Bovine pasteurellosis indirect ELISA detection kit and detection method

Abstract

The invention discloses a bovine pasteurellosis indirect ELISA (enzyme-linked immuno sorbent assay) detection kit and a detection method, and relates to the technical field of biology. The indirect ELISA kit comprises an ELISA plate coated with an antigen, the amino acid sequence of the antigen is as shown in SEQ ID NO. 2. The indirect ELISA detection kit constructed by the invention is strong in specificity and good in sensitivity; the intra-batch variation coefficient ranges from 1.79% to 7.54%, the inter-batch variation coefficient ranges from 2.23% to 6.91%, both the intra-batch variation coefficient and the inter-batch variation coefficient are lower than 10%, and inter-batch and intra-batch repeatability is good; 30 clinical samples are detected, and the total coincidence rate is 96.67% compared with the cattle hemorrhagic septicemia diagnosis technology (GB / T27530-2025). The indirect ELISA detection kit is good in comprehensive evaluation and can be applied to clinical serum sample detection of bovine pasteurella multocida.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an indirect ELISA detection kit and a detection method for bovine Pasteurellosis multocida. Background Art

[0002] Pasteurella multocida (Pm) is a Gram-negative pathogen that is coccobacilli or short rods, lacks flagella, and does not form spores. It is an aerobic or facultative anaerobic bacterium. Serotypes are distinguished based on capsular antigens and somatic antigens, with the former comprising five types and the latter at least 16 types. The bacterium has a wide range of host infections, infecting a variety of animals and humans, causing varying degrees of damage. Although serotype-specific inactivated vaccines and antibiotics such as penicillin and florfenicol can effectively control P. multocida infections, increasing antimicrobial resistance and the risk of cross-host transmission continue to 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, as well as for 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 toxins, pili, and superoxide dismutase. After infection, the host rapidly produces high-titer antibodies against bacterial proteins, making serological testing an important basis for diagnosing the disease. Outer membrane proteins, as surface components of the bacteria, participate in the bacteria's adoptive response to adverse environments, can adhere to host cells to enhance pathogenicity, and facilitate the absorption and transport of nutrients. Due to their strong antigenicity and immunogenicity, they have become important targets for vaccine development and diagnostic methods. The present invention optimizes expression conditions to achieve soluble expression of the OmpW protein, ensuring its native conformation. A new indirect ELISA kit for bovine Pasteurella multocida detection based on the recombinant OmpW protein is proposed, providing technical support for the rapid, sensitive, and accurate diagnosis of bovine Pasteurella multocida. Summary of the Invention

[0004] The present invention aims to provide an indirect ELISA detection kit and method for bovine Pasteurella multocida to address the problems of the prior art. The indirect ELISA detection kit has good specificity, sensitivity, and repeatability and can be used for the detection of bovine Pasteurella multocida in clinical serum samples.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an indirect ELISA kit for detecting antibodies to bovine Pasteurella multocida, comprising an ELISA plate coated with an antigen;

[0007] The antigen is recombinant OmpW protein, and its amino acid sequence is shown in SEQ ID NO.2.

[0008] Furthermore, the preparation method of the recombinant OmpW protein comprises 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 a recombinant expression strain;

[0011] After inducing expression culture on the recombinant expression strain, protein purification is performed to obtain the recombinant OmpW protein;

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

[0013] Furthermore, the method for preparing the antigen-coated ELISA plate comprises the following steps:

[0014] The antigen is diluted with a coating solution to obtain a suspension, and the suspension is added to an enzyme-labeled well for coating treatment. A blocking solution is then added for blocking treatment to obtain the antigen-coated enzyme-labeled plate.

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

[0016] Furthermore, the blocking solution is 10% skim milk powder.

[0017] Furthermore, the indirect ELISA kit also includes an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a stop solution, a negative quality control product, and a positive quality control product.

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

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

[0020] Furthermore, the color developing solution is TMB solution.

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

[0022] The present invention also provides a method for detecting antibodies to Pasteurella multocida in cattle for non-disease diagnosis purposes, comprising the steps of detecting antibodies to Pasteurella multocida in cattle using the above-mentioned indirect ELISA kit:

[0023] Add negative control, positive control and sample solution to the antigen-coated ELISA plate respectively for the first incubation reaction;

[0024] Enzyme-labeled secondary antibody was added to each reaction well for a second incubation reaction;

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

[0026] Add stop solution to each reaction well, measure the absorbance value at a wavelength of 450nm using an enzyme reader, and judge the result.

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

[0028] The present invention cloned the OmpW gene of bovine Pasteurella multocida into the vector pColdII-10His to construct the prokaryotic expression vector pColdII-10His-OmpW. The purified OmpW protein was purified using a nickel column. Conditions were optimized, and an indirect ELISA method for detecting Pasteurella antibodies was established using the purified OmpW protein as a coating antigen. The method was tested for specificity, sensitivity, and reproducibility, and clinical samples were tested. Results showed that after overnight induction with 0.25 mM IPTG at 16°C and 150 rpm for 16 hours, OmpW was soluble, as determined 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°C for 2 h, the test serum was diluted 1:400 and incubated at 37°C for 1 h, the enzyme-labeled secondary antibody was diluted 1:20,000 and incubated at 37°C for 30 min, and the color was developed for 10 min, the positive and negative critical value was 0.3, an indirect ELISA detection method for bovine Pasteurella multocida could be established. The present invention also found that the established indirect ELISA method only reacts with Pasteurella multocida-positive serum, has no cross-reaction with other sera, and has strong specificity; when the serum dilution is 1:1600, the test result is still positive, and the sensitivity is good; the intra-batch coefficient of variation is between 1.79% and 7.54%, and the inter-batch coefficient of variation is between 2.23% and 6.91%, both of which are lower than 10%, and the inter-batch and intra-batch repeatability are good; 30 clinical samples were tested, and compared with the "Diagnostic Technology for Bovine Hemorrhagic Sepsis (GB / T27530-2025)", the overall compliance rate was 96.67%.

[0029] In summary, the indirect ELISA detection kit and method for bovine Pasteurella multocida established in the present invention have a good comprehensive evaluation and can be applied to the detection of bovine Pasteurella multocida in clinical serum samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

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

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

[0033] Figure 3 It is the specific detection result of the ELISA method of the present invention. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting 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 terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

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

[0039] Example 1 Establishment and Optimization of Indirect ELISA Detection Method for Pasteurella multocida in Bovine

[0040] 1 Materials and Methods

[0041] 1.1 Materials

[0042] 1.1.1 Strains, serum, and vectors

[0043] Pasteurella multocida (Pm), clinical sera, bovine Pm-positive quality control, and bovine Pm-negative quality control were provided by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. Serum positive for antibodies to bovine Brucella, Mycobacterium paratuberculosis, Clostridium perfringens, and Escherichia coli, and the pColdII-10His vector were 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.

[0044] 1.1.2 Main reagents and instruments

[0045] PrimeSTAR Max DNA Polymerase was purchased from TAKARA Biotechnology (Beijing) Co., Ltd. (TAKARA), TME.ZNAGel Extraction Kit was purchased from Omega Bio-Tek, USA, ClonExpress MultiS OneStep Cloning Kit, 2×Rapid Taq Master Mix was purchased from Nanjing Novozymes Biotechnology Co., Ltd., plasmid minipreparation and midi kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., IPTG, gel preparation kit, PAGE gel staining solution, and BSA were all purchased from Beijing Solebao Company, purified beads were purchased from Tiandirenhe Company, skim milk powder was purchased from BD Company, primary antibody diluent, His-tagged primary antibody, and HRP-labeled secondary antibody were all purchased from Beyotime Company, and exposure solution, TMB color development solution, and 96-well detachable ELISA plate were all purchased from ThermoFisher Scientific.

[0046] PCR amplifier T100 Thermal Cycler (Bio-Rad), NanoDrop One (ThermoFisherScientific), nucleic acid electrophoresis instrument, vertical electrophoresis instrument, electroblotting instrument (BioRad), exposure instrument (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, SnapGene software was used to design the full-length OmpW amplification primers: His-OmpW-F and His-OmpW-R. These primers 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. A 50 μL PCR reaction system consisted of 25 μL PrimeSTAR Max DNA Polymerase, 20 μL sterile enzyme-free water, 2 μL of each primer, and 1 μL of template. The mixture was then centrifuged. The PCR amplification procedure included 35 cycles of initial denaturation at 98°C for 2 minutes, denaturation at 98°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 30 seconds. The final extension was at 72°C for 5 minutes and stored at 4°C.

[0060] The pColdII-10His empty vector plasmid was double-digested with Kpn I and Hind III. The total digestion system (50 μL) included 5 μL of 10× rCutsmart, 1 μg of the pColdII-10His empty vector plasmid, and 1 μL each of Kpn I and Hind III endonucleases. The volume was brought up to 50 μL with ddH2O. The above components were mixed and centrifuged, and the digestion reaction was carried out in a 37°C water bath for 2 hours. The OmpW gene amplification product and the digestion product were then analyzed by agarose gel electrophoresis. The band of the correct size was excised and DNA was recovered from the agarose gel.

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

[0062] Then, 10 μL of the above ligation product was transformed into Escherichia coli Trans5α competent cells, and a single clone was picked for bacterial culture. PCR identification was performed using a primer pair (M13-F / R, see Table 2), and the positive bacterial sample was sequenced. The plasmid of the bacterial sample with correct sequencing was extracted and named pColdII-10His-OmpW. After the concentration was detected, it was stored at -20°C.

[0063] Table 2 Primer sequences for identifying 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. Then, the positive clones were expanded on a 37°C constant temperature shaker. When the bacterial solution OD 600 When the value reached 0.6, IPTG was added to a final concentration of 0.25 mM to induce protein expression. Induction was carried out overnight at 16°C, 150 rpm, for 16 hours. The induced bacterial suspension was collected and centrifuged at 6000 rpm, 4°C, for 10 minutes, and the supernatant discarded. The bacterial pellet was resuspended and disrupted by sonication at low temperature. The suspension was then centrifuged at 12000 rpm, 4°C, for 20 minutes. The supernatant and pellet were collected, and expression was analyzed by SDS-PAGE and Western blot. The supernatant expressing the recombinant OmpW protein was collected and purified on a nickel column. The purified protein was then collected and its concentration was determined.

[0067] 1.2.4 Optimization of indirect ELISA conditions

[0068] Indirect ELISA method:

[0069] (1) diluting the serum sample with a diluent and mixing thoroughly to obtain a sample solution;

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

[0071] Negative quality control: negative quality control serum containing antibodies that are not infected with Pasteurella multocida;

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

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

[0074] (4) Dilute the enzyme-labeled secondary antibody (Goat Anti-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 of 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. After incubation, measure the absorbance of each reaction well at a wavelength of 450 nm using a microplate reader.

[0077] (7) Result determination: OD was measured using an enzyme-labeled instrument 450 The values ​​were calculated, the mean (X) and standard deviation (SD) were calculated, and the positive critical value (X+3×SD) was calculated. When the S / P value of the test sample was greater than X+3×SD, it was judged to be positive, otherwise it was negative.

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

[0079] The chessboard method was used to find the best reaction conditions by comparing the OD values ​​of positive control products with those of negative control products. 450 The optimal coating concentration (16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL) and serum dilution (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200) of OmpW protein were determined by the ratio (P / N value). The optimal blocking solution (5% skim milk powder, 10% skim milk powder, 1% BSA, 3% fish gelatin) and blocking time (30 min, 60 min) of OmpW protein were determined by the square array titration method. The following parameters were used: incubation time (1:10,000, 1:20,000, 1:30,000, 1:40,000, 1:50,000, 1:60,000), incubation time (30, 60, 90, 120 minutes), primary antibody incubation time (30, 60, 90, 120 minutes), secondary antibody dilution (1:10,000, 1:20,000, 1:30,000, 1:40,000, 1:50,000, 1:60,000), secondary antibody incubation time (30, 60, 90, 120 minutes), and color development time (5, 10, 15, 20 minutes). The protein coating solution was prepared by fully dissolving 0.159 g Na2CO3 and 0.293 g NaHCO3 in 100 mL of deionized water.

[0080] 1.2.5 Establishment of the judgment criteria for the indirect ELISA method

[0081] According to the established optimal reaction conditions, the positive and negative control samples and 30 Pasteurella multocida negative sera were tested, and the OD was measured using a microplate reader. 450The values ​​were calculated, the mean (X) and standard deviation (SD) were calculated, and the positive critical value (X+3×SD) was calculated. When the S / P value of the test sample was greater than X+3×SD, it was judged to be positive, otherwise it was negative.

[0082] 1.2.6 Specificity Detection

[0083] According to the indirect ELISA method established in 1.2.4, the positive control material for bovine Pasteurella multocida, Brucella, Mycobacterium paratuberculosis, Clostridium perfringens, and Escherichia coli antibody-positive serum were tested with duplicate wells to observe whether the OmpW protein cross-reacts with other pathogens to determine the specificity of this method.

[0084] 1.2.7 Sensitivity testing

[0085] Take the positive control sample of Pasteurella multocida in cattle and dilute it in series according to 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, and then test it according to the indirect ELISA method established in 1.2.4. At the same time, set up a negative control. Read the OD value using a microplate reader. 450 By comparing the P / N value (average OD of positive control 450 / Average OD of negative control products 450 ) to determine the sensitivity of the method.

[0086] 1.2.8 Repeatability test

[0087] Repeatability test within the batch: Coat a batch of enzyme-labeled plates according to the indirect ELISA method established in 1.2.4, block and wash the next day, then add 90 μL of 50% glycerol to each well, incubate at room temperature for 10 minutes, shake off the glycerol and store in a vacuum at 4°C. Select positive and negative control products of bovine Pasteurella multocida, and select a preserved enzyme-labeled plate on the first, third and fifth days to test the above serum, and set up duplicate wells. After the test is completed, use an enzyme reader to read the OD of each well. 450 The intra-batch coefficient of variation was calculated according to the formula:

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

[0089] Inter-batch repeatability test: Prepare three different batches of enzyme-labeled plates according to the established indirect ELISA method. Each enzyme-labeled plate is tested with the above-selected bovine Pasteurella multocida positive and negative quality control products and repeated wells are set. After the test is completed, the OD value of each well is read using a microplate reader. 450 The coefficient of variation between batches was calculated according to the formula: coefficient of variation CV = (SD ÷ X) × 100%.

[0090] 2 Results

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

[0092] To amplify the OmpW gene fragment, the genome of Pasteurella multocida was used as a template and the primer pair His-OmpW-F / R was used for PCR amplification. The results showed that an OmpW gene fragment of approximately 600 bp was successfully amplified ( Figure 1 In order to linearize the pColdII-10His empty vector, the pColdII-10His empty vector plasmid was double-digested with KpnⅠ and HindⅢ. The results showed that the vector was successfully linearized ( Figure 1 In order to identify the positive recombinant plasmid, the primer pair (M13-F / R) was used to perform PCR identification on the single clones of the recombinant plasmid. The results showed that clones 1, 2, and 3 were all positive clones ( Figure 1 Middle C).

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

[0094] In order to identify the expression of OmpW protein, the supernatant and precipitate of the prokaryotic expression bacterial solution of pColdII-10His-OmpW 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 was expressed in both soluble and inclusion bodies, with comparable expression levels. To purify the OmpW protein, the supernatant after ultrasonication was passed through a nickel column for purification. SDS-PAGE results confirmed that the OmpW protein was successfully purified.

[0095] 2.3 Optimization of indirect ELISA conditions

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

[0097] Table 3 ELISA optimal protein coating concentration and optimal serum dilution results

[0098]

[0099] 2.4 Determination of the judgment criteria for the indirect ELISA method

[0100] In order to determine the cut-off value of the established indirect ELISA method, positive and negative control samples and 30 bovine Pasteurella multocida negative sera were tested and the OD 450 The average S / P value of 30 Pasteurella multocida sera was 0.112, and the standard deviation was 0.058. The positive critical value was calculated according to the formula Cut-off = X + 3 × SD to be 0.286. Taking into account factors such as error, and on the premise of confirming the reliability of the test results, when the S / P value of the test sample is greater than or equal to 0.3, it is judged as positive, and when it is less than 0.3, it is judged as negative.

[0101] 2.5 Specificity detection

[0102] In order to prove whether this ELISA method is highly specific for Pasteurella multocida antibodies, the positive serum samples of bovine Pasteurella multocida, Brucella, Mycobacterium paratuberculosis, Clostridium perfringens and Escherichia coli antibodies were tested and repeated wells were set. The results showed that OmpW protein reacted strongly only with the positive control sample of bovine Pasteurella multocida, and the S / P values ​​of other sera were lower than the critical value of 0.3 ( Figure 3 ).

[0103] 2.6 Sensitivity testing

[0104] To test the sensitivity of this indirect ELISA method, a positive control sample for Pasteurella multocida in cattle was serially diluted at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800. The sample was then tested according to the established indirect ELISA method, along with a negative control. The results showed that a positive result was still observed at a dilution of 1:1600, indicating that this indirect ELISA method has good sensitivity.

[0105] 2.7 Repeatability test

[0106] To verify the intra-batch reproducibility of the indirect ELISA method, a batch of enzyme-labeled plates were coated at one time, and the positive and negative quality control products of the same batch of bovine Pasteurella multocida were tested on the 1st, 3rd and 5th days, and duplicate wells were set. After the test was completed, the OD value of each well was read using an enzyme-labeled instrument. 450 The intra-batch coefficient of variation calculated according to the formula was between 1.79% and 7.54%, which was less than 10%, proving that the method had good intra-batch repeatability.

[0107] To verify the batch-to-batch reproducibility of the indirect ELISA method, three different batches of ELISA plates were prepared three times, and the positive and negative control samples of bovine Pasteurella multocida selected above were tested simultaneously and repeated wells were set. After the test was completed, the OD value of each well was read using a microplate reader. 450The inter-batch coefficient of variation calculated according to the formula was between 2.23% and 6.91%, which was less than 10%, proving that the method had good inter-batch repeatability.

[0108] Example 2 Assembly of an indirect ELISA kit for bovine Pasteurellosis multocida

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

[0110] ELISA plate: coated with the OmpW protein prepared in Example 1, with a coating 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 solution: PBS buffer containing 0.1% Tween 20;

[0114] Color development solution: TMB solution;

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

[0116] Negative quality control: negative quality control serum containing antibodies that are not infected with Pasteurella multocida;

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

[0118] Example 3 Cattle clinical sample detection

[0119] The indirect ELISA method was used to test 30 clinical serum samples from a cattle farm. The samples were also tested with reference to the method in the Diagnostic Technique for Bovine Hemorrhagic Sepsis (GB / T27530-2025). The compliance rates of the two methods were analyzed. The results showed that among the 30 clinical samples, 4 samples were positive and 26 samples were negative when tested with reference to the method in the Diagnostic Technique for Bovine Hemorrhagic Sepsis (GB / T27530-2025). Three samples were positive and 27 samples were negative when tested with the indirect ELISA method. Only one positive sample was interpreted as negative, resulting in a positive compliance rate of 75.00% and an overall compliance rate of 96.67% (29 / 30).

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

Claims

1. An indirect ELISA kit for detecting antibodies to Pasteurella multocida in cattle, characterized in that: including an ELISA plate coated with an antigen; The antigen is recombinant OmpW protein, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The indirect ELISA kit according to claim 1, wherein The preparation method of the recombinant OmpW protein comprises the following steps: The OmpW gene was homologously recombined 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 a recombinant expression strain; After inducing expression culture on the recombinant expression strain, the recombinant OmpW protein is obtained by separation and purification; The nucleotide sequence of the OmpW gene is shown in SEQ ID NO.

1.

3. The indirect ELISA kit according to claim 1, wherein The method for preparing the antigen-coated ELISA plate comprises the following steps: The antigen is diluted with a coating solution to obtain a suspension, and the suspension is added to an enzyme-labeled well for coating treatment. A blocking solution is then added for blocking treatment to obtain the antigen-coated enzyme-labeled plate.

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

5. The indirect ELISA kit according to claim 3, wherein The blocking solution is 10% skim milk powder.

6. The indirect ELISA kit according to claim 1, wherein The indirect ELISA kit also includes an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a stop solution, a negative quality control product, and a positive quality control product.

7. The indirect ELISA kit according to claim 6, wherein The diluent is PBST buffer; and / or The washing solution is PBS buffer containing 0.1% Tween 20.

8. The indirect ELISA kit according to claim 6, wherein The color developing solution is TMB solution.

9. The indirect ELISA kit according to claim 6, wherein The stop solution is 2 mol / L H2SO4 solution.

10. A method for detecting antibodies to Pasteurella multocida in cattle for non-disease diagnosis purposes, characterized in that: The method comprises the steps of detecting bovine Pasteurella multocida antibodies using the indirect ELISA kit according to any one of claims 1 to 9: Add negative control, positive control and sample solution to the antigen-coated ELISA plate respectively for the first incubation reaction; Enzyme-labeled secondary antibody was added to each reaction well for a second incubation reaction; Add color development solution to each reaction well and continue incubation for 10 min; Add stop solution to each reaction well, measure the absorbance value at a wavelength of 450nm using an enzyme reader, and judge the result.

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