CN113702640A - Indirect ELISA method for clostridium perfringens beta 1 toxin antibody - Google Patents
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Abstract
The invention discloses a clostridium perfringens beta 1 toxin antibody indirect ELISA detection method, which comprises the steps of establishing the clostridium perfringens beta 1 toxin antibody indirect ELISA method, and determining the result judgment standard. The invention belongs to the technical field of pathogenic microorganism detection, and particularly provides an indirect ELISA system for detecting a clostridium perfringens beta 1 toxin antibody, which has the advantages of good specificity, high sensitivity and good repeatability, provides an effective detection means for the clostridium perfringens infection condition of animals, has wide application prospect in clinical serum epidemiology investigation and vaccine immune effect evaluation, and can quickly detect the clostridium perfringens beta 1 toxin antibody.
Description
Technical Field
The invention belongs to the technical field of pathogenic microorganism detection, and particularly relates to an indirect ELISA detection method for clostridium perfringens beta 1 toxin antibody.
Background
Clostridium perfringens (c.perfringens), also known as clostridium welchii (c.welchii), is a opportunistic pathogen that normally inhabits the gastrointestinal tract of humans and animals. Clostridium perfringens is classified into A, B, C, D, E subtypes according to its four major exotoxins (α, β, epsilon, iota) secreted.
Clostridium perfringens (Clostridium perfringens) is an important pathogenic bacterium for both human and livestock, can cause enterotoxemia, gas gangrene, necrotic enteritis and even death of human and various animals, has serious morbidity, can cause shock and even death of infected livestock within a short period of time, and can cause poor elimination of the diseased young livestock even if the diseased young livestock survive and the healthy development of the cattle and sheep breeding industry is seriously influenced because the diseased young livestock are not well eliminated due to growth conditions if the diseased young livestock cannot be diagnosed and treated in time. The beta 1 toxin is produced by clostridium perfringens types B and C, which can cause hemorrhagic mucosal ulcers and small intestinal mucosal gangrene which are lethal to human and animals and even enterotoxemia, and the clostridium perfringens types B and C are common pathogenic plant types in livestock. The toxin is a classical beta-type pore-forming protein, can destroy the ion balance inside and outside cells, and induces an organism to generate inflammatory factors such as IL-1 beta and the like.
At present, relevant detection products are not available for detecting the toxin, and a method which is convenient to operate, high in sensitivity, good in specificity, stable and reliable is urgently needed for epidemiological investigation and antibody level detection of clostridium perfringens in farm animals clinically, so that an indirect ELISA detection method aiming at a clostridium perfringens beta 1 toxin monoclonal antibody is very necessary to establish.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a method and a material for detecting the level of a clostridium perfringens beta 1 toxin antibody in animal serum, which are suitable for clinical serum epidemiological investigation and vaccine immune effect evaluation caused by the clostridium perfringens beta 1 toxin, and particularly provide an indirect ELISA detection method which has strong specificity, high sensitivity and good stability and can quickly detect the clostridium perfringens beta 1 toxin antibody.
The technical scheme adopted by the invention is as follows: the invention relates to a clostridium perfringens beta 1 toxin antibody indirect ELISA detection method, which comprises the following steps:
step one, establishing an indirect ELISA method of a clostridium perfringens beta 1 toxin antibody:
(1) diluting the coated antigen to 10 mu g/mL by using a coating solution, coating the antigen in a 96-hole enzyme label plate at 100 mu L/hole overnight at 4 ℃, wherein the coated antigen is soluble recombinant clostridium perfringens beta 1 toxin protein;
the antigen used in the step is recombinant clostridium perfringens beta 1 toxin protein, and the preparation method comprises the following steps:
s1: taking a recombinant expression strain BL21(DE3) pTIG-cpb1 glycerol frozen strain, streaking on an LB solid plate containing 100 mu g/mL ampicillin, picking a single colony the next day, inoculating into 10mL LB liquid culture medium containing 100 mu g/mL ampicillin, and culturing to obtain a bacterial liquid OD600Up to 1.0 or more;
s2: then inoculating the strain into 200mL LB liquid culture medium containing 100 mug/mL ampicillin in an inoculation amount of 1 percent for culturing for 3 hours, then adding 0.5mM IPTG to induce and express for 16 hours at the temperature of 20 ℃, and centrifuging to obtain thalli;
(2) washing the plate with PBST solution for 3 times, each time for 5 minutes, adding a sealing solution, sealing with 200 mu L/hole, and keeping the temperature at 37 ℃ for 2 hours;
the PBST solution is prepared by adding 2 per mill of Tween-20 into 1L of PBS solution and mixing uniformly;
the confining liquid in the step (2) is 5% skimmed milk powder, and the preparation method of the 5% skimmed milk powder comprises weighing 5g of skimmed milk powder, adding PBS solution and fixing the volume to 100 mL;
(3) washing the plate with PBST solution for 3 times, each time for 5 minutes, adding a serum sample to be detected, taking a positive control as a clostridium perfringens beta 1 toxin monoclonal antibody 1K19 and a negative control as a clostridium perfringens beta 2 toxin monoclonal antibody, keeping the temperature at 37 ℃ for 1 hour, wherein each hole is 100 mu L;
the positive control is clostridium perfringens beta 1 toxin monoclonal antibody 1K19, and the preparation method comprises the following steps: recovering 1K19 hybridoma cells frozen in liquid nitrogen tank, subculturing, and transferring at a ratio of 1:100 to 75cm after cell state is good2In a culture flask of (1), and placing in CO2Transferring the cell culture solution into a 50mL centrifuge tube after the culture solution turns yellow after about 5 days until the cells overgrow to enrich the antibodies in the incubator, centrifuging at room temperature for 10min at 1500G, collecting 1K19 culture supernatant, performing filtration sterilization by using a 0.22 mu m filter, and purifying by using Protein G by using an AKTA purification system to obtain McAb1K 19; performing SDS-PAGE on the purified sample, identifying, quantifying by BCA, adding glycerol, and storing in a refrigerator at-80 deg.C;
the negative control used in this step was a monoclonal antibody to the β 2 toxin.
(4) Washing the plate with PBST solution for 3 times, each time for 5 minutes, adding HRP-goat anti-mouse IgG into a positive control hole and a negative control hole as a secondary antibody, adding a corresponding HRP-labeled secondary antibody into a serum sample hole to be detected, and keeping the concentration at 100 mu L/hole for 1 hour at room temperature;
in the step (4), the secondary HRP-goat anti-mouse IgG antibody is diluted 2000 times by the secondary HRP-goat anti-mouse IgG antibody, and the secondary HRP-labeled corresponding antibody is diluted 2000 times by the secondary HRP-labeled goat anti-animal IgG antibody;
(5) washing the plate with PBST solution for 3 times, each time for 5 min, adding substrate developing solution 100 μ L/well, keeping away from light at room temperature for 10min, adding stop solution H2SO4Stopping the reaction of the solution, measuring an OD450nm value and detecting an absorbance value;
step two, determining a result judgment standard:
positive when the sample OD450nm value > X +3 SD; when the OD450nm value is less than X +2SD, the result is judged to be negative; when the number is between the two, the result is judged to be suspicious;
by statistical analysis, the overall mean value of the negative control was 0.086, the standard deviation SD was 0.028, so the positive cut-off value was 0.17 and the negative cut-off value was 0.142.
Further, the coating solution in the step (1) is 0.85M carbonate buffer solution, the pH value is 9.6, and the preparation method of the coating solution of the 0.85M carbonate buffer solution is to weigh NaHCO32.93g and Na2CO31.59g, adding distilled water to a constant volume of 1L.
Further, the positive control in the step (3) is clostridium perfringens beta 1 toxin monoclonal antibody 1K19, and the positive control is diluted by PBS (phosphate buffer solution) 28Fold (0.25. mu.g/. mu.L).
Further, the substrate color developing solution in the step (5) is a 5% o-phenylenediamine solution, and the preparation method of the 5% o-phenylenediamine solution comprises the steps of weighing 5mg of OPD (o-phenylenediamine), weighing 10mL of substrate buffer solution for dissolving, and adding 3% H2O20.15mL, which is prepared at present, wherein the substrate buffer solution is a citric acid buffer solution, the pH value is 5.0, and the preparation method of the citric acid buffer solution comprises weighing 1.92g of citric acid and Na2HPO42.84g is weighed and added with distilled water to a volume of 100 mL.
Further, the stop solution in the step (5) is 2mol/L of H2SO4The preparation method of the stop solution comprises the steps of measuring 178.3mL of distilled water and dropwise adding 21.7mL of concentrated sulfuric acid with the concentration of 98%.
Further, the construction method of the envelope antigen expression strain of the soluble recombinant clostridium perfringens beta 1 toxin protein comprises the following steps: according to the gene sequence of clostridium perfringens beta 1 toxin reported in literature, a primer is designed:
Forward(5`-3`)CGGAATTCCATATGGATATAGGCAAAACTACTACTAT
Reverse(5`-3`)GAATGCGGCCGCAATAGCTGTTACTTTATG
the cpb1 gene was amplified from a C-type standard strain of Clostridium perfringens, constructed into a pTIG-Trx vector, transformed into BL21(DE3) strain, and labeled as BL21(DE3) pTIG-cpb 1.
Further, the coating antigen is recombinant clostridium perfringens beta 1 toxin protein, and the preparation method of the soluble recombinant protein comprises the following steps:
step a: centrifuging to collect 800mL of induced recombinant expression thallus, washing the thallus once by PBS, adding 20mM Tris-HCL (pH8.0) buffer solution into thallus sediment to resuspend 10mL, adding 100 mu g/mL lysozyme, 1 per thousand TritonX-100 and 0.01mM PMSF, and carrying out ultrasonic crushing for 10 min;
step b: centrifuging at 10000rmp for 10min, and washing the precipitate with 10mL of 20mM Tris-HCl (pH8.0) buffer containing 0.2M urea; centrifuging at 10000rmp for 10min, adding 10mL of 20mM Tris-HCL (pH8.0) denaturation buffer containing 2M urea into the centrifugation sediment, and incubating at 37 ℃ for 2h to completely dissolve and denature the sediment;
step c: transferring the protein solution after dissolving and denaturation into a dialysis bag, and sequentially adding 150mL of 20mM Tris-HCL (pH8.0) renaturation buffer solution containing urea with concentration gradients of 1.5M, 1M, 0.5M and 0M to the liquid outside the dialysis bag, wherein each concentration gradient is treated for 6 h.
Further, the positive control is a clostridium perfringens beta 1 toxin monoclonal antibody 1K19, protein sequences are analyzed and antigen prediction is carried out according to beta 1 toxin amino acid sequences provided by NCBI, sites of escherichia coli, clostridium perfringens beta 2 toxin, alpha toxin and epsilon toxin which are possibly subjected to cross recognition are removed, a peptide section with 14 amino acids and the highest epitope index of a target protein is selected and used as an antigen to be injected into a BalB/C mouse, after three times of immunization, the spleen of the mouse is fused with myeloma cells, and positive clone cell strains are obtained after restrictive gradient dilution and are obtained through screening.
Further, the McAb1K19 purification was performed using 20mM sodium phosphate, pH 7.0 as binding buffer, 0.1M glycine-HCl buffer, pH 2.7 as elution buffer, and 1M Tris-Cl, pH 9.0 as neutralization buffer.
Further, the complete Medium contains 10% by volume of RPMI Medium 1640 of fetal bovine serum, and the complete Medium contains 1% by volume of streptomycin and 1% by volume of glutamine.
Further, the negative control is a monoclonal antibody against clostridium perfringens beta 2 toxin.
The invention with the structure has the following beneficial effects: the scheme provides an indirect ELISA detection method for clostridium perfringens beta 1 toxin antibody, which has the following advantages:
1. the specificity is strong, the method is used for detecting positive serum of clostridium perfringens and collected healthy bovine serum reserved in a laboratory, and the result shows that only the positive serum of the clostridium perfringens is positive, and the healthy bovine serum is negative, so that the established detection system has good specificity;
2. the sensitivity is high, the McAb1K19 is diluted according to a multiple ratio, the sensitivity test is carried out on the established and optimized indirect ELISA detection method, and the experimental result shows that the OD450nm is 0.2235 when the McAb1K19 is diluted to 2 ng/mu L and is still above the positive critical value of 0.17, which indicates that the established and optimized indirect ELISA detection method has high sensitivity;
3. the stability is good, the established indirect ELISA detection method is utilized to carry out batch and batch detection on the clostridium perfringens beta 1 toxin positive antibody (McAb1K19) and the negative antibody, the variation coefficient is calculated, the stability of the method is verified, and the test result shows that the variation coefficient of 4-time batch detection is 3.654% -7.934%, the variation coefficient of 4-time batch detection is 3.725% -6.759%, and the variation coefficients of batch-to-batch and batch-to-batch repetition are both less than 10%, which indicates that the established method has good stability.
Drawings
FIG. 1 is an electrophoresis diagram of the expression and purification protein of the recombinant protein of the clostridium perfringens beta 1 toxin of the indirect ELISA detection method for the clostridium perfringens beta 1 toxin antibody of the invention;
FIG. 2 is a diagram showing the optimal antigen coating concentration optimization result of the clostridium perfringens beta 1 toxin antibody indirect ELISA detection method of the present invention;
FIG. 3 is a graph showing the optimal dilution factor optimization results of McAb1K19 in an indirect ELISA detection method for Clostridium perfringens beta 1 toxin antibody according to the present invention;
FIG. 4 is a graph showing the determination of the cut-off value of the Clostridium perfringens beta 1 toxin antibody indirect ELISA detection method of the present invention.
In FIG. 1, M represents Marker (CST 1420880 kDa (red), 57k Da, 46kDa, 32kDa, 25kDa (green), 22kDa, 17kDa, 11kKDa, 17KDa, 10 KDa); 1 represents a bacterial protein after induction of BL21(DE3) pTIG; 2 represents the cells after induction of BL21(DE3) pTIG-cpb 1; 3 denotes BL21(DE3) pTIG-cpb1 inclusion bodies washed with 0.2M urea and renatured.
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1-4, the present invention provides a clostridium perfringens beta 1 toxin antibody indirect ELISA detection method, comprising:
example 1: expression of soluble recombinant clostridium perfringens beta 1 toxin protein:
designing a primer according to the gene sequence of the clostridium perfringens beta 1 toxin reported by the literature
Forward(5`-3`)CGGAATTCCATATGGATATAGGCAAAACTACTACTAT
Reverse(5`-3`)GAATGCGGCCGCAATAGCTGTTACTTTATG
The cpb1 gene was amplified from a C-type standard strain of Clostridium perfringens, constructed into a pTIG-Trx vector, transformed into BL21(DE3) strain, and labeled as BL21(DE3) pTIG-cpb 1.
BL21(DE3) pTIG-cpb1 glycerol frozen strains are taken, streaked on LB solid plates containing 100 mu g/mL ampicillin, single colonies are picked the next day and inoculated into 10mL LB liquid culture medium containing 100 mu g/mL ampicillin to be cultured until the OD600 of the bacterial liquid reaches more than 1.0, then the bacterial liquid is inoculated into 200mL LB liquid culture medium containing 100 mu g/mL ampicillin in an inoculation amount of 1% to be cultured for 3 hours, and then the induced expression is carried out for 16 hours under the conditions of 20 ℃ and 0.5mM IPTG, and the bacterial liquid is harvested.
Example 2: purification of soluble recombinant clostridium perfringens beta 1 toxin protein:
centrifuging to collect 800mL of induced recombinant expression thallus, washing the thallus once by PBS, adding 20mM Tris-HCL (pH8.0) buffer solution into thallus sediment to resuspend 10mL, adding 100 mu g/mL lysozyme, 1 per thousand TritonX-100 and 0.01mM PMSF, and carrying out ultrasonic crushing for 10 min; centrifuging at 10,000rmp for 10min, washing the pellet with 10mL of 20mM Tris-HCl (pH8.0) buffer containing 0.2M urea; centrifuging at 10,000rmp for 10min, adding 10mL of a denaturation buffer solution of 20mM Tris-HCL (pH8.0) containing 2M urea to the centrifuged precipitate, and incubating at 37 ℃ for 2h to completely dissolve and denature the precipitate; transferring the protein solution after dissolving and denaturation into a dialysis bag, and sequentially adding 150mL of 20mM Tris-HCL (pH8.0) renaturation buffer solution containing urea with concentration gradients of 1.5M, 1M, 0.5M and 0M to the liquid outside the dialysis bag, wherein each concentration gradient is treated for 6 h.
Example 3: determination of soluble clostridium perfringens beta 1 toxin protein antigen coating conditions:
adopting a matrix titration method, taking the beta 1 toxin recombinant protein as a coating antigen, diluting the beta 1 toxin recombinant protein by using a coating buffer solution, diluting according to the concentration of each hole of 2 mu g/mL, 5 mu g/mL, 8 mu g/mL and 10 mu g/mL respectively, adding 100 mu L of each hole of an enzyme label plate, and coating overnight at 4 ℃; using 5% skimmed milk powder, purified McAb1K19 as primary antibody, HRP-goat anti-mouse IgG as secondary antibody, OPD as color development liquid, 2M H2SO4The solution is a stop solution, and the absorbance value is detected by measuring the OD450nm value.
Example 4: determination of the blocking conditions:
adopting a matrix titration method, and taking 10 microgram/mL beta 1 toxin recombinant protein as a coating antigen; respectively incubating 5% BSA or 5% skimmed milk powder for 30min, 60min, 90min and 120min as blocking solution, purifying McAb1K19 as primary antibody, HRP-goat anti-mouse IgG as secondary antibody, OPD as developing solution, and 2M H2SO4The solution was used as a stop solution, and the OD450nm number was measuredThe absorbance values were measured.
Example 5: preparing a clostridium perfringens beta 1 toxin monoclonal antibody 1K 19;
according to the amino acid sequence of the beta 1 toxin provided by NCBI, analyzing and predicting the antigen of the protein sequence, removing the sites of beta 2 toxin, alpha toxin and epsilon toxin of escherichia coli and clostridium perfringens which are possibly subjected to cross recognition, selecting a section of peptide with 14 amino acids with the highest epitope index from target protein, taking the peptide as an antigen to be injected into a BalB/C mouse, after three times of immunization, fusing the spleen of the mouse with myeloma cells, obtaining a positive clone cell strain after restrictive gradient dilution, and screening to obtain the monoclonal antibody.
Example 6: purification of clostridium perfringens beta 1 toxin monoclonal antibody 1K 19:
recovering 1K19 hybridoma cells frozen in liquid nitrogen tank, inoculating into RPMI Medium 1640 containing 1% streptomycin, 1% glutamine and 10% fetal calf serum, subculturing, and transferring at a ratio of 1:100 to 75cm after cell state is good2In a culture flask of (1), and placing in CO2After about 5 days in the incubator until the cells overgrow to enrich the antibody, after the culture became yellow, 1K19 culture supernatant was collected, sterilized by filtration using a 0.22 μ M filter, and purified using Protein G using AKTA purification system using 20mM sodium phosphate as a binding buffer, pH 7.0, 0.1M glycine-hydrochloric acid buffer as an elution buffer, pH 2.7, 1M Tris-Cl as a neutralization buffer, pH 9.0, and the Protein elution peak was harvested to obtain purified McAb1K 19.
Example 7: determination of the dilution ratio of monoclonal antibody 1K 19:
adopting a matrix titration method, and taking 10 microgram/mL beta 1 toxin recombinant protein as a coating antigen; incubation with 5% skimmed milk powder for 120min is used as a blocking condition, and purified McAb1K19 is used as a primary antibody, and the primary antibody is added in a ratio of 1:256, 1:512, 1:1024, 1:2048 and 1: 4096. 1:8192 and 1:16384, setting PBS as a blank control, HRP-goat anti-mouse IgG as a secondary antibody, OPD as a color development solution, and 2M H2SO4The solution is a stop solution, and the absorbance value is detected by measuring the OD450nm value.
Example 8: determination of dilution of HRP-goat anti-mouse IgG:
adopting a matrix titration method, taking 10 mu g/mL beta 1 toxin recombinant protein as a coating antigen, taking 5% skimmed milk powder for incubation for 120min as a closed condition, diluting 2 by using purified McAb1K198The primary antibody is used as a primary antibody, HRP-goat anti-mouse IgG is diluted into a secondary antibody according to the gradient of 1:1000, 1:2000, 1:4000 and 1:8000, OPD is color development liquid, and 2M H2SO4The solution is a stop solution, and the absorbance value is detected by measuring the OD450nm value.
Example 9: determination of the cut-off value of the indirect ELISA detection method:
ELISA assays were performed on 30 negative antibodies using established indirect ELISA detection methods and the mean OD450nm values (X) and Standard Deviation (SD) were calculated for these samples. Positive when the sample OD450nm value > X +3 SD; when the OD450nm value is less than X +2SD, the result is judged to be negative; if the difference is between the two, it is judged to be suspected.
By statistical analysis, the overall mean value of the negative control was 0.086, the standard deviation SD was 0.028, so the positive cut-off value was 0.17 and the negative cut-off value was 0.142.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. An indirect ELISA detection method for clostridium perfringens beta 1 toxin antibody is characterized by comprising the following steps:
step one, establishing an indirect ELISA method of a clostridium perfringens beta 1 toxin antibody:
(1) diluting the coated antigen to 10 mu g/mL by using a coating solution, coating the antigen in a 96-hole enzyme label plate at 100 mu L/hole overnight at 4 ℃, wherein the coated antigen is soluble recombinant clostridium perfringens beta 1 toxin protein;
(2) washing the plate with PBST solution for 3 times, each time for 5 minutes, adding a sealing solution, sealing with 200 mu L/hole, and keeping the temperature at 37 ℃ for 2 hours;
(3) washing the plate with PBST solution for 3 times, and adding a serum sample to be detected 5 minutes each time, wherein the positive control is a clostridium perfringens beta 1 toxin monoclonal antibody 1K19, the negative control is a clostridium perfringens beta 2 toxin monoclonal antibody, the concentration is 100 mu L/hole, and the temperature is kept at 37 ℃ for 1 hour;
(4) washing the plate with PBST solution for 3 times, each time for 5 minutes, adding HRP-goat anti-mouse IgG into a positive control hole and a negative control hole as a secondary antibody, adding a corresponding HRP-labeled secondary antibody into a serum sample hole to be detected, and keeping the concentration at 100 mu L/hole for 1 hour at room temperature;
(5) washing the plate with PBST solution for 3 times, each time for 5 min, adding substrate developing solution 100 μ L/well, keeping away from light at room temperature for 10min, adding stop solution H2SO4Stopping the reaction of the solution, measuring an OD450nm value and detecting an absorbance value;
step two, determining a result judgment standard:
positive when the sample OD450nm value > X +3 SD; when the OD450nm value is less than X +2SD, the result is judged to be negative; when the two are in between, the result is judged to be suspicious.
2. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection of claim 1The method is characterized in that: the coating solution in the step (1) is 0.85M carbonate buffer solution, the pH value is 9.6, and the preparation method of the coating solution of the 0.85M carbonate buffer solution comprises the following steps: weighing NaHCO32.93g and Na2CO31.59g, adding distilled water to a constant volume of 1L; the PBST solution is prepared by adding 2 per mill of Tween-20 into 1L of PBS solution and mixing uniformly; the confining liquid in the step (2) is 5% skimmed milk powder, and the preparation method of the 5% skimmed milk powder comprises weighing 5g of skimmed milk powder, adding PBS solution and fixing the volume to 100 mL; the positive control in the step (3) is a clostridium perfringens beta 1 toxin monoclonal antibody 1K19, and PBS is used for diluting 28Double (0.25. mu.g/. mu.L); in the step (4), the secondary HRP-goat anti-mouse IgG antibody is diluted 2000 times by the secondary HRP-goat anti-mouse IgG antibody, and the secondary HRP-labeled corresponding antibody is diluted 2000 times by the secondary HRP-labeled goat anti-animal IgG antibody; the substrate color developing solution in the step (5) is a 5% o-phenylenediamine solution, and the preparation method of the 5% o-phenylenediamine solution comprises the steps of weighing 5mg of OPD (o-phenylenediamine), weighing 10mL of substrate buffer solution for dissolving, and adding 3% H2O20.15mL, which is prepared at present, wherein the substrate buffer solution is a citric acid buffer solution, the pH value is 5.0, and the preparation method of the citric acid buffer solution comprises weighing 1.92g of citric acid and Na2HPO4Weighing 2.84g, and adding distilled water to a constant volume of 100 mL; the stop solution in the step (5) is 2mol/L of H2SO4The preparation method of the stop solution comprises the steps of measuring 178.3mL of distilled water and dropwise adding 21.7mL of concentrated sulfuric acid with the concentration of 98%.
3. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection method according to claim 1, characterized in that:
the construction method of the envelope antigen expression strain of the soluble recombinant clostridium perfringens beta 1 toxin protein comprises the following steps: according to the gene sequence of clostridium perfringens beta 1 toxin reported in literature, a primer is designed:
Forward(5`-3`)CGGAATTCCATATGGATATAGGCAAAACTACTACTAT
Reverse(5`-3`)GAATGCGGCCGCAATAGCTGTTACTTTATG
the cpb1 gene was amplified from a C-type standard strain of Clostridium perfringens, constructed into a pTIG-Trx vector, transformed into BL21(DE3) strain, and labeled as BL21(DE3) pTIG-cpb 1.
4. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection method according to claim 1, characterized in that: the clostridium perfringens beta 1 toxin protein indirect ELISA detection method specifically comprises the following steps:
s1: taking BL21(DE3) pTIG-cpb1 glycerol frozen strain, streaking on LB solid plate containing 100 mug/mL ampicillin, picking single colony the next day, inoculating into 10mL LB liquid culture medium containing 100 mug/mL ampicillin, culturing to bacterial liquid OD600Up to 1.0 or more;
s2: then, the cells were inoculated in 200mL of LB liquid medium containing 100. mu.g/mL of ampicillin in an inoculum size of 1% and cultured for 3 hours, and then 0.5mM IPTG was added to induce expression at 20 ℃ for 16 hours, followed by centrifugation to harvest the cells.
5. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection method of claim 4, which is characterized in that: the envelope antigen is recombinant clostridium perfringens beta 1 toxin protein, and the preparation method of the soluble recombinant protein comprises the following steps:
step a: centrifuging to collect 800mL of induced recombinant expression thallus, washing the thallus once by PBS, adding 20mM Tris-HCL (pH8.0) buffer solution into thallus sediment to resuspend 10mL, adding 100 mu g/mL lysozyme, 1 per thousand TritonX-100 and 0.01mM PMSF, and carrying out ultrasonic crushing for 10 min;
step b: centrifuging at 10000rmp for 10min, and washing the precipitate with 10mL of 20mM Tris-HCl (pH8.0) buffer containing 0.2M urea; centrifuging at 10000rmp for 10min, adding 10mL of 20mM Tris-HCL (pH8.0) denaturation buffer containing 2M urea into the centrifugation sediment, and incubating at 37 ℃ for 2h to completely dissolve and denature the sediment;
step c: transferring the protein solution after dissolving and denaturation into a dialysis bag, and sequentially adding 150mL of 20mM Tris-HCL (pH8.0) renaturation buffer solution containing urea with concentration gradients of 1.5M, 1M, 0.5M and 0M to the liquid outside the dialysis bag, wherein each concentration gradient is treated for 6 h.
6. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection method according to claim 1, characterized in that: the positive control is a clostridium perfringens beta 1 toxin monoclonal antibody 1K19, protein sequences are analyzed and subjected to antigen prediction according to a beta 1 toxin amino acid sequence provided by NCBI, sites of escherichia coli, clostridium perfringens beta 2 toxin, alpha toxin and epsilon toxin which are possibly subjected to cross recognition are removed, a peptide section with 14 amino acids and the highest epitope index of a target protein is selected and used as an antigen to be injected into a BalB/C mouse, after three times of immunization, the spleen of the mouse is taken to be fused with myeloma cells, and a positive clone cell strain is obtained after restrictive gradient dilution and is obtained through screening.
7. The indirect ELISA detection method for Clostridium perfringens beta 1 toxin antibody according to claim 6, wherein said detection method comprises the following steps: the positive control is clostridium perfringens beta 1 toxin monoclonal antibody 1K19, and the purification method comprises the following steps: recovering 1K19 hybridoma cells frozen in liquid nitrogen tank, subculturing, and transferring at a ratio of 1:100 to 75cm after cell state is good2In a culture flask of (1), and placing in CO2In the incubator until the cells overgrow to enrich the antibody, after 4-5 days, after the culture solution turns yellow, 1K19 culture supernatant was collected, filter sterilized with a 0.22 μm filter, and purified with Protein G using AKTA purification system to obtain McAb1K 19.
8. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection method of claim 7, wherein: the McAb1K19 purification uses 20mM sodium phosphate, pH 7.0, elution buffer 0.1M glycine-hydrochloric acid buffer, pH 2.7, neutralization buffer 1M Tris-Cl, pH 9.0.
9. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection method according to claim 1, characterized in that: the complete Medium contains 10% by volume of RPMI Medium 1640 of fetal bovine serum, and the complete Medium contains 1% by volume of streptomycin and 1% of glutamine.
10. The clostridium perfringens beta 1 toxin antibody indirect ELISA detection method according to claim 1, characterized in that: the negative control is monoclonal antibody of clostridium perfringens beta 2 toxin.
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