An indirect ELISA kit for detecting Salmonella enterica subsp. enterica serovar Indiana antibodies and its application

Abstract

The present invention discloses an indirect ELISA kit for specifically detecting Salmonella enterica serovar Indiana antibodies and its application. The kit includes an antigen-coated enzyme-linked immunosorbent assay (ELISA) plate, enzyme-labeled antibody, negative serum, positive serum, concentrated washing solution, chromogenic solution, and reaction termination solution. Compared with the traditional slide agglutination antigen detection method, the kit of the present invention not only greatly improves the detection throughput, but also has good sensitivity, specificity, and repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of serological detection, and relates to an indirect ELISA kit for detecting Salmonella Indiana antibodies and its application. Background Art

[0002] Salmonella Indiana was first isolated in 1955 from a girl in Indiana, USA, suffering from vomiting, diarrhea and fever. Subsequently, this pathogen has caused multiple outbreaks of human and mammalian infections in North America, Europe and other places. In China, there were few reports on Salmonella Indiana in the early years, but in recent years, it has a relatively high prevalence in different sources including humans, animals, food and the environment, and has become the second most prevalent serotype after Salmonella enteritidis, surpassing several other common serotypes (such as Salmonella typhimurium, Salmonella derby, Salmonella agona, etc.). Many studies have shown that Salmonella Indiana is closely related to symptoms such as vomiting, diarrhea, fever, gastroenteritis and local infections in humans and livestock. At present, the widespread prevalence of Salmonella Indiana in China has attracted high attention in the fields of livestock and poultry breeding, food safety, public health, etc.

[0003] Currently reported detection methods for Salmonella Indiana are all etiological detection techniques and do not involve serological detection techniques. Enzyme-linked immunosorbent assay (ELISA) is an enzyme-labeled solid-phase immunoassay technique. Its basic principle is to bind an antigen (or antibody) to a solid-phase carrier and link the antigen (or antibody) with a certain enzyme to form an enzyme-labeled antigen (or antibody). During detection, the sample to be tested and the enzyme-labeled antigen (or antibody) are reacted with the antigen (or antibody) on the solid-phase carrier according to a certain procedure, and then the unreacted part is removed by washing. After adding the substrate, the substrate is catalyzed by the enzyme bound to the solid-phase carrier to produce a colored substance. By qualitatively or quantitatively detecting the amount of the colored product, the content of the substance to be detected in the sample can be determined. It has high sensitivity, strong specificity, a large number of samples can be detected at one time, and the results can be directly read, which is convenient and fast, and is one of the hotspots in current serological detection. Through a large number of studies, the present invention has successfully screened out specific antigens for detecting Salmonella Indiana antibodies, and through exploration and optimization of reaction conditions, an indirect ELISA detection method for Salmonella Indiana with high sensitivity, strong specificity and good repeatability has finally been established.

[0004] Through literature retrieval of the existing technology, it has been found that there is no report on the indirect ELISA kit for detecting Salmonella Indiana antibodies and its application of the present invention. Summary of the Invention

[0005] In view of the current lack of specific methods for detecting Salmonella indiana antibodies, the present invention provides an indirect ELISA detection kit for Salmonella indiana with high sensitivity, strong specificity and good repeatability, and its application.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides an indirect ELISA kit for specifically detecting Salmonella indiana antibodies and its application. The indirect ELISA kit for Salmonella indiana includes an antigen-coated enzyme-linked immunosorbent assay (ELISA) plate, an enzyme-labeled antibody, a negative serum, a positive serum, a concentrated washing solution, a chromogenic solution and a reaction termination solution.

[0008] Further, the preparation method of the antigen-coated ELISA plate is as follows: Using the genomic DNA of Salmonella indiana strain ATCC51959 as a template, a pair of specific primers P1 / P2 are designed for PCR amplification;

[0009] P1: 5’-CTCGAGATGAGAGTGTCGTTA-3’

[0010] P2: 5’-GGATCCCTAATTATTAGCCAG-3’

[0011] After the PCR amplification product is recovered, it is double-digested with Xho I and BamH I, and the digested fragment is inserted between the Xho I and BamH I restriction sites of plasmid pET-15b to construct a prokaryotic expression plasmid;

[0012] The constructed prokaryotic expression plasmid is transformed into BL21(DE3) to obtain a recombinant expression bacterium, and IPTG is used to induce protein expression;

[0013] The bacteria are centrifuged to collect the precipitate, the precipitate is redissolved in PBS, the bacteria are lysed by sonication, and the supernatant is collected. His-tag column affinity chromatography is used for purification, and finally renaturation is carried out using a density gradient to obtain a recombinant protein as the coated antigen.

[0014] Further, the preparation method of the antigen-coated ELISA plate is as follows: Using the ELISA plate as a solid-phase carrier, by the checkerboard titration method, the recombinant protein prepared above is diluted to 2 μg / mL with a coating solution, added to each well of the ELISA plate, and incubated overnight at 4 °C for coating; after washing with the washing solution, a blocking solution is added and blocked at 37 °C, and finally washed again with the washing solution to obtain an ELISA plate coated with the recombinant protein antigen.

[0015] Further, the coating solution is 0.05 mol / L carbonate buffer solution with a pH of 9.6; the blocking solution is 5% skim milk powder.

[0016] Further, the enzyme-labeled antibody is a goat anti-chicken IgG secondary antibody labeled with HRP; the negative serum and positive serum need to be diluted 1:50 when used; the washing solution is a PBST solution with a pH of 7.4; the reaction termination solution is 2 mol / L H 2 SO 4 solution.

[0017] Further, a method for detecting antibodies using the indirect ELISA kit for Salmonella enterica serovar Indiana includes the following steps:

[0018] S1: Collect chicken blood samples and separate the test serum by static centrifugation;

[0019] S2: Dilute the test serum 1:50, set negative and positive serum controls, add to the antigen-coated enzyme-linked immunosorbent assay (ELISA) plate, 100 μl / well, and incubate at 37 °C for 60 min;

[0020] S3: Wash 5 times with the washing solution, add a 1:1000 dilution of the goat anti-chicken IgG secondary antibody labeled with HRP, 100 μl / well, and incubate at 37 °C for 60 min;

[0021] S4: Wash 5 times with the washing solution, add the TMB chromogenic solution, 100 μl / well, and incubate at 37 °C for 15 min;

[0022] S5: Add 2 mol / L H 2 SO 4 solution as the termination solution, 100 μl / well, and read the OD 450 value.

[0023] Furthermore, the result judgment criterion is as follows: If the OD 450 value of the serum sample is greater than 0.298, it is judged as positive; if the OD 450 value is less than 0.259, it is judged as negative; when 0.259 < OD 450 value < 0.298, it is judged as suspicious.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention screens a specific detection reaction antigen for Salmonella enterica serovar Indiana through a large number of experiments, obtains the coated antigen after protein expression and purification, and establishes an indirect ELISA antibody detection kit for Salmonella enterica serovar Indiana with high specificity and good repeatability through exploration and optimization of reaction conditions. Each antigen-coated plate can detect 96 samples at a time, greatly improving the detection throughput and detection efficiency compared with the traditional slide agglutination antigen detection method. Detailed implementation manners

[0026] The present invention will be further described below in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the relevant art may use the disclosed technical content to make equivalent changes into equivalent embodiments. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0027] Example 1 Preparation of Coated Antigen

[0028] 1.1 Amplification of Target Gene

[0029] According to the gene sequence of Salmonella enterica serovar Indiana A7P63_13855 (GenBank: CP015724.1), a pair of cloning primers P1 / P2 for the A7P63_13855 gene was designed. The restriction enzyme cleavage sites of Xho I and BamH I were respectively contained at the 5'-ends of the primers. The primers were designed using Primer Premier 5.0 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are as follows:

[0030] P1: 5'-CTCGAGATGAGAGTGTCGTTA-3'

[0031] P2: 5'-GGATCCCTAATTATTAGCCAG-3'

[0032] Using the genomic DNA of Salmonella enterica serovar Indiana ATCC51959 strain as a template, pre-denaturation was carried out at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s; extension at 72°C for 25 s; a total of 30 cycles, and finally extension at 72°C for 10 min, obtaining a target gene band with a size of 390 bp.

[0033] 1.2 Construction of Prokaryotic Expression Plasmid

[0034] After subjecting the PCR product to agarose gel electrophoresis, the target band was cut under ultraviolet light, and the target fragment in the gel was recovered using a DNA rapid purification kit. The recovered PCR product was double-digested with Xho I and BamH I, and the plasmid pET-15b was extracted and double-digested. Then the digested products were ligated with T4 DNA ligase. The ligation product was transformed into DH5α Escherichia coli competent cells, and the successful construction of the prokaryotic expression plasmid was confirmed by PCR identification and double-digestion identification.

[0035] 1.3 Induced Expression of Target Protein

[0036] Take 1 μL of the recombinant pET-15b-A7P63_13855 vector and transform it into BL21(DE3). After heat shock at 42°C for 90 s, let it stand on ice for 2 min and then spread it on an LB solid plate containing ampicillin at a final concentration of 50 μg / mL. Incubate at 37°C overnight.

[0037] Pick a single colony of the expression bacteria containing the recombinant plasmid BL21(DE3) into 10 mL of LB medium (ampicillin concentration 50 μg / mL) and culture it overnight at 37°C with shaking at 220 rpm. Inoculate the overnight culture into 10 mL of LB medium at a ratio of 1:100, add ampicillin at a final concentration of 50 μg / mL, and culture at 37°C with shaking at 220 rpm. When the OD value reaches 0.6, add IPTG at a final concentration of 0.5 mM, and induce overnight at 20°C with shaking at 220 rpm and induce for 4 h at 37°C with shaking at 220 rpm. The culture without adding IPTG inducer is used as a negative control. Centrifuge at 4000 rpm for 10 min to collect the bacteria, discard the supernatant, suspend the bacteria in 500 μL of PBS (pH 7.4) buffer, sonicate for 6 min (sonication for 0.5 s and pause for 1.5 s), centrifuge to collect the supernatant and precipitate respectively, dissolve the precipitate in 500 μL of inclusion body lysis solution (8 M Urea, 50 mM Tris-HCl, 300 mM NaCl, pH 8.0), take 40 μL of the sample and mix it with 10 μL of 5× protein loading buffer, and boil in a water bath for 10 min. Perform SDS-PAGE detection to determine the optimal induction conditions and the expression form of the protein.

[0038] After determining the optimal induction conditions, induce the expression of the fusion protein in large quantities. Inoculate the cultured bacterial solution into 3 L of LB liquid medium at a ratio of 1:100, add ampicillin at a final concentration of 50 μg / mL, and culture at 37°C with shaking at 220 rpm. When the OD value reaches 0.6, add IPTG at a final concentration of 0.5 mM and induce overnight at 20°C with shaking at 220 rpm, and then centrifuge to collect the bacterial cells.

[0039] 1.4 Purification and refolding of the target protein

[0040] Dissolve the collected bacteria in lysis Buffer (50 mM Tris, 150 mM NaCl, 7 mM Gua-HCl, 0.1% TritonX-100, pH 8.0), sonicate the bacteria in an ice bath, with a power of 400 W for 20 min (sonication for 2 s and pause for 6 s as a cycle). After sonication, centrifuge at 12000 rpm at 4°C for 20 min, and collect the supernatant for the next purification step.

[0041] Take 5 mL of Ni-NTA and wash and equilibrate the column with 5 column volumes of Binding buffer at a flow rate of 5 mL / min. Add the sample to be filtered to the Ni column at a flow rate of 2 mL / min and collect the flow-through. Wash the column with 5 column volumes of Binding buffer at a flow rate of 5 mL / min. Wash away impurities with Washbuffer at a flow rate of 5 mL / min and collect the eluate. Finally, elute with Elutionbuffer at a flow rate of 2 mL / min and collect the eluate.

[0042] The protein with confirmed complete purification needs to be further renatured. Dialyze the 6th fraction with the best purity into: 2 M urea, 50 mM Tris, 300 mM NaCl, pH 8.0. After 12 h, change the buffer to: 1×PBS, 2 mM DTT, pH 8.5 and continue dialysis for 8 h. After ultrafiltration and concentration, filter through a 0.45 μm membrane, aliquot 1 mL / tube and store at -80 °C. The amino acid sequence of the protein is: MRVSLLLIYFIPFALLADDPCLSIQYSEAVFLCSKKTFEDSDAKLNETYRTLLSTIRKKYNSQPNSGAEFVE KIKLSQRAWINFRDANCTVFSFIIDEESQAYDTSMYSCKNDMTLKRTKELETILANN.

[0043] Establishment of the indirect ELISA detection method in Example 2

[0044] 2.1 Determination of the optimal coating concentration of the antigen and the optimal dilution of the serum

[0045] Using the square matrix titration method, the solution in the dialysis bag collected last in 1.4 was serially diluted 2-fold with the coating solution (0.05 mol / L carbonate buffer at pH 9.6), and the final concentrations were 0.5, 1, 2, 4, 8, 16 μg / mL respectively. 100 uL was added to each well, and two rows were repeated for each dilution. It was placed at 4 °C for overnight coating; then it was washed 5 times with the washing solution (PBST solution at pH 7.4), 3 min each time; 200 μL of the blocking solution (5% skim milk powder) was added to each well and blocked at 37 °C for 2 h; it was washed 5 times with the washing solution, 3 min each time; the negative and positive sera of Salmonella enterica serovar Indiana were serially diluted 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 with the blocking solution, and 100 μL was taken and added to each well respectively, and incubated at 37 °C for 1 h; it was washed 5 times with the washing solution, 3 min each time; the HRP-labeled goat anti-chicken IgG secondary antibody diluted with the blocking solution (diluted 1:1000) was added to each well, 100 μL per well, and incubated at 37 °C for 1 h; it was washed 5 times with the washing solution, 3 min each time; finally, 100 μL of the TMB chromogenic solution was added to each well, and it was developed color at 37 °C in the dark for 15 min, and 50 uL of the reaction termination solution (2 mol / L H 2 SO 4 solution) was added, and the absorbance value (OD 450 ) at a wavelength of 450 nm was read with an enzyme-linked immunosorbent assay reader. Select the antigen concentration and serum dilution when the OD 450 value of the positive serum well is about 1.0, the OD 450 value of the negative serum well is about 0.1, and the P / N value is the largest as the optimal antigen coating concentration and serum dilution.

[0046] When the recombinant protein antigen coating concentration is 2 μg / mL and the serum dilution is 1:50, the OD 450 value of the positive serum is close to 1, the OD 450 value of the negative serum is close to 0.1, and the P / N value is the largest. Therefore, the optimal antigen coating concentration is determined to be 2 μg / mL, and the optimal serum dilution is 1:50.

[0047] 2.2 Determination of the blocking solution

[0048] Coat the enzyme-linked immunosorbent assay plate according to the optimal antigen concentration, overnight at 4 °C and after washing, add 5 groups of different blocking solutions respectively: Group 1 is 1% BSA, Group 2 is 2% BSA, Group 3 is 1% FBS, Group 4 is 5% skim milk powder, and Group 5 is 2% gelatin. After blocking, react with the optimal serum dilution, wash and then add the HRP-labeled goat anti-chicken IgG secondary antibody, and finally add TMB to develop color and add the termination solution. Read the OD 450 value, compare the readings and P / N values of each group, and select the blocking solution with the largest P / N value as the optimal blocking solution.

[0049] After washing, the ELISA plate blocked with 5% skim milk powder had no granular precipitate and had the largest P / N value. Therefore, 5% skim milk powder was used as the optimal blocking solution.

[0050] 2.3 Determination of the optimal reaction time of serum

[0051] Coat the ELISA plate with the optimal antigen concentration, incubate overnight at 4°C and wash. Then add the optimal blocking solution and block for 2 h. Add the negative and positive sera diluted with the optimal dilution factor respectively, and divide them into 4 groups according to different incubation times at 37°C: Group 1 for 15 min, Group 2 for 30 min, Group 3 for 45 min, and Group 4 for 60 min. After washing, add the secondary antibody of HRP-labeled goat anti-chicken IgG, and finally add TMB for color development and then add the stop solution. Read the OD450nm, compare the readings and P / N values of each group, and select the serum reaction time when the P / N value is the largest as the optimal serum reaction time.

[0052] When the primary antibody serum acts at 37°C for 60 min, the P / N value is the highest. Therefore, the optimal action time of the serum to be detected is determined to be 60 min.

[0053] 2.4 Determination of the reaction concentration and time of the enzyme-labeled secondary antibody

[0054] Coat the ELISA plate with the optimal antigen concentration, incubate overnight at 4°C and wash. Then add the optimal blocking solution and block for 2 h. Add the negative and positive sera diluted with the optimal dilution factor respectively and act for the optimal reaction time. Divide them into 4 groups according to different dilution factors of the secondary antibody of HRP-labeled goat anti-chicken IgG: Group 1 for 1:5000, Group 2 for 1:3000, Group 3 for 1:2000, and Group 4 for 1:1000. Keep other conditions unchanged, read and compare the OD450nm values and P / N values of the negative and positive sera in each group, and select the dilution factor when the P / N value is the largest as the optimal reaction concentration of the enzyme-labeled secondary antibody.

[0055] Coat the ELISA plate with the optimal antigen concentration, incubate overnight at 4°C and wash. Then add the optimal blocking solution and block for 2 h. Add the negative and positive sera diluted with the optimal dilution factor respectively and act for the optimal reaction time. Divide them into 4 groups according to different reaction times of the secondary antibody of HRP-labeled goat anti-chicken IgG at 37°C: Group 1 for 15 min, Group 2 for 30 min, Group 3 for 45 min, and Group 4 for 60 min. Keep other conditions unchanged, read and compare the OD 450 values and P / N values of the negative and positive sera in each group, and select the reaction time when the P / N value is the largest as the optimal reaction time of the enzyme-labeled secondary antibody.

[0056] When the dilution of the goat anti - chicken secondary antibody labeled with HRP is 1:1000, its P / N value is the largest. Therefore, 1:1000 is determined as the optimal dilution of the secondary antibody. When the reaction time of the goat anti - chicken secondary antibody labeled with HRP is 60 min, the P / N value is the largest. Therefore, the optimal reaction time of the goat anti - chicken secondary antibody labeled with HRP is determined to be 60 min.

[0057] 2.5 Indirect ELISA operation procedure

[0058] Table 1 Indirect ELISA operation procedure

[0059]

[0060] Example 3 Determination of the positive and negative critical values of the indirect ELISA detection method

[0061] Using the indirect ELISA method established under various previously determined conditions, 20 chicken - sourced negative sera of Salmonella Indiana were detected. According to the obtained OD 450 values, the overall average value (X) and standard deviation (S) were calculated. According to statistical principles, when the OD 450 value ≥ X + 3S, it can be determined as positive; when the OD 450 value ≤ X + 2S, it can be determined as negative; when X + 2S < OD 450 value < X + 3S, the sample is determined to be suspicious.

[0062] According to the calculated average OD 450 value of 0.181 and standard deviation of 0.039. According to the test results, when the OD 450 value of the serum sample is greater than or equal to 0.298, it is determined as positive; when the OD 450 value is less than or equal to 0.259, it is determined as negative; when 0.259 < OD 450 value < 0.298, it is determined as suspicious.

[0063] Example 4 Specificity of the indirect ELISA detection method

[0064] Using the established method, positive sera of 3 common Salmonella serotypes such as Salmonella pullorum, Salmonella typhimurium, and Salmonella enteritidis, positive sera of 3 common pathogenic bacteria belonging to non - Salmonella genera such as Escherichia coli, Pasteurella multocida, and Haemophilus paragallinarum, and positive sera of 3 common viral diseases such as avian influenza H5 subtype, Newcastle disease, and infectious bursal disease of chickens (all the above sera are chicken - sourced) were detected respectively. To determine whether there is cross - reaction to test the specificity of the ELISA method. The results of all 9 sera were determined to be negative. While the sera prepared by the method of artificially infecting with Salmonella Indiana were all positive (a total of 12 were prepared). It can be seen from this that the indirect ELISA detection method for Salmonella Indiana established by the present invention has high specificity.

[0065] Note: The positive sera of Salmonella Indiana, Salmonella typhimurium, Salmonella enteritidis, Escherichia coli, Pasteurella multocida, and Haemophilus paragallinarum used in this experiment were all prepared by ourselves; the positive sera of Salmonella pullorum, avian influenza H5 subtype, Newcastle disease, and infectious bursal disease of chickens were all purchased from Beijing Zhonghai Biotechnology Co., Ltd.

[0066] Example 5 Repeatability of the indirect ELISA detection method

[0067] 5.1 Intra-batch repeatability test

[0068] The same 5 randomly selected serum samples were continuously detected for 5 days using the established ELISA method on the plates coated with the same batch of antigen for intra-batch repeatability test. The coefficient of variation of the detection results was between 2.45% and 5.21%.

[0069] 5.2 Inter-batch repeatability test

[0070] Another 5 randomly selected serum samples were detected using the established ELISA method under the same conditions on the plates coated with different batches for inter-batch repeatability test. The coefficient of variation of the detection results was between 3.04% and 7.56%.

[0071] Example 6 Assembly of the indirect ELISA detection kit

[0072] Table 2 Composition of the indirect ELISA detection kit

[0073]

Claims

1. Use of an indirect ELISA kit for detecting Salmonella indiana antibodies in the preparation of a reagent for detecting Salmonella indiana, said indirect ELISA kit for detecting Salmonella indiana antibodies comprises: a coated antigen ELISA plate, an enzyme-labeled antibody, negative serum, positive serum, concentrated washing solution, chromogenic solution and reaction termination solution, wherein the antigen ELISA plate is loaded with a recombinant coated antigen protein, and the amino acid sequence of the recombinant coated antigen protein is: MRVSLLLIYFIPFALLADDPCLSIQYSEAVFLCSKKTFEDSDAKLNETYRTLLSTIRKKYNSQPNSGAEFVEKIKLSQRAWINFRDANCTVFSFIIDEESQAYDTSMYSCKNDMTLKRTKELETILANN.

2. The use according to claim 1, said use comprising the following steps: S1: Collect chicken blood samples, and use the static centrifugation method to separate the test serum; S2: Dilute the test serum 1:50, set negative and positive serum controls, add it into the antigen ELISA plate, 100 ul / well, and incubate at 37 °C for 60 min; S3: Wash 5 times with the washing solution, add HRP-labeled goat anti-chicken IgG secondary antibody diluted 1:1000, 100 ul / well, and incubate at 37 °C for 60 min; S4: Wash 5 times with the washing solution, add TMB chromogenic solution, 100 ul / well, and incubate at 37 °C for 15 min; S5: Add 2 mol / L of H 2 SO 4 solution terminator, 100 ul / well, and read the OD 450 value.

3. The use according to claim 2, characterized in that, The result judgment criteria are as follows: For the serum sample, if the OD 450 value is greater than 0.298, it is judged as positive; if the OD 450 value is less than 0.259, it is judged as negative; when 0.259 < OD 450 value < 0.298, it is judged as suspicious.

4. The preparation method of the recombinant coated antigen protein in the use according to claim 1 comprises the following steps: (1) Using the genomic DNA of Salmonella indiana ATCC51959 strain as a template, design a pair of specific primers P1 / P2 for PCR amplification; P1: 5’-CTCGAGATGAGAGTGTCGTTA-3’ P2: 5’-GGATCCCTAATTATTAGCCAG-3’ (2) After recovering the PCR amplification product, perform double digestion with Xho I and BamH I, and insert the digested fragment between the Xho I and BamH I digestion sites of plasmid pET-15b to construct a prokaryotic expression plasmid; (3) Transform the constructed prokaryotic expression plasmid into BL21(DE3) to obtain a recombinant expression bacterium, and induce protein expression with IPTG; (4) Centrifuge the bacteria to collect the precipitate, dissolve the precipitate in PBS, collect the supernatant after ultrasonic disruption of the bacteria, purify it by affinity chromatography using a His-tag column, and finally perform renaturation using a density gradient to obtain a recombinant protein as the coated antigen.

5. The use according to claim 1, characterized in that, The preparation method of the coated antigen ELISA plate comprises the following steps: Use the ELISA plate as a solid-phase carrier, adopt the square matrix titration method, dilute the recombinant protein prepared above to 2 μg / mL with the coating solution, add it to each well of the ELISA plate, and incubate at 4 °C overnight; After washing with the washing solution, add the blocking solution and block at 37 °C, and finally wash again with the washing solution to obtain an ELISA plate coated with the recombinant antigen.

6. The indirect ELISA kit for detecting Salmonella enterica serovar Indiana antibodies according to claim 1, characterized in that: The enzyme-labeled antibody is a goat anti-chicken IgG secondary antibody labeled with HRP; the negative serum and positive serum are diluted 1:50 when used; the washing solution is a PBST solution with a pH of 7.4; the reaction termination solution is 2 mol / L H 2 SO 4 solution.

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