CN110376388B

CN110376388B - Haemophilus parasuis antibody detection method and kit thereof

Info

Publication number: CN110376388B
Application number: CN201910756529.9A
Authority: CN
Inventors: 范红结; 张鹏云; 周红; 蔺辉星
Original assignee: Nanjing Changji Biotechnology Co ltd; Nanjing Agricultural University
Current assignee: Nanjing Changji Biotechnology Co ltd; Nanjing Agricultural University
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2021-10-19
Anticipated expiration: 2039-08-16
Also published as: CN110376388A

Abstract

The invention discloses a novel indirect ELISA detection method for a haemophilus parasuis antibody, relates to the field of antibody detection, and particularly relates to an indirect ELISA detection method for detecting the haemophilus parasuis antibody by using a recombinant truncated protein P2 and a kit thereof. The indirect ELISA detection method of the haemophilus parasuis expresses a recombinant haemophilus parasuis outer membrane protein P2 through truncation, takes the protein as a coating antigen, and optimizes ELISA reaction conditions, a detection mode and a critical value determination mode; the method can realize accurate detection of the haemophilus parasuis antibody, and has better specificity and sensitivity; the problem of overhigh background in the antibody detection process is effectively solved; and the detection cost is greatly reduced.

Description

Haemophilus parasuis antibody detection method and kit thereof

Technical Field

The present invention relates to the field of veterinary medicine. Specifically, the invention relates to an ELISA detection method for rapidly detecting Haemophilus Parasuis (HPS) antibodies and a detection kit thereof.

Background

Haemophilus Parasuis (HPS) can cause Haemophilus parasuis disease of pigs, the clinical symptoms of the disease are polyarthritis, serositis and meningitis, the disease is usually caused in the nursery stage of piglets, and the death rate can reach 50%. With the rapid expansion of the pig industry and the change of the pig raising mode, the epidemic trend of the disease becomes more serious in recent years, and huge economic loss is brought to the pig raising industry in China. The haemophilus parasuis serum antibody is detected, so that whether the pig is infected by the haemophilus parasuis serum antibody can be accurately diagnosed, and the immune protection effect of related vaccines can be evaluated.

The indirect ELISA detection method has the characteristics of high sensitivity, strong specificity and the like, and is the most main method for detecting serum antibodies at present. At present, the price of a commercialized kit for detecting haemophilus parasuis antibody produced abroad is extremely high, the average detection cost of each sample is close to 100 yuan, and the kit is forbidden for breeding enterprises. There is no commercial kit for detecting haemophilus parasuis antibodies in China. Meanwhile, the detection results of the studied haemophilus parasuis antibody detection methods reported in the literature are not ideal. Therefore, the method for exploring a novel antibody detection target and establishing the antibody detection method with high accuracy has important significance for the diagnosis of haemophilus parasuis and the evaluation of the immune effect of related vaccines.

The P2 protein is an outer membrane protein of haemophilus parasuis, exists in haemophilus parasuis of all serotypes, and has homology of more than 95 percent in genus and homology of less than 50 percent with other bacteria among the genera. Therefore, the outer membrane protein P2 is an ideal antigen for detecting haemophilus parasuis antibodies.

Based on the consideration, the inventor establishes an indirect ELISA detection method for haemophilus parasuis antibodies by truncating P2 protein expressing different fragment sizes as a coating antigen through a large number of experiments. Through specificity and sensitivity detection and optimization, the preferred P2 protein truncated fragment is screened out. The outer membrane protein P2 gene of haemophilus parasuis is taken as a target, the N end is truncated and amplified to 795bp, prokaryotic expression is carried out by using escherichia coli, and the protein is purified to obtain the recombinant P2 protein truncated protein. And on the basis, the indirect ELISA reaction conditions are optimized, and a Haemophilus parasuis antibody indirect ELISA detection kit is developed.

Disclosure of Invention

The invention aims to provide an indirect ELISA detection method for detecting a haemophilus parasuis antibody and a kit thereof, which can solve the current situation that no commercial haemophilus parasuis antibody detection kit exists in China at present, and can also solve the problem that the specificity of the current haemophilus parasuis antibody detection method is not ideal.

The specific technical scheme of the invention is as follows:

the kit adopts a truncated expressed and purified haemophilus parasuis outer membrane recombinant protein P2 as an enzyme label plate coated by a coating antigen, the nucleotide sequence of the truncated outer membrane recombinant protein P2 of haemophilus parasuis is shown as SEQ ID No.1, and the amino acid sequence of the truncated expressed P2 protein is shown as SEQ ID No. 2.

Further, the preparation method of the recombinant protein P2 comprises the following steps:

(1) a pair of specific primers P2F/P2R is designed by taking the genome DNA of the haemophilus parasuis serotype 5 strain as a template for amplification:

P2F：5’-CGGGATCCACAGTTTATGAAAATGAAG-3’(SEQ ID NO.3)；

P2R：5’-GCGTCGACTCTTGCGCCAGTTCTTACG-3’(SEQ ID NO.4)；

(2) recovering the PCR product, performing double enzyme digestion by using BamH I and Sal I, and inserting the enzyme digestion fragment between BamH I and Sal I enzyme digestion sites of the plasmid pET-28a to construct a prokaryotic expression plasmid pET-28 a-P2;

(3) converting the constructed prokaryotic expression plasmid pET-28a-P2 into BL21 expression bacteria to obtain recombinant expression bacteria, and inducing the recombinant expression bacteria to express a target protein by IPTG;

(4) centrifuging the bacterial liquid, collecting the precipitate, re-dissolving the precipitate in PBS, ultrasonically breaking the bacteria, centrifuging and taking the precipitate to obtain a recombinant protein P2 inclusion body;

(5) and purifying the obtained recombinant protein P2 inclusion body by adopting a His-tag affinity chromatography method to obtain recombinant protein P2 as a detection antigen.

Further, the recombinant protein P2 was coated at a concentration of 4. mu.g/mL, and the coating solution was carbonate buffer (pH 9.6).

Further, the kit also comprises a sealing liquid, wherein the sealing liquid is 2% of gelatin.

Further, the kit also comprises the serum to be detected, positive reference serum (namely, standard positive control serum), negative reference serum (namely, standard negative control serum), enzyme conjugate, washing solution, color development solution and stop solution.

Further, the sample diluent is PBST, the enzyme conjugate is goat anti-pig IgG marked by HRP, the washing solution is PBST, the color development solution is TMB color development solution, and the stop solution is 2mol/L sulfuric acid solution.

The second purpose of the invention is to provide a novel indirect ELISA detection method for haemophilus parasuis antibodies, which comprises the following steps:

(1) coating: diluting the recombinant protein P2 (containing antigen) with amino acid sequence as SEQ ID NO.2 to 4 μ g/mL by using carbonate buffer solution (pH9.6) as coating solution, and adding 100 μ L of diluted recombinant protein P2 into each hole of odd columns of the ELISA plate; meanwhile, 100 mu L of coating solution is added into each hole of even columns of the ELISA plate to serve as a blank control (not containing P2 protein, namely not containing antigen), and the mixture is coated overnight at 4 ℃, specifically for 12-16 h;

(2) and (3) sealing: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 mu L of PBST into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 250 mu L of 2% gelatin sealing solution into each hole, and sealing for 2h at 37 ℃;

(3) incubation of serum to be detected: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 μ L into each hole, standing for 5min, spin-drying the liquid in the holes after the last time,

respectively arranging a standard positive control hole (containing antigen), a standard positive control hole (containing no antigen), a standard negative control hole (containing no antigen), a to-be-detected serum hole (containing antigen) and a to-be-detected serum hole (containing no antigen),

the standard positive control wells are two groups of odd wells and even wells (in certain specific examples, 100 μ L of standard positive control serum diluted 100-fold with PBST is added to a1 (row 1, column 1), a2 (row 1, column 2), and B1 (row 2, column 1), B2 (row 2, column 2), respectively, the two groups of odd wells resulting in two groups of standard positive control wells (containing antigen) (i.e., a1, B1 in certain specific examples), and the two groups of even wells resulting in two groups of standard positive control wells (containing no antigen) (i.e., a2, B2 wells in certain specific examples);

the standard negative control wells were prepared by adding 100 μ L of standard negative control serum 100-fold diluted PBST to C1 (row 3, column 1), C2 (row 3, column 2), and D1 (row 4, column 1), D2 (row 4, column 2), respectively, to two groups of odd wells, in which two groups of standard negative control wells (containing antigen) (i.e., C1, D1 in certain embodiments) and two groups of even wells, in which two groups of standard negative control wells (containing no antigen) (i.e., C2, D2 in certain embodiments) were prepared;

the serum wells to be detected are obtained by adding 100 mu L of corresponding serum to be detected diluted 100 times by PBST into the other odd-numbered wells and even-numbered wells (in some specific embodiments, E1, E2, F1, F2, G1 and G2 … … wells) respectively to obtain the serum wells to be detected (containing antigens) and the serum wells to be detected (not containing antigens);

incubating the sample at 37 ℃ for 1 h;

(4) and (3) secondary antibody incubation: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 mu L of liquid into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 100 mu L of HRP-labeled goat anti-pig IgG diluted by 10000 times of PBST into each hole, and incubating for 1h at 37 ℃;

(5) color development: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 μ L into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 100 μ L of TMB color developing solution into each hole, developing for 15min at 37 ℃ in a dark place, adding 50 μ L of stop solution into each hole, respectively2mol/L sulfuric acid solution, and reading OD on a microplate reader within 15min₄₅₀A numerical value;

(6) and (4) calculating a result: the standard positive control detection values are respectively P [ OD₄₅₀(containing antigen)]And P [ OD ]₄₅₀(antigen-free)]The detected value of the serum to be detected is S [ OD ]₄₅₀(containing antigen)]And S [ OD ]₄₅₀(antigen-free)]，

The P [ OD ]₄₅₀(containing antigen)]OD of two standard positive control wells (containing antigen)₄₅₀Average value, (in some specific examples, A1 well OD₄₅₀And B1 well OD₄₅₀Average value of (d);

the P [ OD ]₄₅₀(antigen-free)]OD of two standard positive control wells (no antigen)₄₅₀Average value; (in certain specific embodiments, A2 pore OD₄₅₀And OD of B2 well₄₅₀Average value).

The S [ OD ]₄₅₀(containing antigen)]For each odd number of wells of serum to be tested₄₅₀(in certain embodiments, the OD of each of the E1, F1, G1 … … wells₄₅₀A value);

the S [ OD450 (antigen free)]For each even number of wells of serum to be tested₄₅₀(ii) a (in certain embodiments, the OD of each of the E2, F2, and G2 … … wells₄₅₀)。

By which the respective correction values are calculated as:

standard positive serocorrected value Δ P [ OD ═₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]

Correction value of serum to be detected as delta S [ OD ═ OD₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]

And then calculating the ratio R of each serum to be detected, which specifically comprises the following steps:

the sample is judged to be positive or negative by the magnitude of the ratio R.

And R is the ratio of the detection correction value of each serum to be detected to the correction value of the standard positive serum, wherein S represents the serum to be detected, and P represents the average value of the standard positive serum.

When the ratio R of the serum to be detected is more than or equal to 0.275, the detection result of the haemophilus parasuis antibody in the serum to be detected is judged to be positive; and when the ratio R of the serum to be detected is less than 0.275, judging the detection result of the haemophilus parasuis antibody in the serum to be detected as negative.

Further, the calculating of the result of the step (6) further includes calculating a ratio R of standard negative sera, specifically:

the standard negative control detection values are respectively N [ OD ]₄₅₀(containing antigen)]And N [ OD ]₄₅₀(antigen-free)]，

The N [ OD ]₄₅₀(containing antigen)]OD of two standard negative control wells (containing antigen)₄₅₀Average value, (in certain specific examples, C1 well OD₄₅₀And D1 pore OD₄₅₀Average value of (d);

the N [ OD ]₄₅₀(antigen-free)]OD of two standard negative control wells (no antigen)₄₅₀Average value, (in certain specific examples, C2 well OD₄₅₀And D2 pore OD₄₅₀Average value of (d);

by which the respective correction values are calculated as:

the standard negative correction value is Δ N [ OD ═ d₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]；

The standard positive correction value ═ Δ P [ OD ═₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]；

The ratio R of standard negative sera was then calculated, specifically:

the ratio R of the standard negative serum should be less than 0.275, otherwise the test results are unreliable and need to be redone.

Furthermore, all the standard negative serum, the standard positive serum and the serum to be detected are used as blank controls without coating antigen holes during detection.

Further, the coating solution in the step (1) is 0.05mol/L carbonate buffer solution (pH9.6), and the coating time is 12-16 h.

The invention has the beneficial effects that:

the detection method and the kit for detecting the haemophilus parasuis antibody can diagnose whether animals are infected by haemophilus parasuis or not, can also judge the immune protection effect of haemophilus parasuis vaccines, have higher specificity and repeatability, can reduce the occurrence rate of false positive to the maximum extent, and avoid the interference of other bacterial antigens of enterobacteriaceae on detection. Compared with the existing method for detecting the agglutination of the haemophilus parasuis antibody glass plate, which is used more in veterinary clinic, the method has higher sensitivity and accuracy, and greatly reduces the detection cost.

Drawings

FIG. 1 PCR amplification of the gene of interest P2.

M: DL2000 DNA marker; 1: negative control; 2,3: PCR amplification product of the target gene P2.

FIG. 2 prokaryotic expression of recombinant P2 protein.

M: protein Maker; 1: pET-28a no-load control; 2: expressing the target protein P2 pronucleus.

FIG. 3 purification of recombinant P2 protein.

M: a protein Marker; 1: purifying the recombinant P2.

FIG. 4ROC curves.

FIG. 5 is a schematic view of an ELISA plate used in the embodiment of the present invention.

Detailed Description

The following examples are provided for a better understanding of the present invention and in no way limit the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any patents, patent applications, and publications cited in this application are hereby incorporated by reference. Experimental procedures without specific conditions noted in the following examples, generally employing conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the methods suggested by the manufacturer.

Example 1: prokaryotic expression of Haemophilus parasuis truncated P2 protein

1.1 amplification of the Gene of interest P2

A pair of cloning primers P2F/P2R aiming at a P2 gene are designed according to a P2 gene sequence of a Haemophilus parasuis Nagasaki strain (GenBank: CP018034.1), the 5' ends of the primers respectively contain restriction sites of restriction enzymes BamH I and Sal I and protective bases, the primers are designed by using Primer Premier 5.0 software and synthesized by Nanjing Kingsry biology company, and the Primer sequences are as follows:

P2F：5’-CGGGATCCACAGTTTATGAAAATGAAG-3’(SEQ ID NO.3)；

P2R：5’-GCGTCGACTCTTGCGCCAGTTCTTACG-3’(SEQ ID NO.4)；

the haemophilus parasuis serotype 5 strain is purchased from commercial approaches such as the Chinese veterinary microbial strain preservation management center and the like by taking the genomic DNA of the haemophilus parasuis serotype 5 strain as a template. In the embodiment, the genome of a haemophilus parasuis clinical isolation strain HPS5-SQ (isolated from a clinically diseased pig in a laboratory) is used as a template, and is subjected to pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45s for 30 cycles; after a PCR cycle of extension at 72 ℃ for 10min, a desired gene band of 795bp was obtained (FIG. 1).

1.2 construction of prokaryotic expression plasmid

And (3) carrying out agarose gel electrophoresis on the PCR product, cutting a target band under an ultraviolet lamp, and recovering a target fragment in the gel by using a DNA rapid purification kit. And carrying out double enzyme digestion on the recovered PCR product by using BamH I and Sal I, extracting a plasmid pET-28a, and carrying out double enzyme digestion. And the products after enzyme digestion are connected by T4 DNA ligase. The ligation product is transformed into DH5 alpha colibacillus competent cells, and the success of constructing the prokaryotic expression plasmid pET-28a-P2 is determined through PCR identification and double restriction enzyme identification.

1.3 inducible expression of the protein of interest

The plasmid pET-28a-P2 is transformed into Escherichia coli BL21, and BL21 expressing bacteria containing the plasmid pET-28a-P2 are obtainedInoculating to LB + Kan solid culture medium, selecting single colony, inoculating to 2mL LB + Kan liquid culture medium, culturing at 37 deg.C overnight, inoculating overnight cultured strain 1:100 to 50mL LB + Kan liquid culture medium, and shake culturing at 37 deg.C to OD₆₀₀0.4-0.6, log phase; adding IPTG (final concentration 1mmol/L) to induce expression, and further culturing at 37 deg.C for 4 hr under shaking; after the end, respectively taking out 500 mu L of fresh bacterial liquid, placing the fresh bacterial liquid in a new 2.0mL EP tube, and centrifuging for 10min at 8000 r/min; the supernatant was removed, 100. mu.L of 1 XProtein loading buffer was added to resuspend the pellet, the pellet was flushed well with a gun, boiled in boiling water for 10min, run off instantaneously, and 10. mu.L of the pellet was finally taken and examined by SDS-PAGE (FIG. 2).

After confirming the strain expression protein, taking the residual IPTG-induced bacterial liquid, centrifuging at 8000r/min for 10min, discarding the supernatant, washing the bacterial liquid with PBS for 3 times, finally resuspending with 5mL PBS, and carrying out ultrasonic bacteria breaking under ice bath (200W, ultrasonic 4s stopping 8s, ultrasonic 40 min). Centrifuging at 12000r/min for 30min after ultrasonication, taking the supernatant, dissolving the precipitate with PBS buffer solution containing 8M urea, and taking the supernatant and the precipitate respectively to perform SDS-PAGE detection to determine the expression form of the protein.

1.4 purification of the protein of interest

After confirming that the recombinant protein P2 existed in the form of inclusion body, the pellet was washed twice with the inclusion body washing solution, and the pellet was dissolved overnight at 4 ℃ with the inclusion body Binding Buffer, and the supernatant was centrifuged and filtered with a 0.45 μm filter. Since the recombinant protein P2 has a polyhistidine (6 XHis) tag, Ni was used²⁺The column is followed by affinity chromatography purification of the recombinant protein. The Ni2+ column is firstly cleaned by ultrapure water with 10 times volume, then balanced by Binding Buffer with the same volume, and the processed protein sample is added into the balanced Ni²⁺In the column, the protein was eluted with the Binding Buffer followed by the Elution Buffer. The complete purification was confirmed by SDS-PAGE. The purified P2 protein was mixed together, 20% glycerol was added, and the mixture was mixed well, and the protein concentration was measured by BCA protein quantification kit and stored at-80 ℃ for further use (FIG. 3).

Example 2: optimization of reaction conditions of indirect ELISA (enzyme-linked immunosorbent assay) detection method for haemophilus parasuis antibody

2.1 determination of optimal coating concentration of antigen and optimal dilution of serum

Adopting a matrix titration method, diluting the purified P2 protein by 2 times with coating solution (0.05 mol/L carbonate buffer solution with pH9.6) to obtain final concentrations of 1, 2, 4, 8 and 16 mug/mL, adding 100 mug/mL into each well, repeating two rows for each dilution, adding a row of control without coating antigen, namely the final concentration of the antigen is 0, and placing at 4 ℃ for coating overnight; then washing with washing solution (PBST solution of pH 7.4) for 4 times, each time with 300 μ L per well, 3min, and drying; adding 250 μ L of blocking solution (2% gelatin) into each well, and blocking at 37 deg.C for 2 hr; washing by the same method; diluting Haemophilus parasuis negative serum and positive serum with PBST at a ratio of 1:50, 1:100, 1:200, and 1:400, respectively, adding 100 μ L into each well, and incubating at 37 deg.C for 1 h; washing by the same method; adding HRP-labeled goat anti-pig IgG secondary antibody (diluted by 1: 10000) diluted by PBST into each well, incubating at 37 ℃ for 1h, wherein each well is 100 mu L; washing by the same method as before; finally, 100. mu.L of TMB developing solution was added to each well, and the mixture was developed in the dark at 37 ℃ for 15min, and 50. mu.L of reaction terminator (2mol/L H) was added to each well₂SO₄Solution), absorbance value (OD) at 450nm wavelength was read with microplate reader₄₅₀)。

Calculating the difference between the correction values of the positive serum and the negative serum at the same dilution, drawing a line graph according to the difference, and selecting the positive serum measurement correction value delta P ═ P [ OD [ ]₄₅₀(With Ag)]-P[OD₄₅₀(Without Ag)]The value was about 1.0, and the correction for negative serum determination Δ N ═ N [ OD [ ]₄₅₀(With Ag)]-N[OD₄₅₀(Without Ag)]The last dilution at which the difference between the positive and negative correction values varies the most between two consecutive dilutions is the optimal dilution, determined as the optimal antigen coating concentration and serum dilution.

The requirements are met when the recombinant protein P2 antigen coating concentration is 4 mug/mL and the serum dilution is 1: 100. Therefore, the optimal antigen coating concentration is determined to be 4 mug/mL, and the optimal serum dilution is 1: 100.

2.2 determination of confining liquid

The elisa plate was coated according to optimal antigen concentration, overnight at 4 ℃ and washed, and 5 different sets of blocking solutions were added: group 1 is1% BSA, 2% BSA in

group

2, 1% FBS in group 3, 5% skim milk powder in

group

4, 2% gelatin in group 5. And (3) carrying out reaction by using the optimal serum dilution after blocking, adding a goat anti-pig IgG secondary antibody marked by HRP after washing, and finally adding TMB for color development and adding a stop solution. Reading OD_450nmThe correction values and the values of Δ P/Δ N of the respective groups are compared, and the most suitable blocking liquid is selected when the value of Δ P/Δ N is the maximum.

The ELISA plates after 2% gelatin blocking had the greatest P/N values after washing, so 2% gelatin was used as the optimal blocking solution.

2.3 determination of optimal reaction time of serum

Coating an enzyme label plate according to the optimal antigen concentration, coating overnight at 4 ℃, washing, adding an optimal sealing liquid, sealing for 2 hours, respectively adding negative serum and positive serum diluted by an optimal dilution factor, and dividing the mixture into 4 groups according to different incubation times at 37 ℃: 15min for group 1, 30min for group 2, 45min for group 3, and 60min for group 4. After washing, adding a goat anti-pig IgG secondary antibody marked by HRP, and finally adding TMB for color development and adding stop solution. Reading OD_450nmThe corrected values and the values of DELTA P/DELTA N of the respective groups are compared, and the optimal serum reaction time is selected as the serum reaction time at which the value of DELTA P/DELTA N is the maximum.

When the antiserum acts for 60min at 37 ℃, the P/N value is the highest, so that the optimal action time of the serum to be detected is determined to be 60 min.

2.4 determination of the reaction concentration and time of enzyme-labeled Secondary antibody

Coating an enzyme label plate according to the optimal antigen concentration, coating overnight at 4 ℃, washing, adding an optimal confining liquid, sealing for 2 hours, respectively adding negative serum and positive serum diluted by an optimal dilution factor for optimal reaction time, and dividing the goat anti-pig IgG secondary antibodies marked by HRP into 4 groups according to different dilution factors of the goat anti-pig IgG secondary antibodies marked by the HRP: group 1 is 1:2500, group 2 is 1:5000, group 3 is 1:10000, group 4 is 1: 20000. And reading and comparing the corrected value of the positive and negative serum of each group and the value of delta P/delta N while keeping other conditions unchanged, and selecting the dilution factor when the value of delta P/delta N is maximum as the optimal reaction concentration of the enzyme-labeled secondary antibody.

Coating an enzyme label plate according to the optimal antigen concentration, coating overnight at 4 ℃, washing, adding an optimal confining liquid, sealing for 2 hours, respectively adding negative serum and positive serum diluted by an optimal dilution factor for optimal reaction time, and dividing goat anti-chicken IgG secondary antibodies marked by HRP into 4 groups according to the different reaction time at 37 ℃: group 1 for 30min, group 2 for 45min, group 3 for 60min, and group 4 for 90 min. And reading and comparing the corrected value of the positive and negative serum of each group and the value of delta P/delta N while keeping other conditions unchanged, and selecting the reaction time when the value of delta P/delta N is maximum as the optimal reaction time of the enzyme-labeled secondary antibody.

When the dilution of the goat-anti-pig secondary antibody of the HRP standard antibody is 1:10000, the value of delta P/delta N is the largest, so that the optimal dilution of the secondary antibody is determined to be 1: 10000. The Δ P/Δ N value was maximized when the reaction time of the HRP-labeled goat-anti porcine secondary antibody was 60min, thus determining that the optimal reaction time of the HRP-labeled goat-anti porcine secondary antibody was 60 min.

2.5 determination of the reaction time of the substrate

Coating an enzyme label plate according to the optimal antigen concentration, coating overnight at 4 ℃, washing, adding an optimal confining liquid, sealing for 2 hours, respectively adding the optimal reaction time of the negative serum and the positive serum diluted by the optimal dilution times, performing experiments according to the determined optimal dilution times and reaction time of the secondary antibody, and dividing the reaction time after adding the TMB color development liquid into 5 groups: group 1 for 5min, group 2 for 10min, group 3 for 10min, group 4 for 20min, and group 5 for 30 min. And reading and comparing the corrected value of the positive and negative serum of each group and the value of delta P/delta N while keeping other conditions unchanged, and selecting the reaction time when the value of delta P/delta N is maximum as the optimal reaction time of the enzyme-labeled secondary antibody.

When the substrate reaction time is 15min, the Δ P/. DELTA.N value is maximized, and thus the substrate optimum reaction time is determined to be 15 min.

2.6 determination of the threshold value

45 portions of haemophilus parasuis immune positive serum and 45 portions of healthy pig serum, namely haemophilus parasuis negative serum, are selected, an established ELISA method is utilized to detect the ratio value, software Medcal is used to draw an ROC curve (receiver operation characteristic curve), the critical value is determined to be positive when the ratio R is larger than or equal to 0.275, and negative when the ratio R is smaller than 0.275. When the cut-off value was defined, the sensitivity of the detection method was 80% and the specificity was 86.67%, and the AUC value of the ROC curve was 0.923, indicating that the detection method was very effective. The ROC curve is shown in fig. 4.

2.7 according to all the optimization results, a working table of detecting the haemophilus parasuis antibody by the P2-ELISA method is drawn.

TABLE 1P 2-ELISA working Table

The following example has the following specific operating steps:

(1) coating: diluting the recombinant protein P2 (containing antigen) to 4 mu g/mL by using 0.05mol/L carbonate buffer solution (pH9.6) as a coating solution, and adding 100 mu L into each hole of the enzyme label plate in odd-numbered rows; meanwhile, 100. mu.L of coating solution is added to each well of the even-numbered rows of the ELISA plate to serve as a blank (containing no P2 protein, namely containing no antigen), and the blank is coated overnight at 4 ℃ (12-16 h).

(2) And (3) sealing: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 μ L of PBST into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 250 μ L of 2% gelatin sealing solution into each hole, and sealing for 2h at 37 ℃.

(3) Incubation of serum to be detected: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 muL into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 100 muL of standard positive control serum diluted by 100 times of PBST into the holes A1 and A2 and the holes B1 and B2, adding 100 muL of standard negative control serum diluted by 100 times of PBST into the holes C1, C2, D1 and D2, and adding 100 muL of standard negative control serum diluted by 100 times of PBST into the holes E1 and E2; f1, F2; corresponding 100. mu.L of serum to be tested diluted 100-fold with PBST was added to wells G1 and G2 … … and incubated at 37 ℃ for 1 h.

(4) And (3) secondary antibody incubation: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 mu L of liquid into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 100 mu L of HRP-labeled goat anti-pig IgG diluted by 10000 times of PBST into each hole, and incubating for 1h at 37 ℃.

(5) Color development: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 μ L into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 100 μ L of TMB color developing solution into each hole, developing in dark at 37 deg.C for 15min, adding 50 μ L of stop solution (2mol/L sulfuric acid) into each hole, and reading OD on the ELISA plate within 15min₄₅₀Numerical values.

(6) And (4) calculating a result:

the standard positive control detection value is P [ OD ]₄₅₀(containing antigen)](A1 pore OD₄₅₀And B1 well OD₄₅₀Average value of) and P [ OD₄₅₀(antigen-free)](A2 pore OD₄₅₀And B2 well OD₄₅₀Average of (d),

the detected value of the serum to be detected is S [ OD ]₄₅₀(containing antigen)](E1, F1, G1 … … well OD₄₅₀Value) and P [ OD₄₅₀(antigen-free)](E2, F2, G2 … … well OD₄₅₀A value),

by which the respective correction values are calculated as:

positive correction value ═ Δ P [ OD₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]

Correction value of serum to be tested (delta S [ OD ]₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]

In addition, the ratio R of standard negative sera has to be calculated to determine whether the test results are reliable, in particular:

The N [ OD ]₄₅₀(containing antigen)]Is C1 pore OD₄₅₀And D1 pore OD₄₅₀Average value of (d);

the N [ OD ]₄₅₀(antigen-free)]C2 pore OD₄₅₀And D2 pore OD₄₅₀Average value of (d);

by which the respective correction values are calculated as:

The ratio R of standard negative sera was then calculated:

Example 3: sensitivity test of haemophilus parasuis antibody indirect ELISA detection kit

A commercial haemophilus parasuis inactivated vaccine is used for immunizing 65 weaned piglets of 3 weeks, secondary immunization is carried out 21 days after the primary immunization, blood is collected from the anterior vena cava of all immunized piglets 14 days after the secondary immunization, serum is separated, and the haemophilus parasuis antibody detection is carried out by using the ELISA method established by the invention. Meanwhile, the separated serum is diluted by 20, 40, 80, 160, 320 and 640 times, and the ELISA method established by the invention is used for detecting the positive serum of the haemophilus parasuis with different dilutions so as to confirm the sensitivity of the detection kit.

The results show that: 63 of 65 positive serum results of haemophilus parasuis show positive results, the positive rate reaches 96.9%, and the results are consistent with the detection results of the commercial antibody detection kit. After the positive serum of the haemophilus parasuis is diluted by different times, the ELISA method established by the invention is used for antibody detection, and the positive serum is still positive when the dilution time is 320. The result shows that the haemophilus parasuis antibody ELISA detection kit established by the invention has good sensitivity.

Example 4: specificity test of haemophilus parasuis antibody indirect ELISA detection kit

Randomly detecting 40 healthy pig sera, performing ELISA detection by using an established method, and displaying the results: the results of all the 40 healthy pig sera are negative; meanwhile, 5 kinds of common virus disease positive serum such as classical swine fever virus (HCV) positive serum, porcine pseudorabies virus (PRV) positive serum, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum, porcine Foot and Mouth Disease Virus (FMDV) positive serum, porcine circovirus type 2 (PCV2) positive serum and the like, and 5 kinds of common porcine bacterial disease positive serum such as porcine escherichia coli (E.coli) positive serum, porcine streptococcus (SS) positive serum, porcine Salmonella (SM) positive serum, porcine mycoplasma pneumoniae (MHP) positive serum, porcine infectious actinobacillus pleuropneumoniae (APP) positive serum and the like are respectively detected by the established method, whether cross reaction exists or not is determined, and the specificity of the detection kit is detected. Results 10 sera were judged negative.

TABLE 2 results of specificity test

Thus, it can be seen that: the kit has high specificity.

Example 5: repeatability test of haemophilus parasuis antibody indirect ELISA detection kit

5.1 in-batch repeatability test

Taking the haemophilus parasuis antibody indirect ELISA detection kit prepared in the same batch to respectively detect the same 5 parts of haemophilus parasuis positive serum and 5 parts of pig negative serum, and carrying out batch repeatability test. The result shows that the coefficient of variation of different serum detection results is between 1% and 13%, and the kit has good repeatability in batches.

TABLE 3 results of the repeatability tests in batches

5.2 run-to-run repeatability test

Taking indirect ELISA detection kits (with the batch numbers of 190427, 190501 and 190504 respectively) for haemophilus parasuis antibodies prepared in different batches, detecting 5 parts of haemophilus parasuis positive serum and 5 parts of pig negative serum under the same condition, and carrying out batch-to-batch repeatability test. The result shows that the coefficient of variation of the detection results of the kits of different batches is between 3% and 11%, and the repeatability of the kit among batches is good.

TABLE 4 results of the repeatability tests between batches

The above research results show that: the kit has good repeatability.

Example 6: comparison test of haemophilus parasuis antibody indirect ELISA detection kit and commercialized imported kit

The kit prepared by the invention is used for detecting 115 parts of clinical pig serum, and the detection result is 51 parts of positive serum and 64 parts of negative serum. Meanwhile, the 115 parts of serum are detected by using a haemophilus parasuis antibody detection kit produced by Biovet company of Canada, and the detection results comprise 49 parts of positive serum and 66 parts of negative serum. Comparing the two detection methods, the coincidence rate of the ELISA detection kit for the haemophilus parasuis antibody prepared by the invention and the imported kit is about 96.5%.

TABLE 5 comparative test for compliance

Sequence listing

<110> Nanjing university of agriculture

NANJING CHANGJI BIOTECHNOLOGY Co.,Ltd.

<120> haemophilus parasuis antibody detection method and kit thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 795

<212> DNA

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 1

acagtttatg aaaatgaagg tacaaaagtt gattttgatg gtcaattgcg tcttctttta 60

gaagaacaag ccacaaaaga gaaaggtcaa tcttcaacac gtggtcacac taacttaaag 120

aataatggtt ctcgtttcgg tatttctatc aaacataata tcaatgagaa tctctacggt 180

tttggtcgtt atgagactcg ccttggccgt aattctgaaa atgctgcagg atggggcgat 240

gttacaacag aatacgctta cgtcggttta ggtggctatg gtcatgaaat ttcttttggt 300

aaacaagctg taatcggtga tagcattggt caagctggtt ttgataaagt atacggtgtt 360

ggtactggtg gaattaaata ttcagcaaac aacacaaaca aaaaaggttt tgatatcctt 420

acttcagatt ctgattcagc aattaactat acctatacag gcattgaagg tttgacgtta 480

ggtgctaact ataatgttgc aaatgagcgt gataagaaga cgggagaagt aaatgtaggt 540

tctactaaat ctggctttgg tttaggtgct aaatacacag ctaagattgc ggaaagtcaa 600

tctgtaactg tggcagcagg ttatactcat gatgactata aatctggagc tgttaataag 660

aaagacaaag atggtgtata ctttggtctt aaatatgtca actctccatt tactgtagct 720

gttgatggtg gtcatggtgt tgtaaaaaca ggtaatgtta aagagaaaat tgacttcgta 780

agaactggcg caaga 795

<210> 2

<211> 265

<212> PRT

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 2

Thr Val Tyr Glu Asn Glu Gly Thr Lys Val Asp Phe Asp Gly Gln Leu

1 5 10 15

Arg Leu Leu Leu Glu Glu Gln Ala Thr Lys Glu Lys Gly Gln Ser Ser

20 25 30

Thr Arg Gly His Thr Asn Leu Lys Asn Asn Gly Ser Arg Phe Gly Ile

35 40 45

Ser Ile Lys His Asn Ile Asn Glu Asn Leu Tyr Gly Phe Gly Arg Tyr

50 55 60

Glu Thr Arg Leu Gly Arg Asn Ser Glu Asn Ala Ala Gly Trp Gly Asp

65 70 75 80

Val Thr Thr Glu Tyr Ala Tyr Val Gly Leu Gly Gly Tyr Gly His Glu

85 90 95

Ile Ser Phe Gly Lys Gln Ala Val Ile Gly Asp Ser Ile Gly Gln Ala

100 105 110

Gly Phe Asp Lys Val Tyr Gly Val Gly Thr Gly Gly Ile Lys Tyr Ser

115 120 125

Ala Asn Asn Thr Asn Lys Lys Gly Phe Asp Ile Leu Thr Ser Asp Ser

130 135 140

Asp Ser Ala Ile Asn Tyr Thr Tyr Thr Gly Ile Glu Gly Leu Thr Leu

145 150 155 160

Gly Ala Asn Tyr Asn Val Ala Asn Glu Arg Asp Lys Lys Thr Gly Glu

165 170 175

Val Asn Val Gly Ser Thr Lys Ser Gly Phe Gly Leu Gly Ala Lys Tyr

180 185 190

Thr Ala Lys Ile Ala Glu Ser Gln Ser Val Thr Val Ala Ala Gly Tyr

195 200 205

Thr His Asp Asp Tyr Lys Ser Gly Ala Val Asn Lys Lys Asp Lys Asp

210 215 220

Gly Val Tyr Phe Gly Leu Lys Tyr Val Asn Ser Pro Phe Thr Val Ala

225 230 235 240

Val Asp Gly Gly His Gly Val Val Lys Thr Gly Asn Val Lys Glu Lys

245 250 255

Ile Asp Phe Val Arg Thr Gly Ala Arg

260 265

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgggatccac agtttatgaa aatgaag 27

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcgtcgactc ttgcgccagt tcttacg 27

Claims

1. An indirect ELISA detection kit for detecting haemophilus parasuis antibodies, which is characterized in that the kit adopts a clinical separation strain HPS5-SQ strain outer membrane recombinant protein P2 of haemophilus parasuis serum 5 type which is expressed in a truncated way and purified as an ELISA plate coated by a coating antigen,

the nucleotide sequence of the Haemophilus parasuis truncated outer membrane recombinant protein P2 is shown as SEQ ID NO.1, and the truncated expressed amino acid sequence is shown as SEQ ID NO. 2.

2. The indirect ELISA detection kit for detecting Haemophilus parasuis antibody according to claim 1, wherein the recombinant protein P2 is prepared by the following steps:

P2F：5’-CGGGATCCACAGTTTATGAAAATGAAG-3’，SEQ ID NO.3；

P2R：5’-GCGTCGACTCTTGCGCCAGTTCTTACG-3’，SEQ ID NO.4；

(5) and purifying the obtained recombinant protein P2 inclusion body by adopting a His-tag affinity chromatography method to obtain recombinant protein P2 serving as a detection antigen.

3. The indirect ELISA detection kit for detecting Haemophilus parasuis antibody of claim 1, wherein the recombinant protein P2 is coated at a concentration of 4 μ g/mL, and the coating solution is carbonate buffer solution with pH of 9.6.

4. The indirect ELISA detection kit for detecting Haemophilus parasuis antibody of claim 1, wherein the coating solution is 0.05mol/L carbonate buffer solution with pH 9.6.

5. The indirect ELISA detection kit for detecting Haemophilus parasuis antibody of claim 1, wherein the kit further comprises a blocking solution, and the blocking solution is 2% gelatin.

6. The indirect ELISA detection kit for detecting Haemophilus parasuis antibodies as claimed in any one of claims 1 to 4, wherein the kit further comprises a serum to be detected, a positive reference serum, a negative reference serum, a sample diluent, an enzyme conjugate, a washing solution, a color development solution and a stop solution.

7. The indirect ELISA detection kit for detecting Haemophilus parasuis antibody of claim 6, wherein the sample diluent is PBST, the enzyme conjugate is goat anti-pig IgG labeled with HRP, the washing solution is PBST, the developing solution is TMB developing solution, and the stop solution is 2mol/L sulfuric acid solution.

8. An indirect ELISA detection method for Haemophilus parasuis antibody based on the indirect ELISA detection kit of claim 1, characterized by comprising the following steps:

(1) coating: diluting the recombinant protein P2 with the amino acid sequence as SEQ ID NO.2 to 4 mu g/mL by using a carbonate buffer solution with the pH of 9.6 as a coating solution, and adding 100 mu L into each hole of the odd-numbered columns of the ELISA plate; meanwhile, only 100 mu L of coating solution is added into each hole of even number rows of the ELISA plate as a blank control, and the mixture is coated overnight at 4 ℃; the coating time is 12-16 h;

respectively arranging a standard positive control hole containing antigen, a standard positive control hole without antigen, a standard negative control hole containing antigen, a standard negative control hole without antigen, a to-be-detected serum hole containing antigen and a to-be-detected serum hole without antigen,

the standard positive control holes are formed by respectively adding 100 mu L of standard positive control serum diluted by 100 times by PBST into two groups of odd-numbered holes and even-numbered holes, two groups of standard positive control holes containing antigens are obtained from the two groups of odd-numbered holes, and two groups of standard positive control holes without antigens are obtained from the two groups of even-numbered holes;

the standard negative control hole is formed by respectively adding 100 mu L of standard negative control serum diluted by 100 times by PBST into the other two groups of odd-numbered holes and even-numbered holes, two groups of standard negative control holes containing antigens are obtained from the two groups of odd-numbered holes, and two groups of standard negative control holes containing no antigens are obtained from the two groups of even-numbered holes;

the serum hole to be detected is formed by respectively adding corresponding 100 mu L of serum to be detected which is diluted by 100 times by PBST into the rest odd-numbered holes and even-numbered holes to obtain a serum hole to be detected containing antigen and a serum hole to be detected containing no antigen;

incubating the sample at 37 ℃ for 1 h;

(5) color development: spin-drying the liquid in the ELISA plate, washing with PBST for 4 times, adding 300 mu L of the liquid into each hole, standing for 5min, spin-drying the liquid in the holes after the last time, adding 100 mu L of TMB color developing solution into each hole, developing for 15min in a dark place at 37 ℃, adding 50 mu L of stop solution into each hole, wherein the stop solution is 2mol/L sulfuric acid solution, and reading the OD on the ELISA plate reader within 15min₄₅₀A numerical value;

The P [ OD ]₄₅₀(containing antigen)]OD of two groups of standard positive control wells containing antigen₄₅₀The average value of the values is calculated,

the P [ OD ]₄₅₀(antigen-free)]OD of two standard positive control wells without antigen₄₅₀AverageA value;

the S [ OD ]₄₅₀(containing antigen)]For each odd number of wells of serum to be tested₄₅₀，

The S [ OD ]₄₅₀(antigen-free)]For each even number of wells of serum to be tested₄₅₀；

By which the respective correction values are calculated as:

standard positive correction value ═ Δ P [ OD₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]

9. The indirect ELISA detection method of Haemophilus parasuis antibody according to claim 8 wherein the coating solution is 0.05mol/L carbonate buffer solution with pH 9.6.

10. The indirect ELISA detection method for Haemophilus parasuis antibody according to claim 8, wherein all standard negative serum, standard positive serum and serum to be detected are blank controls without coating antigen wells.

11. The indirect ELISA detection method for Haemophilus parasuis antibody according to claim 8, wherein when the ratio R of the serum to be detected is not less than 0.275, the detection result of the Haemophilus parasuis antibody in the serum to be detected is determined as positive; and when the ratio R of the serum to be detected is less than 0.275, judging the detection result of the haemophilus parasuis antibody in the serum to be detected as negative.

12. The indirect ELISA detection method of Haemophilus parasuis antibody according to claim 8 wherein the calculation of the result of step (6) further comprises calculating a ratio R of standard negative sera,

The N [ OD ]₄₅₀(containing antigen)]OD of two groups of standard negative control wells containing antigen₄₅₀The average value of the values is calculated,

the N [ OD ]₄₅₀(antigen-free)]OD of two groups of standard negative control wells without antigen₄₅₀The average value of the values is calculated,

by which the respective correction values are calculated as:

the standard negative correction value ═ Δ N [ OD [ ]₄₅₀(containing antigen) -OD₄₅₀(antigen-free)]；

The ratio R of standard negative sera was then calculated:

the method specifically comprises the following steps:

the standard negative serum R should be less than 0.275.