solution, and the blocking solution is 2% BSA solution; the positive-negative cutoff value was 0.25. Clinical serum detection shows that the variation coefficient of the detection results in batches and between batches is less than 10 percent; the method has good specificity; the comparison test of 306 sera with the cELISA kit produced by ID-Vet company shows that the coincidence rate of the two is 92.75%. The PPRV H protein antibody iELISA detection method can be used for detecting the clinical peste des petits ruminants antibody, and the operation is more time-saving and labor-saving than that of cELISA, and is simple, convenient and quick.

Description

Peste des petits ruminants virus H protein antibody iELISA detection method and application

Technical Field

The invention relates to the technical field of animal epidemic disease detection, in particular to a peste des petits ruminants virus H protein antibody iELISA detection method and application.

Background

Peste des petits ruminants (PPR) is a viral disease caused by PPR virus (PPRV), a member of the family of paramyxoviruses of the genus morbillivirus, (Gibbs et al, 1979) that is highly lethal to Peste ruminants, and is a highly contagious infectious disease that is transmitted mainly through the respiratory system by direct contact, and also transmitted through semen, embryos, milk, and the like. The PPRV is mainly used as a host of small ruminants such as goats and sheep, and can also be infected with large ruminants such as camels, cows and buffalos (Balamurgan et al, 2014; Maillard et al, 2008; Zakian et al, 2016), and clinical symptoms after infection are mainly manifested by appetite reduction, fever, increase of oral and nasal secretions, foul breath and the like of early animals; acute stage hyperpyrexia (above 40 ℃), respiratory disturbance, oral mucosa hemorrhage, conjunctival and conjunctival flush, etc.; the later manifestations of necrotic lesions and watery diarrhea in the tongue, cheeks, and mandible etc. (Jagtap et al, 2012; Thang et al, 2014), with morbidity and mortality rates as high as 100%. PPR is listed as a legally reported animal epidemic disease by the world animal health Organization (OIE) (Santhamani et al, 2016), and is also listed as a type of animal epidemic disease in China. Since the PPR was first reported in our country in 2007, it spread in many provinces such as tibetan, Shandong, inner Mongolia, etc. of our country, posing a serious challenge to the healthy and orderly development of animal husbandry in our country (Liu et al, 2018).

PPRV has only one serotype, and is divided into 4 lineages (I, II, III and IV) based on the F protein and N protein gene sequences (Balamurugan et al, 2014; Balamurugan, 2010). The virus is a mononegavirale RNA virus that encodes 8 proteins in total, 6 structural proteins and 2 non-structural proteins (C, V) (Bailey et al, 2005), fusion protein (F), hemagglutinin protein (H), matrix protein (M), nucleocapsid protein (N), phosphoprotein (P) and large protein (L). The gene sequence of the coded N protein is positioned at the 3' end of the PPRV gene sequence, is the protein with the most abundant content and the strongest immunogenicity in the virus, and the PPRV diagnosis technology based on the N protein is also widely developed and applied, but the protein does not induce the organism to generate immune protection. In addition, the N protein plays an important role in the assembly of viruses as well as in replication and transcription (Servan de Almeida et al, 2007). The H protein is a virus envelope protein, is divided into an inner membrane region and an outer membrane region, is one of the proteins with the worst conservation of measles virus, and is a main protein for inducing a host humoral immune response. The H protein is a disulfide-linked homodimer with the signal peptide at the N-terminus on the cytosolic side and the C-terminus extracellular (vongpunswawad et al, 2004). The measles virus H protein is the major determinant of cell tropism, and rinderpest H protein is the major cause of cross-species pathogenesis in the laggard attenuated strain (Yoneda et al, 2002), indicating that H protein is the most important antigenic determinant of measles virus. Current serological assays for PPRV are mostly based on antibodies produced by the N and H proteins. Since the N protein is highly conserved in morbillivirus, detection methods based on the N protein will generate certain cross-reactivity. The H protein is most diverse among all members of the morbillivirus genus, has strong species specificity, and has considerable advantages in DIVA (differentiation of human immunized from naturally infected animals) strategy design.

The accurate diagnosis or detection of PPR and its immune effects is beneficial to global prevention control and elimination of PPR. Therefore, on the basis of screening and identifying PPRV H protein B cell epitope in the early stage of the laboratory, the research selects a B cell epitope fragment with better reactionogenicity with a PPRV specific antibody, then carries out polypeptide synthesis by connecting the B cell epitope fragment in series, establishes a PPRV H antibody iELISA detection method by taking the synthesized polypeptide as a coating antigen, and has the advantages of time and labor saving, simplicity, convenience and rapidness compared with cELISA.

Disclosure of Invention

In order to solve the technical problems, the invention provides a peste des petits ruminants virus H protein antibody iELISA detection kit, which comprises an epitope peptide antigen, peste des petits ruminants standard negative serum, peste des petits ruminants standard positive serum, an enzyme label plate, an enzyme-labeled secondary antibody diluent, a coating solution, a substrate developing solution, a sealing solution, a serum diluent and a stop solution;

the preparation method of the epitope peptide antigen comprises the following steps:

b cell epitope fragments with good reactogenicity with PPRV specific antibodies are selected and then are connected in series to carry out polypeptide synthesis, and the synthesized polypeptide is used as a coating antigen.

Preferably, three bioinformatics software of IEDB, Immunodicine Group and Bepided are used for predicting B cell epitopes of H protein, 27 antigenic epitope peptides are obtained in total and are synthesized into polypeptide fragments, and then the reactogenicity of the polypeptide fragments and PPRV positive serum is identified by an immunological method, and the B cell epitope fragments H123, H138, H185, H362, H368, H487, H569 and H585 have antigen reactogenicity; the peptide segment is synthesized by selecting the fragments H123, H185, H362 and H487 with the best reactogenicity in series.

Preferably, the amount of the coating of the polyepitope antigen peptide is 1.5X 10^-6ug/well.

Preferably, the coating liquid is a carbonate buffer.

Preferably, the blocking solution is a 2% BSA-PBST solution.

Preferably, the standard negative and positive serum and the serum diluent to be detected are PBST solution, and the critical dilution concentration is 1: 100.

Preferably, the enzyme-labeled secondary antibody diluent is a PBST solution, and the dilution of the enzyme-labeled secondary antibody is 1: 80000.

Preferably, the determination standard of the detection result is the average value OD of two wells of the positive control₄₅₀Not less than 0.5, average OD of negative control two wells₄₅₀<The detection result is true when the serum sample OD is 0.25₄₅₀Value of<0.25 is negative, OD₄₅₀The value is more than or equal to 0.30 as positive, and the OD is more than or equal to 0.25₄₅₀<0.30 is suspect.

A Peste des petits ruminants virus H protein antibody iELISA detection method comprises diluting synthesized epitope peptide with coating solution, coating 96-well ELISA plate, and incubating at 4 deg.C overnight; washing the PBST plate for 3-5 times, spin-drying, adding confining liquid, and incubating in an incubator at 37 deg.C for 1 h; washing the PBST for 3-5 times, spin-drying, adding 1:100 diluted standard negative and positive serum and serum sample to be detected, and incubating in 37 deg.C incubator for 1 h; washing the PBST for 3-5 times, spin-drying, adding diluted enzyme-labeled secondary antibody, and incubating in a 37 ℃ incubator for 1 h; washing the PBST for 3-5 times, spin-drying, adding substrate TMB (100 ul per well), and incubating in an incubator at 37 deg.C for 15 min; taking out the enzyme label plate, adding 100ul of stop solution 2M H into each hole₂SO₄Terminating the reaction; enzyme-linked immunosorbent assay (OD) reading₄₅₀An absorbance value;

the preparation method of the epitope peptide antigen comprises the following steps: b cell epitope fragments with good reactogenicity with PPRV specific antibodies are selected and then are connected in series to carry out polypeptide synthesis, and the synthesized polypeptide is used as a coating antigen;

the coating amount of the multi-epitope antigen peptide is 1.5 multiplied by 10^-6ug/hole;

the coating solution is carbonate buffer solution;

the blocking solution is 2% BSA-PBST solution;

the standard negative and positive serum and the serum diluent to be detected are PBST solutions, and the critical dilution concentration is 1: 100;

the enzyme-labeled secondary antibody diluent is a PBST solution, and the dilution of the enzyme-labeled secondary antibody is 1: 80000;

and (3) judging standard: positive control two-well mean OD₄₅₀Not less than 0.5, average OD of negative control two wells₄₅₀<0.25, the detection result is true, otherwise, the detection result is not true; when the serum sample OD₄₅₀Value of<0.25 was judged negative, OD₄₅₀The value is more than or equal to 0.30, the positive is judged, and the OD is more than or equal to 0.25₄₅₀<0.30 is judged suspect.

The Peste des petits ruminants virus H protein antibody iELISA detection method is applied to Peste des petits ruminants virus H protein antibody detection.

The invention selects 4 antigen epitope peptides with better reactogenicity to be connected in series and then synthesized as coating antigen based on the B cell epitope of the H protein of the screened Peste Des Petits Ruminants Virus (PPRV), establishes an iELISA method aiming at the PPRV H protein antibody, and optimizes related conditions. After optimization, the optimal coating amount of the multi-epitope antigen peptide is 1.5 multiplied by 10^-6ug/hole, the critical dilution concentration of the negative and positive serums to be detected is 1:100, and the optimal dilution concentration of the enzyme-labeled secondary antibody is 1: 80000; various buffers were screened for the reaction: the coating solution is carbonate buffer solution, the serum and the secondary antibody diluent are PBST solution, and the blocking solution is 2% BSA solution; the positive-negative cutoff value was 0.25. Clinical serum detection shows that the variation coefficient of the detection results in batches and between batches is less than 10 percent; the positive serum detection of sheep pox, foot-and-mouth disease and bluetongue is negative, which shows that the method has good specificity; the comparison test of 306 sera with the cELISA kit produced by ID-Vet company shows that the coincidence rate of the two is 92.75%. The research proves that the established PPRV H protein antibody iELISA detection method can be used for detecting the clinical peste des petits ruminants antibody, and the operation is more time-saving and labor-saving than that of cELISA, and is simple, convenient and quick.

Drawings

The present invention is described in further detail below with reference to the attached drawings.

Fig. 1 is a clinical serum sample compliance rate detection graph.

Detailed Description

1 materials and methods

1.1 Virus strains, sera and major reagents

The PPRV Nigeria 75/1 vaccine strain is subcultured in the laboratory; PPRV negative and positive serum, foot-and-mouth disease positive serum, capripox positive serum and standard serum are prepared and stored in the laboratory, and the bluetongue disease positive serum is gifted by the doctor of the army administration of Lanzhou veterinary institute of Chinese academy of agricultural sciences. PPRV cELISA detection kit was purchased from French ID-Vet company; horseradish peroxidase-labeled rabbit anti-sheep secondary antibody was purchased from SIGMA (cat # 087M4838V), and TMB substrate solution was purchased from Sumodics (cat # TMBW-1000-01); BSA was purchased from Hewlett-packard (cat # NZ62780101), skim milk was purchased from BD (cat # 0211649), and casein Blocking Buffer was purchased from SIGMA (cat # B6429-500).

1.2 screening of B cell epitope of H protein and design and synthesis of polypeptide

The laboratory predicts the B cell epitope of the H protein by three bioinformatics software of IEDB, Immunodicine Group and Bepided to obtain 27 antigenic epitope peptides in total and synthesize the polypeptide fragments, and then identifies the reactogenicity of the antigenic epitope peptides and PPRV positive serum by an immunological method to find that the B cell epitope fragments H123, H138, H185, H362, H368, H487, H569 and H585 have the antigen reactogenicity (Table 1) (Liangloy, 2017). The fragments H123, H185, H362 and H487 with the best reactogenicity are selected to be connected in series (Table 2), and are sent to Shanghai Qianzhibio Limited to synthesize the peptide fragment.

TABLE 1B cell epitopes with reactogenicity

TABLE 2 tandem B cell epitope fragments

Note: GS (grey fraction) is a linking amino acid between different epitopes

1.3 establishment of PPRV H protein antibody iELISA detection method

1.3.1 iELISA method procedures

Diluting the synthesized epitope peptide with a coating solution, coating the epitope peptide on a 96-well enzyme label plate, and incubating at 4 ℃ overnight; taking out the enzyme label plate, washing the plate with PBST for 3-5 times, then spin-drying, adding the sealing liquid, and placing in a 37 ℃ incubator for incubation for 1 h; taking out the ELISA plate, washing the plate with PBST for 3-5 times, spin-drying, adding 1:100 diluted standard negative and positive serum and the serum sample to be detected, and incubating for 1h in a 37 ℃ incubator; taking out the enzyme-labeled plate, washing the plate with PBST for 3-5 times, spin-drying, adding the diluted enzyme-labeled secondary antibody, and incubating in a 37 ℃ incubator for 1 h; taking out the ELISA plate, washing the plate with PBST for 3-5 times, spin-drying, adding substrate TMB (100 ul per well), and incubating in a 37 ℃ incubator for 15 min; taking out the enzyme label plate, adding 100ul of stop solution 2M H into each hole₂SO₄Terminating the reaction; enzyme-linked immunosorbent assay (OD) reading₄₅₀An absorbance value.

1.3.2 optimization of optimal coating concentration of Multi-epitope peptide antigen

Epitope peptide antigen is expressed according to 3 x 10^-6ug/well, 2.5X 10^-6ug/hole, 2.0X 10^-6ug/well, 1.5X 10^-6ug/hole, 1.0X 10^-6ug/hole, 0.5X 10^-6ug/well 96-well microplate was coated with bicarbonate buffer at pH 9.6, 100ul per well, overnight at 4 ℃. Standard negative-positive serum of peste des petits ruminants was purified with sterile PBST solution at a ratio of 1:25, 1: 50. diluting at a ratio of 1:100 and 1:200, and adding 100ul of the diluted solution into a 96-well enzyme label plate. The assay was performed according to the iELISA protocol, the enzyme-labeled secondary antibody was diluted with sterile PBST solution at a ratio of 1:40000 according to the instructions, 100ul of TMB solution was added to the substrate, and 100ul of 2M sulfuric acid solution was added to the stop solution. Reading OD₄₅₀Selecting the combination pair with larger P/N valueThe antigen coating concentration and serum dilution are used as the optimal antigen coating concentration and serum dilution.

1.3.3 enzyme-labeled Secondary antibody dilution and optimization of dilution

And (3) keeping the substrate and the stop solution unchanged, diluting the enzyme-labeled secondary antibody according to the ratio of 1:40000 and 1:80000 on the basis of optimally determining the epitope peptide coating concentration and the serum dilution concentration, and diluting the enzyme-labeled secondary antibody diluent by using 5% of skimmed milk, 0.5% of BSA and sterilized PBST. And (4) performing an experiment according to an iELISA operation program, reading the experiment data by using an automatic microplate reader, and selecting the value with the maximum P/N value as the optimal dilution concentration of the enzyme-labeled secondary antibody.

1.3.4 screening of coating solution

On the basis of determination of epitope peptide Coating concentration, optimization of secondary antibody dilution concentration and completion of diluent screening, a PBS (pH 7.3) solution, a commercial Coating Buffer (pH 9.6) was diluted by 10-fold and used, and a carbonate Buffer (pH 9.6) was used as a Coating solution to coat a polypeptide antigen. And (3) performing tests according to an iELISA operation program, keeping the substrate and the stop solution unchanged, reading the numerical value by an automatic microplate reader, and selecting the combination with the maximum P/N value as the optimal coating solution.

1.3.5 screening of blocking solutions

Performing polypeptide antigen coating by using the optimal coating solution on the basis of determining the epitope peptide coating concentration, optimizing the secondary antibody dilution concentration and completing the diluent screening; casein diluted by 10 times, 5% skim milk and 2% BSA were used as blocking solutions; and (3) performing tests according to an iELISA operation program, keeping the substrate and the stop solution unchanged, reading the numerical value by an automatic microplate reader, and selecting the combination with the maximum P/N value as the optimal confining solution.

1.3.6 screening of serum dilutions

On the basis of finishing screening of epitope peptide coating concentration, secondary antibody dilution concentration and diluent, optimal coating solution and confining solution, 5% skim milk, 2% BSA and sterilized PBST are used as serum diluent; and (3) performing tests according to an iELISA operation program, keeping the substrate and the stop solution unchanged, reading the numerical value by an automatic microplate reader, and selecting the combination with the maximum P/N value as the optimal serum diluent.

1.3.7 determination of the criteria of the iELISA detection method

After optimization of various conditions, 237 parts of peste des petits ruminants negative serum is detected according to optimal conditions of various steps. Under the condition that the average value of two positive control wells is more than or equal to 0.5 and the average value of two negative control wells is less than or equal to 0.25, calculating the OD of all negative sera₄₅₀Mean value of

And the standard deviation S; to be provided with

Is judged to be negative to

The result is judged to be suspicious,

the result was positive.

2 results and analysis

2.1 Multi-epitope peptide antigen tandem and chemical Synthesis results

The fragments H123, H185, H362 and H487 with the best reactogenicity after screening are selected to be connected in series (Table 2), and then the fragments are sent to Shanghai QiaoYao biology Limited company to be chemically synthesized, the total amount is 10mg,1 mg/tube is subpackaged, and the final synthetic purity is 90.57%.

2.2 optimization and determination of optimal coating concentration and serum dilution of epitope peptide antigen

And (3) coating the epitope peptide antigens with different concentrations on a 96-well enzyme label plate, performing a plurality of matrix titration tests according to a conventional ELISA operation method, and diluting the positive serum and the negative serum according to a ratio of 1:25, a ratio of 1:50, a ratio of 1:100 and a ratio of 1:200 respectively. The results are shown in Table 3. The coating amount of different polypeptide antigens is 1.5 multiplied by 10^-6The enzyme label plate is coated by the concentration of ug/hole, and the P/N value is highest when the serum is diluted according to the ratio of 1: 100. Therefore, it can be 1.5 × 10^-6ug/well was used as the optimal coating concentration and 1:100 was used as the optimal serum dilution.

TABLE 3 optimization of optimal antigen coating concentration and serum dilution

Table 3 Optimization for the concentration of coating antigen and serum dilution

2.3 enzyme-labeled Secondary antibody dilution and determination of dilution

At 1.5X 10^-6The concentration of ug/well was coated on an ELISA plate, serum was diluted at 1:100, and horseradish peroxidase-labeled rabbit anti-sheep secondary antibody was diluted at 1:40000 and 1: 80000. The experiment was performed according to the iELISA protocol, as shown in Table 4, the P/N value was higher when diluted at 1:80000, so 1:80000 was the optimal dilution for the secondary antibody; the assay was performed according to the elisa protocol using PBST solution, 5% Milk and 0.5% BSA as secondary antibody dilutions, respectively. As shown in Table 5, the secondary antibody was used as the optimal diluent because the P/N value was high when PBST was used as the diluent.

TABLE 4 determination of dilution concentration of enzyme-labeled Secondary antibody

Table4 Determination of secondary antibody concentration

TABLE 5 determination of enzyme-labeled secondary antibody dilution

Table 5 Determination of secondary antibody dilution buffer

2.4 determination of coating solution

Polypeptide antigens were coated with three solutions, i.e., a PBS (pH 7.3) solution, a Coating Buffer solution and a carbonate Buffer solution, as Coating solutions, and the iELISA test was performed under the same conditions. As shown in Table 6, since the P/N value was the largest when the carbonate buffer was used as the coating solution, the carbonate buffer was selected as the most suitable coating solution for the method.

TABLE 6 determination of coating solution

Table 6 Determination of coated buffer

2.5 determination of confining liquid

The coated and spin-dried polypeptide antigen was blocked with three solutions, Casin (10-fold diluted), 5% mik-PBST and 2% BSA-PBST, respectively, and subjected to the iELISA test under otherwise unchanged conditions. As shown in Table 7, the P/N value was the largest when blocking was performed with 2% BSA-PBST, so 2% BSA-PBST was selected as the most suitable blocking solution for the present method.

TABLE 7 determination of confining liquids

Table 7 Determination of blocking buffer

2.6 determination of serum dilutions

The negative and positive serum was diluted with PBST solution, 5% mil-PBST solution and 2% BSA-PBST solution as serum dilutions, and the iELISA test was performed under the same conditions. As shown in Table 8, the P/N value was the largest when PBST solution was used as the serum diluent, and therefore PBST solution was selected as the most suitable serum diluent for the method.

TABLE 8 determination of serum dilutions

Table 8 Determination of serum dilution buffer

2.7 determination of the threshold value

The ID-Vet PPRV c-ELISA detection kit detects 237 serum samples, the result is negative serum, and the 237 serum and the negative and positive control serum are mixed according to the ratio of 1:100 was diluted with PBST buffer, duplicate wells were tested for each sample and control, a blank control was set, and the assay was performed according to the optimized iELISA methodThe results of the line tests are shown in Table 9. Positive control two-well mean OD for each assay₄₅₀Not less than 0.5, average OD of negative control two wells₄₅₀<0.25, the detection result is true, otherwise, the detection result is not true. Performing statistical treatment on the obtained detection data, and calculating the OD of the detected negative serum₄₅₀Has an average value of

Standard deviation S is 0.0459,

to be provided with

Judging the result to be negative;

judging the sample to be suspicious;

the result was positive. The criteria for determination of serum samples in this study were therefore: OD₄₅₀Value of<0.25 is negative, OD₄₅₀The value is more than or equal to 0.30 is positive, and the OD is more than or equal to 0.25₄₅₀<0.30 is suspect.

TABLE 9 result of PPRV negative serum detection

Table9 The Detection Results of PPRV negative serums

Example 1

The invention discloses a Peste des petits ruminants virus H protein antibody iELISA detection method, which comprises an epitope peptide antigen, Peste des petits ruminants standard negative serum, Peste des petits ruminants standard positive serum, an enzyme label plate, an enzyme-labeled secondary antibody diluent, a coating solution, a substrate developing solution, a sealing solution, a serum diluent and a stop solution; b cell epitope of H protein is predicted by three bioinformatics software of IEDB, Immunodicine Group and Bepided, 27 pieces of epitope peptide are obtained in total and are synthesized into polypeptide fragments, and then the reactogenicity of the polypeptide fragments and PPRV positive serum is identified by an immunological method, and the B cell epitope fragments H123, H138, H185, H362, H368, H487, H569 and H585 have antigen reactogenicity; the peptide segment is synthesized by selecting the fragments H123, H185, H362 and H487 with the best reactogenicity in series.

The coating amount of the multi-epitope antigen peptide is 1.5 multiplied by 10^-6ug/well.

The coating solution is carbonate buffer solution.

The blocking solution is 2% BSA-PBST solution.

The standard negative and positive serum and the serum diluent to be detected are PBST solutions, and the critical dilution concentration is 1: 100.

The enzyme-labeled secondary antibody diluent is a PBST solution, and the dilution of the enzyme-labeled secondary antibody is 1: 80000.

The substrate color development liquid is TMB.

The stop solution is 2M H₂SO₄And (3) solution.

A Peste des petits ruminants virus H protein antibody iELISA detection method comprises diluting synthesized epitope peptide with coating solution, coating 96-well ELISA plate, and incubating at 4 deg.C overnight; washing the PBST plate for 3-5 times, spin-drying, adding confining liquid, and incubating in an incubator at 37 deg.C for 1 h; washing the PBST for 3-5 times, spin-drying, adding 1:100 diluted standard negative and positive serum and serum sample to be detected, and incubating in 37 deg.C incubator for 1 h; washing the PBST for 3-5 times, spin-drying, adding diluted enzyme-labeled secondary antibody, and incubating in a 37 ℃ incubator for 1 h; washing the PBST for 3-5 times, spin-drying, adding substrate TMB (100 ul per well), and incubating in an incubator at 37 deg.C for 15 min; taking out the enzyme label plate, adding 100ul of stop solution 2M into each hole H₂SO₄Terminating the reaction; enzyme-linked immunosorbent assay (OD) reading₄₅₀An absorbance value;

the coating solution is carbonate buffer solution;

the blocking solution is 2% BSA-PBST solution;

and (3) judging standard: positive control two-well mean OD₄₅₀Not less than 0.5, average OD of negative control two wells₄₅₀<0.25, the detection result is true, otherwise, the detection result is not true; serum sample OD₄₅₀Value of<0.25 was judged negative, OD₄₅₀The value is more than or equal to 0.30, the positive is judged, and the OD is more than or equal to 0.25₄₅₀<0.30 is judged suspect.

1 materials and methods

1.1 identification of reproducibility and specificity

1.1.1 repeatability test determination

Randomly selecting 4 serum samples with different PPRV antibody levels, using the enzyme label plates coated in the same batch, and parallelly repeating each serum sample for 5 times to detect the repeatability in the batch; sera at 2 different antibody levels were tested using 5 different lot coated elisa plates to test for lot-to-lot reproducibility. And calculating the average value, the standard deviation and the variation coefficient after the detection is finished.

1.1.2 specificity test assay

And (3) respectively detecting 3 parts of PPRV negative and positive sample serum, 3 parts of capripox positive sample serum, 3 parts of bluetongue positive sample serum and 3 parts of foot-and-mouth disease positive sample serum according to the optimized iELISA operation program, and judging the specificity of the method.

1.2 clinical serum sample compliance Rate detection

306 clinically collected sheep serum samples are simultaneously detected by the established iELISA method and PPRV c-ELISA kit produced by French ID-Vet company respectively, and the coincidence rate of the detection results of the 2 methods is counted.

2 results

2.1 repeatability analysis

The in-batch repeatability tests are shown in table 10, and the results show that the detected variation coefficients in 5 multiple wells of 4 sera are 8.3% at the maximum and 3.47% at the minimum, which are all less than 10%, indicating that the method has good in-batch repeatability; the repeatability results of different batches are shown in table 11, and the variation coefficients of 2 sera tested by coating antigen of different batches are respectively 4.06% and 6.12%, and are both less than 10%, which indicates that the method has good repeatability among different batches.

TABLE 10 results of the in-batch repeatability experiments

Table 10 Results of batch repeatability experiment

TABLE 11 results of the batch to batch repeatability experiments

Table 11 Results of different batch repeatability experiment

2.2 specific assay

And 3 parts of PPRV negative positive sample serum, 3 parts of foot-and-mouth disease positive sample serum, 3 parts of capripox positive sample serum and 3 parts of bluetongue positive sample serum are detected by the established iELISA. The results are shown in table 12, the PPRV positive sera detected by the ELISA method were all positive, and the PPRV negative sera, foot-and-mouth disease positive sera, capripox positive sera, and bluetongue positive sera detected were all negative, indicating that the established ELISA detection method had no cross-reaction with the foot-and-mouth disease, capripox, and bluetongue positive sera, and had good specificity.

TABLE 12 specific assays

Table12 Specificity detection

2.3 clinical serum sample compliance Rate detection

The established iELISA method and the ID-Vet PPRV cELISA detection kit are used for simultaneously detecting 306 clinically collected sheep serum, and the cELISA detects positive serum to be 69 parts and negative serum to be 237 parts; the iELISA detects 64 positive copies and 234 negative copies. Compared with the cELISA, the sensitivity is 92.6%, the specificity is 90.3%, the coincidence rate is 92.75%, and the clinical serum sample coincidence rate detection is shown in FIG. 1.

Peste des petits ruminants is an acute and virulent national animal infectious disease, and no effective treatment method (vicuna agriculture et al, 2007) is available at present, which increases the difficulty for effective prevention and control of the disease, so that establishment of a rapid and accurate diagnosis and detection method is particularly important for prevention and control of the disease. ELISA is widely applied to detection of PPRV antigen and antibody due to the characteristics of rapidness, sensitivity and the like.

When the multiple antigen epitopes are synthesized in series, flexible amino acids are inserted into the joints for connection, which is favorable for promoting the hydrolysis efficiency of protease and improving the presentation efficiency of the antigen epitopes. Studies have shown that the use of "GPGPG" as a ligation site between two epitopes can avoid the formation of new epitope fragments (Livingston et al, 2002); however, the use of "AAY" as a point of attachment between epitopes can improve the efficiency of epitope cleavage to some extent (Velders et al, 2001). Glycine and serine are used as amino acids connected in series among multi-epitope antigen fragments, and since glycine is the most flexible amino acid among all amino acids, flexible amino acids are inserted among all antigen epitopes to connect the antigen epitopes, so that a new antigen epitope sequence can be prevented from being formed at a joint, and different antigen epitopes can be ensured to be fully exposed, therefore, glycine and serine sequences are added at the joint of each B cell antigen epitope for spacing.

In the research of PPRV detection methods, N and H protein-based diagnostic detection methods are also widely applied to large-scale PPRV serum detection. The epitope has the advantages of easy acquisition, relatively reasonable chemical synthesis price, high purity and the like, and is widely adopted in the development of vaccines, and the epitope vaccine has the advantages of safety, high efficiency, stability, good immune effect, no toxicity, no variation and the like. The multiple antigen epitopes are used for chemical synthesis after being connected in series, and the multiple antigen epitopes are used as coating antigens to establish an iELISA detection method for virus protein antibodies, so that the using amount of the antigens can be reduced, and the preparation cost of the kit in commercialization can be effectively reduced. The determination of the iELISA detection method is a key step of an experiment, and the accuracy of the detection result is directly reflected on the determination of the result. The qualitative determination of the iELISA method is to determine whether the color is developed or not and whether the color is dark or light, while the competitive ELISA method is usually to determine whether the color is not developed or positive. When the P/N ratio is adopted for result judgment, the negative control is influenced by factors such as nonspecific adsorption, temperature and the like, so that the negative value is easily low, and the P/N value is large, so that false positive is caused; on the contrary, when the negative value is too high, false negative is easily caused (gossypium barbadense, 2005). Samples were used in this study

The result is negative, and the result is judged to be negative,

the result is judged to be suspicious,

the method for judging positive is used as a judgment standard, the average value of the negative serum detected by the method is counted on the basis of detecting 237 negative sera with clear backgrounds, the average value is used as a reference for judging the test result, and false negative in the judgment of the result can be effectively avoidedThere are too many sexual or false positives, which are also the criteria used when most elisa methods are set up.

ELISA detection methods based on PPRV-specific monoclonal antibodies have been widely applied to serological detection, epidemiological investigation and clinical diagnosis of PPR. Competitive ELISA and blocking ELISA detection methods based on PPRV H protein neutralizing monoclonal antibodies have also been established (Libeau et al, 1992; Singh et al, 2004), and ELISA detection methods established using viral H protein monoclonal antibodies can effectively improve the accuracy of test results. Research shows that the coincidence rate of an iELISA detection method established by using PPRV H protein expressed by a prokaryotic expression system as an antigen reaches 93.75 percent (Koenchigfang, 2016) compared with a foreign c-ELISA kit. The 454-472 amino acid sequence of the PPRV N protein has very strong immunogenicity aiming at PPRV, the 452-472 amino acid region is a linear B cell epitope of the PPRV, the PPRV can be specifically identified by a polypeptide antibody generated by the region, and a detection method established by utilizing the advantage can be used for differential diagnosis of the PPRV and the RPV; the ELISA detection method established by using the N protein as the antigen needs to rely on the mutual competition between the monoclonal antibody generated by the specific epitope of the protein and the corresponding antibody in the detected sample, and only 45% of the competition level can be observed in the negative control due to the lack of cross reaction with the N protein (Libeau et al, 1995). Corresponding studies have been carried out to map the functional domain of PPRV H protein, and the immunodominant epitopes at amino acid positions 263-368 and 539-609 are the most (Seth and Shaila,2001), and since the H protein is the most diverse among morbillivirus members, it is quite advantageous to design a DIVA assay method using the H protein as a target.

Based on the screened PPRV H protein B cell epitope, 4 sections of epitope with better reactogenicity after identification are synthesized in series to serve as coating antigen to establish an iELISA detection method aiming at H protein antibody. Compared with a PPRV c-ELISA detection kit established abroad, the coincidence rate is 92.75 percent; the established iELISA method is used for detecting positive serums of sheep pox, foot-and-mouth disease and bluetongue, and no cross reaction is found, which shows that the established detection method has better sensitivity and specificity, can be used for clinical serum detection and has the possibility of developing related detection kits.

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Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A peste des petits ruminants virus H protein antibody iELISA detection method is characterized in that:

(1) comprises epitope peptide coating antigen, peste des petits ruminants standard negative serum, peste des petits ruminants standard positive serum, an enzyme label plate, enzyme-labeled secondary antibody diluent, coating liquid, substrate developing liquid, confining liquid, serum diluent and stop solution;

the preparation method of the epitope peptide coating antigen comprises the following steps:

predicting B cell epitope of H protein by three bioinformatics software of IEDB, Immunodicine Group and Bepided, screening and identifying by an immunological method, identifying peptide segments H123, H185, H362 and H487 with best reactionogenicity, and then connecting the peptide segments in series by taking GS as a joint, wherein the sequence after the series connection is as follows: KFLNPDREYDFRDLRGSGTGCLGRTVTRAGSEANWVVPSTDVRDLGSIRGPRGRCH, synthesizing the polypeptide epitope peptide fragment as a coating antigen;

the coating amount of the epitope peptide coating antigen is 1.5 multiplied by 10^-6Microgram per hole;

the coating solution is carbonate buffer solution;

the blocking solution is 2% BSA-PBST solution;

the standard negative serum of peste des petits ruminants is prepared by separating whole blood of healthy sheep which is negative by antibodies of peste des petits ruminants;

the standard positive serum of the peste des petits ruminants is obtained by immunizing healthy sheep with the peste des petits ruminants attenuated vaccine for more than 2 times, collecting whole blood after the titer reaches more than 1:200 through antibody detection, and separating and preparing;

the standard negative and positive serum and the serum diluent to be detected are PBST solution, and the critical dilution concentration of the serum is 1: 100;

(2) the Peste des petits ruminants virus H protein antibody iELISA detection method comprises the following steps: diluting the synthesized epitope peptide coating antigen to 1.5 × 10 with coating solution^-5Coating a 96-well enzyme label plate according to 100 mu L/well after mu g/mL, and incubating overnight at 4 ℃; taking out the enzyme label plate, washing the plate with PBST for 3-5 times, then spin-drying, adding the sealing liquid, and placing in a 37 ℃ incubator for incubation for 1 h; taking out the ELISA plate, washing the plate with PBST for 3-5 times, spin-drying, adding 1:100 diluted standard negative and positive serum and the serum sample to be detected, and incubating for 1h in a 37 ℃ incubator; taking out the enzyme-labeled plate, washing the plate with PBST for 3-5 times, spin-drying, adding the diluted enzyme-labeled secondary antibody, and incubating in a 37 ℃ incubator for 1 h; taking out the enzyme label plate, washing the plate with PBSTSpin-drying after 3-5 times, adding a substrate TMB (Tetramethylbenzidine), placing 100 microliters of substrate TMB in each hole, and incubating for 15min in an incubator at 37 ℃; taking out the enzyme label plate, and adding 100 mu l of stop solution 2M H into each well₂SO₄Terminating the reaction; enzyme-linked immunosorbent assay (OD) reading₄₅₀An absorbance value; judging the detection result according to the judgment standard; and (3) judging standard: positive control two-well mean OD₄₅₀The value is more than or equal to 0.5, and the average OD of two negative control wells₄₅₀Value of<0.25, the detection result is true, otherwise, the detection result is not true; when the serum sample OD₄₅₀Value of<0.25 was judged negative, OD₄₅₀The value is more than or equal to 0.30, the positive is judged, and the OD is more than or equal to 0.25₄₅₀Value of<0.30 is judged suspect.

2. The use of the Peste des petits ruminants virus H protein antibody iELISA detection method of claim 1 in Peste des petits ruminants virus H protein antibody detection.