An indirect ELISA detection kit and method for bovine pasteurellosis

Abstract

This invention discloses an indirect ELISA detection kit and method for bovine pasteurellosis, relating to the field of biotechnology. The indirect ELISA kit includes an enzyme-labeled plate coated with an antigen; the amino acid sequence of the antigen is shown in SEQ ID NO.2. The indirect ELISA detection kit constructed in this invention exhibits high specificity and sensitivity; the intra-assay coefficient of variation is between 1.79% and 7.54%, and the inter-assay coefficient of variation is between 2.23% and 6.91%, both below 10%, demonstrating good inter-assay and intra-assay repeatability. In testing 30 clinical samples, the overall concordance rate with the standard "Diagnostic Techniques for Bovine Hemorrhagic Sepsis (GB / T27530-2025)" was 96.67%. The indirect ELISA detection kit demonstrates good overall performance and can be applied to the detection of bovine pasteurellosis in clinical serum samples.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an indirect ELISA detection kit and method for bovine pasteurellosis. Background Technology

[0002] Pasteurella multocida (Pm) is a Gram-negative pathogenic bacterium, appearing as coccobacilli or short rods, without flagella, and non-spore-forming. It is an aerobic or facultative anaerobic bacterium. Serotypes are distinguished based on capsular antigens and somatic antigens; the former has five serotypes, while the latter has at least 16. This bacterium has a wide host range, infecting various animals and humans, causing varying degrees of damage. Although serotype-specific inactivated vaccines and antibiotics such as penicillin and florfenicol can effectively control Pasteurella multocida infection, increasing antimicrobial resistance and the risk of cross-species transmission still pose challenges to prevention and control. Therefore, establishing rapid, sensitive, and accurate diagnostic methods to monitor its prevalence and antibody levels is of great significance for scientific prevention and control and mitigating economic losses and public health risks.

[0003] The pathogenicity of *Pasteurella multocida* is associated with multiple virulence factors, including lipopolysaccharide, capsule, outer membrane proteins, iron regulatory proteins, *Pasteurella multocida* toxin, fimbriae, and superoxide dismutase. After infection, the host rapidly produces high-titer antibodies to counteract these bacterial proteins, making serological testing a crucial basis for diagnosis. Among these, the outer membrane proteins, as bacterial surface components, participate in the bacterial adoptive response to adverse environments. They can adhere to host cells to enhance pathogenicity, facilitate nutrient absorption and transport, and, due to their strong antigenicity and immunogenicity, serve as important targets for vaccine development and diagnostic methods. This invention optimizes expression conditions to achieve soluble expression of the OmpW protein, ensuring its native conformation. A novel indirect ELISA detection kit for bovine *Pasteurella multocida* infection is proposed based on the OmpW recombinant protein, providing technical support for rapid, sensitive, and accurate diagnosis of bovine *Pasteurella multocida* infection. Summary of the Invention

[0004] The purpose of this invention is to provide an indirect ELISA detection kit and method for bovine pasteurellosis, thereby addressing the problems existing in the prior art. This indirect ELISA detection kit exhibits good specificity, sensitivity, and repeatability, and can be applied to the detection of bovine pasteurellosis in clinical serum samples.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an indirect ELISA kit for detecting antibodies against Pasteurella multocida, comprising an enzyme-labeled plate coated with an antigen;

[0007] The antigen is a recombinant OmpW protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] Furthermore, the method for preparing the recombinant OmpW protein includes the following steps:

[0009] The OmpW gene was homologously recombined with the pColdII-10His plasmid to obtain the recombinant plasmid pColdII-10His-OmpW;

[0010] The recombinant plasmid pColdII-10His-OmpW was transformed into competent Escherichia coli cells to obtain the recombinant expression strain;

[0011] After inducing expression culture of the recombinant expression strain, the recombinant OmpW protein was obtained by protein purification.

[0012] The nucleotide sequence of the OmpW gene is shown in SEQ ID NO.1.

[0013] Furthermore, the method for preparing the enzyme-labeled plate coated with the antigen includes the following steps:

[0014] The antigen was diluted with a coating buffer to obtain a suspension. The suspension was added to the enzyme label wells for coating, and then a blocking buffer was added for blocking to obtain the enzyme label plate coated with the antigen.

[0015] Furthermore, the concentration of the recombinant OmpW protein in the suspension is 8 μg / mL.

[0016] Furthermore, the sealing liquid is 10% skim milk powder.

[0017] Furthermore, the indirect ELISA kit also includes enzyme-labeled secondary antibody, diluent, washing solution, chromogenic solution, stop solution, negative control and positive control.

[0018] Further, the diluent is PBST buffer; and / or

[0019] The washing solution is a PBS buffer containing 0.1% Tween 20.

[0020] Furthermore, the colorimetric solution is a TMB solution.

[0021] Furthermore, the terminating solution is a 2 mol / L H2SO4 solution.

[0022] This invention also provides a method for detecting Pasteurella multocida antibodies for non-disease diagnostic purposes, comprising the steps of detecting Pasteurella multocida antibodies using the aforementioned indirect ELISA kit:

[0023] Add negative control, positive control and test sample solution to the enzyme-labeled plate coated with antigen, and perform the first incubation reaction;

[0024] Add enzyme-labeled secondary antibody to each reaction well and perform a second incubation reaction;

[0025] Add the colorimetric solution to each reaction well and continue incubation for 10 min;

[0026] Add stop solution to each reaction well, measure the absorbance at 450 nm using an ELISA reader, and interpret the results.

[0027] The present invention discloses the following technical effects:

[0028] This invention clones the OmpW gene of *Pasteurella multocida* into the vector pColdII-10His to construct the prokaryotic expression vector pColdII-10His-OmpW. Purified OmpW protein is obtained using a nickel column chromatography method. Conditions are optimized, and an indirect ELISA method for detecting *Pasteurella multocida* antibodies is established using the purified OmpW protein as the coating antigen. Specificity, sensitivity, and repeatability tests are performed on this method, and clinical samples are tested. Results show that after overnight induction with 0.25 mM IPTG at 16°C and 150 rpm for 16 h, OmpW is solublely expressed as detected by Western blotting. After optimization, it was found that when the OmpW protein was coated at 8 μg / mL, blocked with 10% skim milk powder at 37℃ for 2 h, the serum to be tested was diluted 1:400 and incubated at 37℃ for 1 h, the enzyme-labeled secondary antibody was diluted 1:20000 and incubated at 37℃ for 30 min, and the color development was performed for 10 min, and the positive and negative cutoff value was 0.3, an indirect ELISA detection method for bovine pasteurellosis could be established. This invention also found that the established indirect ELISA method reacts only with Pasteurella multocida-positive serum and has no cross-reactivity with other sera, exhibiting high specificity; the detection result remains positive even at a serum dilution of 1:1600, demonstrating good sensitivity; the intra-assay coefficient of variation is between 1.79% and 7.54%, and the inter-assay coefficient of variation is between 2.23% and 6.91%, both below 10%, indicating good inter-assay and intra-assay repeatability; when 30 clinical samples were tested, the overall concordance rate with the "Diagnostic Techniques for Bovine Hemorrhagic Sepsis (GB / T27530-2025)" was 96.67%.

[0029] In summary, the indirect ELISA detection kit and method for Pasteurella multocida established in this invention have received positive comprehensive evaluation and can be applied to the detection of Pasteurella multocida in clinical serum samples. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The results show the identification of the pColdII-10His-OmpW recombinant plasmid construction process; where A is the identification result of PCR amplification of the OmpW gene fragment, M: DNA Marker, 1: negative control, 2: OmpW gene fragment; B is the detection result of double enzyme digestion of the pColdII-10His empty vector, M: DNA Marker, 1: linearized pColdII-10His; C is the PCR identification result of the pColdII-10His-OmpW recombinant plasmid, M: DNA Marker, 1-3: single clones of pColdII-10His-OmpW recombinant plasmid, 4: negative control;

[0032] Figure 2 The results of Western Blot identification of His-OmpW protein expression; where M: Protein Marker; 1: His-OmpW protein expression supernatant; 2: His-OmpW protein expression precipitate; 3: pColdII-10His empty vector expression supernatant; 4: pColdII-10His empty vector expression supernatant.

[0033] Figure 3 This is the specific detection result of the ELISA method of this invention. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1

[0040] 1. Materials and Methods

[0041] 1.1 Materials

[0042] 1.1.1 Strains, serum and vectors

[0043] Pasteurella multocida (Pm), clinical serum, positive and negative controls for Bovine Pasteurella multocida were provided by the Beijing Academy of Agricultural and Forestry Sciences, Institute of Animal Husbandry and Veterinary Medicine. Positive sera for Brucella bovis, Mycobacterium paratuberculosis, Clostridium perfringens, and Escherichia coli antibodies, and the pColdII-10His vector were provided by the Animal Biosafety and Public Health Control Team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0044] 1.1.2 Main Reagents and Instruments

[0045] PrimeSTAR Max DNA Polymerase was purchased from TAKARA Biotechnology (Beijing) Co., Ltd.; TME.ZNAGel Extraction Kit was purchased from Omega Bio-Tek Inc.; ClonExpress MultiS OneStep Cloning Kit and 2×Rapid Taq Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Plasmid mini-preparation kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; IPTG, gel preparation kit, Page gel staining solution, and BSA were all purchased from Beijing Solarbio Co., Ltd.; purified beads were purchased from Tiandi Renhe Co., Ltd.; skim milk powder was purchased from BD Biosciences Co., Ltd.; primary antibody dilution buffer, His-tagged primary antibody, and HRP-labeled secondary antibody were all purchased from Beyotime Biotechnology Co., Ltd.; exposure solution, TMB chromogenic solution, and 96-well detachable microplate were all purchased from ThermoFisher Scientific.

[0046] The T100 Thermal Cycler PCR amplification instrument (Bio-Rad), NanoDrop One (ThermoFisher Scientific), nucleic acid electrophoresis apparatus, vertical electrophoresis apparatus, electrotransfer apparatus (BioRad), exposure apparatus (Tecan), spectrophotometer (ThermoFisher Scientific), etc., were all provided by the Animal Biosafety and Public Health Prevention and Control Team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0047] 1.1.3 Gene and protein sequences

[0048] The nucleotide sequence of the Pasteurella multocida OmpW gene is shown in SEQ ID NO.1, and the amino acid sequence of the Pasteurella multocida OmpW protein is shown in SEQ ID NO.2.

[0049] SEQ ID NO.1:

[0050] ATGAAAAAGACAGTATTAGCATTAGGTGTGATGGCAGCATTAGTTGCAGGTTCGGCAGTGGCACATCAAGCAGGTAGTGTGATTGTGCGAGGTGGTCCAATTTTAGTAGTCCCAAACGCGTCGACGAATCATGATGTGTTTAAGTTTGATGTCAATTCCAACGCACAATTAGGCTTAACCGCCACTTATATGGCAACAGATAATCTTGGTGTTGAGTTATTAGCGGCGACACCATTCAGCCATGAGATTACATTAGGCAATACGCTTGTTGGTAAAACGAAGCATTTACCGCCAAGTTTATATGCACAATATTATTTCTTAGACAAAGATGCGAAAGCTCGTCCTTATGTGGGGGCGGGTGTTAACTACACCACATTCTTTAGTGAAAAAGCCGTATTAAATGGCGTGACTGATCTGAAATTAAAAGATTCTTGGGGACCTGCGTTCAATGCTGGGGTAGATATCCAAGTCGCAGATAACTTATTTTTAAATACAGCTATTTGGTATGCAAAAATCAAAAGTAAGGCCACATTTAAACTTGGCGGTGAAGAGCATAAAGTCAATGTGAAATTAGATCCAACGGTGTTCTTTGTTGGCTTAGGTTATCGTTTTTAA.

[0051] SEQ ID NO.2:

[0052] MKKTVLALGVMAALVAGSAVAHQAGSVIVRGGPILVVPNASTNHDVFKFDVNSNAQLGLTATYMATDNLGVELLAATPFSHEITLGNTLVGKTKHLPPSLYAQYYFLDKDAKARPYVGAGVNYTTFFSEKAVLNGVTDLKLKDSWGPAFNAGVDIQVADNLFLNTAIWYAKIKSKATFKLGGEEHKVNVKLDPTVFFVGLGYRF.

[0053] 1.2 Method

[0054] 1.2.1 Primer Design and Synthesis

[0055] Based on the OmpW gene sequence of Pasteurella multocida, full-length amplification primers for OmpW were designed using SnapGene software: His-OmpW-F and His-OmpW-R, which were synthesized by Beijing Qingke Biotechnology Co., Ltd. The specific sequences are shown in Table 1.

[0056] Table 1 Primer sequences for amplifying the OmpW gene

[0057]

[0058] 1.2.2 Construction of pColdII-10His-OmpW recombinant plasmid

[0059] Using the *Pasteurella multocida* genome as a template, the OmpW gene fragment was amplified using the primer pair His-OmpW-F / R. The PCR reaction system consisted of 50 μL, including 25 μL PrimeSTAR Max DNA Polymerase, 20 μL sterile enzyme-free water, 2 μL each of the primers, and 1 μL of template. After mixing all components, the mixture was centrifuged cis-wise. The PCR amplification program was as follows: pre-denaturation at 98℃ for 2 min, denaturation at 98℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, for 35 cycles, followed by a final extension at 72℃ for 5 min, and storage at 4℃.

[0060] The empty pColdII-10His vector plasmid was digested with KpnI and HindIII in a total digestion volume of 50 μL, including 5 μL of 10×rCutsmart, 1 μg of the empty pColdII-10His vector plasmid, 1 μL each of KpnI and HindIII restriction enzymes, and ddH2O was added to bring the total volume to 50 μL. After mixing the above components, the mixture was centrifuged cis-wise and placed in a 37°C water bath for 2 h of digestion. Subsequently, the OmpW gene amplification product and the digestion product were analyzed by agarose gel electrophoresis. After excising the correctly sized bands, the DNA was recovered from the agarose gel.

[0061] The recovered OmpW gene fragment was ligated with the linearized pColdII-10His empty vector in a 20 μL ligation system, including 4 μL of 5×CE MultiS Buffer, 2 μL of Exnase MultiS, 0.03 pmol each of the linearized vector and the OmpW gene fragment, and ddH2O was added to bring the total volume to 20 μL. After mixing the above components, the mixture was centrifuged cis-centrifuged and placed in a 37°C water bath for 30 min. After the reaction was completed, the mixture was immediately placed on ice to cool.

[0062] Next, 10 μL of the above ligation product was transformed into E. coli Trans5α competent cells. Single clones were picked and cultured. PCR identification was performed using primer pair (M13-F / R, see Table 2). Positive bacterial samples were sequenced. Plasmids were extracted from the correctly sequenced bacterial samples. The extracted plasmid was named pColdII-10His-OmpW. After the concentration was detected, it was stored at -20℃.

[0063] Table 2 Primer sequences for identifying the pColdII-10His vector

[0064]

[0065] 1.2.3 Prokaryotic expression, identification, and purification of OmpW protein

[0066] The pColdII-10His-OmpW plasmid was transformed into E. coli BL21 competent cells, and positive clones were screened. The positive clones were then cultured on a shaker at 37°C until the bacterial culture reached OD500. 600 When the value reached 0.6, IPTG at a final concentration of 0.25 mM was added to induce protein expression. Induction was performed overnight at 16°C and 150 rpm for 16 h. The induced bacterial culture was collected and centrifuged at 6000 rpm and 4°C for 10 min, the supernatant was discarded, the bacterial pellet was resuspended, and the mixture was sonicated at low temperature. Then, it was centrifuged at 12000 rpm and 4°C for 20 min. The supernatant and pellet were collected separately, and their expression was identified using SDS-PAGE and Western blot. A large amount of supernatant expressing the OmpW recombinant protein was collected, purified using a nickel column, and the purified protein was collected and its concentration determined.

[0067] 1.2.4 Optimization of Conditions for Indirect ELISA Method

[0068] Indirect ELISA method:

[0069] (1) Dilute the serum sample with diluent and mix thoroughly to obtain the sample solution;

[0070] (2) Add 100 μL of diluent, negative control and positive control to the enzyme label wells respectively, and perform replicate detection experiments, and then incubate at 37°C;

[0071] Negative control: Negative control serum containing antibodies against Pasteurella multocida that has not been infected;

[0072] Positive control: Pasteurella multocida OmpW protein antibody positive control serum.

[0073] (3) Discard the unbound sample solution and rinse 3 times with 200 μL of washing solution; the washing solution is PBST buffer.

[0074] (4) After diluting the enzyme-labeled secondary antibody (GoatAnti-Mouse IgG(H+L), HRP Conjugate) with diluent, add 100 μL of the diluted enzyme-labeled secondary antibody to each reaction well and repeat step (3) for washing.

[0075] (5) Add 200 μL TMB substrate solution to each reaction well and incubate at 37°C in the dark.

[0076] (6) Add 50 μL of stop solution (2 mol / L H2SO4 solution) to each reaction well, incubate, and then measure the absorbance of each reaction well at 450 nm using an ELISA reader.

[0077] (7) Result interpretation: OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 450 The average (X) and standard deviation (SD) of the sample are calculated. The positive cutoff value (X+3×SD) is calculated. The sample is considered positive when the S / P value is greater than X+3×SD, and negative when it is less than X+3×SD.

[0078] Optimize the different conditions in the above detection method:

[0079] The optimal reaction conditions were determined using a checkerboard method by comparing the OD values ​​of positive and negative control samples. 450 The optimal coating concentrations (16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL) and serum dilutions (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200) of OmpW protein were determined using the P / N ratio. Then, the optimal blocking solutions (5% skim milk powder, 10% skim milk powder, 1% BSA, 3% fish gelatin) and blocking times (30 min, 60 min) of OmpW protein were determined using a square matrix titration method. The incubation times for primary antibodies were 30 min, 60 min, 90 min, and 120 min, respectively. The dilutions for secondary antibodies were 1:10000, 1:20000, 1:30000, 1:40000, 1:50000, and 1:60000. The protein coating buffer was prepared by dissolving 0.159 g of Na₂CO₃ and 0.293 g of NaHCO₃ in 100 mL of deionized water.

[0080] 1.2.5 Establishment of Judgment Criteria for Indirect ELISA Method

[0081] Based on the established optimal reaction conditions, positive and negative control samples and 30 Pasteurella multocida-negative serum samples were tested, and OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 450The average (X) and standard deviation (SD) of the sample are calculated. The positive cutoff value (X+3×SD) is calculated. The sample is considered positive when the S / P value is greater than X+3×SD, and negative when it is less than X+3×SD.

[0082] 1.2.6 Specificity Detection

[0083] The indirect ELISA method established in 1.2.4 was used to detect positive control samples of Pasteurella multocida, Brucella, Mycobacterium paratuberculosis, Clostridium perfringens, and Escherichia coli antibodies, and duplicate wells were set up to observe whether OmpW protein had cross-reactivity with other pathogens in order to determine the specificity of this method.

[0084] 1.2.7 Sensitivity Detection

[0085] Positive control samples of Pasteurella multocida were serially diluted at ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, and then detected using the indirect ELISA method established in section 1.2.4. A negative control was also included. OD values ​​were read using a microplate reader. 450 The value is determined by comparing the P / N value (average OD of positive control samples). 450 Average OD of negative control products 450 Determine the sensitivity of the method.

[0086] 1.2.8 Repeatability Test

[0087] Intra-batch repeatability testing: A batch of ELISA plates were coated according to the indirect ELISA method established in 1.2.4. The plates were blocked and washed the following day. Then, 90 μL of 50% glycerol was added to each well, and the plates were incubated at room temperature for 10 min. The glycerol was then removed, and the plates were vacuum-sealed at 4°C. Positive and negative controls for *Pasteurella multocida* were selected. On days 1, 3, and 5, a stored ELISA plate was used to test the serum samples, with duplicate wells provided. After testing, the OD values ​​of each well were read using an ELISA reader. 450 The value is used to calculate the coefficient of variation within the batch using the formula:

[0088] Coefficient of variation (CV) = (SD ÷ X) × 100%.

[0089] Inter-batch repeatability testing: Three different batches of ELISA plates were prepared according to the established indirect ELISA method. Each plate was used to test for the selected Pasteurella multocida positive and negative controls, and duplicate wells were included. After testing, the OD values ​​of each well were read using an ELISA reader. 450 The value is calculated using the formula: Coefficient of Variation (CV) = (SD ÷ X) × 100%. The inter-batch coefficient of variation is then calculated.

[0090] 2 Results

[0091] 2.1 Construction of pColdII-10His-OmpW recombinant plasmid

[0092] To amplify the OmpW gene fragment, PCR amplification was performed using the *Pasteurella multocida* genome as a template and primer pair His-OmpW-F / R. The results showed that the OmpW gene fragment, approximately 600 bp in size, was successfully amplified. Figure 1 (A). To linearize the empty pColdII-10His vector, the pColdII-10His empty vector plasmid was digested with KpnⅠ and HindⅢ. The results showed that the vector was successfully linearized. Figure 1 (B). To identify positive recombinant plasmids, PCR was performed on single clones of the recombinant plasmids using primer pair (M13-F / R). The results showed that clones 1, 2, and 3 were all positive clones. Figure 1 (C)

[0093] 2.2 Expression, identification and purification of OmpW protein

[0094] To identify the expression of OmpW protein, the supernatant and precipitate of pColdII-10His-OmpW prokaryotic bacterial culture were analyzed after ultrasonic disruption. Western blot results showed that OmpW protein was expressed in both the supernatant and the precipitate. Figure 2 SDS-PAGE results showed that OmpW protein could be expressed both solublely and in inclusion bodies, and the expression levels of both were comparable. To purify the OmpW protein, the supernatant after sonication was purified using a nickel column. SDS-PAGE results confirmed successful purification of the OmpW protein.

[0095] 2.3 Optimization of Conditions for Indirect ELISA Method

[0096] Using the maximum P / N value as the criterion for judging the experimental results, the reaction conditions of the indirect ELISA method were optimized. The optimal coating concentration of OmpW protein was determined to be 8 μg / mL, serum dilution concentration was 1:400, optimal blocking solution was 10% skim milk, blocking time was 2 h, primary antibody incubation time was 1 h, secondary antibody dilution was 1:20000, secondary antibody incubation time was 30 min, and color development time was 10 min.

[0097] Table 3 Results of optimal protein coating concentration and optimal serum dilution for ELISA

[0098]

[0099] 2.4 Determination of Judgment Criteria for Indirect ELISA Methods

[0100] To determine the cut-off value for interpreting the results of the established indirect ELISA method, positive and negative control samples and 30 negative serum samples of Pasteurella multocida were tested, and OD values ​​were read. 450 The average S / P value of 30 Pasteurella multocida sera was calculated to be 0.112, with a standard deviation of 0.058. The positive cutoff value was calculated to be 0.286 according to the formula Cut-off = X + 3 × SD. Considering factors such as error, and on the premise that the test results are reliable, a test sample is considered positive when the S / P value is greater than or equal to 0.3, and negative when it is less than 0.3.

[0101] 2.5 Specificity Detection

[0102] To demonstrate the high specificity of this ELISA method for Pasteurella multocida antibodies, positive controls for Pasteurella multocida, Brucella, Mycobacterium paratuberculosis, Clostridium perfringens, and Escherichia coli antibodies were tested with duplicate wells. The results showed that OmpW protein reacted strongly only with the positive controls for Pasteurella multocida; the S / P values ​​of other sera were all below the critical value of 0.3. Figure 3 ).

[0103] 2.6 Sensitivity Testing

[0104] To test the sensitivity of this indirect ELISA method, positive control samples of Pasteurella multocida were serially diluted at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, and then detected using the established indirect ELISA method. A negative control was also included. The results showed that a positive result was still obtained at a dilution of 1:1600, indicating that the indirect ELISA method has good sensitivity.

[0105] 2.7 Repeatability Test

[0106] To verify the intra-assay reproducibility of this indirect ELISA method, a batch of microplates was coated once, and positive and negative controls of the same batch of Pasteurella multocida were tested on days 1, 3, and 5, with replicate wells included. After the assay, the OD values ​​of each well were read using a microplate reader. 450 The calculated intra-batch coefficient of variation was between 1.79% and 7.54%, less than 10%, demonstrating that the method has good intra-batch repeatability.

[0107] To verify the batch-to-batch reproducibility of this indirect ELISA method, three different batches of ELISA plates were prepared in three separate runs. The selected Pasteurella multocida positive and negative controls were simultaneously tested, with duplicate wells provided. After testing, the OD values ​​of each well were read using an ELISA reader. 450The calculated inter-batch coefficient of variation was between 2.23% and 6.91%, less than 10%, demonstrating that the method has good inter-batch repeatability.

[0108] Example 2

[0109] An indirect ELISA kit for detecting Pasteurella multocida includes the following components:

[0110] ELISA plate: an ELISA plate coated with the OmpW protein prepared in Example 1, with an antigen concentration of 8 μg / mL, a blocking solution of 10% skim milk powder, and a blocking time of 2 h;

[0111] Enzyme-labeled secondary antibody: Goat Anti-Mouse IgG (H+L), HRP Conjugate;

[0112] Diluent: PBST buffer;

[0113] Washing buffer: PBS buffer containing 0.1% Tween 20;

[0114] Colorimetric solution: TMB solution;

[0115] Stop solution: 2 mol / L H2SO4 solution;

[0116] Negative control: Negative control serum containing antibodies against Pasteurella multocida that has not been infected;

[0117] Positive control: Pasteurella multocida OmpW protein antibody positive control serum.

[0118] Example 3

[0119] Thirty clinical serum samples from a cattle farm were tested using this indirect ELISA method. Simultaneously, samples were also tested using the method described in "Diagnostic Techniques for Bovine Hemorrhagic Septicemia (GB / T27530-2025)". The concordance rate between the two methods was analyzed. Results showed that among the 30 clinical samples, 4 were positive and 26 were negative when tested using the method described in "Diagnostic Techniques for Bovine Hemorrhagic Septicemia (GB / T27530-2025)". Using this indirect ELISA method, 3 were positive and 27 were negative, with only 1 positive sample being interpreted as negative. The positive concordance rate was 75.00%, and the overall concordance rate was 96.67% (29 / 30).

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An indirect ELISA kit for detecting antibodies against Pasteurella multocida, characterized in that, Enzyme-labeled plates including those coated with antigens; The antigen is a recombinant OmpW protein, the amino acid sequence of which is shown in SEQ ID NO.2; The method for preparing the recombinant OmpW protein includes the following steps: Will OmpW The gene was homologously recombinated with the pColdII-10His plasmid to obtain the recombinant plasmid pColdII-10His-OmpW; The recombinant plasmid pColdII-10His-OmpW was transformed into competent Escherichia coli cells to obtain the recombinant expression strain; After inducing expression culture of the recombinant expression strain, the recombinant OmpW protein was obtained through isolation and purification. The induction culture was performed using IPTG at a final concentration of 0.25 mM to induce protein expression. The induction conditions were 16°C and 150 rpm overnight for 16 h. The OmpW The gene nucleotide sequence is shown in SEQ ID NO.

1.

2. The indirect ELISA kit according to claim 1, characterized in that, The method for preparing the enzyme-labeled plate coated with the antigen includes the following steps: The antigen was diluted with a coating buffer to obtain a suspension. The suspension was added to the enzyme label wells for coating, and then a blocking buffer was added for blocking to obtain the enzyme label plate coated with the antigen.

3. The indirect ELISA kit according to claim 2, characterized in that, The concentration of the recombinant OmpW protein in the suspension was 8 µg / mL.

4. The indirect ELISA kit according to claim 2, characterized in that, The sealing liquid is 10% skim milk powder.

5. The indirect ELISA kit according to claim 1, characterized in that, The indirect ELISA kit also includes enzyme-labeled secondary antibody, diluent, washing solution, chromogenic solution, stop solution, negative control and positive control.

6. The indirect ELISA kit according to claim 5, characterized in that, The diluent is PBST buffer; and / or The washing solution is a PBS buffer containing 0.1% Tween 20.

7. The indirect ELISA kit according to claim 5, characterized in that, The colorimetric solution is a TMB solution.

8. The indirect ELISA kit according to claim 5, characterized in that, The terminating solution is a 2 mol / L H2SO4 solution.

9. A method for detecting Pasteurella multocida antibodies for non-disease diagnostic purposes, characterized in that, The steps include detecting Pasteurella multocida antibodies using the indirect ELISA kit according to any one of claims 1-8: Add negative control, positive control and test sample solution to the enzyme-labeled plate coated with antigen, and perform the first incubation reaction; Add enzyme-labeled secondary antibody to each reaction well and perform a second incubation reaction; Add the colorimetric solution to each reaction well and continue incubation for 10 min; Add stop solution to each reaction well, measure the absorbance at 450 nm using an ELISA reader, and interpret the results.

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