A kind of indirect ELISA detection kit and detection method of Mycoplasma synoviae

Abstract

The invention discloses an indirect ELISA detection kit and a detection method for Mycoplasma synoviae. An indirect ELISA detection kit and a detection method for Mycoplasma synoviae are constructed using the Mycoplasma synoviae antigen protein MS-eLPD of the amino acid sequence shown in SEQ ID NO.1 as a coating antigen, which is used to detect the antibody level of Mycoplasma synoviae, has no cross reaction with Mycoplasma gallisepticum positive serum, chicken infectious bronchitis positive serum, chicken infectious bursal disease positive serum, avian leukosis positive serum, avian reticuloendotheliosis positive serum, and chicken infectious rhinotracheitis positive serum, has good specificity and repeatability, and provides an effective means for the clinical diagnosis, epidemiological research and immune detection of Mycoplasma synoviae.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an indirect ELISA detection kit and a detection method for Mycoplasma synoviae. Background Art

[0002] Mycoplasma synoviae (MS) is a common extracellular pathogen that can cause chronic respiratory disease, eggshell tip abnormalities, infectious synovitis, and arthritis in poultry. Infection in chickens can cause severe inflammatory responses and evade immune recognition, causing serious economic losses to the global poultry industry.

[0003] Since Mycoplasma synoviae is mostly an invisible infection with subclinical symptoms, laboratory diagnosis of the disease is extremely important. At present, the detection methods of MS antibodies are mainly serological detection methods, including serum plate agglutination test (SPA), hemagglutination inhibition test (HI) and enzyme-linked immunosorbent assay (ELISA). SPA and HI have low sensitivity and are difficult to be applied to the detection of large quantities of clinical samples. ELISA is a sensitive, simple and rapid detection method with high specificity and sensitivity, and can detect a large number of antibody samples at the same time. The commercialized MS antibody detection kits on the market now come from BioChek and IDEXX, both of which are imported kits and are expensive. Therefore, it is of great significance to develop a more economical MS antibody ELISA detection kit to replace imported products for the monitoring of MS infection in my country.

[0004] Previous studies have found that the key to the successful attachment and colonization of MS in the host epithelium is that its membrane contains abundant lipid-associated membrane proteins (LAMPs), which have strong immunogenicity. In this study, referring to the synoviae WVU1853 strain in GenBank, the structure and function of DLD, LP78, LDH, and ENO proteins were comprehensively analyzed through a variety of bioinformatics tools, and peptides with better parameters were selected, appropriately optimized, synthesized, and prokaryotic expression was performed. The target protein (MS-eLPD) was induced and purified by IPTG, and its reactivity was verified by Western blot and ELISA. Using the purified MS-eLPD protein as the coating antigen, an indirect ELISA detection method for MS antibodies was established. This provides an opportunity for the combined application of multiple proteins and the development of a new method with high sensitivity for detecting MS antibodies. Summary of the invention

[0005] The purpose of the present invention is to provide an indirect ELISA detection kit and a detection method for Mycoplasma synoviae.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention firstly uses bioinformatics software and related websites to analyze the dominant linear epitopes of MS structural proteins, obtains the antigenic epitope sequence, analyzes the antigenic site situation by Protean software, and names it as MS-eLPD; then deduces the amino acid sequence of the multi-epitope peptide into a nucleotide sequence by genetic codons, selects the dominant codons of Escherichia coli as much as possible, sends the antigenic epitope peptide nucleotide sequence to a biological company for gene synthesis, and connects the target gene and plasmid by ligase; transforms the MS-eLPD recombinant prokaryotic expression plasmid into DH5α competent cells, and extracts the plasmid by using a plasmid extraction kit; takes the recombinant plasmid as a template, and uses EcoRΙ and Hind III. Perform double enzyme digestion and nucleic acid electrophoresis on the recombinant plasmid; send the recombinant plasmid identified as positive by enzyme digestion to the detection sequence, and name the correctly constructed recombinant prokaryotic expression plasmid pET-32a(+)-MS-eLPD; transform the recombinant plasmid pET-32a(+)-MS-eLPD into BL21, culture overnight and pick out single dispersed colonies to continue culturing in LB liquid culture medium, induce protein expression with IPTG, and purify to obtain chicken synoviae Mycoplasma antigen protein MS-eLPD. The amino acid sequence of chicken synoviae Mycoplasma antigen protein MS-eLPD is shown in SEQ ID NO.1, and the molecular weight is 54KDa.

[0008] The present invention further provides the use of the Mycoplasma synoviae antigen protein MS-eLPD in the preparation of an indirect ELISA detection kit for Mycoplasma synoviae.

[0009] The present invention further provides an indirect ELISA detection kit for Mycoplasma synoviae. The kit comprises an enzyme label plate with an amino acid sequence of Mycoplasma synoviae antigen protein MS-eLPD as shown in SEQ ID NO.1 as a coating antigen, and the kit further comprises a coating buffer, a blocking solution, a washing solution, Mycoplasma synoviae positive serum, Mycoplasma synoviae negative serum, an enzyme-labeled secondary antibody, a substrate color developing solution and a stop solution; the coating buffer is a carbonate buffer with a pH value of 7.4 and a concentration of 0.01 mol / L; the blocking solution is a mixture of a phosphate buffer with a concentration of 0.01 mol / L and a pH value of 7.4, Tween 20 and fetal bovine serum BSA, wherein the volume concentration of Tween 20 in the blocking solution is 0.1%, and the mass concentration of fetal bovine serum BSA is 5%; the washing solution is a mixture of a phosphate buffer with a concentration of 0.01 mol / L and a pH value of 7.4 and Tween 20, wherein the Tween 20 in the washing solution is 0.1%. The volume concentration of 20 is 0.1%; the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-chicken secondary antibody; the substrate color developing solution is TMB substrate color developing solution; and the stop solution is a H2SO4 solution with a concentration of 2 mol / L.

[0010] The present invention also provides a method for using the above-mentioned Mycoplasma synoviae indirect ELISA detection kit, which is as follows:

[0011] (1) Antigen coating of ELISA plate: Dilute the chicken synoviae Mycoplasma antigen protein MS-eLPD to 0.5 μg / ml with coating buffer, add 100 μl / well to the ELISA plate, incubate overnight at 4°C, wash the plate 3 times with washing buffer, each time for 5 min, and pat dry;

[0012] (2) Blocking of ELISA plate: Add 200 μl of blocking solution to each well of the coated ELISA plate and block at 37°C for 2 h. Wash the plate three times with washing solution for 5 min each time and pat dry.

[0013] (3) Add diluted serum: add 100 μl / well of the serum to be tested diluted 1:100 with washing solution, and use Mycoplasma gallisioides positive serum and Mycoplasma gallisioides negative serum diluted 1:100 with washing solution as positive and negative controls, respectively. Incubate at 37°C for 30 min. Wash the plate 3 times with washing solution for 5 min each time, and pat dry.

[0014] (4) Add diluted enzyme-labeled secondary antibody: add 100 μl of enzyme-labeled secondary antibody diluted 1:2000 in washing solution to each well, incubate at 37°C for 60 min, wash the plate three times with washing solution, 5 min each time, and pat dry;

[0015] (5) Add substrate for color development: add 100 μl of TMB substrate color development solution to each well and develop color at 37°C in the dark for 5 min;

[0016] (6) Add stop solution to complete the assay: Add 50 μl of stop solution to each well and measure OD on a microplate reader within 15 min. 450 Value; serum OD to be tested 450 A value equal to or higher than 0.2785 was considered positive, and a value lower than 0.2785 was considered negative. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Western blot detection of MS-eLPD recombinant protein. DETAILED DESCRIPTION

[0018] The specific implementation methods of the present invention are described below in conjunction with the accompanying drawings. For the specific methods or materials used in the examples, those skilled in the art can make conventional replacement choices based on the technical ideas of the present invention and according to existing technologies, and are not limited to the specific records of the embodiments of the present invention. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0019] In the present invention, the concentration of the carbonate buffer is 0.01 mol / L and the pH value is 7.4.

[0020] In the present invention, the PBST solution is prepared by mixing a phosphate buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 with Tween 20, and the volume concentration of Tween 20 in the PBST solution is 0.1%.

[0021] In the present invention, the BSA-PBST solution is composed of a phosphate buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4, Tween 20 and fetal bovine serum BSA. The volume concentration of Tween 20 in the BSA-PBST solution is 0.1%, and the mass concentration of fetal bovine serum BSA is 5%.

[0022] In the present invention, TMB colorimetric solution was purchased from Bio-Tech Biotechnology Co., Ltd., item number P0209.

[0023] In the present invention, the stop solution is a H2SO4 solution with a concentration of 2 mol / L.

[0024] Example 1

[0025] 1.1 Prediction and screening of B / T cell antigen epitopes of Mycoplasma synoviae

[0026] Antigenic epitope prediction was performed using the ABCpred website and the SYFPEITHI website. The parameters related to B cell epitopes (antigenicity, hydrophilicity, flexibility, etc.) of proteins such as DLD, LP78, VLHA, LDH, and ENO of Mycoplasma synoviae were analyzed and predicted using the DNAStar software Protean program. Log in to the IEBD website (http: / / tools.immuneepitope.org / main / ) and the SYFPEITHI database website (http: / / www.syfpeithi.de / bin / mhcserver.dll / ) and enter the amino acid sequences of potential dominant epitopes predicted by the above analysis. Evaluate the T cell sites with strong correlation (correlation>85%) in each fragment.

[0027]

[0028]

[0029] 1.2 Design and synthesis of MS-eLPD nucleotide sequence

[0030] The antigenic epitopes were arranged in series through 4 glycine residues (GGGG) from the selected antigenic epitopes to obtain the expected multi-epitope peptide sequence, and the antigenic site of the multi-epitope peptide was analyzed by Protean software, and the multi-epitope peptide was named MS-eLPD. The amino acid sequence (SEQ ID NO.1) of the multi-epitope peptide MS-eLPD was derived as a nucleotide sequence by genetic codons, and the dominant codon of Escherichia coli was selected as much as possible, and the tripeptide bursa was used as a carrier protein. At the same time, EcoRΙ and HindIII restriction sites were added at both ends of the sequence, respectively, to obtain a total of 1113 bases (SEQ ID NO.2). The designed multi-epitope peptide nucleotide sequence was sent to a biological company for gene synthesis.

[0031] 1.3 Construction and identification of MS-eLPD prokaryotic expression plasmid

[0032] The target gene shown in SEQ ID NO.2 and the pET-32a(+) plasmid were connected by T4 DNA ligase to obtain the recombinant plasmid pET-32a(+)-MS-eLPD. The ligation product was transformed into Escherichia coli (E.coli) competent cells DH5α, reacted on ice for 30 minutes; heat shock at 42°C for 90 seconds, placed on ice for 2 minutes after the heat shock, and then added 1mL of LB liquid culture medium without Amp, and cultured at 25°C for 45 minutes; 100μL of bacterial liquid was evenly spread on a solid LB culture medium plate containing 0.1wt% Amp, and placed upside down in a 37°C incubator for overnight culture. The next day, single colonies were randomly selected and cultured in LB liquid medium containing 0.1 wt% Amp. After 3 h, the bacterial solution was taken as a template and PCR reaction was performed using T7 universal primers (F: 5'-TAATACGACTCACTATAGGG-3', R: 5'-GCTAGTTATTGCTCAGCGG-3'), and agarose gel electrophoresis was performed.

[0033] 1.4 Prokaryotic expression and SDS-PAGE analysis of MS-eLPD

[0034] The correctly identified recombinant plasmid pET-32a(+)-MS-eLPD was transferred into competent cells E. coli BL21(DE3). A single colony was picked the next day and incubated at 37°C overnight. 200 μL of the bacterial solution was inoculated into 20 ml of liquid LB medium containing 0.1 wt% Amp and cultured until OD 600When the value reached 0.6-0.8, 20 μL 1.0 mmol / L IPTG was added and induced at 25°C for 2h, 4h, 6h, 8h, and 10h, and 1 ml of bacterial solution was collected for each time. At the same time, empty vector and recombinant bacteria without IPTG induction were set as negative controls. The cells were centrifuged at 12000 r / min for 1 min, and the supernatant and precipitate were taken for SDS-PAGE electrophoresis analysis to determine the relative expression levels of soluble protein and inclusion body of MS-eLPD in E. coli.

[0035] 1.5 Purification of MS-eLPD recombinant protein

[0036] The MS-eLPD recombinant protein was purified, subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and Western blot was performed using clinical chicken positive serum as the primary antibody. The results showed ( Figure 1 ), the MS-eLPD recombinant protein showed good reactivity with serum antibodies in the Western blot method.

[0037] Example 2

[0038] This example uses the Mycoplasma synoviae antigen protein MS-eLPD (SEQ ID NO. 2) obtained in Example 1 to establish an indirect ELISA detection kit for Mycoplasma synoviae, and describes the corresponding detection method.

[0039] 2.1 Determination of optimal reaction conditions

[0040] 2.1.1 Determination of the optimal antigen coating concentration and the optimal serum dilution multiple

[0041] Array titration method was used to take a 96-well ELISA plate. The chicken synoviae Mycoplasma antigen protein MS-eLPD was diluted with carbonate buffer to 8μg / ml, 4μg / ml, 2μg / ml, 1μg / ml, 0.5μg / ml and 0.25μg / ml, and 100μl / well was added to the ELISA plate, incubated at 4℃ overnight, washed 3 times with PBST solution the next day, each time for 5 minutes, and patted dry; 200μl was added to each well of the coated ELISA plate The cells were blocked with BSA-PBST solution at 37°C for 2 h, then washed three times with PBST solution for 5 min each time, and patted dry. 100 μl / well of Mycoplasma synoviae positive serum diluted with PBST solution at 1:25, 1:50, 1:100, 1:200, 1:400 and 1:800 was added as a positive control, and 100 μl / well of Mycoplasma synoviae positive serum diluted with PBST solution at 1:25, 1:50, 1:100, 1:200, 1:400 and 1:800 was added as a positive control. 100 μl / well of Mycoplasma synoviae negative serum was used as negative control, and the serum action condition was set at 37°C for 30 min, then washed 3 times with PBST solution, 5 min each time, and patted dry; the enzyme-labeled antibody (HRP-labeled rabbit anti-chicken secondary antibody) was diluted 1:2000 with PBST solution, added to the ELISA plate at 100 μl / well, and acted at 37°C for 1 h, then washed 3 times with PBST solution, 5 min each time, and patted dry; 100 μl of TMB substrate colorimetric solution was added to each well, and color was developed at 37°C in the dark for 5 min; 50 μl of stop solution was added to each well, and OD was measured on an ELISA reader within 15 min. 450 Value, select positive OD 450 Value and negative OD 450 The antigen coating concentration and serum dilution ratio when the ratio of the values ​​(P / N value) is the largest is taken as the optimal working concentration.

[0042] The results of the matrix titration method showed (Table 1) that the P / N value was the largest when the chicken synoviae Mycoplasma antigen protein MS-eLPD was coated at a concentration of 0.5 μg / ml and the serum was diluted at 1:100. Therefore, 0.5 μg / ml was determined to be the optimal antigen coating concentration and 1:100 was determined to be the optimal serum dilution multiple.

[0043] Table 1 ELISA test results of different protein coating concentrations and serum dilution ratios

[0044]

[0045]

[0046] 2.1.2 Determination of the action time of serum and enzyme-labeled antibodies

[0047] Using the square array titration method, take a 96-well ELISA plate. Dilute the chicken synoviae Mycoplasma antigen protein MS-eLPD to 0.5μg / ml with carbonate buffer, add 100μl / well to the ELISA plate, incubate at 4℃ overnight, wash with PBST solution 3 times, 5min each time, and pat dry the next day; add 200μl to each well of the coated ELISA plate BSA-PBST solution, blocked at 37℃ for 2h, then washed 3 times with PBST solution, 5min each time, and patted dry; 100μl / well of chicken synoviae positive serum diluted 1:100 with PBST solution was added as positive control, and 100μl / well of chicken synoviae negative serum diluted 1:100 with PBST solution was added as negative control, and the serum action condition was set to 37℃ for 30min and 60min, then washed 3 times with PBST solution, 5min each time, and patted dry; enzyme-labeled antibody (HRP-labeled rabbit anti-chicken secondary antibody) was diluted 1:2000 with PBST solution, added to the ELISA plate at 100μl / well, acted at 37℃ for 30min and 60min, then washed 3 times with PBST solution, 5min each time, and patted dry; 100μl TMB substrate colorimetric solution was added to each well, and color was developed at 37℃ in the dark for 5min; 50μl stop solution was added to each well, and OD was measured on the ELISA instrument within 15min 450 Value, select positive OD 450 Value and negative OD 450 The serum action time and enzyme-labeled antibody action time when the ratio of the values ​​(P / N value) is the largest is taken as the optimal working time.

[0048] The results of the matrix titration method showed (Table 2) that the P / N value was the largest when the serum action time was 30 minutes and the enzyme-labeled antibody action time was 60 minutes. Therefore, 30 minutes was determined to be the optimal serum action time, and 60 minutes was determined to be the optimal enzyme-labeled antibody action time.

[0049] Table 2 ELISA test results of serum and enzyme-labeled antibodies at different action times

[0050]

[0051]

[0052] 2.1.3 Determination of the optimal working concentration of enzyme-labeled secondary antibody

[0053] Using the square array titration method, take a 96-well ELISA plate. Dilute the chicken synoviae Mycoplasma antigen protein MS-eLPD to 0.5μg / ml with carbonate buffer, add 100μl / well to the ELISA plate, incubate at 4℃ overnight, wash with PBST solution 3 times, 5min each time, and pat dry the next day; add 200μl to each well of the coated ELISA plate BSA-PBST solution, blocked at 37℃ for 2h, then washed 3 times with PBST solution, 5min each time, and patted dry; 100μl / well of chicken synoviae positive serum diluted 1:100 with PBST solution was added as a positive control, and 100μl / well of chicken synoviae negative serum diluted 1:100 with PBST solution was added as a negative control. The serum action condition was set to 37℃ for 30min, then washed 3 times with PBST solution, 5min each time, and patted dry; enzyme-labeled antibody (HRP-labeled rabbit anti-chicken secondary antibody) was diluted 1:1000, 1:2000, 1:3000 and 1:5000 with PBST solution, added to the ELISA plate at 100μl / well, acted at 37℃ for 60min, then washed 3 times with PBST solution, 5min each time, and patted dry; 100μl was added to each well TMB substrate colorimetric solution was used for color development at 37°C in the dark for 5 min. 50 μl of stop solution was added to each well and the OD was measured on a microplate reader within 15 min. 450 Value, select positive OD 450 Value and negative OD 450 The enzyme-labeled antibody dilution ratio when the ratio of the values ​​(P / N value) is the largest is taken as the optimal working concentration.

[0054] The results of the matrix titration test showed (Table 3) that the P / N value was the largest when the serum was diluted 1:2000. Therefore, 1:2000 was determined to be the optimal enzyme-labeled antibody dilution ratio.

[0055] Table 3 ELISA test results of different dilution ratios of enzyme-labeled antibodies

[0056]

[0057]

[0058] 2.1.4 Determination of the critical value of yin and yang

[0059] According to the most suitable reaction conditions determined above, ELISA test was carried out to detect 30 negative sera of chicken synoviae Mycoplasma antibody. 450 The mean (X) and standard deviation (SD) of the values ​​were calculated, and the positive and negative critical value point C = X + 3SD = 0.2785 was determined, that is, the OD of the serum to be tested 450 A value equal to or higher than 0.2785 is considered positive, and a value lower than 0.2785 is considered negative.

[0060] Table 4 Determination of positive and negative critical values

[0061] Sample No. <![CDATA[OD 450 ]]> Sample No. <![CDATA[OD 450 ]]> Sample No. <![CDATA[OD 450 ]]> 1 0.088 11 0.101 21 0.097 2 0.132 12 0.234 22 0.105 3 0.080 13 0.113 23 0.133 4 0.137 14 0.175 24 0.210 5 0.070 15 0.093 25 0.148 6 0.183 16 0.136 26 0.075 7 0.112 17 0.148 27 0.087 8 0.145 18 0.064 28 0.124 9 0.136 19 0.262 29 0.217 10 0.132 20 0.082 30 0.136

[0062] 2.2 Specificity test

[0063] According to the most suitable reaction conditions determined above, ELISA test was carried out to detect Mycoplasma gallisepticum (MG) positive serum, infectious bronchitis (IBV) positive serum, infectious bursal disease (IBDV) positive serum, avian leukosis (ALV) positive serum, reticuloendotheliosis (REV) positive serum, infectious rhinotracheitis (ART) positive serum. Each serum sample was repeated 3 times, and the average value of the 3 times was compared with the positive and negative critical value.

[0064] The results showed (Table 5) that the OD of IBV, IBDV, ALV, REV and ART positive serum 450 The values ​​were all less than 0.2785 and were judged as negative, indicating that the established ELISA method had good specificity.

[0065] Table 5 Indirect ELISA specificity experimental results

[0066] Serum type MG IBV IBDV ALV REV ART Positive Control Negative control <![CDATA[OD 450 Mean]]> 0.228 0.203 0.175 0.184 0.098 0.169 1.6273 0.0877 Result determination — － － － － － + －

[0067] 2.3 Intra-batch and inter-batch replication tests

[0068] The chicken synoviae Mycoplasma antigen protein MS-eLPD purified from the same batch (within the batch) and the chicken synoviae Mycoplasma antigen protein MS-eLPD purified from different batches (between batches) were coated with ELISA enzyme labeling plates, and the ELISA test was carried out according to the most suitable reaction conditions determined above. Four chicken synoviae Mycoplasma positive and four chicken synoviae Mycoplasma negative sera were taken for ELISA detection, and each sample was repeated 3 times. According to the obtained OD 450 Inter-batch repeatability test: Randomly select one ELISA plate from different batches, take the same positive and negative serum for ELISA test, and calculate the coefficient of variation based on the test results: CV = (SD ÷ X) × 100%.

[0069] The results showed (Table 6) that the intra-batch coefficient of variation of negative serum (sample numbers 1-4) was 0.761%-3.406%, less than 10%; the inter-batch coefficient of variation was 1.295%-4.714%. The intra-batch coefficient of variation of positive serum (sample numbers 5-8) was

[0070] The coefficient of variation between batches was 1.786%-4.526% and 0.637%-5.081%, respectively. The coefficient of variation was less than 10%, indicating that the established antibody ELISA method had good repeatability and stability between batches.

[0071] Table 6 Intra-batch and inter-batch replication of the indirect ELISA method

[0072]

[0073] 2.4 Clinical sample test results

[0074] The ELISA test was performed according to the most suitable reaction conditions determined above, and 92 serum samples were tested simultaneously with the IDEXX test kit. The results showed a compliance rate of 91.33% (84 / 92), including a positive compliance rate of 82.6% (28 / 34) and a negative compliance rate of 96.25% (56 / 58).

Claims

1. An application of a Mycoplasma synoviae antigen protein MS-eLPD in the preparation of an indirect ELISA detection kit for Mycoplasma synoviae, characterized in that: The amino acid sequence of the chicken synoviae Mycoplasma antigen protein MS-eLPD is shown in SEQ ID NO.1, and the molecular weight is 54kD.

2. An indirect ELISA detection kit for Mycoplasma synoviae, characterized in that: The kit comprises an ELISA plate with the chicken synoviae Mycoplasma antigen protein MS-eLPD having an amino acid sequence as shown in SEQ ID NO.1 as a coating antigen.

3. The indirect ELISA detection kit for Mycoplasma synoviae according to claim 2, characterized in that: The kit also includes a coating buffer, a blocking solution, a washing solution, a positive serum of Mycoplasma synoviae, a negative serum of Mycoplasma synoviae, an enzyme-labeled secondary antibody, a substrate color developing solution and a stop solution.

4. The indirect ELISA detection kit for Mycoplasma synoviae according to claim 3, characterized in that: The coating buffer is a carbonate buffer with a pH value of 7.4 and a concentration of 0.01 mol / L; the blocking solution is a mixture of a phosphate buffer with a concentration of 0.01 mol / L and a pH value of 7.4, Tween 20 and fetal bovine serum BSA, wherein the volume concentration of Tween 20 in the blocking solution is 0.1%, and the mass concentration of fetal bovine serum BSA is 5%; the washing solution is a mixture of a phosphate buffer with a concentration of 0.01 mol / L and a pH value of 7.4 and Tween 20, wherein the volume concentration of Tween 20 in the washing solution is 0.1%; the enzyme-labeled secondary antibody is an HRP-labeled rabbit anti-chicken secondary antibody; the substrate color developing solution is a TMB substrate color developing solution; and the stop solution is a H2SO4 solution with a concentration of 2 mol / L.

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