An indirect ELISA method

Abstract

The present invention discloses an indirect ELISA method for detecting the level of ApxI toxin antibody in porcine serum samples, and the indirect ELISA method comprises the following steps: Step S1: Prepare the antigen; Step S2: Determine the conditions of the ELISA method; Step S3: Measure the positive standard product to obtain the average value X and the standard deviation S of OD 450nm , and determine the positive and negative critical values of the indirect ELISA method; Step S4: Measure the sample to be tested. By optimizing conditions such as the antigen coating concentration, serum dilution ratio and action time, enzyme-labeled secondary antibody dilution concentration and action time, antigen-antibody binding time, blocking time, substrate chromogenic time, etc., the present invention can effectively detect the serum antibody after immunizing with the APP vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an indirect ELISA method. Background Art

[0002] Actinobacillus pleuropneumoniae (APP) is a Gram-negative pathogenic bacterium that causes porcine contagious pleuropneumonia (PCP) and only infects pigs. According to the structural differences of the capsular polymers on the surface of APP, Actinobacillus pleuropneumoniae is currently divided into 18 serotypes, and serotype 19 has also been documented in the literature. The prevalent serotypes and ranges in different countries and regions are not exactly the same. In China, it is prevalent throughout the country, and the main strain serotypes are 3, 7, 1, and 5.

[0003] Early laboratory diagnostic methods for APP were indirect hemagglutination (IHA) and complement fixation test (CFT), with low sensitivity; bacterial isolation and PCR identification can type APP, but it is inefficient and time-consuming; ApxIV can only be induced to express when APP infects pigs, does not express in vitro culture, and pigs vaccinated with inactivated APP vaccine cannot induce antibodies against ApxIV toxin. Therefore, the ApxIV-ELISA detection method is used to identify whether it is wild virus infection.

[0004] Apx toxins belong to the RTX toxin protein family, including four toxins: ApxI, ApxII, ApxIII, and ApxIV, and are important protective antigen candidate factors for APP. All serotypes of APP can secrete ApxIV and secrete one or two of ApxI-III. Among them, ApxI has strong hemolytic activity and cytotoxicity, and serotypes 1, 5, 9, 10, 11, 14, and 16 can secrete it and have relatively stronger virulence. Protein AI2 is a truncated protein of the dominant determinant of ApxI. The indirect ELISA method established with the truncated protein AI2 as the antigen can lay a foundation for the early detection of APP strong strains and detect ApxI antibodies in the sera of pigs immunized with APP vaccine, so as to evaluate the immune effect of APP vaccine.

[0005] Since the cross-protection between different serotypes of APP is poor, inactivated vaccines of different serotype combinations or subunit vaccines containing recombinant proteins of three toxins ApxI-III are generally used clinically to prevent porcine contagious pleuropneumonia. Therefore, a simple and rapid serum detection method is needed to evaluate the immune effect of the vaccine, which is very important for screening out APP vaccines with good immune effects and formulating correct immunization programs, and then preventing and purifying porcine contagious pleuropneumonia. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an indirect ELISA method, which can effectively detect serum antibodies after immunization with APP vaccine by optimizing conditions such as antigen coating concentration, serum dilution ratio and action time, enzyme-labeled secondary antibody dilution concentration and action time, antigen-antibody binding time, blocking time, substrate chromogenic time, etc.

[0007] To achieve the above purpose, the present invention provides an indirect ELISA method, characterized in that the indirect ELISA method is used to detect the ApxI toxin antibody level in porcine serum samples, and the indirect ELISA method includes the following steps:

[0008] Step S1: Prepare the antigen, which is obtained by purification after expression of Rosetta-pColdI-sumo-AI2;

[0009] Step S2: Determine the conditions of the ELISA method, and the conditions include: antigen coating concentration is 1 μg / mL, serum dilution ratio is 1:160, coating condition is overnight at 4 °C, blocking solution is 5% skim milk, blocking condition is 3 h at 37 °C, serum action condition is 1 h at 37 °C, enzyme-labeled antibody dilution ratio is 1:15000, enzyme-labeled antibody action condition is 30 min at 37 °C, and chromogenic time is 10 min at 37 °C;

[0010] Step S3: Measure the positive standard product to obtain the average value X and standard deviation S of OD 450nm , and determine the positive and negative critical values of the indirect ELISA method. The positive and negative critical values include a positive critical value and a negative critical value. The positive critical value is X + 3S, and the negative critical value is X + 2S;

[0011] Step S4: Measure the sample to be tested. If the OD 450nm value of the sample to be tested is greater than or equal to the positive critical value, then it is determined that the sample to be tested is positive; if the OD 450nm value of the sample to be tested is less than the positive critical value and greater than or equal to the negative critical value, then it is determined that the sample to be tested is suspicious; if the OD 450nm value of the sample to be tested is less than the negative critical value, then it is determined that the sample to be tested is negative.

[0012] Preferably, the step S1 includes:

[0013] Inoculate the Rosetta-pColdI-sumo-AI2 bacterial solution into the LB liquid medium containing ampicillin;

[0014] Cultivate with shaking at 200 rpm on a shaker at 37 °C;

[0015] When OD 600nmWhen the value reaches the range of 0.6 to 0.8, the bacterial solution is placed at 15°C and left standing for 30 min. Then, IPTG with a final concentration of 1.0 mmol / L is added to the bacterial solution, and induction expression is carried out in a shaker at 15°C and 160 rpm for 24 h;

[0016] The bacterial solution after induction expression is centrifuged, washed twice with PBS buffer solution, and the thalli are resuspended with binding buffer solution and broken by a high-pressure cell crusher;

[0017] The broken liquid is centrifuged, filtered through a 0.22-μm filter, purified by a Ni-NTA column, and stored at -70°C for later use.

[0018] Preferably, the amino acid residue sequence of the antigen is SEQ ID NO.2.

[0019] The beneficial effect of the present invention is that through the optimization of conditions such as antigen coating concentration, serum dilution ratio and action time, enzyme-labeled secondary antibody dilution concentration and action time, antigen-antibody binding time, blocking time, substrate chromogenic time, etc., it can effectively detect serum antibodies after immunization with APP vaccine and has good specificity. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is a schematic diagram of the expression and purification of the truncated protein AI2 in the embodiment of the present invention;

[0022] Figures 2 to 4 It is a test result diagram of the coincidence rate test in the embodiment of the present invention. Detailed Embodiments

[0023] The core of the present invention is to provide an indirect ELISA method. Through the optimization of conditions such as antigen coating concentration, serum dilution ratio and action time, enzyme-labeled secondary antibody dilution concentration and action time, antigen-antibody binding time, blocking time, substrate chromogenic time, etc., it can effectively detect serum antibodies after immunization with APP vaccine.

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] The following materials will be used in this embodiment:

[0026] Rosetta-pColdI-sumo-AI2, Bordetella bronchiseptica (Bb), Pasteurella multocida (Pm), Escherichia coli (E. coli), Streptococcus suis (SS), positive sera of Haemophilus parasuis (HPS), positive sera of porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Mycoplasma hyopneumoniae (M. hyopneumoniae), positive sera after immunization with Merck APP vaccine, and APP negative sera can be obtained through conventional channels; isopropyl β-D-thiogalactoside (IPTG) is purchased from Solarbio Science & Technology Co., Ltd.; ampicillin is purchased from Shengxing Biotechnology Co., Ltd.; SDS-PAGE kit is purchased from Shanghai Yaen Biotechnology Co., Ltd.; HRP-staphylococcal protein A and HRP-goat anti-rabbit IgG are purchased from Wuhan Boster Biological Engineering Co., Ltd.; Immuno detachable transparent enzyme-linked immunosorbent assay (ELISA) plate is purchased from Thermo Fisher Scientific (China) Co., Ltd.; TMB is purchased from Beyotime; P003 Ni NTA μSphere is purchased from Tianyan Biotechnology Co., Ltd., and the rest of the conventional reagents are of domestic or imported analytical grade and can be obtained through conventional channels.

[0027] The expression and purification of the truncated protein AI2 (the amino acid residue sequence of which is shown in SEQ ID NO.2, and the corresponding nucleotide sequence is shown in SEQ ID NO.1) are as follows Figure 1 shown in the figure, where M: protein Marker; 1: Rosetta / pColdI-sumo (N-his)-AI2 induced whole bacteria, 2: AI2 supernatant, 3: eluate, 4: purified AI2 protein.

[0028] The indirect ELISA method disclosed in this embodiment includes the following steps:

[0029] Step S1: Prepare the antigen, which is obtained by purification after being expressed by Rosetta-pColdI-sumo-AI2.

[0030] Step S2: Determine the conditions of the ELISA method, including: the antigen coating concentration is 1 - 1.5 μg / mL, the serum dilution is 1:160, the coating condition is overnight at 4°C, the blocking solution is 5% skim milk, the blocking condition is 3 h at 37°C, the serum incubation condition is 1 h at 37°C, the dilution of the enzyme-labeled antibody is 1:15000, the incubation condition of the enzyme-labeled antibody is 30 min at 37°C, and the color development time is 10 min at 37°C.

[0031] Step S3: Measure the positive standard product to obtain the average value X and the standard deviation S of OD 450nm , and determine the positive and negative critical values of the indirect ELISA method. The positive and negative critical values include the positive critical value and the negative critical value. The positive critical value is X + 3S, and the negative critical value is X + 2S.

[0032] Step S4: Measure the test sample. If the OD 450nm value of the test sample is greater than or equal to the positive critical value, then the test sample is determined to be positive; if the OD 450nm value of the test sample is less than the positive critical value and greater than or equal to the negative critical value, then the test sample is determined to be suspicious; if the OD 450nm value of the test sample is less than the negative critical value, then the test sample is determined to be negative.

[0033] In this embodiment, Step S1 includes: inoculating the Rosetta-pColdI-sumo-AI2 bacterial solution into an LB liquid medium containing ampicillin;

[0034] Culturing with shaking at 200 rpm on a shaker at 37°C;

[0035] When the OD 600nm value reaches the range of 0.6 - 0.8, let the bacterial solution stand at 15°C for 30 min, then add IPTG with a final concentration of 1.0 mmol / L to the bacterial solution, and induce expression at 160 rpm on a shaker at 15°C for 24 h;

[0036] Centrifuge the induced bacterial solution, wash it twice with PBS buffer, resuspend the cells with binding buffer, and break them using a high-pressure cell disruptor;

[0037] Centrifuge the broken liquid, filter it through a 0.22 μm filter, purify it using a Ni-NTA column, and store it at -70°C for later use.

[0038] In step S2, the square titration method is used to determine the optimal coating concentration of the antigen and the optimal dilution of the serum. The antigen is diluted with carbonate buffer at pH 9.6 to final concentrations of 4.0 μg / mL, 3.0 μg / mL, 2.5 μg / mL, 2.0 μg / mL, 1.5 μg / mL, 1.0 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, respectively. The standard negative and positive sera are diluted with PBST in a serial dilution method at ratios of 1:80, 1:120, 1:160, 1:200, 1:240, and 1:280. Each antigen coating concentration and serum dilution is repeated in three wells. Other steps are performed according to the indirect ELISA protocol. Finally, when the OD value of the positive serum is close to 1.0 and the P / N value is the largest, the corresponding antigen coating concentration and serum dilution are the optimal ones. 450nm value is close to 1.0 and the P / N value is the largest, the corresponding antigen coating concentration and serum dilution are the optimal ones.

[0039] As shown in Table 1, when the antigen coating concentration is 1 μg / mL and the serum dilution is 1:160, the OD value of the positive serum is close to and does not exceed 1.0, and the P / N value is the largest at this time. Therefore, these are used as the optimal antigen coating concentration and the optimal serum dilution. 450nm value is close to and does not exceed 1.0, and the P / N value is the largest at this time. Therefore, these are used as the optimal antigen coating concentration and the optimal serum dilution.

[0040] Table 1 Determination of the optimal coating concentration of recombinant protein and

[0041] Table 1 Determination of the optimal coating concentration of recombinant protein and

[0042] serum dilution

[0043]

[0044]

[0045] The method for determining the optimal antigen coating conditions is based on the determined optimal antigen coating concentration and optimal serum dilution. The antigen coating conditions are set as a single variable. Six coating conditions are set: 37°C for 2 h plus overnight at 4°C, 37°C for 1 h plus overnight at 4°C, overnight at 4°C, 37°C for 3 h, 37°C for 2 h, and 37°C for 1 h. Other steps are performed according to the indirect ELISA protocol. Each condition is tested using one standard negative serum and four different standard positive sera, with 3 wells repeated for each. Finally, when the P / N value is the largest, the corresponding antigen coating condition is the optimal one.

[0046] The test results are shown in Table 2. Six groups of antigens coated under different conditions were compared. The results show that (Table 2) when coated overnight at 4°C, the P / N value of the test results is the highest. Therefore, the selected optimal antigen coating time is overnight at 4°C.

[0047] Table 2 Determination of the optimal coating antigen condition

[0048]

[0049]

[0050] The method for determining the optimal blocking solution is to apply the optimal conditions screened above, set the blocking solution as the single variable, and set five blocking solutions: 2% gelatin, 0.1% BSA, 1% BSA, 5% skim milk, and 10% skim milk (all diluted with PBST). Other steps are the same as the indirect ELISA procedure. Each blocking solution is tested with one standard negative serum and four different standard positive sera, and each is set with 3 replicates. Finally, when the P / N value is the largest, the corresponding blocking solution is the best.

[0051] The test results are shown in Table 3. Five blocking solutions were compared. The results show that (Table 3) when blocked with 5% skim milk, the P / N value of the test results is the highest. Therefore, the selected optimal blocking solution is 5% skim milk.

[0052] Table 3 Determination of optimal sealing solution

[0053]

[0054] The method for determining the optimal blocking time is to apply the optimal conditions screened above, set the blocking time as the single variable, and set three blocking times: 1 h, 2 h, and 3 h at 37°C. Other steps are the same as the indirect ELISA procedure. Each blocking time is tested with one standard negative serum and four different standard positive sera, and each is set with 3 replicates. Finally, when the P / N value is the largest, the corresponding blocking time is the best.

[0055] The test results are shown in Table 4. Three groups of blocking conditions were compared. The results show that (Table 4) when blocked at 37°C for 3 h, the P / N value of the test results is the highest. Therefore, the selected optimal blocking time is 3 h.

[0056] Table 4 Determination of optimal sealing condition

[0057]

[0058] The method for determining the optimal action time is to apply the optimal conditions screened above, set the serum action time as a single variable, set four serum action times of 0.5 h, 1.0 h, 1.5 h, and 2.0 h at 37 °C, and the other steps are the same as the indirect ELISA method. One standard negative serum and four different standard positive serums are used for detection at each action time, and 3 replicates are set for each. Finally, when the P / N value is the largest, the corresponding serum action time is the best.

[0059] The test results are shown in Table 5. Comparing the 3 groups of serum action times, the results show that (Table 5) when the serum action time is 1 h at 37 °C, the P / N value of its test results is the highest. Therefore, the selected optimal serum action time is 1 h.

[0060] Table 5 Determination of optimal serum reaction time

[0061]

[0062]

[0063] The method for determining the optimal dilution of the enzyme-labeled antibody is to apply the optimal conditions screened above, set the dilution of the enzyme-labeled antibody as a single variable, and set four dilutions of 1:5000, 1:10000, 1:15000, and 1:20000. One standard negative serum and four different standard positive serums are used for detection at each dilution, and 3 replicates are set for each. Finally, when the P / N value is the largest, the corresponding dilution of the enzyme-labeled antibody is the best.

[0064] The test results are shown in Table 6. Comparing the working concentrations of the 4 groups of enzyme-labeled antibodies, the results show that (Table 6) when the dilution of the enzyme-labeled antibody is 1:15000, the P / N value of its test results is the highest. Therefore, the selected optimal dilution of the enzyme-labeled antibody is 1:15000.

[0065] Table 6 Determination of optimal dilution of HRP-SPA

[0066]

[0067] The method for determining the optimal reaction time of the enzyme-labeled antibody is to apply the optimal conditions screened above. Set the reaction time of the enzyme-labeled antibody as a single variable, with four reaction times: 15 min, 30 min, 45 min, and 60 min at 37°C. For each reaction time, one standard negative serum and four different standard positive sera are used for detection, and three replicates are set for each. Finally, when the P / N value is the largest, the corresponding reaction time of the enzyme-labeled antibody is the optimal one.

[0068] The test results are shown in Table 7. By comparing the reaction times of the four groups of enzyme-labeled antibodies, the results show that (Table 7) when the reaction time of the enzyme-labeled antibody is 30 min at 37°C, the P / N value of its detection result is the highest. Therefore, the selected optimal reaction time of the enzyme-labeled antibody is 30 min.

[0069] Table 7 Determination of optimal reaction time of HRP-SPA

[0070]

[0071] The method for determining the optimal color development time is to apply the optimal conditions screened above. Set the color development time of the substrate as a single variable, with four color development times: 5 min, 10 min, 15 min, and 20 min at 37°C. For each color development time, one standard negative serum and four different standard positive sera are used for detection, and three replicates are set for each. Finally, when the P / N value is the largest, the corresponding color development time of the substrate is the optimal one.

[0072] The test results are shown in Table 8. By comparing the color development times of the three groups of TMB, the results show that (Table 8) when the color development time is 10 min at 37°C, the P / N value of its detection result is the highest. Therefore, the selected optimal color development time is 10 min.

[0073] Table 8 Determination of optimal reaction time of substrate

[0074]

[0075] In step S3, the method for determining the positive and negative critical values of the indirect ELISA method is to screen 69 negative sera by Western blot and perform ELISA detection according to the established detection method, and calculate the average value (X) and standard deviation (S) of the OD values of the negative serum samples. Determine the critical value according to statistical principles, that is, when OD 450nm ≥X + 3S, it is judged as positive; when OD 450nm ≥X + 3S, it is judged as positive; when OD 450nmWhen it is ≤ X + 2S, it is judged as negative; those in between are judged as suspicious

[0076] The test results are shown in Table 9. Among the 69 sera, the OD 450nm The maximum value is 0.294, the minimum value is 0.112, the average value X is 0.166, and the standard deviation S is 0.0478. Therefore, the lower limit of detection for positive samples is X + 3S = 0.3093. To reduce the probability of false positives and false negatives, the critical value plus or minus one standard deviation is set as the suspicious area, that is, when OD 450nm ≥ 0.3093, it is judged as positive; when OD 450nm < X + 2S, that is, when it is 0.2615, it is judged as negative, and those in between are judged as suspicious (Table 9)

[0077] Table 9 Determination of the threshold for indirect ELISA

[0078]

[0079] Specificity test

[0080] According to the established detection method, positive sera infected with 11 porcine pathogen species including Pm, Bb, HPS, SS, E. coli, Erysipelothrix rhusiopathiae, PCV2, PRV, PRRSV, CSFV, and Mycoplasma hyopneumoniae were detected simultaneously to verify whether there was cross-reaction with other porcine pathogen species

[0081] The test results of the specificity test are shown in Table 10. Using the established indirect ELISA method, positive sera of 11 porcine pathogen species including Pm, Bb, HPS, SS, E. coli, PCV2, PRV, PRRSV, CSFV, and Mycoplasma hyopneumoniae were detected simultaneously. The results showed (Table 10) that the PRRSV positive serum was detected as suspicious, and the rest were negative

[0082] Table 10 Specificity test for indirect ELISA

[0083]

[0084] Repeatability test

[0085] Intra-assay repeatability test: The ELISA plates were coated with the recombinantly expressed and purified truncated protein AI2 from the same batch. Five positive serum samples with different antibody levels and one negative serum sample were detected according to the established ELISA reaction conditions. Each sample was set with 8 replicate wells, and 2 replicate wells were set on each of the 4 ELISA plates from the same batch. The results were statistically analyzed.

[0086] Inter-assay repeatability test: The ELISA plates were coated with the recombinantly expressed and purified truncated protein AI2 from 4 different batches. Five positive serum samples with different antibody levels and one negative serum sample were detected according to the established ELISA reaction conditions. Each sample was set with 8 replicate wells, and 2 replicate wells were set on each of the 4 ELISA plates from different batches. The results were statistically analyzed.

[0087] The test results of the repeatability test are shown in Table 11. The results of the indirect ELISA repeatability test showed that the coefficients of variation of the intra-assay repeatability test were 0.62% - 4.50%, and those of the inter-assay repeatability test were 1.48% - 8.08%, both less than 10%. The results indicated that the ELISA method had good repeatability (Table 11).

[0088] Table 11 Intraassay and Interassay repeating test of the Indirect ELISA

[0089]

[0090] Sensitivity test

[0091] Four positive sera after APP immunization were serially diluted at ratios of 1:100, 1:200, 1:400, ……, 1:12800, and the antibody titers were detected according to the established indirect ELISA method to evaluate the sensitivity of the method.

[0092] The test results of the sensitivity test are shown in Table 12. The sera from 4 different dilutions of APP vaccine immunization were detected by the established indirect ELISA method. The results showed that the detection result was still positive after the positive serum with the highest titer was diluted 1:3200. It indicated that the indirect ELISA antibody detection method established in this study had high sensitivity (Table 12).

[0093] Table 12 Sensitivity test for Indirect ELISA

[0094]

[0095] Coincidence rate test

[0096] For APP5 type streaking, pick single colonies and culture them in TSB. After cultivation, adjust the OD of the APP1 type bacterial liquid 600nm to 0.6 - 0.8, inoculate into LB liquid medium at an inoculation amount of 1%, and add 10 mM CaCl2 and NAD coenzyme, then culture at 37°C and 180 rpm for 6 - 12 h. Centrifuge to obtain the supernatant, slowly add ammonium sulfate to the supernatant until the concentration reaches 60%, perform salting out overnight; centrifuge to collect the precipitate, dissolve it with PBS, and concentrate it using an ultrafiltration tube to obtain ApxI toxin.

[0097] Use the AI2 indirect ELISA method to detect 30 sera to be tested to determine positive and negative. At the same time, transfer ApxI by SDS - PAGE. Immerse the transferred NC membrane into PBST containing 5% skim milk and block it overnight at 4°C; use the sera to be tested (1:160) as the primary antibody and incubate it on a shaker at room temperature for 1 h; after washing 3 times with PBST, use HRP - SPA (1:15000) as the secondary antibody and incubate for 30 min; after washing, evenly coat the luminescent solution and perform exposure analysis to compare the coincidence rate of the two methods.

[0098] The results of the coincidence rate test are as Figures 2 to 4 (In the figure, M: protein Marker; 1: irrelevant protein, 2: ApxI toxin) and shown in Table 13. Use the AI2 indirect ELISA method and ApxI toxin Western blot to detect 30 sera to be tested to determine positive and negative. The detection results are shown in the following figure. ELISA detected 12 positive and 18 negative, and Western blot detected 15 positive and 15 negative. It can be seen from this that the positive coincidence rate of this method is 80%, the negative coincidence rate is 100%, and the total coincidence rate is 90%.

[0099] Table 13 Comparison of results between indirect ELISA and Western blot

[0100]

[0101] Porcine contagious pleuropneumonia is a highly contagious respiratory disease of pigs characterized by hemorrhagic and fibrinous necrotic pleuropneumonia. Due to the large number of APP serotypes and the weak cross-protection among different serotypes, the prevention and control of this disease are relatively difficult. In recent years, its prevalence trend has been on the rise in China, causing huge losses to the pig farming industry in China. Currently, the APP vaccines on the domestic and international markets are multi-serotype mixed inactivated vaccines or subunit vaccines containing recombinant proteins of three toxins, ApxI, ApxII, and ApxIII. For example, Shandong Huahong inactivates serotypes 1 and 7 of APP, Huapai Biology inactivates serotypes 1 and 2 of APP, Wuhan Keqian inactivates serotypes 1, 2, and 7 of APP, and MSD Animal Health uses toxins ApxI to III plus the 42kD outer membrane protein. Currently, most of the relevant detections for porcine pleuropneumonia are ApxIV-ELISA, which can only be used to identify whether pigs are infected with wild virus and cannot be used to evaluate the immune effect of vaccines. For example, Dreyfus et al. established an indirect ELISA method using recombinant ApxIV protein [Dreyfus A, Schaller A, Nivollet S, et al. Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA[J]. Vet Microbiol, 2004, 99(3-4):227-238.], and Wuhan Keqian Biological Co., Ltd. has produced an indirect ELISA kit for Actinobacillus pleuropneumoniae ApxIV. In addition, Jia Fan et al. developed an ApxⅡ-ELISA antibody detection kit for Actinobacillus pleuropneumoniae, which can be used to monitor the level of ApxⅡ antibodies [Jia Fan, Wang Jianyin, Hu Junyong, et al. Development and application of an ApxⅡ-ELISA antibody detection kit for Actinobacillus pleuropneumoniae[J]. Chinese Journal of Veterinary Science, 2009, 29:283-287.].

[0102] The virulence of APP strains is closely related to the types of Apx toxins. Among them, the strains that can secrete ApxI toxin have relatively stronger virulence. Therefore, the antibody level against ApxI toxin after immunization with the contagious pleuropneumonia vaccine is an important indicator to measure the vaccine effect. In the previous laboratory, ApxI was truncated and expressed in segments, and finally it was determined that its dominant antigenic determinant was located at the C-terminus. The C-terminus is the region where Ca2+ binds to exert hemolytic activity, which is closely related to its pathogenicity [Burdychova R, Rychtera M, Horvath R, et al. Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli [J]. FEMS Microbiol Lett, 2004, 230(1): 9-12.]. Liu Jianjie et al. preliminarily established an ApxI-ELISA detection method [Liu Jianjie, He Qigai, Chen Huanchun, et al. Cloning, expression of Actinobacillus pleuropneumoniae toxin I gene and establishment of its ELISA detection method [J]. Scientia Agricultura Sinica, 2004: 148-151.]. Hu Yaofang et al. established a toxin neutralization test using the hemolytic characteristics of ApxI [Hu Yaofang, Wu Hao, Jiang Changsheng, et al. Establishment and preliminary application of ApxI toxin neutralization test for Actinobacillus pleuropneumoniae [J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43: 158-164.], which requires purification of ApxI toxin and preparation of fresh red blood cells, and the process is relatively cumbersome. The ELISA method established with the truncated protein AI2 as the antigen in this example can detect the antibody of toxin ApxI, so as to evaluate the immune effect, and can also be used for the early detection of APP virulent strains.

[0103] In the establishment of the indirect ELISA method, in this example, by optimizing conditions such as antigen coating concentration, serum dilution ratio and incubation time, enzyme-labeled secondary antibody dilution concentration and incubation time, antigen-antibody binding time, blocking time, and substrate chromogenic time, it can effectively detect the serum antibody after immunization with the APP vaccine. The coefficient of variation of intra-batch and inter-batch repeated tests in this example is less than 10%, showing good repeatability and facilitating the wide promotion of this test method. In the specificity test, the positive sera of 11 porcine disease pathogens, including Pm, Bb, HPS, SS, E. coli, PCV2, PRV, PRRSV, CSFV, and Mycoplasma hyopneumoniae, were detected simultaneously. Except for the suspicious detection of the PRRSV positive serum, the others were negative, proving good specificity. Because there is currently no commercial diagnostic product for ApxI toxin antibody, the established ELISA method was compared with ApxI toxin Western blot, and the positive coincidence rate was 80% and the negative coincidence rate was 100%.

[0104] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0105] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An indirect ELISA method, characterized in that, The indirect ELISA method is used to detect the level of ApxI toxin antibody in porcine serum samples, and the indirect ELISA method comprises the following steps: Step S1: Prepare the antigen, which is obtained by purification after the expression of Rosetta-pColdI-sumo-AI2; the amino acid residue sequence of the antigen is SEQ ID NO. 2; Step S2: Determine the conditions of the ELISA method, which include: the antigen coating concentration is 1 μg / mL, the serum dilution is 1:160, the coating condition is overnight at 4 °C, the blocking solution is 5% skim milk, the blocking condition is 3 h at 37 °C, the serum action condition is 1 h at 37 °C, the enzyme-labeled antibody dilution is 1:15000, the action condition of the enzyme-labeled antibody is 30 min at 37 °C, and the color development time is 10 min at 37 °C; Step S3: Measure the positive standard product to obtain the average value X and the standard deviation S of OD450nm, and determine the positive and negative critical values of the indirect ELISA method. The positive and negative critical values include the positive critical value and the negative critical value. The positive critical value is X + 3S, and the negative critical value is X + 2S; Step S4: Measure the sample to be tested. If the OD450nm value of the sample to be tested is greater than or equal to the positive critical value, it is determined that the sample to be tested is positive; if the OD450nm value of the sample to be tested is less than the positive critical value and greater than or equal to the negative critical value, it is determined that the sample to be tested is suspicious; if the OD450nm value of the sample to be tested is less than the negative critical value, it is determined that the sample to be tested is negative.

2. The indirect ELISA method according to claim 1, wherein The step S1 includes: Inoculate the Rosetta-pColdI-sumo-AI2 bacterial solution into the LB liquid medium containing ampicillin; Cultivate with shaking at 200 rpm at 37 °C; When the OD600nm value reaches the range of 0.6 - 0.8, let the bacterial solution stand at 15 °C for 30 min, then add IPTG with a final concentration of 1.0 mmol / L to the bacterial solution, and induce expression at 160 rpm at 15 °C for 24 h; Centrifuge the induced bacterial solution, wash it twice with PBS buffer, resuspend the thallus with the binding buffer, and break it through a high-pressure cell crusher; Centrifuge the broken liquid, filter it through a 0.22 μm filter, purify it with a Ni-NTA column, and store it at -70 °C for later use.

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