CN114231496A

CN114231496A - Salmonella abortus competition ELISA antibody detection kit and application thereof

Info

Publication number: CN114231496A
Application number: CN202111444112.2A
Authority: CN
Inventors: 胡哲; 郭奎; 王晓钧
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-03-25
Anticipated expiration: 2041-11-30
Also published as: CN114231496B

Abstract

The invention discloses a salmonella equiabortus competition ELISA antibody detection kit and application thereof. The kit contains a monoclonal antibody of anti-Salmonella abortus FljB protein secreted and produced by a hybridoma cell strain with the preservation number of CGMCCNo.21990. In addition, the invention also establishes a competition ELISA antibody detection method for the salmonella abortus equi, and compared with the TAT method, the competition ELISA method established by the invention has higher sensitivity: the maximum dilution detectable for standard positive sera was 32-fold dilution and the maximum dilution detectable for TAT was original fold. The competitive ELISA method is more specific: the antibody does not react with the serum antibodies of common viral diseases such as equine infectious anemia, equine influenza, equine rhinopneumonitis, equine arteritis and the like and bacterial diseases such as equine strangles, Salmonella typhimurium infection, Salmonella dublin infection and the like, and only reacts with the specific antibody of the Salmonella abortus of the horse. Therefore, the invention provides a new technical means for detecting the antibody of the salmonella abortus equi.

Description

Salmonella abortus competition ELISA antibody detection kit and application thereof

Technical Field

The invention relates to a salmonella abortus equi antibody detection kit and application thereof. In particular to a salmonella abortus equi competition ELISA antibody detection kit and application thereof. The invention belongs to the field of biotechnology.

Background

Salmonella abortus (Equise abortus Salmonella) is an Equine infectious disease of animals of the genus Equis, which is primarily characterized by abortion in pregnant horses, and is also known as Equine paratyphoid (Equis paratyphoid) or Salmonella abortus. The disease is susceptible to primary-birth dams, most of the dams have no obvious clinical symptoms before abortion, the most of the dams are dead fetuses, and a few of the surviving fetuses also die after several days of birth. During abortion of pregnant mares, germs are discharged out of the body along with aborted fetuses, fetal membranes, amniotic fluid and vaginal secretions, and sick male horses (donkeys) can discharge germs along with semen.

In recent years, the disease has outbreak epidemic in China, such as east China, North China, northwest China and the like, and mainly causes abortion of horses and donkeys, and the abortion rate is 20-100%. In the early laboratory study, the visceral organs and tissues of fetuses of abortion foals are separated and identified as Salmonella abortus (s.abortus. equi). However, the disease is rarely researched at home and abroad, and a specific detection technology is lacked, the serological detection method for the disease mentioned in the industry standard (the standard) of salmonellosis equi diagnostic technology (NY/T570-2002) in China is test Tube Agglutination Test (TAT), the method can be used for detecting antibodies of the salmonellosis equi, but in individual cases, other serological antibodies of the salmonellosis, such as antibodies of the salmonellosis typhimurium (S.typi), the salmonellosis enteritidis (S.enteritidis), the salmonellosis dublin (S.dublin), and the like, have weak specificity; the test tube is adopted for operation, and the sample needs a plurality of dilutions, so that the requirement of large-scale sample detection cannot be met.

Therefore, in order to cope with the continuous outbreak of the epidemic situation, a detection method with strong specificity, high sensitivity and high flux needs to be established urgently.

Disclosure of Invention

One of the purposes of the invention is to provide a hybridoma cell strain secreting anti-Salmonella abortus (S.abortus. equi) FljB protein monoclonal antibody and a monoclonal antibody secreted by the hybridoma cell strain;

the invention also aims to provide the application of the monoclonal antibody in a reagent for detecting the antibody of the salmonella abortus equi.

The invention also aims to provide a salmonella abortus competition ELISA antibody detection kit and a detection method thereof.

In order to achieve the purpose, the invention adopts the following technical means:

the invention expresses FljB protein through in vitro fusion to obtain a plurality of monoclonal antibodies of anti-FljB protein, and finally establishes a competitive ELISA (C-ELISA) antibody detection method through screening competitive monoclonal antibodies, optimizing reaction conditions and reaction strategies. The reaction principle of the method is as follows: coating with monoclonal antibody, adding the serum to be detected and the FljB protein marked by HRP into a reaction hole of an ELISA plate at the same time, acting for 30 minutes at 37 ℃, reading the light absorption value of OD450, and judging the detection result by calculating the inhibition rate.

The Hybridoma cell strain secreting monoclonal antibody against Salmonella abortus (S.abortus. equi) FljB protein obtained by screening is named as S-FljB-1A10 and classified as Hybridoma cell strain (Hybridoma cell line), is preserved in the general microbiological center of China Committee for culture Collection of microorganisms (CCC), and is addressed to the institute of microbiology, academy of sciences in North West Lu No. 1 institute of Shangyang, Beijing, and the strain preservation numbers are as follows: CGMCC No.21990, with a preservation date of 2021, 4 months and 1 days.

The monoclonal antibody of anti-Salmonella abortus FljB protein secreted by the hybridoma cell strain also falls within the protection scope of the invention.

Furthermore, the invention also provides application of the monoclonal antibody in preparing a reagent for detecting the antibody of the salmonella abortus equi. Preferably, the reagent is a competitive ELISA antibody detection reagent.

Furthermore, the invention also provides a salmonella abortus competition ELISA antibody detection kit, wherein the kit contains the monoclonal antibody.

Among them, the monoclonal antibody is preferably used as a coating antibody.

Preferably, the kit further comprises 5% w/v of NCS blocking solution, HRP-labeled FljB protein, washing solution, two-component TMB developing solution and stop solution.

Wherein, preferably, when the kit is used for detecting the antibody of the salmonella abortus equi, the method comprises the following steps:

(1) coating an enzyme label plate with 2 mu g/mL of the monoclonal antibody at 4 ℃ overnight, washing with a washing solution, blocking with 5% w/v NCS at 37 ℃ for 2h, and washing the plate;

(2) mixing the HRP-labeled FljB protein and a control or to-be-detected serum sample according to the volume ratio of 1:1, adding the mixture into a plate, incubating the mixture for 30min at 37 ℃, and washing the plate;

(3) adding two-component TMB developing solution, developing at 37 deg.C for 10min, adding stop solution to terminate reaction, and measuring OD_450nmValue, calculating the inhibition rate, when the inhibition rate is>When 41.5%, the test piece was judged to be positive.

Compared with the prior art, the invention has the beneficial effects that:

compared with TAT method, the competitive ELISA method established by the invention has higher sensitivity: the maximum dilution detectable for standard positive sera was 32-fold dilution and the maximum dilution detectable for TAT was original fold. The competitive ELISA method has better specificity, does not react to the common virus diseases such as equine infectious anemia, equine influenza, equine rhinopneumonitis, equine arteritis and the like and the serum antibodies of bacterial diseases such as equine strangles, Salmonella typhimurium infection, Salmonella dublin infection and the like, and only reacts to the specific antibodies of the Salmonella abortus of the horse. The TAT method has non-specific cross reaction with other salmonella.

At present, no specific antibody detection method aiming at different pathogens of the disease exists at home and abroad, namely antibodies generated by pathogens such as salmonella typhimurium, salmonella dublin, salmonella enteritidis, salmonella abortus equi and the like are all likely to be detected to be positive in TAT. In order to more scientifically and objectively evaluate the competitive ELISA method established by the people, positive serum and negative serum detected by TAT are further verified, namely, Western Blot (WB) test is carried out by specific protein of the Salmonella abortus equi, the serum with positive TAT and WB is determined as the Salmonella abortus equi positive serum, and the serum with negative TAT and WB is determined as the Salmonella abortus equi negative serum. And each of the positive serum and the negative serum of the equine abortion salmonella is 20 parts. The 20 sera were tested by competition ELISA and showed 100% positive and negative agreement. 1 positive serum sample with TAT detection maximum dilution being the original time is selected as standard positive serum, and the sensitivity of the serum detected by a competitive ELISA method is 32-time dilution, thereby showing that the sensitivity of the competitive ELISA method is higher. 860 parts of clinical serum samples are detected by a competitive ELISA method, and the positive detection rate is 37.7 percent.

Drawings

FIG. 1 is the measurement of the affinity of monoclonal antibodies of different strains;

FIG. 2 is the analysis of the binding force between different antibodies and enzyme-labeled antigen under the same concentration;

FIG. 3 is a screen of best-packaged monoclonal antibodies;

FIG. 4 shows the result of SDS-PAGE electrophoresis of the monoclonal antibody 1A 10;

wherein: m: a protein Marker; 1: antibody elution tube 1; 2: antibody elution tube 2; 3: antibody elution tube 3; 4: antibody elution tube 4; 5: antibody elution tube 5; 6: antibody elution tube 6; 7: antibody elution tube 7;

FIG. 5 shows the specific detection results of the monoclonal antibody against Salmonella abortus 1A 10;

wherein: 1. equine influenza virus; 2. equine herpes type I virus; 3. equine herpes type VI virus; 4. equine arteritis virus; 5. equine infectious anemia virus; 6. salmonella typhimurium; 7. salmonella enteritidis; 8. salmonella dublin; 9. salmonella abortus in equi;

FIG. 6 shows the reaction strategy of the competitive ELISA method;

FIG. 7 shows the one-step optimization of reaction conditions in the competitive ELISA method;

FIG. 8 shows the two-step reaction condition optimization of competitive ELISA;

FIG. 9 shows the optimization of substrates for the competitive ELISA method;

FIG. 10 is the determination of the time of the action of the serum and the enzyme-labeled antigen;

FIG. 11 shows the specificity of competitive ELISA;

FIG. 12 shows the results of the competitive ELISA (C-ELISA) and Indirect ELISA (iELISA) methods for detecting the sensitivity of positive sera from Salmonella abortus in horse;

FIG. 13 shows the results of detecting the sensitivity of the positive serum of paratyphoid donkey by competitive ELISA;

FIG. 14 is a schematic diagram of the reaction principles of competition ELISA (C-ELISA) and Indirect ELISA (iELISA) and the effect of non-specific reaction on the detection results.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 expression and purification of recombinant FljB protein of Salmonella abortus equi

The expression and purification of the recombinant FljB protein was carried out according to the method described in the patent application having publication No. CN111458501A entitled "Indirect ELISA kit for detecting antibodies against Salmonella abortus, method of preparation and use thereof", which is incorporated herein by reference in its entirety.

EXAMPLE 2 preparation of monoclonal antibodies

1 Material

1.1 immunogen purified Salmonella abortus FljB protein (diluted content 1mg/ml), prepared from example 1.

1.2 cellular myeloma cells SP2/0, supplied by Harbin veterinary research institute of Chinese academy of agricultural sciences.

1.3 test animal 6-8 week old female Balb/c mice, by Chinese agricultural academy of sciences Harbin veterinary research institute experimental animal center.

1.4 cell culture medium containing 20% fetal calf serum and 100U (mug) of streptomycin each/ml 1640 medium, and storing at 2-8 deg.C. 1640 medium, purchased from Sigma; fetal bovine serum, purchased from Ausbian.

1.5 taking 10ml of dimethyl sulfoxide (DMSO) from the cell frozen stock solution, dissolving in 90ml of fetal calf serum, uniformly mixing, and placing at 2-8 ℃ for later use.

1.6 kits

1.6.1BCA kit, purchased from Novagen.

1.6.2SBA Clonotyping^TMSystem/HRP, purchased from southern Biotech.

1.7 other reagents PEG4000, Freund's complete adjuvant, Freund's incomplete adjuvant and HRP-labeled goat anti-mouse IgG, all purchased from Sigma; TMB color developing solution purchased from Thermo company.

2 method

2.1 construction of hybridoma cells

2.1.1 animal immunization

2.1.1.1.1 one-time immunization, emulsifying the purified Salmonella abortus FljB protein with equal volume of Freund's complete adjuvant to be used as immunogen, and injecting 200 mul (containing 100 mug FljB protein) per mouse with 4-6 weeks old by back subcutaneous injection.

2.1.1.2 after the first immunization, the purified Salmonella abortus FljB protein is mixed and emulsified with equal volume of Freund incomplete adjuvant to be used as immunogen, and the intraperitoneal injection is adopted to carry out the second immunization according to the immunization mode and dosage of the first immunization.

And 2.1.1.3, 3 weeks after the third immunization for the second immunization, mixing and emulsifying the purified Salmonella abortus FljB protein and an equivalent volume of Freund incomplete adjuvant to serve as immunogen, and carrying out third immunization according to the immunization mode and dosage of the second immunization.

2.1.1.4 day before the fourth immune cell fusion, 200. mu.l (containing 100. mu.g of FljB protein) of mice are inoculated with purified Salmonella abortus FljB protein as immunogen by intraperitoneal injection.

2.1.2 myeloma cells are cultured by expanding them to be in the logarithmic growth phase and to have a good growth state 1 to 2 days before the preparation of fusion. On the day of fusion, the medium was discarded and gently rinsed twice with serum-free 1640 medium, and the cells were gently blown off the walls of the flask with 15ml of 1640 base medium. A small amount of the osteoma cell suspension was taken, counted by a cell counting plate, and prepared for fusion.

2.1.3 preparation of immune splenocytes

2.1.3.1 before the fusion, the blood of the eyeballs of the mice with the strengthened immunity is collected and killed, and positive serum is prepared. Soaking in 75% alcohol for 5min, and placing on a clean bench.

2.1.3.2 mice were fixed on a mouse rack, the abdominal cavity was aseptically opened, the spleen was taken out by separating connective tissue, the spleen was placed in a dish containing 15ml 1640 basic culture solution, the culture solution was aspirated with a sterile syringe and the spleen cells were gently blown off, and the operation was repeated 3 to 4 times.

2.1.3.3 the spleen cell suspension is transferred into a 50ml centrifuge tube, about 1640 ml of basal medium is added, and the mixture is mixed evenly. The cell suspension was counted on a cell counting plate and kept ready for use.

2.1.4 fusion of spleen cells with myeloma cells

2.1.4.1HAT culture solution, 1640 culture solution and 1ml PEG4000 water bath, and 500ml of sterilized water preheated at 42 deg.C is prepared.

2.1.4.2 the SP2/0 cells and the splenocytes from the immunized mice in the logarithmic growth phase prepared above were added to a 50ml centrifuge tube at a ratio of 1: 8, and mixed by gentle inversion. Centrifuging at 800r/min for 10min, sucking up the supernatant to avoid affecting the fusion efficiency, and flicking the bottom of the centrifuge tube to ensure that the cells are uniformly paved on the bottom of the centrifuge tube as much as possible.

2.1.4.3 the fusion process is carried out in a fusion cup containing 42 ℃ water, 1ml of PEG4000 solution preheated at 37 ℃ is dropwise added into a 50ml centrifuge tube, the centrifuge tube is slowly rotated while adding, the addition is completed within 90 seconds, and the mixture is kept stand at 37 ℃ for 1-2 minutes.

2.1.4.4 Add 1640 basic culture medium slowly and then quickly to stop the reaction, 1ml of 1640 is added dropwise in 1 minute, 1ml of 1640 is added dropwise in 2 minutes, 3ml of 1640 is added dropwise in 3 minutes, 10ml of 1640 is added dropwise in 4 minutes, and 10ml of 1640 is added dropwise in 5 minutes. The mixture was allowed to stand for 2 minutes, gently inverted 2 times, left to stand for 7 minutes, centrifuged at 800r/min for 10 minutes, and the supernatant was discarded. Gently resuspending the cells in 110ml HAT medium, inoculating 200. mu.l/well of the suspension in a 96-well plate containing feeder cells, and culturing in 5% CO at 37 ℃₂Culturing in an incubator.

And (3) changing the liquid after 2.1.4.53 days, changing the liquid after 6 days, taking the supernatant when the cells grow to the bottom area of the holes 1/4-1/3, carrying out screening detection, and changing the supernatant into an HT culture solution.

2.1.5 screening and cloning of Positive hybridoma cell lines hybridoma cell culture supernatants were detected by indirect ELISA, and Positive clones were screened by 2-3 consecutive limiting dilution cloning, expanded culture and frozen storage of the cell lines. The indirect ELISA method was as follows:

2.1.5.1 coating with carbonate buffer solution (0.05mol/L, pH 9.6), adding FljB protein of Salmonella abortus to 1 μ g/ml, adding into 96-well enzyme-linked reaction plate, 100 μ L/well, and standing at 2-8 deg.C overnight.

2.1.5.2 washing to remove the liquid from the wells, the plates were washed 3 times with PBST (0.01mol/L, pH 7.4) 250. mu.l/well, and the liquid was blotted dry by flipping the plates over dry filter paper for each wash.

2.1.5.3 blocking and adding 5% skim milk-containing PBS (0.01mol/L, pH 7.4), 200. mu.l/well, and blocking at 37 ℃ for 2 hours.

2.1.5.4 the washing method is the same as 2.1.5.2.

2.1.5.5 the sample to be tested is added to 100. mu.l/well and acted at 37 ℃ for 1 hour.

2.1.5.6 the washing method is as same as 2.1.5.2

2.1.5.7 addition of secondary antibody 1: the HRP-labeled goat anti-mouse IgG diluted in 10000, 100. mu.l/well, was allowed to act at 37 ℃ for 30 min.

2.1.5.8 the washing method is as same as 2.1.5.2

2.1.5.9 adding TMB developing solution for developing color, 100 mul/hole, and placing at room temperature (15-25 ℃) for developing color (incubation in dark place) for 5 minutes.

2.1.5.10 stopping adding stop solution 50 μ l/well, slightly shaking, mixing, and reading OD with microplate reader at wavelength of 450nm_450nmValues (readings should be completed within 5 minutes after addition of stop solution) and the results recorded.

2.1.5.11 decision

When the test conditions are satisfied, if the value of the sample to be tested OD450nm is more than 1.0 and the value of P/N (P/N is the value of the sample to be tested OD450 nm/the value of the negative control OD450 nm) is more than 2.1, the test is judged to be positive, and the corresponding maximum dilution is the titer.

2.1.5.12 screening

(1) The different antibodies were serially diluted from 2.5. mu.g/ml in a 2-fold gradient. Diluted antibodies were added to ELISA plates coated with 1 μ g and 0.1 μ g FljB protein for 1h, followed by 1: HRP-labeled anti-mouse secondary antibody diluted 4000 times was allowed to act for 30min, and developed for 5 min. And calculating the affinity of the monoclonal antibodies of different strains.

(2) Diluting different monoclonal antibodies to coat with the same concentration (2 mug/ml), adding enzyme-labeled antigen with the same concentration after sealing, directly acting for 30min, and evaluating the binding condition of different antibodies and the enzyme-labeled antigen under the same concentration.

(3) Screening of coated antibody negative serum was mixed with HRP-FljB protein (1:3000) and added to ELISA plates coated with different concentrations of MAb (4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml) for 30min or 1h to screen for the best coated monoclonal antibody. Because no specific antibody of the salmonella abortus equi in the negative serum reacts with the enzyme-labeled antigen, the enzyme-labeled antigen directly reacts with the coated MAb, and the higher the OD450nm value is, the better the MAb effect is under the same negative serum and the same MAb coating concentration.

(4) The screening standard of the coating antibody selects positive hybridoma cells with strong affinity and strong antigen capturing capability for subsequent tests.

2.1.6 subcloning of Positive hybridoma cell line subcloning of positive hybridoma cell by limiting dilution method, cloning for 3 times until the antibody positive rate in cloning hole reaches 100%, enlarging culturing the obtained positive hybridoma cell, and freezing in liquid nitrogen.

2.2 preparation and purification of monoclonal antibodies

2.2.1 preparation of ascites A6-8 week old Balb/c mouse was injected with 0.5ml Freund's incomplete adjuvant to the abdominal cavity of each mouse, and after 7-10 days of inoculation, 1-2.5X 10 mice were injected to the abdominal cavity of each mouse⁶0.5ml of hybridoma cells with good growth. After the abdomen of the mouse is obviously enlarged, the ascites is collected. The harvested ascites is placed at 4 ℃ and centrifuged at 10000r/min for 10 minutes, and the supernatant is taken and stored for later use.

2.2.2 purification of monoclonal antibodies

2.2.2.1 preparation of sample the prepared ascites fluid was dissolved at room temperature, mixed with 4-5 times volume of binding/washing buffer (20mmol/L sodium phosphate, pH 7.0), and filtered through 0.45 μm filter.

2.2.2.2 column equilibration HiTrap protein G packing (approx. 2ml bed volume) was loaded onto a suitable chromatography column and equilibrated with 10 column volumes of binding/washing buffer (20mmol/L sodium phosphate, pH 7.0) at a flow rate of 1 ml/min.

2.2.2.3 loading, adding the sample into the well balanced chromatographic column at a flow rate of 0.2-1 ml/min, collecting the effluent, and repeatedly flowing the sample through the column for 3-5 times.

2.2.2.4 washing with 10-15 times column volume of binding/washing buffer (20mmol/L sodium phosphate, pH 7.0) to remove non-specifically adsorbed hybrid proteins.

2.2.2.5 eluting with an elution buffer solution (0.1mol/L glycine, pH 2.7) for 5-6 times, wherein the elution volume is 1ml each time, and collecting the effluent. Immediately after elution, neutralization buffer (1mol/L Tris-HCl, pH 9.0) was used, and about 120. mu.l of neutralization buffer (1mol/L Tris-HCl, pH 9.0) was added to each 1ml of the elution effluent.

2.3 identification of monoclonal antibodies

2.3.1 monoclonal antibody subclass identification the 1A10 strain to be screened was SBA Clonotyping^TM

And identifying the selected monoclonal antibody subclasses by using a System/HRP kit according to the instruction.

2.3.2 potency assay ascites purified from hybridoma cell line 1A10 was diluted 10-fold with PBS (0.01mol/L, pH 7.4) and detected by indirect ELISA as described in 2.1.5.1-2.1.5.11.

2.3.3 determination of protein concentration the protein concentration of the purified monoclonal antibody was determined according to the detection method in the BCA kit instructions.

2.3.4SDS-PAGE

2.3.4.1 sample treatment 2.5. mu.l of 5 XSDS-PAGE loading buffer was added to 10. mu.l of the harvested purified monoclonal antibody, boiled at 100 ℃ for 10 minutes, and gently centrifuged for use.

2.3.4.2SDS-PAGE analysis 10. mu.l of the treated sample and protein molecular weight Marker were subjected to SDS-PAGE electrophoresis according to Bio-Rad instructions, and the gel image was analyzed by the software BandScan5.0 to determine the purity of the monoclonal antibody.

2.3.5 affinity assay the affinity constant of the monoclonal antibody was determined by indirect ELISA. The 96-well plate was coated with purified FljB protein at concentrations of 0.1. mu.g/ml and 1.0. mu.g/ml per well. The purified monoclonal antibody was 2-fold diluted from 2.5. mu.g/ml to 0.001221. mu.g/ml. Single antibody was added to 2 antigen concentrations in coated wells and incubated at 37 ℃ for 1 hour. After washing 3 times with PBST, HRP-conjugated goat anti-mouse IgG (1: 10000) was used as a secondary antibody and incubated at 37 ℃ for 30 min. TMB substrate was added to carry out a color reaction. After termination, the OD is read_450nmAnd plotting the curve according to the concentration of the monoclonal antibody used. The monoclonal antibody affinity constant was then calculated using the formula using the mab concentration corresponding to 50% of the highest OD value (ODmax). K ═ n-1)/2(nAb '-Ab) Ab and Ab' represent antibody concentrations corresponding to half the maximum OD values at coating antigen concentrations of 0.1 and 1.0 μ g/ml, respectively, and n is the fold difference between the coating concentrations.

Specific detection of 2.3.61A 10 monoclonal antibody

Equine influenza virus, equine herpes type I virus, equine herpes type VI virus, equine arteritis virus, equine infectious anemia virus, Salmonella typhimurium, Salmonella enteritidis, Salmonella dublin, and Salmonella abortus proteins were subjected to SDS-PAGE electrophoresis using 1A10 as a primary antibody and a mouse-resistant secondary antibody for Western blot analysis to observe the specificity of the 1A10 monoclonal antibody.

3 results

3.1 selection of hybridoma cells

(1) The affinity calculation results are shown in fig. 1, indicating that: the 1A10 monoclonal antibody has the highest affinity constant of 1.19X10⁹1C5F10 times with an affinity constant of 9.22x10⁸1H9B4 minimum 1.05x10⁸。

(2) The binding force between different antibodies and enzyme-labeled antigen under the same concentration is analyzed as shown in figure 2, which shows that: when different monoclonal antibodies are coated at the same concentration, the OD value obtained after the enzyme-labeled antigen and the 1A10 monoclonal antibody are acted is the largest, which shows that the 1A10 monoclonal antibody has the strongest capacity of capturing the enzyme-labeled antigen under the condition of the coating of the monoclonal antibodies at the same concentration.

(3) The results of the screening for the best-coated monoclonal antibody are shown in FIG. 3, and show that: the enzyme-labeled antigen and the negative serum act for 30min or 1h in the ELISA plate pre-coated by the MAb, and the reaction hole OD of the 1A10MAb is coated₄₅₀The highest value, therefore 1A10 is the best coating MAb.

The characteristics of different monoclonal antibodies are comprehensively analyzed, and compared with other 7 monoclonal antibodies, the 1A10 antibody has the advantages of strong affinity, strong antigen capturing capability and the like, and is used for carrying out subsequent tests.

The hybridoma cell strain secreting the monoclonal antibody 1A10 is named as S-FljB-1A10, classified and named as hybridoma cell strain, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is located at the institute of microbiology, Zhongkou institute of Zhonglu 1 institute of China, Beijing, the rising area, wherein the strain preservation numbers are as follows: CGMCC number 21990 with a preservation date of 2021, 4 months and 1 day.

3.2 identification of monoclonal antibody 1A10

3.2.1 monoclonal antibody 1A10 subclass identification the selected monoclonal antibody subclass was identified according to the SBA clonotyping TMSystem/HRP kit instructions, and the results showed that the 1A10 monoclonal antibody subclass was IgG1 and the light chain was kappa-type.

TABLE 1 monoclonal antibody 1A10 subclass identification results

3.2.2 titer determination the titer of ascites after purification of hybridoma cell strain 1A10 was determined by indirect ELISA method to be 1: 6.6X 10⁶See table 2 for details.

TABLE 21A 10 monoclonal antibody potency assay results

3.2.3 determination of the concentration of monoclonal antibody 1A10 the concentration of the purified monoclonal antibody was determined to be 7 mg/ml.

3.2.4SDS-PAGE analysis 7 tubes of the monoclonal antibody 1A10 eluted were subjected to SDS-PAGE. The results show that the purified monoclonal antibody shows two specific bands by SDS-PAGE electrophoresis, wherein the heavy chain is about 50kDa, the light chain is about 25kDa, and the purity is about 98%, and the purified monoclonal antibody is mainly concentrated in the 3 rd, 4 th and 5 th tubes, as shown in detail in FIG. 4.

3.31A 10 specific test results

The results of the specificity test are shown in FIG. 5, and show that 1A10 reacts only with the protein of Salmonella abortus equi, and has no cross reaction with other pathogenic bacteria, indicating that the specificity is good.

Example 3 establishment of competitive ELISA antibody detection method for Salmonella abortus equi

1. Design of optimal reaction strategy

Four different reaction conditions and strategies were designed according to the competitive ELISA principle (fig. 6), one-step: directly adding the serum to be detected and HRP-labeled FljB protein (enzyme-labeled antigen) into an ELISA plate to act for 30 minutes or 1 hour; a two-step method: the serum to be detected is firstly acted on the HRP-labeled FljB protein for 30 minutes or 1 hour, and then the mixture of the two is transferred into an ELISA plate to be acted for 30 minutes.

2. Screening of optimal envelope monoclonal antibody and enzyme-labeled antigen working concentration

Reaction strategies of a one-step method (figure 6) and a two-step method (figure 6) are adopted to respectively optimize and screen the reaction conditions of the concentration of the coated antibody and the concentration of the enzyme-labeled antigen, determine the optimal reaction conditions of each reaction strategy, detect the same positive serum by using the optimal reaction conditions, and further compare the sensitivity difference of different reaction strategies.

The optimization results of the one-step method and the two-step method for 30min and 1h respectively under different concentrations of the coated antibody and the enzyme-labeled antigen are shown in fig. 7 (one-step method) and fig. 8 (two-step method), and the optimal reaction conditions obtained by screening are shown in table 3. Under the optimal reaction conditions, the result of detecting the maximum dilution of the same positive serum is shown in Table 3, if the inhibition rate is more than 41.5 percent and is taken as the positive result judgment standard, the result shows that the detection sensitivity is 16-fold dilution under different reaction strategies, which indicates that the detection effect of each reaction strategy is equivalent, so that the monoclonal antibody coating concentration is low, and the OD of the negative serum is equivalent_450nmThe value is 1.0-2.0, and the subsequent experiments are respectively carried out by selecting a one-step method for 30min and 1h under the condition that the operation is simple and convenient as a screening principle.

The 2 optimal reaction conditions for screening were: 1) the coating concentration of the monoclonal antibody is determined to be 2 mug/ml, the concentration of the FljB protein marked by HRP is determined to be 3000 times dilution (3000x), and the monoclonal antibody directly acts for 30 min; 2) the monoclonal antibody coating concentration was determined to be 2. mu.g/ml and the HRP-labeled FljB protein concentration was determined to be 6000-fold dilution (6000 ×). Directly acting for 1 h.

TABLE 3 optimal reaction conditions and comparison of the four reaction strategies

3. Screening of substrate developing solution

Because the OD values of the negative serum under the optimized 4 reaction conditions are all between 1.5 and 1.8, in order to improve the OD value of the negative serum detection and improve the detection sensitivity, the color development liquid is screened: the substrate is replaced by a two-component (a developing solution with two components of the substrate A and the substrate B separated independently) developing solution from a single component (a premixed developing solution with two components of the substrate A and the substrate B). As shown in FIG. 9, the two-component developing solution is adopted, and the OD values of the negative serum are increased to 2.0-3.0 under the reaction conditions of direct action for 30min and 1h, while the change of the positive serum is very small, so that the screened substrate developing solution is the two-component developing solution.

4. Screening of action time of serum and enzyme-labeled antigen

The optimal time for the action of the serum and the enzyme-labeled antigen is screened out by applying the reaction conditions and the two-component developing solution for experimental optimization screening and performing sensitivity detection analysis on the same positive serum.

The results are shown in fig. 10, the results of the inhibition detection by direct action for 30 minutes and 1 hour are relatively close, if the inhibition is greater than 41.5% as the positive result judgment standard, the maximum dilution times of the positive serum detected under the two reaction conditions are 32 times and 16 times respectively, which shows that the serum and the enzyme-labeled antigen directly act for 30 minutes by using the two-component developing solution, and the sensitivity of the detection can be improved by 2 times by comparing the experimental result (i.e., the maximum dilution time of the detection is 16 times) of the single-component developing solution in table 3. Therefore, the time for the serum to react with the enzyme-labeled antigen was determined to be 30 minutes.

5. Determination of the threshold for competitive ELISA

The 413 parts of negative serum are detected by an optimized competitive ELISA method, the average value (X value) of all inhibition rates is 2.79 percent, the standard deviation (SD value) is 0.1498, and the critical value (X +3SD) of positive and negative serum judgment in clinical serum detection is calculated to be 41.5 percent by a biological statistical analysis method, so that the serum sample to be detected is judged to be positive when the inhibition rate value is greater than or equal to the critical value (Cutoff), and is judged to be negative when the inhibition rate value is lower than the critical value.

6. Test steps of equine abortion salmonella competitive ELISA antibody detection method

By optimizing the reaction conditions of the competitive ELISA method, the optimal reaction conditions were determined as follows: mu.g/mL of 1A10MAb was coated overnight at 4 deg.C, 5% w/v N-chlorosuccinimide (NCS) blocked for 2h at 37 deg.C, serum was mixed with 1:3000 fold dilution of HRP-FljB and applied to ELISA plates for 30min at 37 deg.C, and TMB developed for 10min (37 deg.C).

The specific operation steps are as follows:

(1) coating the ELISA plate with 2 mu g/mL 1A10MAb at 4 ℃ overnight (>12h), washing with a washing solution, blocking with a blocking solution 5% w/v NCS at 37 ℃ for 2h, and washing the plate;

(2) mixing HRP-labeled FljB protein with a control or serum sample to be detected (1:1), adding the mixture into a plate, incubating at 37 ℃ for 30min, and washing the plate;

(3) adding two-component TMB developing solution, developing at 37 deg.C for 10min, adding stop solution to terminate reaction, and measuring OD_450nmValue, calculate inhibition ([ negative control OD)₄₅₀Value-sample OD₄₅₀Value of]/[ negative control OD₄₅₀value-Positive control OD₄₅₀Value of]X 100%), when the inhibition rate is not less than 41.5%, the test piece is judged to be positive, and when the inhibition rate is less than 41.5%, the test piece is judged to be negative.

7. Competitive ELISA specificity assays

Positive sera such as salmonella equi (s.abortus equi), Equine Infectious Anemia Virus (EIAV), Equine Arteritis Virus (EAV), Equine Herpes Virus (EHV), Equine Influenza Virus (EIV), equine theileria (t.equi), equine babesia (b.caballi), streptococcus equi (s.equi), salmonella typhimurium, salmonella dublin, salmonella enteritidis and the like are detected by using the optimized optimal reaction conditions, and the specificity of the competitive ELISA antibody detection method for salmonella equi is evaluated, and the result shows that only the detection result of the salmonella equi serum is positive, and the detection results of other pathogenic positive sera are negative (as shown in fig. 11). Thus demonstrating good specificity of the method.

8. Comparison of competitive ELISA with tube agglutination assay, Indirect ELISA (iELISA) for detection of horse serum sample sensitivity

For 2 parts of horse abortion positive serum (serum 1 and serum 2), the test Tube Agglutination (TAT) titer is 1:1600 (according to the specification of the industry standard of NYT-570-2002-horse abortion salmonella disease diagnosis technology, the test tube agglutination titer is more than or equal to 1:1600, the test is positive), the serum to be detected is diluted by 2 times, then the TAT test is carried out, the detection result is negative, and therefore the maximum dilution detectable by the serum 1 and the serum 2 in the TAT test is the original time (1 x).

The detection is carried out by adopting competitive ELISA and indirect ELISA methods respectively, wherein the indirect ELISA method is carried out according to the method described in the patent application with the publication number of CN111458501A and the invented name of 'indirect ELISA kit for detecting the antibody of the salmonella abortus equi, the preparation method and the application thereof'.

Results as shown in fig. 12, the 2 positive samples were tested by competition ELISA with 32-fold dilutions (32 ×) for maximum dilution versus test tube agglutination test results: namely, the result of original double serum is positive, the result of serum diluted by 2 times is negative, and the maximum dilution of TAT detection is original double. Therefore, the competitive ELISA assay is 32 times more sensitive than the tube agglutination assay.

The maximum dilution of the 2 samples tested by the indirect ELISA was also 32 x, indicating that the competitive ELISA is comparable to the detection sensitivity of the indirect ELISA.

9. Comparison of competitive ELISA with test tube agglutination assay (TAT) for sensitivity detection of donkey serum samples

2 (serum 3 and serum 4) TAT positive serum samples with the maximum dilution being the original time are detected by competitive ELISA, as shown in figure 13, the 2 positive samples with the maximum dilution being 32 times (32 x) are detected by competitive ELISA, and the sensitivity is higher than that of the test tube agglutination test.

10. The influence of animal species on the serum samples tested by competitive ELISA is less than that of indirect ELISA

From the experimental principle, the competitive ELISA judges the result through the result condition of the monoclonal antibody and the enzyme-labeled antigen, and the judgment of the result is not influenced by the animal serum of different animal sources (horse or donkey); and the indirect ELISA method adopts an anti-horse secondary antibody for detection, if donkey serum is detected, an anti-donkey secondary antibody is adopted to reestablish a detection method, otherwise, the obtained data is not scientific.

11. The specificity of competitive ELISA was better than indirect ELISA

In indirect ELISA detection, horse serum reacts with enzyme-labeled anti-horse secondary antibody after non-specific binding with an ELISA plate, a coating antigen and the like to generate a non-specific detection result; in the competitive ELISA method, if the enzyme-labeled antigen is bound to an ELISA plate or a coated antigen, the binding between the enzyme-labeled antigen and the coated monoclonal antibody is not affected, and the determination of the result is not greatly affected. The schematic is shown in fig. 14.

12. Detection of clinical samples

12.1 competitive ELISA detection of Standard serum

20 parts of each of positive and negative samples of the test Tube Agglutination Test (TAT) were tested by the competitive ELISA method established in the present invention, and the results are shown in Table 4. The positive and negative match rates for the competitive ELISA and TAT were 100%.

TABLE 4 test results of clinical samples

Note: "+" represents a positive result, and "-" represents a negative result

12.2 competitive ELISA assay of clinical samples

860 clinical sera were tested by competitive ELISA, 380 of which were positive and the detection rate was 37.7%.

Claims

1. A hybridoma cell strain secreting anti-Salmonella abortus (S.abortus. equi) FljB protein monoclonal antibody is named as S-FljB-1A10 and is preserved in China general microbiological culture Collection center, and the strain preservation numbers are as follows: CGMCC No.21990, with a preservation date of 2021, 4 months and 1 days.

2. The hybridoma cell strain of claim 1 secreting a monoclonal antibody against FljB protein of Salmonella abortus.

3. Use of the monoclonal antibody of claim 2 in the preparation of a reagent for detecting antibodies to salmonella abortus equi.

4. The use of claim 3, wherein the reagent is a competitive ELISA antibody detection reagent.

5. A kit for detecting competitive ELISA antibodies of Salmonella abortus equi, which comprises the monoclonal antibody of claim 2.

6. The kit of claim 5, wherein the monoclonal antibody acts as a coating antibody.

7. The kit according to claim 5, further comprising 5% w/v of NCS blocking solution, HRP-labeled FljB protein, washing solution, two-component TMB developing solution, and stop solution.

8. The kit of claim 7, wherein the kit is used for detecting antibodies against Salmonella abortus equi according to the following steps:

(1) coating an ELISA plate with 2 μ g/mL of the monoclonal antibody of claim 2 at 4 ℃ overnight, washing with a washing solution, blocking with 5% w/v NCS at 37 ℃ for 2h, and washing the plate;