An ELISA antibody detection method for serotype 2 Riemerella anatipestifer and its application

Abstract

The present invention relates to an ELISA antibody detection method for serotype 2 Riemerella anatipestifer and an application thereof. The method is based on a bacterial lipopolysaccharide coated plate, and an indirect ELISA method for serotype 2 Riemerella anatipestifer is established through optimization of optimal reaction conditions, determination of critical values, specificity tests, sensitivity tests, repeatability tests and clinical trials. Compared with the prior art, the present invention extracts serotype 2 Riemerella anatipestifer lipopolysaccharide by hot phenol water method and coats the ELISA plate. By optimizing reaction conditions such as antigen coating conditions, dilution multiple and incubation conditions of serum to be tested / rabbit anti-duck enzyme-labeled antibodies, and substrate color development time, an indirect ELISA method for detecting serotype 2 Riemerella anatipestifer antibodies is successfully established.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an ELISA antibody detection method for serotype 2 Riemerella anatipestifer and an application thereof. Background Art

[0002] Riemerella anatipestifer (RA) is a small Gram-negative rod that causes infection and pathogenicity in a variety of poultry such as ducks and geese. It is spore-free and has a capsule. It appears singly or in pairs, and occasionally in chains and filaments. It cannot grow in ordinary culture medium or MacConkey medium, but grows well on blood agar, chocolate agar, and tryptic soy agar (TSA) agar. RA infection is also known as duck infectious serositis, duck septicemia, duck plague syndrome, etc. It is more common in ducklings aged 1-8 weeks, especially ducklings aged 2-3 weeks are the most susceptible, and often cause large-scale illness and death in ducklings. The disease is a contact infectious disease, mainly transmitted through contaminated feed, drinking water, dust, droplets, etc., through the respiratory tract, digestive tract or skin wounds (especially the skin of the webbed feet). Since the first report of Riemerella anatipestifer disease in my country in 1982, [1] Since 1990, the disease has been prevalent in duck-raising areas, especially in Guangxi, Guangdong, Hunan, Hubei, Hainan and other places, causing serious impacts on the health of duck flocks and the economic benefits of the breeding industry. In addition, there have been reports of RA being isolated from chickens and geese. [2，3] . The disease occurs all year round, and is mainly manifested as acute or chronic sepsis. Clinically, it is mainly manifested by increased eye and nasal secretions, wheezing, coughing, diarrhea, ataxia and head and neck tremors. A few chronic cases have symptoms such as head and neck tilt. The lesions are characterized by fibrinous pericarditis, perihepatitis, airbag inflammation, meningitis and arthritis in some cases. RA infection is mainly diagnosed by clinical symptoms and autopsy changes, and confirmed by bacterial isolation and culture, biochemical identification and PCR [4] RA serotypes are relatively complex, with 21 RA serotypes reported. The dominant serotypes prevalent in my country are mainly serotypes 1, 2, 6, 7, and 10, of which serotype 2 is widely prevalent both at home and abroad. There is a lack of effective cross-protection between different RA serotypes. [5，6] Therefore, it is of great significance to prepare serotype-specific vaccines for RA infection with different serotypes for immune prevention in the prevention and treatment of Riemerella anatipestifer disease. [7，8] .

[0003] In addition, testing the titer of serotype-specific antibodies produced by the vaccine can be used to evaluate the effectiveness of the vaccine. To date, there has been no research report on the detection method of serotype 2 RA specific antibodies, nor has there been a commercialized RA serotype-specific antibody detection kit.

[0004] There are many methods for detecting RA antibodies, such as slide agglutination test, tube agglutination test, agar diffusion precipitation test, indirect hemagglutination test, immunofluorescence antibody technology, etc. [10-14] . These methods have their own advantages, disadvantages and scope of application. The slide agglutination test, tube agglutination test and agar diffusion precipitation test are mainly used for the identification of RA isolates and the detection of bacteria in diseased tissues. Although the tube agglutination test and precipitation test have the ability to quantitatively detect antibodies, their application scenarios are relatively limited. They are generally only used to determine the standard strains of each serotype and to conduct in-depth detection of isolates that are difficult to type using the slide agglutination test. Due to their low sensitivity and cumbersome operating procedures, these two methods are not suitable for large-scale serum sample testing and are difficult to meet the requirements of high efficiency and accuracy for large sample volume testing. Enzyme-linked immunosorbent assay (ELISA) is suitable for testing a large number of serum samples, and is more suitable for evaluating the immune effect of vaccines from the perspective of antibody levels.

[15] . Wu Caiyan

[16] et al. used the whole inactivated RA bacteria as antigen coating and established an indirect ELISA method. This method can be used for clinical diagnosis of RA infection and evaluation of immune effects, but it cannot detect serotype-specific RA antibodies.

[17] et al. selected recombinant outer membrane protein (P45N') as the coating antigen to detect RA.

[18] etc. et al. respectively expressed RA outer membrane protein A in prokaryotes, used OmpA recombinant protein as coating antigen to initially establish ELISA method and optimized indirect ELISA method, which can detect RA antibodies.

[20] et al. established an indirect immunofluorescence antibody test to detect RA antibodies.

[21] et al. successfully established a rapid, sensitive, and specific ELISA for detecting antibodies to type 1 RA. However, these methods lack specificity and cannot accurately measure the in vivo antibody levels of different serotypes of RA. Summary of the invention

[0005] The purpose of the present invention is to provide an ELISA antibody detection method for serotype 2 Riemerella anatipestifer and its application. The ELISA detection method for serotype 2 RA-specific antibodies can effectively detect specific antibodies in the serum of ducks infected with type 2 RA and immunized with RA, and can be used for epidemiological surveys of RA and evaluation of vaccine effectiveness.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The invention discloses an ELISA antibody detection method for serotype 2 Riemerella anatipestifer. The method is based on a bacterial lipopolysaccharide coated plate. Through optimization of optimal reaction conditions, determination of critical values, specificity tests, sensitivity tests, repeatability tests and clinical trials, an indirect ELISA detection method for serotype 2 Riemerella anatipestifer antibodies is established.

[0008] Furthermore, the optimization of the best reaction conditions includes optimizing the coating solution, coating time, coating concentration, dilution of the serum to be tested / enzyme-labeled antibody and incubation time, blocking time and color development time.

[0009] Furthermore, the optimal reaction condition is evaluated by the P / N value, where P is the OD of the serum to be tested. 450 Value, N is negative serum OD 450 value.

[0010] Furthermore, the specific method for determining the critical value is: calculate 80 negative serum OD 450 The mean (X) and standard deviation (SD) of the data were taken, with X+3SD as the critical value.

[0011] Furthermore, in the specificity test, serotype 2 NJ3, Yb2, RA serotype 1 CH3, RA serotype 6 SDJ-80, RA serotype 7 SDJ-69, serotype 10 HXb2, avian pathogenic Escherichia coli-positive duck serum, avian Pasteurella-positive duck serum, avian Salmonella-positive duck serum, reovirus-positive duck serum and duckling hepatitis-positive duck serum were selected, and duck positive serum and negative serum controls were set up.

[0012] Furthermore, in the sensitivity test, two positive sera of serotype 2 RA duck were selected and diluted 1:100-1:51200 times, and the standard positive sera of different dilution multiples were tested.

[0013] Furthermore, in the repeatability test, 5 positive sera and 5 negative sera of serum type 2 RA ducks were randomly selected, and the intra-batch repeatability test was performed using ELISA plates coated with the same batch and the inter-batch repeatability test was performed using ELISA plates coated with different batches, with 3 replicates set for each serum.

[0014] Furthermore, in the clinical trial, duck sera at 14, 28, 42, 56 and 70 days after immunization with RA serotype 2 NJ3 inactivated vaccine, and duck sera at 7 days after infection with RA serotype 1 WJ4, type 2 Yb2, type 10 HXb2 and undetermined serotype WH6 were tested.

[0015] Furthermore, the specific steps of the ELISA antibody detection method for serotype 2 Riemerella anatipestifer are as follows:

[0016] S1, extracting lipopolysaccharide of Riemerella anatipestifer serotype 2 strain by hot phenol water method, and using lipopolysaccharide of Riemerella anatipestifer serotype 2 strain as coating antigen;

[0017] S2, dilute the coating antigen with coating solution, add it into the ELISA plate for coating, and then block it with blocking solution;

[0018] S3, discard the blocking solution in step S2, add primary antibody for incubation, and then incubate with secondary antibody;

[0019] S4, discard the secondary antibody incubation solution in step S3, use TMB single-component colorimetric solution to develop color, add colorimetric stop solution to terminate the reaction;

[0020] S5, using an ELISA reader to measure the OD of the liquid in the ELISA plate well of step S4 450 Value, according to OD 450 The critical value determines negative or positive.

[0021] Furthermore, in step S2, the primary antibody is serum type 2 RA duck positive serum, and the secondary antibody is rabbit anti-duck enzyme-labeled antibody.

[0022] Furthermore, in step S5, the OD 450 The critical value is 0.151.

[0023] In addition, the present invention also provides an ELISA antibody detection kit for serotype 2 Riemerella anatipestifer, the kit comprising: an antigen-coated plate, a sample diluent, a washing solution, an enzyme-labeled antibody, a color developing solution, a stop solution, a positive control and a negative control; wherein,

[0024] The antigen-coated plate is an ELISA plate coated with lipopolysaccharide of Riemerella anatipestifer serotype 2 strain;

[0025] The sample diluent is PBS containing 5% skimmed milk powder;

[0026] The washing solution is PBST solution;

[0027] The enzyme-labeled antibody shown is a 10,000-fold diluted rabbit anti-duck enzyme-labeled antibody;

[0028] The color developing solution is a TMB single-component solution;

[0029] The stop solution is 2M sulfuric acid.

[0030] Furthermore, the coating concentration of lipopolysaccharide of Riemerella anatipestifer serotype 2 strain on the antigen-coated plate is 5 μg / mL.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] RA is a Gram-negative bacterium, and the lipopolysaccharide on its outer membrane surface is an important virulence factor and the main antigenic component. [22，23] Lipopolysaccharide is a substance composed of a carbohydrate part and a lipid part, lipid A, which are covalently linked. The carbohydrate part includes core polysaccharide and O-specific polysaccharide. O-specific polysaccharide appears as a ladder-like band composed of different molecular weights in SDS-PAGE silver staining.

[22] The core polysaccharide is genus-specific. The core polysaccharide of the same genus is basically the same, but the type, position, arrangement order and spatial configuration of the monosaccharide of the O-antigen are very different in different species.

[23] , which is an important component that determines the serotype specificity. The present invention uses the purified LPS of the serotype 2 RA strain as an antigen to coat the ELISA plate, which can specifically detect the serotype 2 RA antibodies. This method can be used to detect specific antibodies in the serum of ducks infected with the serotype 2 RA strain in clinical practice, serving clinical diagnosis and epidemiological surveys; and this method can be used to detect specific antibodies in the serum of ducks immunized with the serotype 2 RA inactivated vaccine, providing strong support and scientific basis for evaluating vaccine-induced immune responses.

[0033] The invention uses a hot phenol water method to extract lipopolysaccharide of Riemerella anatipestifer serotype 2 strain, and coats the ELISA plate. By optimizing the reaction conditions such as the antigen coating conditions, the dilution multiple and incubation conditions of the serum to be tested / rabbit anti-duck enzyme-labeled antibody, and the color development time of the substrate, an indirect ELISA method for detecting antibodies of Riemerella anatipestifer serotype 2 is successfully established.

[0034] The present invention detects 80 healthy non-immune duck sera and establishes a positive judgment standard of OD according to the average value (X) + 3 standard deviations (SD). 450 ≥0.151. The results of specificity test showed that the method was positive for the detection of Riemerella anatipestifer serotype 2 positive serum; the detection of Riemerella anatipestifer serotype 1, 6, 7 and 10 duck positive serum, avian pathogenic Escherichia coli positive duck serum, avian Pasteurella positive duck serum, avian Salmonella positive duck serum, reovirus positive duck serum and duckling hepatitis positive duck serum were all negative. The results of sensitivity test showed that the detection limit for positive duck serum was >50000 times dilution. The results of repeatability test showed that the inter-batch and intra-batch variation rates of the detection method were less than 10%. The detection results of the detection method for the antibody in the duck serum 7 days after infection with Riemerella anatipestifer serotype 2 were 100% positive; the antibody detection results after immunization with Riemerella anatipestifer serotype 2 inactivated vaccine showed that the positive rate of serum antibody was 80% 14 days after immunization and 100% 21 days after immunization.

[0035] The establishment of the detection method of the present invention fills the gap in the ELISA detection method of Riemerella anatipestifer serum type 2 specific antibody, provides effective data support and scientific basis for the epidemiological investigation and vaccine development of Riemerella anatipestifer, and has important theoretical and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the identification of purified LPS, where A is LPS silver staining identification, B is LPS Coomassie Brilliant Blue staining identification, Note: M is PageRuler TM Prestained protein molecular weight standards, 1 to 5: LPS sample amounts are 500 ng, 250 ng, 125 ng, 50 ng and 25 ng, respectively. DETAILED DESCRIPTION

[0037] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0038] In the following examples, the sources of the bacterial strains and duck positive serum are as follows:

[0039] RA serotype 2 strain 2019XZ012 was isolated, identified and preserved by Jiangsu Provincial Key Laboratory of Veterinary Biopharmaceutical High Technology Research, Jiangsu Agricultural and Animal Husbandry Science and Technology Vocational College (Dong Hongyan et al., Analysis of biological characteristics of 28 Riemerella anatipestifer isolates in Xuzhou area, Chinese Journal of Zoonotic Infectious Diseases, 2023, 31(2):73-79);

[0040] RA serotype 1, 2, 6, 7, and 10 duck positive sera were kindly donated by the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Feng Yating et al., Isolation, Identification and Biological Characteristics of the Epidemic Strains of Riemerella anatipestifer, Microbiology Bulletin, 2022, 49(11): 4778-4785);

[0041] Duck Pasteurella -positive serum, duck Escherichia coli -positive serum, and duck Salmonella -positive serum were prepared, identified, and stored by Yangzhou Youjiachuang Biotechnology Co., Ltd.;

[0042] The duckling hepatitis positive serum was kindly donated by Cheng Anchun from Sichuan Agricultural University (Zhang Yize, The growth and decline of ELISA antibodies in ducklings immunized with duck viral hepatitis bivalent (1+3 types) attenuated vaccine, Sichuan Agricultural University, Master's thesis, 2017);

[0043] The duck reovirus-positive serum was kindly donated by Professor Wu Yantao of Yangzhou University (Isolation, identification and development of oil-emulsion inactivated vaccine of reovirus ZY / AH / 1 / 2011 strain causing decreased egg production in ducks, Yang Rui, Master's thesis of Yangzhou University, 2013).

[0044] The preparation method of duck Pasteurella positive serum is as follows: the preserved strain PM-11 (Liu Dan et al., Isolation, Identification and Pathogenicity of Duck Pasteurella, Chinese Journal of Zooinfectious Diseases, first published online: 2022-11-25) was thawed at room temperature and inoculated into tryptic soy broth (TSB) at 37°C and 200r / min. The strain was cultured to the logarithmic growth phase and the OD was measured. 600 Count the number of bacteria; collect the bacteria by centrifugation and concentrate the bacterial solution to 3×10 10 CFU / ml; add 0.4% formaldehyde, inactivate at 37℃, 100rpm / min for 8 hours; after passing the sterility test, mix the inactivated bacterial solution and adjuvant (Montanide ISA71VG adjuvant, Seppic) at a volume ratio of 3:7, and emulsify at 3000r / min for 15 minutes to prepare the vaccine. 7-day-old healthy non-immunized Cherry Valley ducks were injected subcutaneously in the neck, 0.3ml / bird (bacterial content = 10 9 CFU), and serum was collected 14 days after immunization to determine the ELISA titer of antibodies. When the titer was greater than 1:4000, duck Pasteurella positive serum was obtained.

[0045] The preparation method of duck Escherichia coli positive serum is as follows: the preserved strain 2019JS001 (Dong Hongyan et al., Isolation, Identification and Pathogenicity Analysis of Duck Escherichia coli, Chinese Journal of Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1977-1984) was thawed at room temperature and inoculated into LB medium, cultured at 37°C, 200r / min until the logarithmic growth phase, and the OD was measured. 600 Count the number of bacteria; collect the bacteria by centrifugation and concentrate the bacterial solution to 3×10 10 CFU / ml; add 0.4% formaldehyde, inactivate at 37℃, 100rpm / min for 8 hours; after passing the sterility test, mix the inactivated bacterial solution and adjuvant (Montanide ISA 71VG adjuvant, Seppic) at a volume ratio of 3:7, and emulsify at 3000r / min for 15 minutes to prepare the vaccine. 7-day-old healthy non-immunized Cherry Valley ducks were injected subcutaneously in the neck, 0.3ml / bird (bacterial content = 10 9 CFU), and serum was collected 14 days after immunization to determine the ELISA titer of the antibody. When the titer was greater than 1:4000, duck Escherichia coli positive serum was obtained.

[0046] The preparation method of duck Salmonella positive serum is as follows: the preserved strain of Salmonella typhimurium strain SAT52 (Xin Suhua et al., Construction of rtsB gene deletion strain of Salmonella typhimurium and its biological characteristics, Microbiology Bulletin, 2019, 46(11):3006-3012) was thawed at room temperature and then cultured in LB medium at 37°C and 200r / min until the logarithmic growth phase, and the OD was measured. 600 Count the number of bacteria; collect the bacteria by centrifugation and concentrate the bacterial solution to 3×10 10 CFU / ml; add 0.4% formaldehyde, inactivate at 37℃, 100rpm / min for 8 hours; after passing the sterility test, mix the inactivated bacterial solution and adjuvant (Montanide ISA71VG adjuvant, Seppic) at a volume ratio of 3:7, and emulsify at 3000r / min for 15 minutes to prepare the vaccine. 7-day-old healthy non-immunized Cherry Valley ducks were injected subcutaneously in the neck, 0.3ml / bird (bacterial content = 10 9 CFU), serum was collected 14 days after immunization to determine the ELISA titer of the antibody, and the duck Salmonella positive serum was obtained when the titer was greater than 1:4000.

[0047] In the following examples, the sources of the main consumables, reagents and instruments are as follows:

[0048] The 96-well ELISA plate was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0049] Skim milk powder was purchased from Shanghai Shenggong Biotechnology Co., Ltd.;

[0050] Rabbit Anti-Duck IgG (H&L)-HRP was purchased from Abmart;

[0051] TMB colorimetric solution was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0052] TSB medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.;

[0053] The constant temperature oscillator (IS-RDS3L) was purchased from Seiki Co., Ltd., USA;

[0054] The CO2 incubator (CLM-170 / 240) was purchased from Thermmo Fisher;

[0055] The multifunctional microplate reader was purchased from BioTek;

[0056] Protein electrophoresis apparatus and protein transfer tank were purchased from Bio-Rad.

[0057] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0058] Example 1

[0059] This embodiment provides a method for extracting and identifying LPS, and the specific steps are as follows:

[0060] S1. Take the RA serotype 2 strain 2019XZ012 stored at -80°C, thaw at room temperature, and then streak culture on a tryptic soy agar medium (TSA, Qingdao Haibo Biotechnology Co., Ltd.) at 37°C and 5% CO. 2 Culture in incubator for 24 to 48 hours.

[0061] S2. Pick a typical single colony and inoculate it into tryptic soy bean liquid medium (TSB, Qingdao Haibo Biotechnology Co., Ltd.), incubate it in a shaking incubator at 37° and 180 r / min for 14-16 h, then harvest the bacterial liquid and extract bacterial lipopolysaccharide (LPS) using the hot phenol water method according to the literature method. [9] .

[0062] S3. The collected bacteria by centrifugation were washed twice with 95% ethanol (50 m / 10 g wet weight of bacteria), acetone (50 m / 10 g wet weight of bacteria), and petroleum ether (25 ml / 10 g wet weight of bacteria), respectively, and then air-dried in a fume hood at room temperature.

[0063] S4. Resuspend the dried cells in sterile water, add an equal volume of phenol and mix well. Incubate in a 68°C water bath for 20 min, then centrifuge at 4°C, 6000 rpm to collect the aqueous phase. Repeat 2-3 times and combine the aqueous phases.

[0064] S5. Add sodium acetate and acetone to the aqueous phase, stir, centrifuge, wash with 95% ethanol, and dry again.

[0065] S6. Dissolve the precipitate with sterile water, add DNase and RNase (0.01 mg / mL) and place in a 37°C water bath for 30 min to remove nucleic acid contamination; then add proteinase K (0.15 mg / mL) and place in a 55°C water bath for 3 h to remove protein contamination.

[0066] S7. After ultracentrifugation at 40,000 r / min for 4 hours, the LPS precipitate was collected and freeze-dried to obtain purified LPS.

[0067] S8. The purified LPS was diluted with sterile deionized water to a concentration of 500 ng, 250 ng, 125 ng, 50 ng, and 25 ng / ml. 80 μL was mixed with 20 μL 5×Loading and boiled in boiling water for 15 minutes before SDS-PAGE, silver staining, and Coomassie brilliant blue staining to identify the purity.

[0068] The purity of the extracted LPS samples was determined by silver staining and Coomassie Brilliant Blue staining. Figure 1As shown in Figure 2, a silver-stained LPS band appeared near 13 kDa, and its band intensity became weaker as the sample amount decreased ( Figure 1 A), no bands in Coomassie Brilliant Blue staining ( Figure 1 B), indicating that no protein contamination was detected in the LPS sample and the quality was good.

[0069] Example 2

[0070] This embodiment provides a method for establishing an indirect ELISA detection method, and the specific steps are as follows:

[0071] S1. Selection of the best coating solution and coating time

[0072] According to the chessboard titration test principle, determine the best coating solution and coating time. The detailed steps are as follows:

[0073] Purified LPS was used as coating antigen, and LPS was diluted to 5 μg / ml using PBS (pH 7.2), carbonate coating solution (pH 9.6), and deionized water, and 100 μL / well was coated on the ELISA plate. The coating temperature and time were set at 4°C for 16h, 4°C for 12h, and 37°C for 4h, respectively.

[0074] After coating, wash 3 times with PBST, add 200μL of PBS containing 5% skim milk powder to each well, block at 37℃ for 2h, and wash twice. Serum type 2 RA duck positive serum and negative serum were diluted 1:400 with PBST, 100μL / well were added to the coated ELISA plate, incubated at 37℃ for 2h, and washed 5 times with PBST. Add Rabbit Anti-Duck IgG (H&L) HRP (1:10000 dilution), 100μL / well, incubate at 37℃ for 1.5h, and wash 5 times. Add TMB single-component colorimetric solution under light-proof conditions, 100mL / well, react at 37℃ for 5min; add 50μL 2M H 2 SO 4 Stop the reaction and measure OD with a microplate reader 450 The test was repeated 3 times, P is the OD of the serum to be tested 450 Value, N is negative serum OD 450 The reaction condition with the largest P / N value is considered the optimal condition.

[0075] S2. Selection of the optimal coating concentration and serum dilution concentration to be tested

[0076] Also according to the chessboard titration test principle, determine the optimal coating concentration and the dilution multiple of the serum to be tested. According to the above optimized coating solution and coating time, dilute LPS to 5, 10, 15, 20 μg / mL, and coat at 100 μL / well. The serum to be tested, serum type 2 RA duck positive serum and duck negative serum were diluted 1:100, 1:200, 1:400, 1:800, respectively. The experiment was repeated 3 times, and the average value was taken to calculate the P / N value. The reaction condition with the largest P / N value was judged to be the optimal condition.

[0077] S3. Selection of the optimal dilution of Rabbit Anti-Duck IgG (H&L)-HRP

[0078] Rabbit anti-duck enzyme-labeled antibody was incubated at dilutions of 1:1000, 1:2000, 1:5000, and 1:10000, respectively. Other conditions were the same as the previously optimized optimal reaction conditions. The negative OD 450 The value is less than 0.2 and the P / N is the highest as the judgment standard.

[0079] S4. Selection of the best closure time

[0080] Add 200 μL of PBS containing 5% skim milk powder to each well and block at 37℃ for 1h, 37℃ for 2h, and 37℃ for 2.5h. Perform the test according to the above optimized optimal reaction conditions. The highest P / N is used as the judgment standard.

[0081] S5. Selection of the optimal incubation time for positive serum

[0082] According to the above optimized conditions, the positive serum of serotype 2 RA duck was incubated at 37°C for 1 h, 37°C for 1.5 h, and 37°C for 2 h, respectively, and the highest P / N ratio was used as the judgment standard.

[0083] S6. Selection of the optimal incubation time for enzyme-labeled antibodies

[0084] According to the above optimized conditions, the incubation time of enzyme-labeled antibody was 37℃0.5h, 37℃1h and 37℃1.5h respectively. The highest P / N average value was used as the judgment standard.

[0085] S7, color development time

[0086] According to the above optimized conditions, the color development time was 5min, 10min, and 15min respectively, and the highest P / N average value was used as the judgment standard.

[0087] S8. Determination of critical value

[0088] According to the ELISA method established above, 80 healthy non-immune duck sera were tested. The OD of serum was determined. 450values, and calculate their mean (X) and standard deviation (SD), with X+3SD as the critical value, i.e. OD 450 When >X+3SD, it is positive, otherwise it is negative.

[0089] The results of the indirect ELISA test method are shown below:

[0090] 1) Establishment of optimal reaction conditions: The results are shown in Tables 1, 2 and 3.

[0091] Table 1 Selection of optimal coating solution and coating conditions

[0092]

[0093] Table Notes: *, P / N ratio is maximum.

[0094] Table 2 Selection of optimal coating concentration and positive serum dilution

[0095]

[0096] Table Notes: *, P / N ratio is the largest.

[0097] Table 3 Selection of blocking conditions, positive serum incubation time, enzyme-labeled antibody dilution / incubation time and color development time

[0098]

[0099] Table Notes: *, P / N ratio is the largest.

[0100] The results are shown in Tables 1, 2 and 3. When PBS was used as the coating solution, the coating time was 4°C for 12 h, the coating antigen mass concentration was 5 μg / mL, the blocking solution was 5% skim milk, the blocking conditions were 37°C for 2 h, the test serum dilution was 1:400, the incubation time was 1 h, the secondary antibody dilution was 1:10000, the incubation time was 1 h, and the color development time was 5 min, the P / N average value was the largest, which was determined to be the optimal reaction condition.

[0101] 2) Establishment of critical value: According to the optimized ELISA detection method, the OD values ​​of the corresponding wells of 80 healthy non-immune duck sera were tested. 450 The calculation results show that X = 0.089, SD = 0.021, X + 3SD = 0.151. 450 When it is greater than 0.151, it is judged as positive, otherwise it is negative.

[0102] Example 3

[0103] This embodiment provides an ELISA antibody detection method for serotype 2 Riemerella anatipestifer, and the specific steps are as follows:

[0104] S1. Extract lipopolysaccharide of Riemerella anatipestifer serotype 2 strain by the hot phenol water method disclosed in Example 1, and use the purified lipopolysaccharide (LPS) of Riemerella anatipestifer serotype 2 strain as coating antigen.

[0105] S2. Use coating solution PBS (pH 7.2) to dilute the coating antigen LPS to 5 μg / ml, add it into a 96-well ELISA plate (100 μL / well) for coating, and set the coating temperature and time to 4°C for 12 h.

[0106] S3. After coating, wash with PBST for 3 times, add 200 μL of PBS containing 5% skimmed milk powder to each well, block at 37°C for 2 h, and then wash twice.

[0107] S4. Discard the blocking solution in step S3, dilute the serum type 2 RA duck positive serum and negative serum with PBST at 1:400, add 100 μL / well to the coated ELISA plate, incubate at 37°C for 2 h, and wash 5 times with PBST.

[0108] S5. Add secondary antibody Rabbit Anti-Duck IgG (H&L) HRP (1:10000 dilution), 100 μL / well, incubate at 37°C for 1.5 h, and then wash 5 times.

[0109] S6. Discard the secondary antibody incubation solution in step S3, add TMB single-component colorimetric solution (100 mL / well) in dark conditions, and react at 37°C in dark for 5 min; add 50 μL 2M H 2 SO 4 The reaction was terminated and the experiment was repeated 3 times.

[0110] S7, using an ELISA reader to measure the OD of the liquid in the ELISA plate well of step S6 450 Value, according to OD 450 The critical value determines negative or positive.

[0111] Example 4

[0112] This embodiment provides a method for specific detection, and the specific steps are as follows:

[0113] According to the ELISA method established by optimizing the conditions described in Example 3, serotype 2 NJ3, Yb2, RA serotype 1 CH3, RA serotype 6 SDJ-80, RA serotype 7 SDJ-69, serotype 10 HXb2, duck serum positive for avian pathogenic Escherichia coli, duck serum positive for avian Pasteurella, duck serum positive for avian Salmonella, duck serum positive for reovirus, and duckling hepatitis positive were tested. Each serum was tested three times. The OD 450 The results were determined based on the determined critical value, thereby analyzing the specificity of the ELISA method.

[0114] The specificity test results are shown in Table 4. The specificity test was performed according to the optimized ELISA method described above, with 3 replicates set up and the OD 450 The value ≥ 0.151 is the standard for positive serum. The results show that the OD of serum of serotype 2 NJ3 and Yb2 positive ducks 450 The value is greater than 0.151, which is considered positive. 450 The values ​​were all less than 0.151, which was considered negative, indicating that the detection method had good specificity.

[0115] Table 4 Specificity test results

[0116]

[0117] Example 5

[0118] This embodiment provides a sensitivity detection method, and the specific steps are as follows:

[0119] Two serum samples of type 2 RA duck positive serum were selected and diluted in multiple ratios (1:100-1:51200) as samples to be tested. The ELISA method established in Example 3 was used for detection. Each serum sample was tested in duplicate for 3 wells and the OD was read. 450 The average value was calculated and the sensitivity of the method was analyzed.

[0120] The sensitivity test results are shown in Table 5. When the dilution ratio of NJ3 positive serum reached 1:51200, the OD 450 The values ​​all exceeded the critical value of 0.151. The results showed that the established indirect ELISA method has high sensitivity.

[0121] Table 5 Sensitivity test results

[0122]

[0123]

[0124] Example 6

[0125] This embodiment provides a method for repeatability detection, and the specific steps are as follows:

[0126] Randomly select 5 positive sera and 5 negative sera from serotype 2 RA ducks. According to the ELISA detection method established in Example 3, the same batch of coated ELISA plates were used for intra-batch repeatability test and different batches of coated ELISA plates were used for inter-batch repeatability test. Three replicates were set for each serum, and the OD 450 , calculate the mean and coefficient of variation within and between batches.

[0127] Intra-batch repeatability test: Three different 96-well ELISA plates from the same batch were used for repeated experiments, and 5 samples were randomly selected from the positive and negative samples for testing; inter-batch repeatability test: Three 96-well ELISA plates from three different batches were used for repeated experiments, and 5 samples were randomly selected from the positive and negative samples for testing. The repeatability test results are shown in Table 6. The intra-batch coefficient of variation is between 0.73% and 6.34%, and the inter-batch coefficient of variation is maintained in the range of 2.08% to 9.25%, both below 10%. The results show that the established ELISA method has good repeatability.

[0128] Table 6 Repeatability test results

[0129]

[0130] Example 7

[0131] This embodiment provides a method for detecting clinical samples, and the specific steps are as follows:

[0132] The antibody levels were detected by the ELISA method established in Example 3 for duck sera 14, 28, 42, 56 and 70 days after immunization with the RA serotype 2 NJ3 inactivated vaccine, and duck sera 7 days after infection with RA serotype 1 WJ4, type 2 Yb2, type 10 HXb2 and undetermined serotype WH6, and the number of positive samples was counted.

[0133] A total of 77 inactivated vaccine immune serum samples were tested using the optimized ELISA method. 450 ≥0.151 was used as the standard, and the test results of the inactivated vaccine immune serum samples were shown in Table 7, indicating that the established ELISA method can effectively detect the antibody level in the serum of ducks immunized with serum type 2 RA inactivated vaccine.

[0134] Table 7 Antibody detection results of inactivated vaccine immunized duck serum samples

[0135]

[0136] The optimized ELISA method was used to detect serum samples collected 7 days after infection with different serotypes of RA strains. 450 ≥0.151 was used as the standard. The test results of clinical infection serum samples (Table 8) showed that the ELISA detection method could specifically detect serum type 2 RA (Yb2) antibodies in duck serum 7 days after infection; the test results of duck serum 7 days after infection with serum type 1 strain WJ4, type 10 strain HXb2 and type 6 strain WH6 were all negative, indicating that this method can be used for clinical diagnosis and epidemiological investigation.

[0137] Table 8 Test results of duck serum samples 7 days after infection

[0138]

[0139] The present invention extracts high-purity RA LPS by hot phenol water method, and uses it as coating antigen to establish an indirect ELISA method for detecting serum type 2 RA antibodies. After a series of condition optimizations, the best reaction parameters are established, including coating solution, coating time, coating concentration, blocking time, dilution and incubation time of serum / enzyme-labeled antibody to be tested, and color development time, thereby ensuring the high sensitivity, specificity and repeatability of the ELISA method. The ELISA antibody detection method can be used for clinical infection detection, epidemiological investigation and inactivated vaccine immune antibody determination of serum type 2 RA.

[0140] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

[0141] The references of the present invention are as follows:

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Claims

1. An ELISA antibody detection method for serotype 2 Riemerella anatipestifer, characterized in that: The method is based on bacterial lipopolysaccharide coated plates. Through optimization of optimal reaction conditions, determination of critical values, specificity tests, sensitivity tests, repeatability tests and clinical trials, an indirect ELISA detection method for serum type 2 Riemerella anatipestifer antibodies was established.

2. The ELISA antibody detection method for serotype 2 Riemerella anatipestifer according to claim 1, characterized in that: The optimization of the optimal reaction conditions includes optimizing the coating solution, coating time, coating concentration, dilution and incubation time of the serum / enzyme-labeled antibody to be tested, blocking time and color development time; The optimal reaction condition is evaluated by P / N value, where P is the OD of the serum to be tested. 450 Value, N is negative serum OD 450 value.

3. The ELISA antibody detection method for serotype 2 Riemerella anatipestifer according to claim 1, characterized in that: The specific method for determining the critical value is: calculate 80 negative serum OD 450 The mean and standard deviation of the data are given in Table 1, with X+3SD as the critical value.

4. The ELISA antibody detection method for serotype 2 Riemerella anatipestifer according to claim 1, characterized in that: In the specificity test, serotype 2 NJ3, Yb2, RA serotype 1 CH3, RA serotype 6 SDJ-80, RA serotype 7 SDJ-69, serotype 10 HXb2, avian pathogenic Escherichia coli positive duck serum, avian Pasteurella positive duck serum, avian Salmonella positive duck serum, reovirus positive duck serum and duckling hepatitis positive duck serum were selected, and duck positive serum and negative serum controls were set up.

5. The ELISA antibody detection method for serotype 2 Riemerella anatipestifer according to claim 1, characterized in that: In the sensitivity test, two positive sera of serotype 2 RA duck were selected and diluted 1:100-1:51200 times respectively, and the standard positive sera of different dilution multiples were tested.

6. The ELISA antibody detection method for serotype 2 Riemerella anatipestifer according to claim 1, characterized in that: In the repeatability test, 5 positive sera and 5 negative sera of serotype 2 RA ducks were randomly selected, and the same batch of ELISA plates were used for intra-batch repeatability test and different batches of ELISA plates were used for inter-batch repeatability test. Three replicates were set for each serum. In the clinical trial, duck sera were tested 14, 28, 42, 56 and 70 days after immunization with RA serotype 2 NJ3 inactivated vaccine, as well as duck sera 7 days after infection with RA serotype 1 WJ4, type 2 Yb2, type 10 HXb2 and undetermined serotype WH6.

7. The ELISA antibody detection method for serotype 2 Riemerella anatipestifer according to claim 1, characterized in that: The specific steps of the ELISA antibody detection method for serotype 2 Riemerella anatipestifer are as follows: S1, extracting lipopolysaccharide of Riemerella anatipestifer serotype 2 strain by hot phenol water method, and using lipopolysaccharide of Riemerella anatipestifer serotype 2 strain as coating antigen; S2, dilute the coating antigen with coating solution, add it into the ELISA plate for coating, and then block it with blocking solution; S3, discard the blocking solution in step S2, add primary antibody for incubation, and then incubate with secondary antibody; S4, discard the secondary antibody incubation solution in step S3, use TMB single-component colorimetric solution to develop color, add colorimetric stop solution to terminate the reaction; S5, using an ELISA reader to measure the OD of the liquid in the ELISA plate well of step S4 450 Value, according to OD 450 The critical value determines negative or positive.

8. The ELISA antibody detection method for serotype 2 Riemerella anatipestifer according to claim 7, characterized in that: In step S2, the primary antibody is serum type 2 RA duck positive serum, and the secondary antibody is rabbit anti-duck enzyme-labeled antibody; In step S5, the OD 450 The critical value is 0.

151.

9. An ELISA antibody detection kit for serotype 2 Riemerella anatipestifer, characterized in that: The kit comprises: an antigen-coated plate, a sample diluent, a washing solution, an enzyme-labeled antibody, a color developing solution, a stop solution, a positive control and a negative control; wherein, The antigen-coated plate is an ELISA plate coated with lipopolysaccharide of Riemerella anatipestifer serotype 2 strain; The sample diluent is PBS containing 5% skimmed milk powder; The washing solution is PBST solution; The enzyme-labeled antibody shown is a 10,000-fold diluted rabbit anti-duck enzyme-labeled antibody; The color developing solution is a TMB single-component solution; The stop solution is 2M sulfuric acid.

10. The ELISA antibody detection kit for serotype 2 Riemerella anatipestifer according to claim 9, characterized in that: The coating concentration of Riemerella anatipestifer serotype 2 strain lipopolysaccharide on the antigen-coated plate is 5 μg / mL.

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