CN118064377A - Hybridoma cell strain secreting anti-mycoplasma ovipneumoniae monoclonal antibody, monoclonal antibody thereof and application - Google Patents
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Abstract
The invention discloses a hybridoma cell strain secreting an anti-mycoplasma ovipneumoniae monoclonal antibody, and monoclonal antibody and application thereof, wherein the hybridoma cell strain is hybridoma cell strain 1A11, and is preserved in China Center for Type Culture Collection (CCTCC) NO: C202408. the monoclonal antibody secreted by the hybridoma cell strain 1A11 is the monoclonal antibody 1A11, and the monoclonal antibody 1A11 provided by the invention can be used for detecting mycoplasma ovipneumoniae, has strong specificity and does not react with common animal mycoplasma such as mycoplasma hyopneumoniae, mycoplasma gallisepticum, mycoplasma bovis and the like. The double-antibody sandwich ELISA method established based on the monoclonal antibody 1A11 has the advantages that the inter-batch and intra-batch variation coefficients are lower than 10%, the specificity, the repeatability and the sensitivity are good, the positive correlation with the CCU content is realized, and the method is a rapid, simple and accurate pathogen detection method and has important significance for rapid quantification of mycoplasma ovipneumoniae and vaccine research.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a hybridoma cell strain secreting an anti-mycoplasma ovipneumoniae monoclonal antibody, and monoclonal antibodies and application thereof.
Background
Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae, mo) can cause mycoplasma ovipneumoniae (Mycoplasma pneumoniae of sheep and goat, MPSG), which is a chronic respiratory infectious disease of sheep and goats, and is characterized by asthma as a clinical feature, which is widely prevalent worldwide, and is more susceptible to both goats and sheep, and the clinical features are hyperthermia, runny nose, cough and cellulose pleuropneumonia; the disease has strong infectivity, can be infected in four seasons, is especially frequent in spring and winter, is most susceptible to lambs, has an infection rate of up to 95.3 percent and has a fatality rate of 27.5 percent. Infection with this pathogen is prone to induce other bacterial pneumonia, particularly pasteurellosis and viral infections, which exacerbate the pathological process and lead to death, resulting in significant economic losses.
Vaccines are the most economical means of controlling the disease. However, the efficacy test of the vaccine is difficult because of no standard experimental animal sheep, and the test and research and development of the vaccine are affected. Meanwhile, the current mycoplasma content measurement still adopts classical Color Change Units (CCU), the method can only measure living mycoplasma, but can not measure insufficient activity or dead mycoplasma, and meanwhile, the method takes 7-10 days and needs mycoplasma culture, so the CCU has various defects as an antigen quantification method in the generation of inactivated vaccines. Currently, the existing antigen ELISA method of the international inactivated vaccine antigen quantification method is applied to the market. However, the ELISA method for mycoplasma ovipneumoniae antigen is not reported.
The method for detecting mycoplasma ovipneumoniae pathogens in mycoplasma ovipneumoniae diagnosis is currently reported to comprise separation culture, PCR and fluorescent quantitative PCR, wherein the pathogen separation culture requires professional laboratory and experimental techniques, the PCR method requires operations such as nucleic acid extraction, the operation is complicated, the cost is high, and expensive instruments are required; the pathogenic ELISA detection method has the characteristics of simple method, low condition requirement and high flux. Thus, it is clinically desirable to provide a pathogen ELISA detection method for Mo pathogen detection of a large number of samples.
Therefore, the double-antibody sandwich ELISA detection method for detecting Mo, which is established by utilizing the monoclonal antibody, achieves the purpose of rapidly, accurately and sensitively detecting Mo, and has important significance for quantitative determination of mycoplasma ovis in vaccine production and Mo detection of clinical samples.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a hybridoma cell strain secreting an anti-mycoplasma ovipneumoniae monoclonal antibody, and monoclonal antibodies and application thereof, aiming at the defects of the prior art.
In order to solve the technical problems, the invention discloses a hybridoma cell strain secreting an anti-mycoplasma ovipneumoniae monoclonal antibody, and monoclonal antibodies and application thereof. The specific technical scheme is as follows:
In a first aspect, the invention provides a hybridoma cell strain secreting an anti-mycoplasma ovipneumoniae monoclonal antibody, wherein the hybridoma cell strain is hybridoma cell strain 1A11 (Hybridoma cell line A11), and is preserved in university of Wuhan, china, and the preservation number is CCTCC NO: c202408, the preservation time is 2024, 1 month and 9 days.
The preparation method of the hybridoma cell strain 1A11 according to the first aspect comprises the following steps: the mycoplasma ovipneumoniae whole mycoprotein is used as an immune antigen to immunize a mouse, spleen cells of the immunized mouse are fused with myeloma cells of the mouse, and then 3 times of subcellular cloning screening are carried out to obtain a hybridoma cell strain capable of stably secreting monoclonal antibodies against mycoplasma ovipneumoniae.
The preparation method of the mycoplasma ovipneumoniae whole mycoprotein comprises the following steps: culturing mycoplasma ovipneumoniae (Mo) NJ01 strain with KM2 mycoplasma culture medium, performing amplification culture, harvesting culture, centrifuging, washing and performing ultrasonic treatment to obtain Mo whole mycoprotein as immune antigen, and freeze preserving. The mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae, mo) NJ01 strain has a preservation number of CCTCC NO: m2020907, the preservation date is 12 months and 14 days in 2020, the preservation address is the university of Wuhan in China, and the detailed information of the M2020907 is disclosed in the Chinese patent with application number 2021103419159.
The method for immunizing the mice comprises the following steps: the Mo immunogen was subcutaneously injected into BALB/c mice (purchased from the university of dulcimer comparative medical center) 3 times in total, each at 2 week intervals. First immunization an immunogen was prepared by emulsification with 40 μg Mo whole mycoprotein and an equal volume of complete freund's adjuvant (purchased from Sigma); the immunogen was prepared in the latter two times by emulsification with 40 μg Mo whole mycoprotein and an equal volume of incomplete freund's adjuvant (purchased from Sigma); blood is collected after the third immunization for 2 weeks, immune serum titer of the mice is detected, the mice with ELISA antibody titer of >10 6 are selected, the immunization is enhanced once 4d before cell fusion, and 100 mug Mo protein is adopted for intraperitoneal injection.
The cell fusion method comprises the following steps: the PEG cell fusion method was performed by taking mouse myeloma cells (SP 2/0) and immunized Balb/c mouse spleen cells in a ratio of 1:10 or 1:5. After 7d of fusion, when the cells grow to 1/10-1/5 of the bottom area of the 96-well plate, taking the supernatant, and carrying out antibody detection according to an indirect ELISA method. And selecting hybridoma cell strains with positive detection results from the two times for subcloning for 3 times until all cloned cell strain supernatants after cloning are positive in detection and OD 450 nm values detected by each hole are relatively close. And (3) performing expansion culture and freezing storage on the cloned anti-Mo specific monoclonal antibody hybridoma cell strain. As a result, 1A11, 1A12 strains and 5C1 mab cell strains were obtained. Monoclonal antibodies secreted by hybridoma cell lines 1a11, 1a12 and 5C1 were designated monoclonal antibodies 1a11, 1a12 and 5C1, respectively. Hybridoma cell line 1A11 is preferred.
In a second aspect, the present invention provides an anti-mycoplasma ovipneumoniae monoclonal antibody 1a11 secreted by the hybridoma cell line 1a11 according to the first aspect.
Wherein, the subclass heavy chain of the monoclonal antibody 1A11 is IgG1, and the subclass light chain is kappa.
Wherein the monoclonal antibody 1A11 does not react with other animal mycoplasma. The other animal mycoplasma include, but are not limited to, any one or more of mycoplasma hyopneumoniae, mycoplasma gallisepticum, mycoplasma bovis, and mycoplasma hyopneumoniae.
Wherein the monoclonal antibody 1A11 recognizes that the antigen is a Mycoplasma ovipneumoniae about 54kDa protein.
The preparation method of the monoclonal antibody 1A11 according to the second aspect comprises the following steps: injecting the hybridoma cell strain 1A11× 6～3×106 in the first aspect into the abdominal cavity of a mouse, collecting ascites of the mouse after 7-10 d, and centrifuging to obtain the supernatant.
In a third aspect, the invention provides an application of the hybridoma cell strain 1A11 according to the first aspect or the monoclonal antibody 1A11 according to the second aspect in preparation of a reagent and/or a kit for detecting mycoplasma ovipneumoniae.
Wherein the detection comprises, but is not limited to, indirect immunofluorescence detection, immunoblotting detection, double-antibody sandwich ELISA detection, antibody indirect ELISA and the like.
In a fourth aspect, the invention provides a double-antibody sandwich ELISA kit for detecting mycoplasma ovipneumoniae antigen, wherein the kit comprises a capture antibody, a detection antibody and an enzyme-labeled secondary antibody;
The capture antibody is monoclonal antibody 1a11 according to the second aspect above;
The detection antibody is mycoplasma ovipneumoniae hyperimmune rabbit serum;
The enzyme-labeled secondary antibody is horseradish peroxidase (HRP) labeled goat anti-rabbit IgG.
Wherein, the detection antibody mycoplasma ovipneumoniae hyperimmune rabbit serum is obtained by immunizing rabbits with mycoplasma ovipneumoniae. Preferably, the mycoplasma ovipneumoniae Y98 strain hyperimmune rabbit serum or the IK3-3 strain hyperimmune rabbit serum is obtained by immunizing rabbits with mycoplasma ovipneumoniae Y98 strain or the IK3-3 strain. More preferably IK3-3 strain hyperimmune rabbit serum. Wherein the mycoplasma ovipneumoniae Y98 strain is derived from the national veterinary microorganism (toxin) species collection (with the collection number of CVCC 384). The mycoplasma ovipneumoniae IK3-3 strain is derived from the national veterinary microorganism (toxin) species collection (with the preservation number of CVCC 380).
Wherein the kit also comprises a washing liquid, a sealing liquid, a substrate liquid and a stopping liquid.
Wherein the washing solution is PBST (ph=7.4). The blocking solution is any one of casein, bovine Serum Albumin (BSA), skimmed milk powder or gelatin; preferably, the volume ratio concentration of the casein is 0.5-4%; the volume ratio concentration of the BSA is 0.5-4%; the volume ratio concentration of the skimmed milk powder is 5-20%; the volume concentration of the gelatin is 1%. More preferably, the blocking solution is BSA, and the volume ratio concentration is 1%. The substrate liquid is TMB color developing liquid; the stop solution is 2M sulfuric acid.
In a fifth aspect, the present invention provides the use of a kit as described in the fourth aspect above for the detection of mycoplasma ovipneumoniae for non-diagnostic purposes.
In a sixth aspect, the invention provides a double-antibody sandwich ELISA method for detecting mycoplasma ovipneumoniae for non-diagnostic purposes, comprising the following conditions: the dilution factor of the monoclonal antibody 1A11 is 1:2.5-1:320 v/v, the coating condition is 37 ℃, and after 60min, the temperature is 4 ℃ overnight or directly 4 ℃ overnight; the sealing condition of the sealing liquid is 37 ℃ for 1h; the antigen to be detected has the action condition of 37 ℃ for 30-120 min; the dilution factor of the detection antibody is 1:256-1:16384 v/v, and the action condition is 37 ℃ for 30-120 min; the dilution multiple of the enzyme-labeled secondary antibody is 1:1000-1:8000 v/v, and the action condition is 37 ℃ for 30-120 min; the color development time is 5-20 min. Preferably, the dilution factor of monoclonal antibody 1A11 is 1:20v/v, and the coating condition is that the temperature is 37 ℃ for 1h and then 4 ℃ is overnight; blocking solution is 1% v/v BSA, blocking condition is 37 ℃ for 1h; the antigen to be detected has the action condition of 37 ℃ for 90min; the detection antibody is mycoplasma ovipneumoniae IK3-3 strain high immunity rabbit serum, the dilution factor is 1:1024v/v, and the action condition is 37 ℃ for 120min; the dilution multiple of the enzyme-labeled secondary antibody is 1:4000v/v, and the acting condition is 37 ℃ for 90min; the development time was 10min.
The method specifically comprises the following steps: diluting the monoclonal antibody 1A11 in a volume ratio of 1:2.5-1:320, and coating the ELISA plate at 37 ℃ overnight at 4 ℃ or directly at 4 ℃ overnight after 60 min; sealing with sealing liquid, and reacting at 37deg.C for 1 hr; adding antigen to be detected, and reacting at 37 ℃ for 30-120 min; adding the detection antibody diluted according to the volume ratio of 1:256-1:16384, and placing the mixture into a temperature box to act for 30-120 min at 37 ℃; adding enzyme-labeled antibody diluted in the volume ratio of 1:1000-1:8000, and placing the mixture into a temperature box to act for 30-120 min at 37 ℃; adding a substrate solution TMB to develop color for 5-20 min; adding a stop solution 2M H 2SO4, and measuring an OD 450 value; preferably, the monoclonal antibody 1A11 is diluted in a volume ratio of 1:20 and then coated on an ELISA plate, and the coating is carried out at 4 ℃ overnight; blocking with blocking solution 1% v/v BSA at 37deg.C for 1 hr; adding an antigen to be detected, reacting for 90min at 37 ℃, adding a detection antibody mycoplasma ovipneumoniae IK3-3 strain of high immunity rabbit serum diluted in a volume ratio of 1:1024, and placing into an incubator to act for 120min at 37 ℃; adding the enzyme-labeled antibody diluted in the volume ratio of 1:4000, and placing the mixture into an incubator to act for 90min at 37 ℃; adding a substrate solution TMB to develop a color substrate for 10min; stop solution 2M H 2SO4 was added and OD 450 was measured.
The antigen to be detected is mycoplasma ovipneumoniae culture stock solution or a mixture system of the stock solution treated by any one of ultrasonic pyrolysis, merthiolate inactivation or formaldehyde inactivation. Preferably, the culture stock solution is subjected to ultrasonic cleavage treatment.
The double-antibody sandwich ELISA method has good specificity, sensitivity and repeatability.
Wherein, the double antibody sandwich ELISA method has obvious positive correlation with the content of CCU 50.
The kits or assays described above may be used for assays including, but not limited to, assaying Mo content in fresh Mo cultures, in chilled or cryopreserved cultures, in inactivated or concentrated Mo content, in vaccines or in formulations or mixtures that may contain Mo.
The person skilled in the art can add other auxiliary reagents into the above-mentioned kit, reagent or test paper according to conventional technical means, and can prepare the kit, reagent or test paper by adopting conventional methods.
The man skilled in the art can add other auxiliary reagents based on the key parameters of the method according to the conventional technical means, and can prepare the mycoplasma ovipneumoniae ELISA antigen detection method by adopting the conventional method.
The person skilled in the art can process other objects to be tested on the basis of the key parameters of the method according to the conventional technical means, and expand the application to the detection of other samples.
The invention has the beneficial effects that:
(1) The specificity is strong: the monoclonal antibody 1A11 obtained by screening and the double-antibody sandwich ELISA kit established by the invention specifically react with mycoplasma ovipneumoniae, and do not react with mycoplasma hyopneumoniae, mycoplasma hyorhinis, mycoplasma gallisepticum, mycoplasma bovis and other common animal mycoplasma.
(2) The sensitivity is high: the minimum detectable mycoplasma ovipneumoniae of 1.95X10 4CCU50/mL of the kit provided by the invention is obviously positively correlated with the content of CCU 50.
(3) Repeatability: the kit has good intra-batch and inter-batch repeatability, and the variation coefficient between batches and between batches is lower than 10%.
(4) Simple and rapid: the kit has low technical difficulty, does not need expensive instruments, is simple and convenient to operate, and has the whole detection process of not more than 21 hours.
(5) High flux: the kit can be used for detecting a large number of samples.
Therefore, the monoclonal antibody of the invention has strong specificity and high antibody titer; the invention provides a rapid, simple, accurate and high-flux Mo qualitative and quantitative detection method, provides technical means in aspects of vaccine evaluation, antigen quantification, pathogen detection, immune function research and the like, and has important significance for Mo prevention and control and laboratory research.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a protein gel diagram of Western blot detection of the reaction of monoclonal antibody 1A11 strain with Mo.
M: protein molecular mass standard; 1-3: mo IK3-3, mo NJ01 and Mo Y98 strain whole mycoprotein
Detailed Description
The invention will be further illustrated, but is not limited, by the following examples. Unless otherwise specified, the method is a conventional method; the reagents and materials, unless otherwise specified, are commercially available.
In the following examples, the goat anti-rabbit IgG (h+l) -HRP (i.e., HRP-labeled goat anti-rabbit IgG) was purchased from beijing full gold biotechnology inc; the substrate solution (TMB developing solution) was purchased from Huzhou Ind biosciences Co., ltd; the ELISA plate is a 96-well ELISA plate purchased from costar company.
In the following examples, mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae, mo) NJ01 strain, with a preservation number of cctccc NO: m2020907, the preservation date is 12 months and 14 days in 2020, the preservation address is the university of Wuhan in China, the China center for type culture Collection, and the detailed information thereof is disclosed in China patent application No. 2021103419159.
In the following examples, the Mycoplasma ovipneumoniae Y98 strain is derived from the national collection of veterinary microorganisms (toxin) species (accession number CVCC 384).
The mycoplasma ovipneumoniae IK3-3 strain is derived from the national veterinary microorganism (toxin) species collection (with the preservation number of CVCC 380).
In the following examples, the PBS is a PBS solution at ph=7.4. The PBST is a PBST solution with ph=7.4.
EXAMPLE 1 selection of Mycoplasma ovipneumoniae-resistant monoclonal antibody hybridoma cell lines
1. Antigen preparation
Culturing mycoplasma ovipneumoniae (Mo) NJ01 strain with KM2 mycoplasma culture medium at 37 ℃, performing amplification culture, harvesting culture, centrifuging, washing and performing 200W, performing ultrasonic treatment for 10min to obtain Mo whole mycoprotein as an immune antigen, and performing freeze preservation.
2. Immunization of Balb/c mice
Mo immunogen was injected subcutaneously into 4 points of the back for a total of 3 immunizations of 6-8 week old female BALB/c mice (purchased from the university of dulcimer comparative medical center) at 2 week intervals. First immunization an immunogen was prepared by emulsification with 40 μg Mo whole mycoprotein and an equal volume of complete freund's adjuvant (purchased from Sigma); the immunogen was prepared in the latter two times by emulsification with 40 μg of whole cell Mo protein and an equal volume of incomplete freund's adjuvant (purchased from Sigma); blood is collected after the third immunization for 2 weeks, immune serum titer of the mice is detected (detection method is referred to literature Xie Qin and the like. Establishment of anti-mycoplasma ovipneumoniae monoclonal antibody hybridoma cell strain [ J ]. Chinese livestock and poultry infectious diseases, 1995 (04): 48-50.) the mice with ELISA antibody titer of >10 6 are selected, 4d before cell fusion is used for enhancing immunity once, and 100 mug of Mo holotoxin is adopted for intraperitoneal injection.
3. Cell fusion and hybridoma cell selection
The PEG cell fusion method was performed by taking mouse myeloma cells (SP 2/0) and immunized Balb/c mouse spleen cells in a ratio of 1:10 or 1:5. After 7d of fusion, cells were grown to 1/10 to 1/5 of the bottom area of the 96-well plate, and the supernatant, reference (Du Gaimei, et al, establishment of mycoplasma ovipneumoniae antibody ELISA assay [ J ] animal husbandry and veterinary, 2020,52 (06): 107-111.), was used for antibody detection by an indirect ELISA method. And selecting hybridoma cell strains with positive detection results from the two times for subcloning for 3 times until all cloned cell strain supernatants after cloning are positive in detection and OD 450 values detected by each hole are relatively close. And (3) performing expansion culture and freezing storage on the cloned anti-Mo specific monoclonal antibody hybridoma cell strain. As a result, 1A11, 1A12 and 5C1 mab cell lines were obtained. Monoclonal antibodies secreted by hybridoma cell lines 1a11, 1a12 and 5C1 were designated monoclonal antibodies 1a11, 1a12 and 5C1, respectively.
Example 2 analysis of antibody Properties
1. Monoclonal antibody Western-blot analysis
The whole mycoproteins of Mo Y98 strain, IK3-3 strain and NJ01 strain were separated by SDS-PAGE gel electrophoresis, transferred to nitrocellulose membrane (NC membrane), and Western-blot analysis was performed by using hybridoma cell line 1A11 monoclonal antibody as primary antibody and goat anti-mouse IgG (H+L) -HRP antibody as secondary antibody.
As shown in FIG. 1, the monoclonal antibody 1A11 secreted by hybridoma cell line 1A11 and Mycoplasma ovipneumoniae strain 3 have a distinct band at about 54kDa, indicating that monoclonal antibody 1A11 immunoreacts with Mo protein.
2. Monoclonal antibody subclass identification
The results of the detection by the monoclonal antibody subclass identification kit (purchased from bloolon corporation) showed that subclasses 1a11 and 1a12 have heavy chains of IgG1 and light chains of κ (table 1).
TABLE 1 monoclonal antibody subclass identification
EXAMPLE 3 preparation and purification of monoclonal antibodies
The ascites preparation method is adopted: sterilized liquid paraffin was intraperitoneally injected into 10-12 week old Balb/c mice (purchased from university of Yangzhou comparative medical laboratory center) at 0.3 mL/mouse, and after 7d, hybridoma cell line 1A11 was injected into the intraperitoneally of the mice at 0.2mL each (2X 10 6～3×106 hybridoma cells). Collecting ascites of the mice with obviously bulged abdomen after 7-10 d, centrifuging for 20min at 3 000r/min, collecting supernatant of the ascites, sub-packaging, marking and preserving to-20 ℃ for standby. The monoclonal antibodies 1A11 used in the subsequent examples were all prepared as ascites.
Example 4 Mycoplasma ovipneumoniae Indirect immunofluorescence assay
1. Mycoplasma ovipneumoniae infected cells
After digestion of MDBK monolayer cells (from China veterinary drug monitoring institute), they were diluted to 5X 10 4/ml with DMEM+10% Fetal Bovine Serum (FBS) cell culture medium, 100. Mu.l were added to each well of each 96-well plate, incubated at 37℃in a CO 2 incubator, and after cell confluency reached 80%, mo adhesion test was performed.
Mo adherent cells
The MDBK monolayer cells were aspirated into the cell supernatant, and after the Mo bacterial liquid was diluted with the cell maintenance medium, 100. Mu.l of the diluted bacterial liquid was added to each well, and the mixture was adhered to the well for 6 hours at 37 ℃. Meanwhile, control cells not infected with Mo were set. After Mo adhered to the cells for 6h, both groups of cells were discarded from the original supernatant and the cells were gently washed with PBS. Slowly adding 100 μl of absolute ethanol into each hole, and fixing at 4deg.C for 30min; discard absolute ethanol, 100 μl PBS per well, wash 3 times, 30s each; 100 μl of blocking solution containing 1% v/v BSAPBS per well, blocked at 37deg.C for 1h; discarding the blocking solution, and washing 3 times with PBS; mu.l of mouse ascites monoclonal antibody 1A11 is added into each hole and incubated for 1h at 37 ℃; discarding the primary antibody, washing 3 times with PBS; 100 μl FITC-labeled goat anti-mouse IgG was added and incubated at 37deg.C in the dark for 1h; the secondary antibody was discarded, washed 3 times with PBS and observed under a fluorescence microscope.
As a result, no fluorescence was observed in the cells of the control group, and the amount of fluorescence increased significantly in the Mo-infected group as the concentration of Mo infection increased. This suggests that mab 1a11 can be used in indirect immunofluorescence assays for Mo.
Example 5 preparation and screening method of high-immune Rabbit serum for detecting antibody mycoplasma ovipneumoniae
1. Preparation method of mycoplasma ovipneumoniae hyperimmune rabbit serum
Fresh cultures of mycoplasma ovipneumoniae Y98 strain or IK3-3 strain were centrifuged at 12000r/min for 20min, the precipitated cells were resuspended in PBS, and centrifuged at 12000r/min for 20min, the cells were washed 3 times in this manner, and the mycoplasma ovipneumoniae obtained was resuspended in PBS at 1/100 (v/v) and sonicated at 200W for 10 min. The antigen references (Lv Jingang, preparation of anti-sheep mycoplasma high-immune serum, livestock and poultry industry, 8 months in 2013, total 292 th stage, pages 11-12) obtained are used for immunizing rabbits to obtain mycoplasma ovipneumoniae Y98 strain high-immune rabbit serum and IK3-3 strain high-immune rabbit serum. The method comprises the following steps: the obtained antigen (Mo whole mycoprotein) was subcutaneously injected in multiple spots to immunize rabbits for a total of 4 immunizations, each at2 week intervals. First immunization an immunogen was prepared by emulsification with 400 μg Mo whole mycoprotein and an equal volume of complete freund's adjuvant (purchased from Sigma); the immunogen was prepared by emulsification with 200 μg Mo whole mycoprotein and an equal volume of incomplete freund's adjuvant (purchased from Sigma company) in the last three times; blood is collected and separated after the fourth immunization for 2 weeks, and sheep pneumonia mycoplasma high immune rabbit serum is obtained.
2. Screening method of mycoplasma ovipneumoniae hyperimmune rabbit serum
2.1 Metabolic inhibition assay
According to the method of reference (Sun Yabo, et al, research on the metabolic inhibition effect of different animal hyperimmune serum on mycoplasma hyopneumoniae, journal of Chinese veterinary medicine, 2016.) the mycoplasma hyopneumoniae (such as Y98 strain or IK3-3 strain) is used for carrying out a metabolic inhibition test on mycoplasma hyopneumoniae hyperimmune serum, and as a result, both the mycoplasma hyopneumoniae Y98 strain hyperimmune serum and the IK3-3 strain hyperimmune rabbit serum prepared can inhibit mycoplasma hyopneumoniae.
2.2 Mycoplasma ovipneumoniae double-antibody sandwich ELISA method detection test
Diluting the Mo monoclonal antibody 1A11 (the diluent is coating buffer solution), taking 100 mu l of coating ELISA plate, and washing by using PBST; blocking with 200 μl blocking solution 1% v/v BSAPBS, and allowing to act at 37deg.C for 1 hr; after PBST washing, 100. Mu.l of the antigen to be detected, namely Mo culture (positive control, P), was added, while 1% BSAPBST (blank control) and mycoplasma culture medium (negative control, N) were established, and the reaction was carried out at 37℃for 1h; after washing with PBST, 100. Mu.l (1% BSAPBST as diluent) of the diluted detection antibody (mycoplasma ovipneumoniae IK3-3 strain high-immune rabbit serum or mycoplasma ovipneumoniae Y98 strain high-immune rabbit serum) prepared in the example was added, and the mixture was put into an incubator at 37 ℃ for 1h; washing with PBST, adding diluted enzyme-labeled secondary anti-goat anti-rabbit IgG (H+L) -HRP (1% BSA PBST as diluent), and placing into an incubator at 37 ℃ for a certain time; after washing, 100. Mu.l of substrate solution was added for color development, after 10min, 50. Mu.l of stop solution 2M H 2SO4 was added, and the OD 450 value was determined by reading. The P/N value is more than or equal to 2.1 and positive, and the P/N is less than 2.1 and negative.
According to the method, the prepared different sheep pneumonia mycoplasma high-immune rabbit serum is used as a detection antibody to detect Mo culture (positive, P) and mycoplasma culture medium (negative, N) respectively, and the detection antibody is screened according to the P/N maximum.
TABLE 2 detection of antibody screening results
As can be seen from Table 2, the P/N value of the serum of the IK3-3 strain of hyperimmune rabbit was higher than that of the serum of the Y98 strain of hyperimmune rabbit, and therefore, the serum of the IK3-3 strain of hyperimmune rabbit was selected as a detection antibody.
Example 6 double antibody sandwich ELISA kit for detection of mycoplasma ovipneumoniae antigen
The embodiment discloses a double-antibody sandwich ELISA kit for detecting mycoplasma ovipneumoniae antigen, which comprises a capture antibody (monoclonal antibody 1A 11), a detection antibody (mycoplasma ovipneumoniae immune serum) and an enzyme-labeled secondary antibody (goat anti-rabbit IgG (H+L) -HRP).
The kit further comprises:
washing liquid: PBST, ph=7.4;
Sealing liquid: BSA;
Substrate solution: TMB color development liquid;
Stop solution: 2M H 2SO4.
Example 7 establishment of Mycoplasma ovipneumoniae double-antibody sandwich ELISA method
The specific method of the double-antibody sandwich ELISA in the embodiment is as follows:
Diluting the Mo monoclonal antibody 1A11 (the diluent is coating buffer solution), taking 100 mu l of coating ELISA plate, and washing by using PBST; blocking with 200 μl blocking solution 1% v/v BSAPBS, and allowing to act at 37deg.C for 1 hr;
after PBST washing, 100. Mu.l of the antigen to be detected, namely Mo culture (positive control, P), was added while 1% BSA PBST (blank control) and mycoplasma culture medium (negative control, N) were established, and the reaction was carried out at 37℃for 1h;
After washing with PBST, 100 μl (1% BSAPBST as diluent) of diluted detection antibody mycoplasma ovipneumoniae IK3-3 strain high immunity rabbit serum prepared in example 5 is added, and the mixture is put into an incubator to act for 1h at 37 ℃;
Washing with PBST, adding diluted enzyme-labeled secondary anti-goat anti-rabbit IgG (H+L) -HRP (1% BSA PBST as diluent), and placing into an incubator at 37 ℃ for a certain time;
After PBST washing, 100. Mu.l of the substrate solution was added for color development, and after 10 minutes, 50. Mu.l of stop solution 2M H 2SO4 was added.
OD 450 values were determined. The P/N value is more than or equal to 2.1 and positive, and the P/N is less than 2.1 and negative.
1. Determination of coating conditions
Mo mab 1a11 was diluted in a ratio of 1:20 (v/v) and coated onto an ELISA plate, one group was allowed to act at 37 ℃ for 1h, then at 4 ℃ overnight, the other group was allowed to act at 4 ℃ directly overnight, experiments were performed according to the specific method of the double antibody sandwich ELISA, and appropriate coating conditions were selected.
TABLE 3 coating conditions
As shown in Table 3, coating was performed at 37℃overnight after 1h at 4℃according to the P/N value.
2. Determination of optimal coating concentration of monoclonal antibody and dilution of detection antibody
Experiments were performed according to the double antibody sandwich ELISA method described above, wherein Mo 1A11 mab was added to the ELISA plate at a volume ratio of 1:2.5, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 diluted with a factor of 1:80, 37℃for 1h and then allowed to act overnight at 4 ℃. Wherein, the detection antibody is diluted in the volume ratio of 1:256, 1:1024, 1:4096 and 1:16384.
OD 450 values were determined and the P/N maximum was taken as the optimal coating antibody dilution and the optimal detection antibody dilution.
TABLE 4 antibody coating concentration and detection antibody action concentration
As shown in Table 4, the optimal coating concentration of the monoclonal antibody 1A11 was selected to be 1:20, and the optimal dilution of the detection antibody was 1:1024 according to the P/N value.
3. Screening of blocking solutions
Casein (4%, 2%, 1% and 0.5%), bovine serum albumin BSA (4%, 2%, 1% and 0.5%), skimmed milk powder (20%, 10% and 5%) and gelatin (1%) were selected as blocking solutions for blocking according to the above-described double antibody sandwich ELISA method and screening conditions.
TABLE 5 blocking fluid screening
As shown in Table 5, 1% BSA was selected as the optimal blocking solution based on the P/N value.
4. Determination of the time of action of the sandwich test antigen
According to the method of the double-antibody sandwich ELISA and the screening conditions, the sandwich action time of the antigen to be detected is selected to be 30, 60, 90 and 120min, and the optimal action time of the antigen to be detected is selected according to the P/N value.
TABLE 6 time to sandwich test antigen
As shown in Table 6, 90min was chosen as the optimal sandwich antigen action time based on the P/N value.
5. Detection of optimal time of antibody action
According to the method of the double-antibody sandwich ELISA and the screening conditions, the acting time of the detection antibody is selected to be 30, 60, 90 and 120min, and the optimal acting time of the detection antibody is selected according to the P/N value.
TABLE 7 detection of antibody action time
As shown in Table 7, 120min was selected as the optimal detection antibody action time based on the P/N value.
6. Dilution and action time of enzyme-labeled secondary antibody
According to the conditions of the screening, the enzyme-labeled secondary antibody action time is selected for 30, 60, 90 and 120min, other conditions are consistent, the enzyme-labeled secondary antibody optimal action time is selected according to the P/N value, the enzyme-labeled secondary antibody dilution is selected according to the enzyme-labeled secondary antibody optimal action time, the four dilutions of 1:1000, 1:2000, 1:4000 and 1:8000 are selected for detection, and the enzyme-labeled antibody optimal dilution is selected according to the P/N value.
TABLE 8 time of action of second enzyme-labeled antibody
As shown in Table 8, 90min was selected as the optimal time for the second enzyme-labeled antibody according to the P/N value.
TABLE 9 concentration of enzyme-labeled secondary antibodies
As shown in Table 9, 1:4000 was selected as the optimal concentration of the second enzyme-labeled antibody according to the P/N value.
7. Substrate development time
According to the conditions of the screening, the substrate color reaction is carried out for four times of 5, 10, 15 and 20 minutes, and the optimal color development time of the substrate is selected according to the P/N value.
TABLE 10 color development time
As shown in Table 10, 10min was selected as the optimal development time.
In summary, the optimal action conditions are as follows: mo 1a11 mab was diluted in a volume ratio of 1:20, coated overnight at 4 ℃ after 60min at 37 ℃ and blocked with 1% v/v BSA. The antigen to be detected reacts for 90min at 37 ℃, the detection antibody is diluted according to the volume ratio of 1:1024, and the reaction is carried out for 120min at 37 ℃; the enzyme-labeled antibody is diluted according to the volume ratio of 1:4000, acts for 90min at 37 ℃, and the substrate is developed for 10min.
Example 8 Mycoplasma ovipneumoniae double-antibody sandwich ELISA method for detection of samples treated by different methods
According to the conditions of the screening, the antigen to be detected selects four treatment modes of stock solution of culture, ultrasonic cracking, merthiolate inactivation and formaldehyde inactivation for detection, and selects the optimal sample treatment method.
TABLE 11 sample processing conditions
As can be seen from Table 11, the Mycoplasma ovipneumoniae double-antibody sandwich ELISA method can be used for detecting samples treated by different methods, and the detection is most sensitive after the samples are subjected to ultrasonic treatment according to the P/N value.
Example 9 evaluation of Properties of Mycoplasma ovipneumoniae double-antibody sandwich ELISA method
1. Specificity test
According to the conditions of the screening, mycoplasma hyopneumoniae, mycoplasma gallisepticum, mycoplasma bovis, mo, escherichia coli, KM2 culture medium, MTB culture medium and pig serum are selected for detection.
TABLE 12 specificity
The established double-antibody sandwich ELISA is used for detecting Mo, mycoplasma hyopneumoniae, mycoplasma bovis, escherichia coli, KM2 culture medium, MTB culture medium and pig serum, and the result shows that the P/N of Mo detection is only 2.1, and the rest P/N is all <2.1, thus indicating that the established method has better specificity.
2. Sensitivity determination
Freshly harvested NJ01 strains, CCU 5X 10 6CCU50/mL, were sonicated and tested using the double antibody sandwich ELISA method established above.
TABLE 13 sensitivity
As can be seen from Table 13, when the dilution factor of the NJ01 strain with a CCU of 5X 10 6CCU50/mL was 1:256, the P/N value was still greater than 2.1 as measured by the double antibody sandwich ELISA method, and the concentration of the NJ01 strain was 1.95X 10 4CCU50/mL. Therefore, the minimum detection limit of the double-antibody sandwich ELISA method for detecting mycoplasma ovipneumoniae established by the invention is below 1.95 multiplied by 10 4CCU50/mL.
3. Repeatability test
According to the above screening conditions, 6 batches of cultures were selected for plate-to-plate and plate-to-plate repetition.
Table 14 plate-to-plate and plate-in-plate replicates
The variation Coefficient (CV) in the plates is 2-6.87%, the variation coefficient between the plates is 0.65-4.18%, which shows that the established ELISA method has good repeatability and stable detection result.
Example 10 application of Mycoplasma ovipneumoniae double-antibody sandwich ELISA method
The method was used to examine 10 batches of freshly cultured Mo cultures, which were simultaneously assayed for CCU content, and the results were subjected to correlation analysis.
Table 15 culture assays
The results of the established double antibody sandwich ELISA method and CCU 50 content measurement on fresh cultures are analyzed by Pearson correlation, and the results show that the Pearson correlation coefficient is 0.986 and has obvious positive correlation, thereby establishing a calculation formula.
The content of the cultured 5 batches of cultures is measured by applying the double-antibody sandwich ELISA method established by the invention, and CCU 50 content is measured at the same time, and the results are shown in Table 16, and the result shows that CCU 50 calculated by the detection result of the double-antibody sandwich ELISA method has smaller difference from the actually measured CCU 50 and the minimum difference is 0.027. The results show that the detection result of the established double-antibody sandwich ELISA method has good linear relation with the measurement of CCU 50, so that the double-antibody sandwich ELISA method can be used for measuring the relative content of CCU 50 of Mo instead of the CCU 50 method. Table 16 Mycoplasma ovipneumoniae double-antibody sandwich ELISA and CCU 50 assay results.
TABLE 16 Mycoplasma ovipneumoniae double-antibody sandwich ELISA and CCU 50 assay results
In recent years, serological investigation results of mycoplasma pneumonia of sheep in China show that Mo has infection in various regions of the country, and the positive rate of the high-infection region is even more than 70%, which means that Mo exists in sheep groups in China as a large number. Mo is easily mixed with other pathogens or secondarily infects, leading to an increase in mortality, and Mo infection seriously hinders the development of sheep farming in China. Therefore, the quantitative detection of Mo is very important, and the current quantitative culture of mycoplasma mainly uses CCU and CFU to count viable bacteria, and the CCU and CFU count has the conditions of difficult grasping of harvest time points, time and labor waste, insufficient standardization and easy pollution, so that a rapid, accurate and sensitive ELISA method for detecting Mo antigen is explored, and the method has important significance for detection and diagnosis of mycoplasma. In practical applications, a double-antibody sandwich ELISA has been used for detection of various mycoplasma, including mycoplasma hyopneumoniae, and mycoplasma hyopneumoniae. In addition, the method is also used for detecting novel coronavirus antigens, shows high sensitivity and applicability, and can detect mycoplasma antigens more accurately by optimizing experimental conditions and establishing standardized experimental procedures, thereby improving the diagnosis level of mycoplasma infection.
The invention provides a hybridoma cell strain secreting an anti-mycoplasma ovipneumoniae monoclonal antibody, and ideas and methods for monoclonal antibodies and applications thereof, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. The hybridoma cell strain secreting the anti-mycoplasma ovipneumoniae monoclonal antibody is characterized in that the hybridoma cell strain is hybridoma cell strain 1A11, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCCNO: c202408, the preservation time is 2024, 1 month and 9 days.
2. An anti-mycoplasma ovipneumoniae monoclonal antibody 1a11 secreted by the hybridoma cell line of claim 1.
3. Use of the hybridoma cell line of claim 1 or the monoclonal antibody 1a11 of claim 2 for preparing a reagent and/or a kit for detecting mycoplasma ovipneumoniae.
4. A double-antibody sandwich ELISA kit for detecting mycoplasma ovipneumoniae, which is characterized by comprising a capture antibody, a detection antibody and an enzyme-labeled secondary antibody;
the capture antibody is monoclonal antibody 1a11 of claim 2;
The detection antibody is mycoplasma ovipneumoniae hyperimmune rabbit serum;
The enzyme-labeled secondary antibody is horseradish peroxidase labeled goat anti-rabbit IgG.
5. The kit of claim 4, wherein said mycoplasma ovipneumoniae hyperimmune rabbit serum is obtained by immunizing rabbits with mycoplasma ovipneumoniae.
6. The kit of claim 4, further comprising a wash solution, a blocking solution, a substrate solution, and a stop solution.
7. The kit of claim 6, wherein the wash solution is PBST; the sealing liquid is any one of casein, bovine serum albumin, skim milk powder or gelatin; the substrate liquid is TMB color developing liquid; the stop solution is 2M sulfuric acid.
8. Use of a kit according to any one of claims 4 to 7 for the detection of mycoplasma ovipneumoniae for non-diagnostic purposes.
9. A double-antibody sandwich ELISA method for the non-diagnostic detection of mycoplasma ovipneumoniae, characterized in that it comprises the following conditions: the dilution factor of the monoclonal antibody 1A11 is 1:2.5-1:320 v/v, the coating condition is 37 ℃, and after 60min, the temperature is 4 ℃ overnight or directly 4 ℃ overnight; the sealing condition of the sealing liquid is 37 ℃ for 1h; the antigen to be detected has the action condition of 37 ℃ for 30-120 min; the dilution factor of the detection antibody is 1:256-1:16384 v/v, and the action condition is 37 ℃ for 30-120 min; the dilution multiple of the enzyme-labeled secondary antibody is 1:1000-1:8000 v/v, and the action condition is 37 ℃ for 30-120 min; the color development time is 5-20 min.
10. The double antibody sandwich ELISA method of claim 9, wherein the antigen to be detected is mycoplasma ovipneumoniae culture stock solution or a mixture system of the stock solution treated by any one of ultrasonic cleavage, merthiolate inactivation or formaldehyde inactivation.
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