CN110261606B - Clostridium perfringens beta toxin antibody capture ELISA detection method - Google Patents
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Abstract
The invention belongs to the technical field of animal immunology, and particularly relates to an antibody capture ELISA detection method for clostridium perfringens beta toxin. The invention uses monoclonal antibody of anti-clostridium perfringens beta toxin to coat an enzyme label plate, uses natural clostridium perfringens beta toxin as capture antigen, and establishes a clostridium perfringens beta toxin antibody capture ELISA detection method. The invention has the characteristics of rapidness, simplicity, convenience, strong specificity, high sensitivity and the like, and is suitable for the detection of clostridium perfringens beta toxin antibody, epidemiological investigation, vaccine immune effect evaluation and the like.
Description
Technical Field
The invention relates to the technical field of animal immunology, in particular to a clostridium perfringens beta toxin antibody capture ELISA detection method.
Background
Clostridium perfringens (c.perfringens), also known as clostridium welchii (c.welchii), is widely found in the natural environment and found in the digestive tract of almost all warm-blooded animals, and is a member of the normal flora in the intestinal tracts of humans and animals. The clostridium perfringens can cause diseases such as lamb dysentery, necrotic enteritis and enterotoxemia of lambs, calves, piglets, rabbits and chicks, has acute morbidity and high mortality, is an important disease seriously harming the breeding industry, is a main pathogen of sudden death disease of domestic animals in recent years, and brings huge economic loss to the development of livestock industry of various countries. The clostridium perfringens type C mainly produces beta toxin (CPB), the bacterium can cause human gas gangrene and enterotoxemia and necrotic enteritis of various animals, and beta toxin antibodies are produced in livestock after the livestock are infected, so the beta toxin antibodies are one of important bases for diagnosing clostridium perfringens type C.
At present, methods for detecting clostridium perfringens beta toxin antibodies mainly comprise neutralization tests, enzyme-linked immunosorbent assays (ELISA) and the like, wherein the classic method for detecting clostridium perfringens toxin antibodies by the neutralization tests is simple and reliable, but is time-consuming and labor-consuming; therefore, neutralization assays are not suitable for large scale sample detection and substrate use. The ELISA detection has the advantages of simple and quick operation, low cost, capability of simultaneously detecting a large number of samples and the like, so that the development of the ELISA detection method for the clostridium perfringens beta toxin antibody can provide an effective tool for the diagnosis of clostridium perfringens diseases, epidemiological investigation, vaccine immune effect evaluation and the like.
The method comprises the steps of converting prokaryotic expression recombinant plasmid pET-28 alpha-beta into escherichia coli BL21 (DE 3) competent cells, performing induction expression by IPTG (isopropyl-beta-thiogalactoside) and the like, performing Western blot identification after nickel column affinity chromatography purification and urea gradient dialysis renaturation on expression products, using renatured beta toxin recombinant protein as a coating antigen, and optimizing indirect ELISA test conditions to establish the indirect ELISA detection method for the clostridium perfringens beta toxin antibody. However, in the method, prokaryotic expression recombinant protein is used as an antigen, antigenic determinants are poorly exposed and displayed, and the detection sensitivity to the beta toxin antibody is to be further improved.
Disclosure of Invention
Aiming at the prior art, the invention aims to establish a clostridium perfringens beta toxin antibody capture ELISA detection method for detection of clostridium perfringens beta toxin antibody, epidemiological investigation, vaccine immune effect evaluation and the like.
In order to realize the purpose, the invention adopts the following technical scheme:
the invention provides a hybridoma cell strain with the preservation number of CGMCC NO.14894.
The hybridoma cell strain is prepared by the following method:
the method comprises the steps of immunizing a Balb/C mouse by using purified natural clostridium perfringens beta toxin as an antigen, fusing splenocytes of the immunized mouse with SP2/0 cells, screening positive clones and subclones, finally screening a hybridoma cell 2C8A capable of stably secreting a monoclonal antibody, classifying and naming the hybridoma cell as clostridium perfringens beta toxin, storing the hybridoma cell in China general microbiological culture Collection center (CGMCC) at 12-8 days in 2017, and storing the hybridoma cell in the microbial culture Collection center (CGMCC) with the address of No. 3 of Ministry of Western Lu 1 in the morning of the Yangtze district in Beijing city, and the microbial research institute of China academy of sciences with the preservation number of CGMCC NO.14894.
The second aspect of the invention provides a monoclonal antibody which is secreted and produced by a hybridoma cell strain with the preservation number of CGMCC NO.14894.
The monoclonal antibody is obtained from a culture solution of a hybridoma cell strain; or inoculating hybridoma cell strain to abdominal cavity of experimental animal to generate ascites.
The monoclonal antibody prepared by the hybridoma cell strain has strong specificity, good sensitivity and high titer. Based on the above, the third aspect of the invention provides an application of the hybridoma cell strain or monoclonal antibody in preparation of a kit for detecting clostridium perfringens beta toxin antibody.
In a fourth aspect of the invention, an ELISA kit for detecting clostridium perfringens beta toxin antibody is provided; the kit comprises a monoclonal antibody secreted by a hybridoma cell strain with the preservation number of CGMCC NO.14894.
Further, the ELISA kit further comprises: antigen coating liquid, confining liquid, capture antigen, secondary antibody, positive control serum and negative control serum.
Preferably, the antigen coating solution is: 0.1M carbonate buffer, pH 9.6.
Preferably, the composition of the sealing liquid is as follows: 5g of skim milk powder was dissolved in 100ml of PBST.
Preferably, the capture antigen is prepared by the following method:
inoculating a C-type clostridium perfringens strain on a blood plate culture medium for resuscitation, carrying out anaerobic culture, selecting a single colony subjected to anaerobic culture, and inoculating the single colony to a liquid sulfur-ethanol culture medium for bacterium-increasing culture; then inoculating the enriched bacterial liquid into the improved Gordon soup, culturing at 45 ℃ for 4-6h to produce toxin, centrifuging the culture, filtering and sterilizing to obtain sterilized C-type clostridium perfringens exotoxin; and purifying the exotoxin to prepare the capture antigen.
Further, the blood plate culture medium comprises the following components: 100ml deionized water, 3.7g bean flour agar, 1g glucose and 5% volume ratio defibrinated sheep blood;
the improved Gordon soup toxigenic culture medium comprises the following components: 2g of peptone, 1g of dextrin, 2g of yeast extract, 1.2g of l-arginine and 1g of glucose, finally diluting the volume to 100ml with PBS, adjusting the pH value to 7.5 with concentrated hydrochloric acid, and sterilizing at high temperature to obtain the oral liquid.
Preferably, the secondary antibody is HRP-labeled goat anti-rabbit IgG.
In a fifth aspect of the present invention, there is provided a clostridium perfringens beta toxin antibody capture ELISA detection method, comprising the following steps:
(1) Coating: diluting the monoclonal antibody secreted by the hybridoma cell strain with the preservation number of CGMCC NO.14894 to 0.3 mu g/ml by using an antigen coating solution, adding 100 mu L of the antigen coating solution into a 96-hole enzyme label plate, and sealing overnight at 4 ℃;
(2) And (3) sealing: washing the coated ELISA plate with PBST washing solution, adding sealing solution after washing, sealing at 4 ℃ overnight with 200 mu L of each hole;
(3) Adding an antigen: washing the sealed elisa plate by PBST washing liquid, drying, adding 100 mu L of capture antigen diluted to 2 mu g/ml by PBS buffer solution into each hole, and incubating for 1h at 37 ℃;
(4) Adding serum to be detected: washing the ELISA plate treated in the step (3) with a PBST washing solution, drying, adding 100 mu L of serum to be detected diluted by PBS buffer solution in a volume ratio of 1 to 160 into each hole, and simultaneously setting a negative control, a positive control and a blank control, wherein the negative control is added with the negative control serum, the positive control is added with the positive control serum, and the blank control is added with the PBS buffer solution; incubating for 1h at 37 ℃;
(5) Adding a secondary antibody: washing the ELISA plate treated in the step (4) with a PBST washing solution, drying, adding HRP-labeled goat anti-rabbit IgG diluted by a PBS buffer solution according to a volume ratio of 1;
(6) Color development: washing the elisa plate processed in the step (5) by using a PBST washing solution, drying, adding 100 mu L/hole of TMB substrate color development solution, and reacting for 15min in a dark place at 37 ℃;
(7) And (4) terminating: add 100. Mu.L of 2M H per well 2 SO 4 Stopping the reaction of the solution, and reading a light absorption value at 450 nm;
(8) And (4) judging a result: calculating the mean value of negative samples
And the Standard Deviation (SD) are determined>
Is a positive threshold value>
Is a negative critical value; determination of OD of serum to be tested 450nm If the serum to be tested is->
The protein is positive, and the protein is positive,
is negative, or>
Is a suspicious sample.
The invention has the beneficial effects that:
(1) The hybridoma cell strain is obtained through multiple screening and cloning, can stably and efficiently secrete the monoclonal antibody, and can realize large-scale batch production. The monoclonal antibody has the advantages of strong specificity, good sensitivity and high titer, can be used for detecting clostridium perfringens beta toxin antibody by enzyme-linked immunosorbent assay (ELISA), and has the characteristics of simple and convenient sample operation, low cost, quick reaction, high sensitivity, strong specificity and the like.
(2) The ELISA kit for detecting the clostridium perfringens beta toxin antibody, which is prepared by using the monoclonal antibody secreted by the hybridoma cell strain, has the advantages of strong specificity, high sensitivity, good repeatability and the like. Wherein: the ELISA kit has stronger reactivity with a natural beta toxin serum antibody, is negative with the detection results of escherichia coli, salmonella and riemerella anatipestifer antibody serum, shows no cross reaction and has stronger specificity; compared with indirect method, the indirect ELISA method of C-type exotoxin coating detects a plurality of toxin antibodies, the ELISA kit only detects beta toxin antibodies, and the specificity is strong.
The serum titers of the prepared positive sera were compared to those of the classical mouse neutralization experiments. Positive sera were expressed as 1:10,1:20,1:40,1:80,1:160,1:320,1:640 serial fold dilutions were followed by ELISA assay to detect serum titers of 1. The result of mouse neutralization experiment shows that 0.1ml of serum can neutralize the minimum lethal dose of 400 toxins to the mouse, and the result shows that the constructed ELISA method has higher sensitivity.
1 piece of serum was randomly extracted from each of the 3 coated 96-well microplate, and 10 clinically known backgrounds were examined. Each serum was repeated 3 times, and 1-well negative and positive control was set for each time. The OD values of the negative and positive serums are compared, and the repeatability results of the detection samples in batches and among batches show that the intra-batch variation coefficient is 1.11% -6.6%, the inter-batch variation coefficient is 1.56% -6.85%, and the intra-batch variation coefficient and the inter-batch variation coefficient are both less than 10%, so that the variation degree of the same sample in the tests of the same batch and different batches is small, and the method has good repeatability. Because individual difference of animal experiment is avoided, the stability of the experiment is good, and the animal welfare protection is facilitated.
Drawings
FIG. 1: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis chart of a C-type clostridium perfringens crude exotoxin protein; in the figure, M is marker, and lanes 1, 2 and 3 are samples; the sample lane had a significant band of the major exotoxin (beta toxin) of clostridium perfringens at 38 KD.
FIG. 2: SDS-PAGE analysis of mouse ascites after purification; in the figure, M is marker, and lanes 1 and 2 are samples; two distinct protein bands are seen in the sample lanes: the heavy chain of about 53kD and the light chain of about 23kD have no other miscellaneous bands, and the results show that the purification effect is better, and the purity of the heavy chain and the light chain reaches more than 90 percent.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, the existing ELISA detection method for clostridium perfringens beta toxin antibody uses prokaryotic expression recombinant protein as antigen, the exposure and display of antigenic determinant are poor, and the detection sensitivity to beta toxin antibody is insufficient. Based on the hybridoma cell strain, the monoclonal antibody secreted by the hybridoma cell strain is strong in specificity, good in sensitivity and high in titer, and can be used for detecting the clostridium perfringens beta toxin antibody by an enzyme-linked immunosorbent assay (ELISA).
In one embodiment of the invention, the method for obtaining the hybridoma cell strain is given as follows: the purified natural clostridium perfringens beta toxin is used as an antigen to immunize a Balb/C mouse, spleen cells of the immunized mouse are fused with SP2/0 cells, then screening of positive cloning and subcloning is carried out, and finally a hybridoma cell capable of stably secreting monoclonal antibodies is screened out.
In the process of obtaining hybridoma cell strains, the fused hybridomas are most complicated and complicated to screen, after the fused hybridomas are cultured for one week, cell supernatants in each pore plate are taken out, ELISA is used for detecting whether the fused hybridomas secrete specific antibodies or not and whether the secretion capacity of the antibodies is strong, and if positive and strong secretion capacity cells are detected, the positive cells are required to be subjected to multiple subcloning to obtain positive monoclonal hybridoma cell strains. In the process, strong positive cells are likely to be missed due to cell variation and competition, so that the hybridoma cell strain capable of secreting the monoclonal antibody continuously, stably and efficiently is difficult to obtain, and has randomness and unpredictability. Therefore, the hybridoma cell strain obtained by the invention is subjected to biological preservation, and the preservation number is CGMCC NO.14894.
The monoclonal antibody secreted by the hybridoma cell strain 2C8A can be specifically combined with clostridium perfringens beta toxin, and the titer of the monoclonal antibody in ascites can reach 1.
The monoclonal antibody prepared by the hybridoma cell strain has strong specificity, good sensitivity and high titer, so that the monoclonal antibody can be used for preparing a kit for detecting the clostridium perfringens beta toxin antibody.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
Reagents and sources thereof used in the examples: freund's complete adjuvant was purchased from Sigma, USA, together with Freund's incomplete adjuvant, tween 20; the goat anti-rabbit IgG-HRP and the goat anti-mouse IgG-HRP are purchased from Hangzhou Huaan biotechnology, inc.; 96-well elisa plates were purchased from Solarbio; anaerobic pork liver soup and liquid thioglycollate culture medium are purchased from Qingdao Haibo organism; the soluble TMB substrate developing solution is purchased from Tiangen Biochemical technology Co., ltd; all other chemicals used were analytical grade.
The reagents involved in the examples are as follows:
the blood plate culture medium comprises the following components: 100ml deionized water, 3.7g bean flour agar, 1g glucose and 5% by volume defibrinated sheep blood.
The improved Gordon soup toxigenic culture medium comprises the following components: 2g of peptone, 1g of dextrin, 2g of yeast extract, 1.2g of L-arginine, 1g of glucose, and finally setting the volume to 100ml by PBS, adjusting the pH value to 7.45 by concentrated hydrochloric acid, and sterilizing at high temperature.
The antigen coating solution is as follows: 0.1M carbonate buffer, pH 9.6.
The PBS buffer comprises the following components: 1L deionized water, potassium dihydrogen phosphate (KH) 2 PO 4 ): 0.27g disodium hydrogen phosphate (Na) 2 HPO 4 ): 1.42g, sodium chloride (NaCl): 8g. Potassium chloride (KCl) 0.2g, pH 7.4.
The PBST washing solution comprises the following components: mu.L of Tween-20 was dissolved in 1L of PBS.
The sealing liquid comprises the following components: 5g of skimmed milk powder was dissolved in 100ml of PBST.
Stop solution (2M H 2 SO 4 ) Comprises the following steps: measuring concentrated H 2 SO 4 27.6ml of the solution is added into 450ml of deionized water, and the solution is mixed evenly and then is added to 1L of the solution.
Example 1: preparation and purification of clostridium perfringens type C exotoxin
Clostridium perfringens Type C (obtained from the National Collection of Type Cultures, accession number: NCTC 3180) was inoculated on a blood plate medium for resuscitation, anaerobically cultured at 37 ℃ for 36 hours, and individual colonies were picked up in a thioglycollate nutrient broth enrichment medium at a gas concentration of 88N 2 、7％H 2 、5％CO 2 Under the anaerobic environment condition of (2) for 12h at 38 ℃. Inoculating 5mL of C-type clostridium perfringens enrichment liquid into 100ml of a toxin-producing culture medium with pH7.5 (2 g of peptone, 1g of dextrin, 2g of yeast extract, 1.2g of L-arginine and 1g of glucose are dissolved in 100mL of PBS buffer solution), carrying out shake culture in an anaerobic environment, carrying out high-efficiency toxin production at 43 ℃ for 5h, then carrying out centrifugation at 8000r/min at 4 ℃ for 15min, and carrying out filtration sterilization by using a Seitz filter with the aperture of 0.22 mu m to obtain the sterilized exotoxin of C-type clostridium perfringens. Final determination of exotoxin LD 50 ＝2 4.25 That is, the exotoxin is diluted 19.03 times and 1ml is injected into the abdominal cavity, so that half of the mice can die.
Slowly adding the sterilized toxin solution into a saturated ammonium sulfate solution, finally enabling the volume concentration of ammonium sulfate in the mixed solution to reach 50%, and standing overnight at 4 ℃; the next day, centrifuging the solution 8000r/min for 20min, discarding supernatant, dissolving the precipitate with appropriate amount of 0.05M Tris-HCL buffer solution, slowly adding saturated ammonium sulfate solution to make final concentration of ammonium sulfate reach 40%, repeating the above operation, and finally dissolving the precipitate in Tris-HCL; then carrying out desalting purification and concentration on the exotoxin by Sephadex-G25 column chromatography; performing SDS-PAGE electrophoresis on the toxin protein after crude extraction and purification, analyzing the protein purity, and showing the resultA distinct band (as shown in FIG. 1) at molecular weight 38KD, which is the main pathogenic exotoxin beta toxin; measuring absorbance values at 260nm and 280nm by using a spectrophotometer, and determining the concentration of the toxin protein as follows: (1.45 XA) 280 －0.74×A 260 ) X dilution factor.
Example 2: resuscitation of anti-clostridium perfringens beta toxin hybridoma cells and preparation and purification of monoclonal antibodies;
performing conventional resuscitation and culture on a hybridoma cell strain 2C8A (preserved in China center for culture Collection of microorganisms with the preservation number of CGMCC NO. 14894) of clostridium perfringens beta toxin; selecting 10 female BALB/C mice of 6-8 weeks old, injecting Freund type incomplete adjuvant into abdominal cavity for 0.5 mL/mouse for sensitization, injecting positive hybridoma cell strain 2C8A after one week, and making dosage 0.5-1 × 10 6 And each mouse is used for collecting ascites of the mice after 7 to 10 days to obtain a large amount of clostridium perfringens beta toxin monoclonal antibodies, the ascites is purified by an octanoic acid-ammonium sulfate method, and the purification effect is detected by SDS-PAGE, and the result is shown in figure 2, wherein two obvious protein bands can be seen in samples in lanes 1 and 2 in the figure: the heavy chain of about 53kD and the light chain of about 23kD, and no other miscellaneous bands, the result shows that the purification effect is better, the purity of the monoclonal antibody reaches more than 90%, the spectrophotometer is used for measuring the absorbance values of the purified monoclonal antibody under 260nm and 280nm, and the concentration of the toxin protein is determined as follows: (1.45 XA) 280 －0.74×A 260 ) X dilution factor.
Example 3: preparation of polyclonal antibody against clostridium perfringens beta toxin
Adding formaldehyde into the sterilized exotoxin of clostridium perfringens type C obtained in the embodiment 1 to enable the final concentration of the exotoxin to be 0.3vt%, fully and uniformly mixing, inactivating at 37 ℃ for 96 hours, and oscillating and shaking once every 5-6 hours;
selecting 1.0-1.5Kg of healthy, non-vaccinated rabbits 5, mixing and emulsifying 2mL of inactivated beta toxin and Freund's complete adjuvant according to the volume of 1; after two weeks of initial immunization, 2mL of inactivated exotoxin and freund's incomplete adjuvant were mixed and emulsified according to volume 1; after 2 weeks, the same method is used for three-time immunization; 2 weeks after the three-immunization, the antigen without adjuvant is used for boosting the immunity, the dosage and the method are the same, and the obtained hyperimmune serum is used as capture ELISA positive control serum; the control group is injected with normal saline, and the same immunization program as the experimental group is ensured, the experiment is synchronously carried out, and the obtained serum is used as capture ELISA negative control serum.
Example 4: establishment of capture ELISA detection method
The optimal dilution concentrations of the coated antibody (obtained in example 2) and the antigen (obtained in example 1), the optimal dilution concentration of the antibody to be detected and the optimal dilution concentration of the enzyme-labeled antibody are determined by a square matrix titration method, and ELISA conditions are searched and optimized to finally determine the following system:
(1) Determination of optimal concentration of partner antibody and antigen
The monoclonal antibody against clostridium perfringens beta toxin recovered and purified in example 2 was selected primarily as the capture antibody, and the exotoxin extracted and purified in example 1 was selected as the binding antigen. In order to determine the optimal concentration of the coating antibody and the antigen, the optimal antibody matching is selected through a square matrix experiment under the condition of determining the optimal dilution multiple of the serum to be detected and the optimal dilution multiple of the enzyme-labeled secondary antibody. The capture antibody is diluted with the coating solution according to the volume ratio of 1 to 100 and added to the first column of the 96-well enzyme label plate, and diluted with the coating solution to the tenth column by a ratio of 1 time to 2 times, 100 mu L/well, coated overnight at 4 ℃, washed 3 times with PBST, 3min each time, and tapped dry. Add blocking solution 200. Mu.L per well, block overnight at 4 deg.C, wash 3 times with PBST, 3min each time, pat dry. The prepared clostridium perfringens type C beta toxin protein is diluted with PBS at volume 1 to 100 in the first row of a 96-well microplate and diluted 1-fold down with PBS to 100 μ L/well in the eighth row, incubated at 37 ℃ for 1h, pbst washed 3 times, 3min each time, and blotted dry. And (3) using PBS buffer solution to remove the positive antibody serum in a volume ratio of 1:100 μ L of 96-well microplate was added at 100 dilution and a negative control was set. Incubate at 37 ℃ for 1h, wash with PBST solution 3 times, 3min each time, and pat dry. HRP-labeled goat anti-rabbit IgG diluted in PBS buffer at 1:8000 vol/vol was added and incubated at 37 ℃ for 1h. Adding soluble TMB substrate developing solution, keeping out of the sun, developing at 37 deg.C for 15min, adding 100 μ L of 2M H per well 2 SO 4 The reaction was stopped and the OD in each well was measured at 450nm using a microplate reader. Selecting antibody and antigen with P/N valueThe basis for the optimal pairing concentration.
Final determination of capture antibody: the optimal dilution multiple of the monoclonal antibody against clostridium perfringens beta toxin is 1; the optimal working concentration of antigen is 2. Mu.g/mL.
(2) Selection of optimal coating solution concentration
The first and second columns of 96-well ELISA plate are coated with 0.01mol/L carbonate buffer solution, the third and fourth columns are coated with 0.05mol/L carbonate buffer solution, the fifth and sixth columns are coated with 0.1mol/L carbonate buffer solution, and the seventh and eighth columns are coated with 0.2mol/L carbonate buffer solution. 1. Positive samples were added in rows 3, 5, and 7, and negative sample controls were added in rows 2, 4, 6, and 8. And determining the optimal coating condition according to the P/N value.
Finally, the best effect can be ensured by using 0.1mol/L carbonate buffer solution as the coating solution.
(3) Determination of optimal incubation time for coated antibodies
The antibody is diluted to the working concentration by using the coating solution, and negative controls are arranged at the same time and are respectively incubated at the conditions of 1h +4 ℃ at 12h, 12h at 4 ℃ and 2h at 37 ℃. And determining the optimal antibody coating condition according to the P/N value.
The optimal incubation time for the coated antibody at 4 ℃ for 12h can be obtained by comparison and analysis.
(4) Determination of optimal sealing conditions
The 96-well ELISA plate was supplemented with 5% skim milk powder in the first and second columns, 5% fetal bovine serum in the third and fourth columns, 1% BSA in the fifth and sixth columns, and positive control in rows 1, 3 and 5 and negative serum control in rows 2, 4 and 6. And determining the optimal sealing condition according to the P/N value.
The 5% skimmed milk powder has the best effect as the sealing liquid.
(5) Determination of optimal closure time
Three enzyme-labeled plates are respectively coated, positive control is added in the first four rows of each enzyme-labeled plate, negative control is added in the last four rows of each enzyme-labeled plate, and the enzyme-labeled plates are respectively incubated under the conditions of 4 ℃ of 12h,37 ℃ of 2h and 37 ℃ of 1h +4 ℃ of 12h. And determining the optimal encapsulation and closure time of the antibody according to the P/N value.
Finally, the optimal sealing time was determined to be 12h at 4 ℃.
(6) Determination of optimal incubation time for antigen
Three enzyme label plates are respectively coated, positive control is added into the first four rows of each enzyme label plate, negative control is added into the last four rows of each enzyme label plate, and the three enzyme label plates are respectively incubated under the conditions of 30min at 37 ℃, 1h at 37 ℃ and 2h at 37 ℃. And determining the optimal incubation time of the antigen according to the P/N value.
Finally, it was determined that 1h at 37 ℃ is the optimal incubation time.
(7) Determination of optimal dilution of serum to be tested
To determine the optimal concentration of serum, positive and negative sera were diluted separately in 1,20, 1. Measuring OD value, and determining the serum to be detected according to the P/N value.
The P/N is the largest when the tested serum dilution multiple is 1:160 is the optimal dilution factor.
(8) Determination of optimal reaction time of serum to be tested
Diluting the positive serum and the negative serum according to the optimal dilution, adding the diluted positive serum and the diluted negative serum into a coated 96-well enzyme label plate, and respectively incubating the diluted positive serum and the diluted negative serum under the conditions of 30min at 37 ℃, 2h at 37 ℃ and 1h at 37 ℃. And determining the optimal encapsulation and closure time of the antibody according to the P/N value.
Finally, the optimal incubation time of the detected serum can be determined to be 37 ℃ for 1h through the positive-negative ratio.
(9) Determination of optimal dilution factor of enzyme-labeled secondary antibody
And (3) respectively carrying out plate coating according to the searched optimal conditions, adding the positive serum and the negative serum into a coated 96-well enzyme label plate according to the optimal dilution, and after incubation, respectively diluting the enzyme-labeled secondary antibody according to the ratio of 1, 2000, 1.
Finally, the optimal dilution factor of the enzyme-labeled secondary antibody can be determined to be 1 by the positive-negative ratio.
(10) Determination of incubation time of enzyme-labeled Secondary antibody
And (3) respectively carrying out plate wrapping according to the searched optimal conditions, adding the positive serum and the negative serum into a coated 96-well enzyme label plate according to the optimal dilution, and incubating, adding the enzyme-labeled secondary antibody with the optimal dilution factor, and respectively placing the mixture under the conditions of 30min at 37 ℃, 1h at 37 ℃ and 2h at 37 ℃. And determining the optimal incubation time of the antibody according to the P/N value.
The optimal incubation time of the enzyme-labeled secondary antibody can be determined to be 37 ℃ for 1h through the positive-negative ratio.
(11) Determination of optimal color development time
Performing plate wrapping incubation and other processes according to the optimal conditions, adding color development solution, and incubating at room temperature for 10min,15min,20min, and at 37 ℃ for 10min,15min, and 20min in dark. And setting a negative control, and determining the optimal incubation time of the antibody according to the P/N value.
The optimal color development time can be determined to be 15min at 37 ℃ through the positive-negative ratio.
The capture ELISA detection conditions are optimized by using a matrix titration experiment, and the following reaction system is finally determined:
(1) Coating: diluting the obtained clostridium perfringens beta toxin monoclonal antibody to 0.3 mu g/ml by using an antigen coating solution, adding the monoclonal antibody into a 96-hole enzyme label plate with each hole of 100 mu L, and sealing the enzyme label plate at 4 ℃ overnight;
(2) And (3) sealing: washing the enzyme-labeled plate with PBST washing solution for 3 times, each for 3min, adding PBST solution containing 5% Tween-20 skimmed milk powder as blocking solution, blocking at 4 deg.C for overnight at 200 μ L per well;
(3) Adding an antigen: washing the enzyme label plate with PBST solution for 3 times, 3min each time, patting dry, adding 100 μ L capture antigen diluted to 2 μ g/ml with PBS into each hole, and incubating for 1h at 37 ℃;
(4) Adding serum to be detected: washing with PBST solution for 3 times for 3min each time, drying, adding the test serum diluted with PBS buffer solution at a volume ratio of 1:160 into each well, setting positive and negative controls at the same time, taking PBS buffer solution as a blank control, and incubating at 37 ℃ for 1h;
(5) Adding a secondary antibody: washing 3 times with PBST solution for 3min each time, beating to dry, adding HRP-labeled goat anti-rabbit IgG diluted with PBS buffer at a volume ratio of 1;
(6) Color development: washing with PBST solution for 3 times for 3min each time, drying, adding soluble TMB substrate color development solution 100 μ L/hole, and reacting at 37 deg.C in dark for 15min;
(7) And (4) terminating: add 100. Mu.L of 2M H per well 2 SO 4 The reaction was stopped and absorbance was read at 450 nm.
Example 5: determination of cut-off values for capture ELISA detection methods
33 negative sera were measured with coated 96-well plates, and ELISA was performed according to the optimized conditions. Measuring OD value, calculating average value and standard deviation to obtain
Is a positive threshold value>
Negative cutoff. OD450nm, determination in conjunction with an established capture ELISA>
Is positive and/or positive>
If the result is negative, the test result is negative,
is a suspicious sample.
Calculating the average value thereof
Based on the standard deviation SD =0.018, the value is determined->
A value of 0.281, is present>
The value is 0.299, so that the OD450nm of the serum to be tested is<0.281 is negative, OD450nm>Positive at 0.299, 0.281<OD450nm<0.299 is suspicious.
Example 6: capture ELISA method Performance evaluation
(1) And (3) specificity test: the established capture ELISA method is used for detecting that the clostridium perfringens beta toxin antibody has stronger reactivity, and has no cross reaction with the serum obtained by respectively immunizing rabbits with escherichia coli, salmonella and riemerella anatipestifer.
(2) Sensitivity test: the serum titer of the positive serum prepared above was measured and compared with the classical mouse neutralization experiment. Positive sera were expressed as 1:10,1:20,1:40,1:80,1:160,1:320,1: after 640,1: 640.
diluting the beta exotoxin purified by desalting with physiological saline by multiple times, and injecting 0.5mL into abdominal cavity to make the toxin content at the maximum dilution time of death of all mice be the minimum lethal dose of the mice. The MLD of the prepared toxin to mice was measured to be 2. Mu.L.
Mixing 2-fold lethal toxin with 0.2mL hyperimmune serum diluted by 1: 50, 1:100, 1:200, 1:400, 1: 600, 1:800 and 1:1000, diluting to 1mL by using physiological saline, placing in a 37 ℃ incubator for 2h, taking 0.5mL of intraperitoneal injection mice, and observing the survival condition of the mice. The result gave a minimum lethal dose of 0.1mL serum that neutralized 400 toxins for mice.
From the above results, it can be seen that: the ELISA detection method provided by the invention has good sensitivity.
(3) And (3) repeatability experiment: 1 piece of serum was randomly extracted from each of the 3 coated 96-well microplate, and 10 clinically known backgrounds were examined. Each serum was repeated 3 times, and 1-well negative and positive control was set for each time. The OD values of the negative and positive serums are compared, and the repeatability results of the detection samples in batches and among batches show that the intra-batch variation coefficient is 1.11% -6.6%, the inter-batch variation coefficient is 1.56% -6.85%, and the intra-batch variation coefficient and the inter-batch variation coefficient are both less than 10%, so that the variation degree of the same sample in the tests of the same batch and different batches is small, and the method has good repeatability.
Example 7: specific application
1. The established capture ELISA method is used for detecting the clostridium perfringens beta toxin antibody from 100 parts of serum in a rabbit farm in Tai' an town of Shandong, and the indirect ELISA method for detecting the clostridium perfringens exotoxin antibody is used as a control (Sun Jia Zhi, 2014). And (3) displaying a detection result: the indirect ELISA detects that 85 rabbits have negative serum samples, 15 rabbits have positive serum samples, and the positive rate is 15 percent; the capture ELISA method detects that the serum samples of 90 rabbits are negative, 10 rabbits are positive, and the positive rate is 10%.
2. The serum titer of 10 positive sera obtained by the above method was compared with that of a classical mouse neutralization experiment. Positive sera were expressed as 1:10,1:20,1:40,1:80,1:160,1:320,1: after 640 serial two-fold dilutions, capture ELISA assays were performed to detect serum titers in a range of 1.
Diluting the beta exotoxin purified by desalting with physiological saline by multiple times, and injecting 0.5mL into abdominal cavity to make the toxin content at the maximum dilution time of death of all mice be the minimum lethal dose of the mice. 2 times of lethal dose toxin (MLD has been measured to be 2 ul) is mixed with 0.2mL of hyperimmune serum diluted by 1: 50, 1:100, 1:200, 1:400, 1: 600, 1:800 and 1:1000, the volume is fixed to 1mL by using physiological saline, the mixture is placed in a 37 ℃ incubator for 2h, 0.5mL of intraperitoneal injection mouse is taken, and the survival condition of the mouse is observed. The results showed that 0.1mL of serum was able to neutralize 200, 100, 200, 200 minimal lethal dose to mice.
In conclusion, compared with an indirect ELISA method, the detection positive rate of the method is slightly low, but the specificity of the method for detecting the clostridium perfringens beta toxin antibody is consistent with the fact that the positive rate is low, the specificity is strong, the method has no reaction to other exotoxins, the indirect ELISA method for detecting the clostridium perfringens exotoxin antibody aims at a target substance which is a mixture of multiple antibodies, the positive rate is high, but the specificity is poor, and therefore, the result is reasonable; compared with the classical mouse neutralization experiment, the sensitivity is higher; the results show that: the established method for detecting the clostridium perfringens beta toxin antibody by capturing ELISA can be used for detecting mass samples, provides a faster and effective tool for the diagnosis and research of the clostridium perfringens disease of livestock and poultry, and lays a solid foundation for the research of vaccine quality and food safety monitoring.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (6)
1. A monoclonal antibody is characterized by being secreted and produced by a hybridoma cell line with the preservation number of CGMCC NO.14894.
2. Use of the monoclonal antibody of claim 1 for the preparation of a kit for the detection of clostridium perfringens beta toxin antibodies.
3. An ELISA kit for detecting a clostridium perfringens beta toxin antibody is characterized by comprising a monoclonal antibody secreted by a hybridoma cell strain with the preservation number of CGMCC NO. 14894;
the ELISA kit also comprises: antigen coating liquid, confining liquid, capture antigen, secondary antibody, positive control serum and negative control serum;
the capture antigen is prepared by the following method:
inoculating a C-type clostridium perfringens strain on a blood plate culture medium for resuscitation, carrying out anaerobic culture, selecting a single bacterial colony subjected to anaerobic culture, inoculating the single bacterial colony to a liquid sulfur-ethanol culture medium, and carrying out bacteria increasing culture; then inoculating the enriched bacterial liquid into the improved Gordon soup, culturing at 45 ℃ for 4-6h to produce toxin, centrifuging the culture, filtering and sterilizing to obtain sterilized C-type clostridium perfringens exotoxin; purifying the exotoxin to prepare a capture antigen;
the blood plate culture medium comprises the following components: 100ml deionized water, 3.7g bean flour agar, 1g glucose and 5% by volume defibrinated sheep blood;
the improved Gordon soup toxigenic culture medium comprises the following components: 2g of peptone, 1g of dextrin, 2g of yeast extract, 1.2g of L-arginine and 1g of glucose, and finally diluting the volume to 100ml with PBS, adjusting the pH value to 7.5 with concentrated hydrochloric acid, and sterilizing at high temperature to obtain the oral liquid.
4. The ELISA kit of claim 3, wherein the antigen coating is: 0.1M carbonate buffer, pH 9.6.
5. The ELISA kit of claim 3 wherein the blocking solution has the composition: 5g of skim milk powder was dissolved in 100ml of PBST.
6. A clostridium perfringens beta toxin antibody capture ELISA detection method for non-disease diagnosis purposes is characterized by comprising the following steps:
(1) Coating: diluting monoclonal antibody secreted by hybridoma cell strain with preservation number of CGMCC NO.14894 to 0.3 mug/ml with antigen coating solution, adding 100 muL of antigen coating solution into 96-hole enzyme label plate, and sealing overnight at 4 ℃;
(2) And (3) sealing: washing the coated ELISA plate with PBST washing solution, adding sealing solution after washing, sealing at 4 ℃ overnight with 200 mu L of each hole;
(3) Adding an antigen: washing the sealed elisa plate by PBST washing liquid, drying, adding 100 mu L of capture antigen diluted to 2 mu g/ml by PBS buffer solution into each hole, and incubating for 1h at 37 ℃;
(4) Adding serum to be detected: washing the ELISA plate treated in the step (3) with a PBST washing solution, drying, adding 100 mu L of serum to be detected diluted by PBS buffer solution in a volume ratio of 1 to 160 into each hole, and simultaneously setting a negative control, a positive control and a blank control, wherein the negative control is added with the negative control serum, the positive control is added with the positive control serum, and the blank control is added with the PBS buffer solution; incubating at 37 ℃ for 1h;
(5) Adding a secondary antibody: washing the elisa plate treated in the step (4) with a PBST washing solution, drying, adding HRP-labeled goat anti-rabbit IgG diluted by a PBS buffer solution according to a volume ratio of 1;
(6) Color development: washing the elisa plate processed in the step (5) by using a PBST washing solution, drying, adding 100 mu L/hole of TMB substrate color development solution, and reacting for 15min in a dark place at 37 ℃;
(7) And (4) terminating: add 100. Mu.L of 2M H per well 2 SO 4 Stopping the reaction of the solution, and reading a light absorption value at 450 nm;
(8) And (4) judging a result: calculating the mean value of negative samples
And the Standard Deviation (SD) are calculated>
Is positive cut-off value>
Is a negative critical value; determination of OD of serum to be detected 450nm If the serum to be tested is->
If the number of the cells is positive,
is negative, or>
Is a suspect sample. />
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