CN111825764B - Brucella canicola monoclonal antibody and application thereof - Google Patents

Abstract

The invention relates to a brucella canis monoclonal antibody and application thereof. The monoclonal antibody (monoclonal antibody) of the brucella canis has specific immunological reaction with antigen of reference strain and clinical isolate of the brucella canis, and does not react with yersinia enterocolitica, escherichia coli and salmonella dublin which have strong cross reaction with the brucella and salmonella irrelevant to the cross reaction. The kit has high specificity, can be widely used for serological detection of the Brucella canis, and fills the blank that a serological method of the Brucella canis lacks a specific monoclonal antibody; meanwhile, a cELISA method for accurately diagnosing the Brucella canicola with high flux is established based on the monoclonal antibody, the method has the characteristics of quickly and accurately diagnosing the Brucella canicola serum antibody with high flux, effectively solves the technical problem of interference diagnosis of Yersinia enterocolitica, Escherichia coli and Salmonella dublin, and enriches serological detection methods of the Brucella canicola.

Description

Brucella canicola monoclonal antibody and application thereof

The invention relates to a brucella canis monoclonal antibody hybridoma cell strain, a preparation method of the brucella canis monoclonal antibody, and a cELISA method or a product established by using the monoclonal antibody, belonging to the field of veterinary microbiology diagnosis.

Background

Brucellosis (hereinafter referred to as "brucellosis") is a serious chronic allergic infectious disease caused by brucella, is one of 16 domestic animal epidemics which are preferentially prevented and treated in the long-term animal epidemic prevention and treatment plan (2012-2020) in China, and is classified as a legal report animal epidemic disease by the world animal health Organization (OIE). Human infection with brucellosis is mainly caused by diseased animals and products thereof (Zhuliangyen et al. report in microbiology 2015(01): 171-177; Mao Kai Rong, et al. animal brucellosis diagnosis technology, Chinese agricultural Press 2014.).

The brucella is divided into 6 classical species according to antigenicity and main host, including brucella melitensis (b.abortus), brucella bovis (b.abortus), brucella suis (b.suis), brucella canis (b.canis), brucella epididymis (b.ovis), and brucella suis sarin (b.neotomae). The main pathogenicity of human is Brucella melitensis, Brucella bovis, Brucella suis and Brucella canis. The Brucella melitensis of sheep, cattle and pig species belongs to smooth Brucella melitensis, has strong pathogenicity to human, is a main pathogen of Brucella melitensis of livestock, and is widely concerned; however, Brucella canicola belongs to a rough strain, is weak in pathogenicity to human, and has relatively few research reports for a long time (Mao Kai Rong, et al.

With the economic development, the number of raised dogs is increased and the dogs are in close contact with human beings, the incidence number of brucellosis of the dogs is rapidly increased, and the potential harm to human health and public health safety is becoming serious. To date, tens of cases of human brucellosis caused by brucella canicola have been reported worldwide. Brucella infection commonly exists in dog groups in China, the brucella infection of dogs in China is reported to have a positive rate of 1.68-13.4%, and the antibody positive rate of brucella disease of dogs in partial areas can reach about 20% (Zhou Guilan, et al, China veterinary journal, 2001 (11): 60, Liu Shi, et al, microbiological report, 2019,46(12): 3325) 3334, yellow extraction et al, biological disaster science, 2018,41(04): 290-); in 2018, the national animal brucellosis reference laboratory detects the serum of pet dogs in several pet hospitals such as Beijing and Shandong, and the brucella melitensis seropositivity of the dogs is up to 2% (unpublished data). The brucellosis infection source of dogs is mainly sick dogs and dogs with bacteria, a large amount of pathogenic bacteria are discharged from infected pregnant bities along with fetuses and fetal membranes during abortion and delivery, and vaginal secretion and milk after abortion also contain pathogenic bacteria. When infected by a male dog and suffering from orchitis, the semen contains pathogenic bacteria which can be transmitted to a female dog through mating (Huang extract et al, biological disaster science, 2018,41(04): 290-.

For the canine brucellosis, no vaccine and ideal drug prevention, control and treatment exist at present, and the strategy of 'quarantine and elimination' is mainly adopted. Although the isolation and identification of bacteria are diagnostic gold standards, serological methods are commonly used due to their long handling time, low isolation rate, and limited biosafety concerns and technical conditions.

Common serological detection methods for Brucella canis include rapid plate agglutination assay (RSAT), rapid plate agglutination assay with 2-mercaptoethanol (ME-RSAT), test tube agglutination assay (SAT), and Agar Gel Immunodiffusion (AGID). The canine Brucella is different from smooth Brucella of cattle, sheep and pig in antigen phenotype, is rough, relatively weak in immunogenicity and has self-coagulation property, so that serological agglutination reactions such as RSAT, ME-RSAT and SAT are not high in specificity, and cross reactions are easy to occur with Yersinia enterocolitica, Escherichia coli, Salmonella dublin and the like, and false positive exists; AGID is commonly used abroad for confirmation, but its sensitivity is relatively low (Fernandes, A.R.F.et al, Brazilian Journal of Microbiology (2011)42: 1405-1408; Ana Laura Bello de Oliveira, et al, Brazilian Journal of Microbiology (2019)50: 307-312; R.Bruce Hollett, Theriogenology 66(2006) 575-587); sharma Barkha, et al, Asian Pacific Journal of clinical Medicine (2011) 857-; tory V Whitten et al, previous Veterinary Medicine 168(2019) 90-94). In view of the fact that the domestic and foreign reports of the preparation of the Brucella monoclonal antibody are almost unsuccessful, the monoclonal antibody hybridoma cell strain which has high affinity and can specifically identify the Brucella antigen of the dog is prepared, and the monoclonal antibody is utilized to establish a serological method for quickly, highly-flux and accurately diagnosing the Brucella of the dog, so that the monoclonal antibody has important significance for widely developing the epidemiological investigation and epidemic disease prevention and control of the Brucella of the dog.

Disclosure of Invention

The purpose of the invention is as follows: mainly aims at the defects of the traditional method for diagnosing the brucellosis of the dogs, such as long operation time, low separation rate, and limitation on biosafety factors and technical conditions when bacteria are separated, cultured and identified; the traditional serum agglutination reaction has poor specificity; the monoclonal antibody hybridoma cell strain of the Brucella canis is established, the Brucella canis monoclonal antibody is obtained, and the cELISA detection method established by the monoclonal antibody is utilized to realize rapid and high-flux diagnosis of the Brucella canis serum and timely prevention and control of epidemic diseases.

Technical scheme of the invention

1. A Brucella canicola monoclonal antibody, characterized by that the monoclonal antibody is IgG2b subclass, its light chain type should be k; the monoclonal antibody respectively generates obvious immunological reactions with brucella Canitis antigens, and does not react with yersinia enterocolitica O9, Escherichia coli O157 antigen, Salmonella dublin antigen and antigen without cross reaction which have strong serological cross reaction with brucella;

the brucella Canitis monoclonal antibody is prepared from a brucella Canitis hybridoma cell 4H3 strain which is delivered to China general microbiological culture Collection center (CGMCC No. 19964) in microbial research institute of China academy of sciences, No. 3 of Beijing West Lu 1 institute of North Chen West province, Tokyo, No.1, in 2020, 08 and 17 days.

2. The invention relates to a brucella canis monoclonal antibody, which is characterized in that the preparation steps of the monoclonal antibody are as follows: (1) immunizing a 6-8-week-old Balb/c mouse by using a Brucella strain RM6/66 strain lysis antigen protein as an immunogen; (2) separating splenocytes of an immune mouse with ELISA titer more than or equal to 1:20000, and fusing the splenocytes with myeloma cells to obtain hybridoma cells; (3) indirect ELISA screening established by the whole-bacterium split protein antigen of the Brucella canis and subcloning culture to obtain hybridoma cells; (4) monoclonal antibodies were isolated and purified from cell culture broth or ascites of mice inoculated with hybridoma cells.

3. The invention relates to a Brucella canis monoclonal antibody, which is characterized in that the monoclonal antibody is used for establishing a cELISA detection method of a Brucella canis serum antibody;

the cELISA detection method for the brucella canis serum antibody mainly comprises the steps of coating an ELISA plate with a split protein antigen containing a brucella canis strain, adding a specific brucella canis monoclonal antibody and brucella canis serum to be detected, establishing a blank control without serum, and then performing normal cELISA program operation, wherein the absorbance value of positive serum is specifically inhibited, the absorbance value of negative serum is not inhibited, and the absorbance value is not different from the blank control.

Novel technical effects of the invention

The invention relates to a brucella canis monoclonal antibody and application thereof. The monoclonal antibody of the Brucella canis has specific immunological reaction with reference strain and clinical isolate antigen of the Brucella canis, and does not react with Yersinia enterocolitica, Escherichia coli, Salmonella dublin which have strong cross reaction with the Brucella and Salmonella unrelated to the cross reaction. The kit has high specificity, can be widely used for serological detection of the Brucella canis, and fills the blank that a serological method of the Brucella canis lacks a specific monoclonal antibody; meanwhile, a cELISA method for accurately diagnosing the Brucella canicola with high flux is established based on the monoclonal antibody, the method has the characteristics of quickly and accurately diagnosing the Brucella canicola serum antibody with high flux, effectively solves the technical problem of interference diagnosis of Yersinia enterocolitica, Escherichia coli and Salmonella dublin, and enriches serological detection methods of the Brucella canicola.

Drawings

FIG. 1 results of different antigenic proteins and monoclonal antibody 4H3 Westernblot

FIG. 2 ROC graph

Information on microbial resources related to the present invention

Brucella canicola CVCC70701(RM6/66) strain is from China veterinary microbial cultures collection management center, please see "China veterinary pharmaceuticals institute, China veterinary microbial cultures collection management center editions, China veterinary bacterial catalogues, second edition, p28 (China agricultural science and technology publishers, 2002).

Brucella melitensis hybridoma cell 4H3 strain, which has been preserved in China general microbiological culture Collection center (CGMCC No. 19964) in the microbiological research institute of China academy of sciences, national institute of sciences, No.1, west way, north Cheng, Chaoyang, Beijing, and 2020, 08, 17 days.

Detailed description of the invention

1. A strain of hybridoma cell is successfully screened by continuously immunizing a Balb/C mouse with the age of 6-8 weeks for 5 times by using the Brucella melitensis cleavage protein, fusing and then continuously subcloning for 3 times, and is named as a Brucella melitensis hybridoma cell strain 4H 3. (1) The ELISA titer of the monoclonal antibody produced by the strain cell reaches 1: 25000; identifying the subtype, wherein the heavy chain type result is IgG2b subclass, and the light chain type is k; (2) the monoclonal antibody respectively carries out plate agglutination test, indirect ELISA and Westernblot with canine brucella antigen, yersinia enterocolitica O9, escherichia coli O157 antigen, Dublin salmonella antigen and salmonella antigen which has strong serological cross reaction with brucella, and as a result, the 4H3 monoclonal antibody is not agglutinated with the Dublin salmonella antigen, the Yersinia enterocolitica O9, the escherichia coli O157 antigen and the salmonella antigen which has no cross reaction, and is specifically agglutinated with the canine brucella antigen. Meanwhile, the lysis antigen is coated on an ELISA plate with the concentration of 1 mu g/mL, indirect ELISA is used for detecting cell supernatant, and the result shows obvious reaction to canine brucella antigen, but no reaction is generated with small enterocolitis Yersinia O9, Escherichia coli O157 antigen, Dublin salmonella antigen and salmonella antigen without cross reaction; this was further confirmed by subjecting the above antigens to SDS-PAGE and Westernblot using hybridoma culture supernatant as a primary antibody (see FIG. 1).

2. Brucella melitensis CVCC70701(RM6/66) is selected and cultured by tryptone agar (TSA)Culturing at 37 deg.C for 48 hr, performing amplification culture, inactivating the obtained thallus in 80 deg.C water bath for 2 hr, and adjusting to 3.0 × 10 with distilled water⁹CFU/mL, adopting ultrasonic wave treatment, the frequency is 30Hz, 15 min/time, repeating for 3-4 times. The lysed supernatant was centrifuged at 8000r/min for 10min, the precipitate was discarded, and the supernatant was diluted to about 0.2mg/mL after quantification. Then emulsifying the immunogen with Freund's adjuvant according to the concentration ratio of 1:1 to serve as a primary immune immunogen, and then emulsifying the immunogen with Freund's incomplete adjuvant according to the ratio of 1:1 to serve as a subsequent reinforcing immunogen; after three times of immunization, detecting the serum titer of the mice by an indirect ELISA method in each blood collection, and fusing when the serum titer reaches more than 1: 20000; injecting double dose of antigen protein without adjuvant into abdominal cavity for enhancing immunity 3 days before fusion, then taking splenocytes of immunized mice to fuse with SP2/0 cells, sequentially carrying out subcloning 3 times by adopting a limiting dilution method, carrying out ELISA screening on cell culture supernatant generated by each subcloning by using a Brucella canicola RM6/66 strain lysis antigen coated plate, taking hybridoma cell strains which can realize single-specificity recognition on Brucella canicola RM6/66 strain antigens and have the highest ELISA titer, and carrying out passage and preservation until the fourth subcloning to obtain hybridoma cell strains 4H 3.

3. Continuously transmitting the hybridoma cell strain for 5 generations, preparing monoclonal hybridoma cell strain 4H3 ascites, purifying by using a chromatographic column method, then coating an ELISA plate with 1ug/mL brucella canicola split protein, simultaneously adding a specific brucella canicola monoclonal antibody 4H3 and brucella canicola serum to be detected, setting a blank control without serum, and then operating according to a normal ELISA program, wherein the absorbance value OD450nm of the positive serum is less than 0.2; the absorbance values of the negative serum are all more than or equal to 1.8, and are not different from the blank control. 70 parts of brucella Canitis serum (positive 40 parts and negative 30 parts) with clear background is adopted to carry out cELISA detection, an ROC curve (shown in figure 2) is established, and the Cut-off value is determined, namely the inhibition rate PI is more than or equal to 30 percent and is judged to be positive.

4. The built cELISA method and the RSAT, ME-RSAT, SAT and AGID methods are used for detecting 80 clinical dog serum, the cELISA and the AGID have the highest coincidence rate, and the coincidence rate with the RSAT, ME-RSAT, SAT and AGID methods is 75.0%, 87.5%, 85.0% and 96.2% respectively. The clinical application effect is good. Meanwhile, positive serum with the antibody agglutination valence of 1:1000 is used, and after serial dilution, cELISA and AGID are respectively used, and the cELISA sensitivity is found to be obviously higher than that of the AGID.

Examples

The following examples are intended to further illustrate the invention and are not to be construed as limiting the invention.

Example 1 hybridoma cell lines and monoclonal antibodies produced thereby

1. Antigen preparation is carried out by taking 1 strain of Brucella canicola CVCC70701(RM6/66) strain glycerol bacteria (obtained from China veterinary medical microorganism culture preservation management center and prepared, identified and stored in reference laboratory of Brucella animal in China veterinary medical supervision), thawing, inoculating into 10mL TSB culture medium (soybean casein peptone broth culture medium, purchased from BD company) small tube according to the proportion of 1%, and carrying out shaking culture at 37 ℃ and 200r/min for 48 h. Dipping a small amount of bacterial liquid by using an inoculating loop, carrying out single colony streak pure culture on a TSA (soybean casein peptone agar culture medium purchased from BD company), after culturing for 48h at 37 ℃, selecting a typical single colony on the TSA plate, carrying out dense streak, culturing for 48h at 37 ℃, washing bacterial lawn by using 5mL of physiological saline on each plate, mixing uniformly, inactivating in 80 ℃ water bath for 2h, adjusting the inactivated bacterial strain to about 3 multiplied by 10 by using distilled water⁹CFU/mL, adopting ultrasonic treatment, with the frequency of 30Hz, 15 min/time, repeating for 3-4 times. Centrifuging at 8000r/min for 10min, discarding precipitate, and diluting the supernatant to about 0.2mg/mL after quantification.

2. Animal immunization the above concentration RM6/66 Brucella melitensis antigen is mixed with Freund's complete adjuvant or Freund's incomplete adjuvant at a ratio of 1:1, and the antigen is emulsified until water-in-oil state is achieved. 0.2mL of BALB/c mice at 6 weeks of age were injected subcutaneously. Complete adjuvant is added for the first immunization, and incomplete adjuvant is adopted for the subsequent immunization of the second immunization, the third immunization and the like after 2 weeks; after three times of immunization, the serum titer of the mice is detected by an indirect ELISA method, and the fusion is carried out until the serum titer reaches more than 1: 20000.

3. Feeder cells are generally prepared 1-2 days before fusion, and the immunized Balb/c mouse is killed at the dislocation of cervical vertebra after exsanguination, and is soaked in 75% alcohol for 5min for disinfection; placing mouse on super clean bench sterile plateCarefully cutting off the skin of the abdomen, separating the skin from the peritoneum, exposing the abdominal wall, and wiping the abdominal wall with an alcohol cotton ball for disinfection; sucking 5mL of DMEM nutrient solution by using a sterile syringe, injecting the DMEM nutrient solution into the abdominal cavity of the mouse, enabling abdominal cavity cells to fully enter the nutrient solution, slightly sucking back the nutrient solution, and adding the nutrient solution into a sterile centrifugal tube; repeatedly washing abdominal cavity for three to four times, centrifuging the sucked culture medium for 10min at 1000r/min, removing supernatant, suspending the precipitate with HAT selection culture medium (1% HAT + 10% bovine serum + 88% DMEM + 1% streptomycin double antibody), mixing uniformly, adding 3-6 blocks of 96-well cell culture plates with each well being 100 mu L, placing at 37 ℃ and 5% CO₂Culturing for later use.

And 4, subculturing SP2/0 cells 24-48 h before preparation and fusion of the SP2/0 cells. On the day of fusion, SP2/0 cells which are good in shape and grow logarithmically are selected, gently blown down from the bottle wall by using a proper amount of DMEM basic culture medium, collected in a 50mL centrifugal tube, and counted; centrifuging at 1000r/min for 10 min.

5. Preparing immune spleen cells, injecting a double dose of protein antigen without adjuvant into abdominal cavity 3 days before fusion to strengthen immunity, taking an immune mouse 3 days after strengthening immunity, removing eyeball bleeding and separating serum to serve as antibody positive control; killing the mice, soaking the mice in 75% alcohol for 5min, taking out the spleen of the mice aseptically, putting the mice in a plate containing 10mL of DMEM nutrient solution, and rinsing gently; transferring the spleen into another dish containing about 20mL of DMEM nutrient solution, sucking the nutrient solution by a disposable syringe to insert into one end of the spleen, washing out cells in the spleen, and repeatedly washing for several times until the color of the spleen is white; the washed splenocytes are transferred into a 50mL centrifuge tube, the cells are counted, centrifuged at 1000r/min for 10min, the supernatant is discarded, and the cell sediment is resuspended for later use.

6. Cell fusion appropriate amounts of spleen cells and myeloma cells (in a quantity ratio of about 10:1) were aspirated separately, added to a 50mL centrifuge tube, and gently mixed. Centrifuging at 1000r/min for 10min, discarding supernatant, flicking tube bottom with finger to make precipitated cells loose uniformly, rotating centrifuge tube with one hand uniformly, sucking 1mL PEG2000 with the other hand, slowly adding along the wall of the rotating centrifuge tube for 60s, sucking cell suspension into the pipette (time controlled at about 30 s), standing for 30ss, lightly blowing the mixture into a centrifuge tube (the time is controlled to be about 30 s); the effect of PEG2000 was then stopped by the addition of 25mL of DMEM. The specific method comprises the following steps: slowly adding 1mL of DMEM solution in the 1 st min, adding 4mL of DMEM solution in the 2 nd min, then adding 20mL of DMEM solution in the 3 rd min, and standing for 10min after the addition is finished; the fused cells are centrifuged at 1000r/min for 7min, the supernatant is discarded, and an appropriate amount of HAT culture solution (1% HAT + 10% bovine serum + 88% DMEM + 1% streptomycin double antibody) is added for gentle aspiration, so that the precipitated cells are uniformly mixed. Adding the suspension into 4 feeder cells-plated 96-well plates (100 μ L per well), and placing in CO₂Culturing in an incubator; cell growth was recorded daily, and on day 4 after fusion, medium was half-changed with 1% HAT (1% HAT + 10% bovine serum + 88% DMEM + 1% streptomycin cyanohydrin) selection medium, half-changed with 1% HT selection medium (1% HT + 10% bovine serum + 88% DMEM + 1% streptomycin cyanohydrin) on day 8, and then cultured with normal 10% bovine serum DMEM medium after day 14.

7. Screening and subtype identification of positive hybridoma cells when cell clones to be fused grow to the bottom 1/3 of a covered cell hole, detecting culture supernatants in which all cells with the clones grow by using an established indirect ELISA method, carrying out expanded culture on the cells which are detected to be positive, identifying the subclasses of antibodies, and identifying the cells as IgG positive holes for cloning.

8. Cloning of positive hybridoma cells feeder cells were prepared the day before cloning by limiting dilution method as described above; gently sucking the cell holes detected as strong positive by using a suction pipe to disperse the cells uniformly, sucking the cells into 1mL of DMEM solution containing 10% fetal calf serum, and taking a small amount of suspension to count the cells; diluting positive hybridomas by about 10/mL with 1% HAT selection medium; adding the diluted cell suspension into a 96-well culture plate with feeder cells, wherein each well is 100 mu L; cell growth was observed daily. On the fourth day, half of the culture solution is changed by 1% HT selection, the supernatant is detected, and a positive hole with only one clone growing is selected for cloning again; after several subclones, when the monoclonal positive rate reaches 100%, obtaining the hybridoma cell strain which can stably secrete the antibody, and performing timely amplification culture and cryopreservation.

9. Preparation of ascites and determination of potency

(1) Preparation of ascites selecting Balb/c-producing mother mouse, injecting sterilized paraffin oil or Freund incomplete adjuvant into abdominal cavity of the mother mouse 0.5 mL/mouse. After 10 days, the expanded hybridoma cells were blown down and counted. l000r/min for 10min, discarding the supernatant, washing once with sterile PBS, resuspending with appropriate amount of PBS, adjusting cell concentration to about 1X 10⁷one/mL. 10 mice were inoculated per hybridoma cell line and 0.5mL of each mouse was inoculated into the abdominal cavity. And after 7-10 days, collecting ascites when the abdominal distension of the mouse is close to saturation. Then the ascites is centrifuged at 5000r/min for 10min, and the supernatant is collected and stored at-20 ℃ for later use.

(2) Potency assay

1) The results of antibody monitoring after immunization are shown in table 1.

TABLE 1 ELISA antibody titer test results after boosting immunization

	1:10	1:100	1:1000	1:5000	1:10000	1:20000	Positive control (+)	Negative control (-)
									Sanwu	3.234	2.03	1.553	0.4013	0.153	0.108	3.124	0.102
Exempt from	3.348	3.134	2.5878	0.6329	0.321	0.219	3.013	0.127
									Wu exempt	3.785	3.321	2.6975	0.7764	0.415	0.316	3.254	0.116

According to the judgment that the P/N is more than or equal to 2.1, the ELISA titer reaches 1:20000 after the five-immunization as shown in the table 1.

2. The fusion and subclone screening results are shown in Table 2.

TABLE 2 fusion and subclone screening of cell line supernatants for test results

	Rough dog species ZG	Smooth type S2	Positive control (+)	Negative control (-)
					Fusion	3.3621	0.4013	3.024	0.202
First subcloning	2.3878	0.2329	2.813	0.127
					Second subcloning	2.2975	0.1764	2.926	0.216
Third subcloning	2.1976	0.187	2.845	0.136
					Fourth subcloning	2.218	0.165	2.937	0.222

The results in Table 2 show that the monoclonal antibody cell strains specifically recognizing the canine species are selected from 4 times of fusion and subcloning by a limiting dilution method, the positive values are high, and 100% positive clones can be stably obtained.

Example 2 determination of subtype of monoclonal antibody against canine species and agglutination reaction

And identifying the monoclonal antibody according to the standard procedure operation of a subtype identification kit of Thermo company. The subtype of the 4H3 cell culture supernatant was determined by using a monoclonal antibody subtype determination Kit (Pierce Rapid ELISA Mouse mAb Isotyping Kit, Thermo, 37503) according to the following protocol 1). 2) After 50. mu.L of diluted antibody to be detected was added to each well of each column (A-H), 50. mu.L of HRP-labeled goat anti-mouse (IgG + IgA + IgM) mixture was added to each well. After mixing, incubation was carried out for 1 hour and the liquid in the wells was poured off. Adding 250 μ L of washing solution, washing for 2 times, and drying by spin-drying. 3) Adding 75 μ L TMB color developing solution into each well, developing for 15min in dark place, adding 75 μ L stop solution, and measuring OD_450nmThe value is obtained. 4) The result was determined as OD_450nmThe antibody was judged to be positive by a value of > 0.2, wherein the antibody types/subclasses were determined in the A-F wells and the antibody light chain types were determined in the G-H wells, and the types are listed in Table 3.

TABLE 3 detection correspondence table for A-H holes in ELISA plate and antibody types and light chain types

Number of holes

A

B

C

D

E

F

G

H

Type (B)

IgG1

IgG2a

IgG2b

IgG3

IgA

IgM

Kappa

Lambda

The results of the determination show that the heavy chain type detection result of the monoclonal antibody of the 4H3 strain is IgG2b subclass, and the light chain type is k.

Example 3 Brucella Canitis hybridoma cell count

Continuously transferring the above hybridoma cells for 5 generations, and counting the hybridoma cells from the 1 st, 3 rd and 5 th generations by colchicine method. The specific procedures are as follows:

1. the hybridoma cells are subcultured 48-36 hours before colchicine is added.

2. Colchicine treatment: colchicine (100. mu.g/mL, sterilized, stored at-20 ℃) was added to the flask to a final concentration of 0.1-0.4. mu.g/mL. And continuously culturing for 4-6 hours, then blowing the cells, transferring the cells into a centrifuge tube, centrifuging 800g to precipitate the cells for 5min, and discarding the supernatant.

3. 5mL of 0.075mol/L KCl solution pre-warmed to 37 ℃ is added, the precipitated cells are suspended and mixed evenly, and the mixture is subjected to water bath at 37 ℃ for 15-20 min.

4. Adding 1mL of newly prepared stationary liquid (methanol and glacial acetic acid 3: 1), mixing, centrifuging 800g to precipitate cells for 5min, and discarding the supernatant.

5. Adding 5mL of fixing solution, suspending and uniformly mixing the cells, standing at room temperature for 20-30 min, then centrifuging 800g to precipitate the cells for 5min, and removing supernatant; repeating the operation once; then 5mL of the fixative solution was added, the cells were suspended and mixed well, sealed at the mouth of the tube and left overnight at 2-8 ℃.

6. Taking out the centrifuge tube, centrifuging and precipitating the cells for 5min at 800g, slightly sucking the supernatant, leaving 0.5-1 mL of fixing solution according to the amount of the packed cells, suspending and uniformly mixing the cells, sucking 1-2 drops of cell suspension, dropping the cell suspension on a glass slide just taken out of ice water, blowing the cell suspension by a mouth, passing the cell suspension on a flame for several times to enable the cells to be flatly laid on the glass slide, and naturally drying the cell suspension.

7. Dyeing with newly prepared 10% Giemsa dye liquor for 10-20 min, washing off the dye liquor with water, and naturally drying. (Giemsa dye solution formula: 0.5g of Giemsa powder, 33mL of glycerol, heat preservation at 55-60 ℃ for 2 hours, adding 33mL of methanol, mixing uniformly, storing in a brown bottle to serve as stock solution, and taking 1 part of the stock solution, adding 1/15mol/L of 9 parts of PBS (pH6.8), thereby obtaining 10% Giemsa dye solution).

8. Microscopic examination: and selecting cells with well dispersed chromosomes, no overlapping and no scattering for observation and analysis. Each specimen should count 100 whole metaphase cells and pay attention to see if there is a marker chromosome.

As a result:

TABLE 43 chromosome number and morphological characteristics of hybridoma cells of different generation strains

As shown in the results in Table 4, after 5 generations, the cell chromosomes are relatively consistent in morphology and well dispersed, and the average value of the number of chromosomes is in the range of 102-111, which is in accordance with the sum of the number of chromosomes (40) of spleen cells and the number of chromosomes (62-72) of SP2/0 cells.

EXAMPLE 4 determination of ascites titer of monoclonal antibody

The collected ascites fluid was centrifuged at 10000g for 5min to remove cell debris and lipids, purified by a column chromatography, and measured by the cELISA method for measuring ascites titer, as follows:

1. dilution of ascites the prepared ascites was diluted with 1:10000, 1:15000, 1:20000, 1:25000, 1:30000, 1:35000 and 1:40000 in 1 XPBS solution.

2. The preparation of the antigen coated plate uses a coating buffer solution (0.05M carbonate buffer solution, pH9.6) to dilute the Brucella canicola lysis antigen to 1 mu g/mL, and the Brucella canicola lysis antigen is added into an ELISA plate, each hole is 100 mu L, and the Brucella canicola lysis antigen is coated for 16 hours at the temperature of 2-8 ℃. Washing with 1 × washing solution 1 time, adding 200 μ L of blocking solution (0.01M PBS (pH7.4) containing 3% gelatin) to each well, and blocking at 2-8 deg.C for 24 hr.

3. The plate was washed 1 time with 1 Xwashing solution 300. mu.L/well and the washing solution was discarded. Diluted positive control serum and negative control serum were added to the ELISA plate at 50 μ L/well, with 2 replicates for each positive and negative control serum. After the sample adding is finished, the diluted monoclonal antibody is added, 50 mu L of the monoclonal antibody is added into each hole, and the mixture is shaken and mixed evenly for 5 min. After incubation at 37 ℃ for 30min, the reaction plate was removed, the reaction solution was discarded, 300. mu.L of 1 Xwashing solution was added to each well, and after washing 3 times, the plate was spin-dried.

4. Adding enzyme-labeled antibody, diluting goat anti-mouse IgG labeled with HRP with corresponding diluent at a ratio of 1:100, adding 100 μ L into each well, incubating at 37 deg.C for 30min, washing 3 times according to the method in item 2, and spin-drying.

5. Color development and termination the substrate solutions A and B were mixed at 1:1(V/V) and immediately added to an ELISA reaction plate at 100. mu.L/well, and after color development at room temperature in the dark for 15min, 50. mu.L of stop solution was added to each well to terminate the reaction.

6. Determining the result, determining OD within 15min by enzyme labeling instrument after reaction termination_450nmThe value is obtained.

7. Formula for calculation

PI (inhibition ratio) ═ negative control OD_450nmPositive control OD_450nm) Negative control OD_450nm×100％

8. The titer was determined at 1. the highest dilution in each dilution at which the PI mean of the positive control serum was not less than 90% was taken as the titer of ascites.

As a result: the ascites cELISA titers prepared by F1, F3 and F5 generation cells are respectively 1:25000, 1:30000 and 1: 25000. Indicating that the titer (1:25000) of ascites production by the continuous 5 generations was not changed much and was more stable than that of the previous primary cells.

Example 6 monoclonal antibody specificity identification

1. Indirect ELISA for antibody reactivity detection

The method comprises the steps of preparing respective split protein antigens from a canine Brucella antigen, Yersinia enterocolitica O9, an Enterobacter coli O157 antigen, a Dublin salmonella antigen and a Salmonella antigen 1791 which has strong serological cross reaction with Brucella according to a preparation method of the canine Brucella split protein antigen, respectively diluting the split protein antigens to 1 mu g/mL by 0.05M carbonate buffer solution (1.59 g of sodium carbonate and 2.93g of sodium bicarbonate, dissolving the dissolved split protein antigens to 1000mL by using sterilized deionized water, containing 0.05% of proclin 300 and having the pH value of 9.6), coating a 96-hole enzyme label plate (Nunc, 468667) according to 100 mu L/hole, and acting for 14-18 hours at the temperature of 2-8 ℃. The plate was washed 4 times for 3min with 300. mu.L of 1 XPBS-Tween wash. Add 3% fish skin gelatin (Sigma, G7041) at 150. mu.L/well and block for 2 hours at 37 ℃. The coating solution was discarded and the plate was washed 4 times with 300. mu.L of 1 XPBS-Tween wash. After the positive control serum and the negative control serum were diluted 1:50, 100. mu.L of the diluted control serum was added to each well and allowed to react at 37 ℃ for 30 min. The reaction plate was removed, the reaction solution was discarded, 300. mu.L of 1 Xwashing solution was added to each well, washed 3 times, and finally spun-dried or patted dry 1 time. HRP-labeled goat anti-mouse IgG was diluted 1:200 with a diluent, and 100. mu.l of the diluted IgG was added to each well and allowed to react at 37 ℃ for 30 min. Washing for 3 times and drying. The substrate solutions A and B were mixed in a ratio of 1:1(V/V)After mixing, the mixture was immediately added to an ELISA plate at 100. mu.L/well, and after development in dark at room temperature for 15min, the reaction was terminated by adding 50. mu.L of stop solution per well. After the reaction is terminated, OD is measured by a microplate reader within 15min_450nmThe value is obtained.

ELISA (enzyme-Linked immuno sorbent assay) result for detecting different antigen proteins by using culture supernatant of 53 hybridoma generations

	Dog ZG	Dog 6/66	Dog QD	Canine SL	Large intestine O157	Yersin O9	Dublin	Salmon	1791	Positive control	Negative control
												F1	2.2888	1.6842	1.7837	1.8142	0.0659	0.0596	0.0641	0.0569	2.3012	0.031
F3	2.2049	1.5012	1.7332	1.7983	0.06215	0.0773	0.06231	0.07326	/	/
											F5	2.062	1.4799	1.611	1.8299	0.0727	0.0699	0.06453	0.0804	/	/

As seen from the results in Table 5, ascites produced by the F1, F3 and F5 generation cells can specifically recognize Brucella canicola.

2. Characteristics of agglutination reaction

The antigen concentration of salmonella dublin, yersinia enterocolitica O9, escherichia coli O157, standard brucella canicola strain RM6/66, clinical isolate ZG of brucella canicola, clinical isolate QD of brucella canicola, clinical isolate SL of brucella canicola and brucella is adjusted by using 0.5% phenol normal saline through turbidimetry, so that the turbidity of each antigen is consistent with the turbidity of national reference antigen (Chinese veterinary medicine inspection institute, 202001 batches) for brucella disease test tube agglutination test, and the antigen is further diluted by 1:20 to be used as test tube agglutination test antigen. And (4) performing agglutination test when the single antibody ascites is diluted to the indirect ELISA titer of 1:100, and judging the reaction intensity according to the agglutination condition.

Agglutination test results of culture supernatants of hybridoma cells of generation 63 and different antigens

As seen from the results in Table 6, ascites produced by the F1, F3 and F5 generation cells recognized specifically with Brucella canis antigen.

3.Westernblot

Quantitatively adding a sample buffer solution into the partial lysis antigen protein sample with the concentration according to the proportion of 1:6, boiling for 5 minutes, and transferring the sample to a nitrocellulose membrane after polyacrylamide gel electrophoresis; blocking with 5% skimmed milk prepared with TBS at 37 deg.C for 1.5 hr; after being sealed, the cells are respectively incubated for 1.5 hours by diluted ascites, washed for 4 times by TBST and incubated for 1 hour by goat anti-mouse IgG marked by HRP at 37 ℃; TBST was washed 5 times, the reaction was terminated in a chemiluminescent substrate and the results were obtained by scanning with a chemiluminescence instrument as shown in FIG. 1.

As can be seen from the results in FIG. 1, the mAb immunologically reacted with Brucella antigen, but not with other bacterial antigens.

Test samples used in the examples of the present invention: the Brucella melitensis 16M strain (CVCC70002 strain), Escherichia coli O157 strain (CVCC1489 strain), Salmonella dublin (CVCC78352 strain), Salmonella CVCC1791 strain, Brucella canis RM6/66 strain (CVCC70701) are from the China veterinary culture collection center; yersinia enterocolitica O9 strain (from chinese medical bacterial collection management center) brucella canicola clinical isolate: ZG strain, QD strain and SL strain are identified and stored in animal brucellosis reference laboratory in China where veterinary medicine is monitored.

Example 7 establishment of cELISA

Taking 40 parts of positive serum and 30 parts of negative serum which are detected by a rapid plate agglutination test, a test tube agglutination test and an agar diffusion test containing 2-mercaptoethanol, diluting according to a ratio of 1:20, performing cELISA, and determining OD_450nmAnd calculating PI. The specific ELISA procedure was as follows: preparing the extracted brucella abortus lysis antigen protein into 1 mu g/mL by using carbonate buffer solution (pH9.6), adding the solution into a 96-hole enzyme label plate according to 100 mu L/hole, and incubating overnight at 4 ℃; blocking with 5% skim milk prepared with PBST for 3 hours at 37 ℃; after the sealing is finished, washing for 2 times by using PBST, adding 50 mu L of diluted monoclonal antibody and 50 mu L of 1:20 diluted sample to be detected into each hole, setting a negative control and a positive control, fully mixing uniformly, and incubating for 30min at 37 ℃; PBST washing 4 times, each hole is added with 100 u L HRP labeled goat anti mouse IgG, 37 degrees C were incubated for 30 min; PBST was washed 4 times, added with a substrate developing solution and allowed to react at room temperature for 10min, and then terminated with 1M HCl. Reading OD_450nmAnd according to the formula: inhibition ratio (PI) ═ negative control OD_450nmDetection of OD in sample_450nm) Negative control OD_450nmX 100%. Inhibition ratio (PI) was calculated. The results were then analyzed using SPSS 17.0 software. And (3) constructing an ROC curve by using a nonparametric method, and taking the tangent point with the maximum Youden index as a critical point for judging whether the product is negative or positive.

As a result:

the test detects 70 serum, the calculation and statistics results are shown in table 7, the ROC curve drawn according to the results is shown in table 2, and the correlation coefficient of the curve is shown in table 8.

TABLE 7 calculation of serum test

The Area Under the ROC Curve (Area Under the Curve) is generally used to reflect the accuracy of the diagnostic system. Theoretically, this index takes values in the range of 0.5 to 1, with a completely worthless diagnosis of 0.5 and a perfect diagnosis of 1. Generally, when Az is 0.5-0.7, the diagnosis accuracy is low; when the value is 0.7-0.9, the diagnosis accuracy is moderate; a value of 0.9 or more indicates high diagnostic accuracy. In the test, the area under the curve is 0.986 (see table 8, between Lower Bound and Upper Bound), which indicates that the diagnostic method has higher accuracy, and the kit has good diagnostic value.

TABLE 8 ROC Curve-related parameters

From the above results, it was found that the louden index is highest when PI is 31.21%, and accordingly PI of 30% was determined as the critical point of the present cselisa method.

Example 8 cELISA reaction characteristics

3 parts of canine brucella positive serum, 2 parts of canine brucella negative serum, Yersinia enterocolitica O9 antiserum, Escherichia coli O157 positive serum and rough brucella positive serum are respectively detected by the built cELISA method, the inhibition rate is calculated according to the formula, and the result is shown in Table 9. From the experimental results, the inhibition rates of 3 detected Brucella positive sera are respectively 97.4%, 96.7% and 93.8%, while the inhibition rates of 2 Brucella negative sera are respectively less than 10%, 9.97% and 0.85%, which indicates that the cELISA method established in the research can be primarily used for detecting Brucella sera. In addition, 1 part of positive serum of yersinia enterocolitica O9, 1 part of positive serum of escherichia coli O157, 1 part of positive serum of brucella abortus and 1 part of salmonella dublin were detected. The results showed that the inhibition was 27.0%, 19.8%, 19.2% and 18.8%, respectively, all less than 30%. The cELISA detection method established based on the monoclonal antibody 4H3 is feasible, and can effectively eliminate false positives brought by Escherichia coli O157, Yersinia enterocolitica O9 and Salmonella dublin.

TABLE 9 results of cELISA reaction characteristics established based on 4H3

Example 9 clinical sample detection and sensitivity test

1. 80 clinical canine serum samples were tested using the established cELISA, RSAT, ME-RSAT, SAT, and AGID methods. The compliance rate of the cELISA method with other methods was compared. As seen from tables 10 to 13, cELISA showed the highest rates of agreement with AGID, 75.00%, 87.5%, 85.0% and 96.2% of the rates of agreement with the methods of RSAT, ME-RSAT, SAT and AGID, respectively.

TABLE 10 comparison of Brucella species cELISA with fast plates

TABLE 11 comparison of Brucella species cELISA and test tube agglutination test

TABLE 12 comparison of Brucella melitensis cELISA with 2-mercaptoethanol-containing rapid plates for dogs

TABLE 13 comparison of Brucella melitensis cELISA and agar diffusion assay for dogs

2. The cELISA and the AGID were used to detect the smallest amount of detection for a known positive sample (antibody agglutination valence about 1: 1000).

TABLE 14 comparison of Brucella species cELISA and agar diffusion assay for dogs

	Qiongzhu (Qiongzhu)	cELISA
			Original multiple	+	+
1:10	+	+
			1:50	(+)	+
1:100	—	+
			1:200	—	+
1:1000	—	+
			1:5000	—	—

As can be seen from Table 14, the sensitivity of the cELISA for positive samples was higher than that of the agarose.

Claims (2)

1. A Brucella canicola monoclonal antibody, characterized by that the monoclonal antibody is IgG2b subclass, its light chain type should be k; the monoclonal antibody respectively generates obvious immunological reactions with brucella Canitis antigens, and does not react with yersinia enterocolitica O9, Escherichia coli O157 antigen, Salmonella dublin antigen and antigen without cross reaction which have strong serological cross reaction with brucella;

the brucella Canitis monoclonal antibody is prepared from a brucella Canitis hybridoma cell 4H3 strain which is delivered to China general microbiological culture Collection center (CGMCC No. 19964) in microbial research institute of China academy of sciences, No. 3 of Beijing West Lu 1 Hotel, Chaoyang, and the Beijing area, 08-17 days, 2020, and the preservation number is CGMCC No. 19964.

2. The Brucella canis monoclonal antibody of claim 1, wherein the monoclonal antibody is used to establish a cELISA assay for Brucella canis serum antibodies;

the cELISA detection method for the brucella canis serum antibody mainly comprises the steps of coating an ELISA plate with a split protein antigen containing a brucella canis strain, adding a specific brucella canis monoclonal antibody and brucella canis serum to be detected, establishing a blank control without serum, and then performing normal cELISA program operation, wherein the absorbance value of positive serum is specifically inhibited, the absorbance value of negative serum is not inhibited, and the absorbance value is not different from the blank control.

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