CN117777256A - ELISA kit for clostridium perfringens toxin antibody of cattle A - Google Patents
ELISA kit for clostridium perfringens toxin antibody of cattle A Download PDFInfo
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- 241000193468 Clostridium perfringens Species 0.000 title claims abstract description 65
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an enzyme-linked immunosorbent assay kit for clostridium perfringens toxin antibody of cattle A. The kit comprises an ELISA plate, positive control serum, negative control serum, an ELISA secondary antibody, a sample diluent, 20-time concentrated washing liquid, substrate liquid A, substrate liquid B and stop solution, wherein the ELISA plate is coated with a clostridium perfringens alpha toxin epitope polypeptide of bovine A. The antigen epitope polypeptide composition is one or more than two of polypeptide shown in a sequence 1 in a sequence table, polypeptide shown in a sequence 2 in the sequence table or polypeptide shown in a sequence 3 in the sequence table. The kit uses the chemically synthesized antigen peptide coated ELISA plate, has less antigen consumption and high sensitivity and specificity, and can efficiently detect whether the clostridium perfringens type-A bovine toxin antibody exists. The kit disclosed by the invention has the advantages of high sensitivity, good specificity, convenience in operation and good market prospect.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an enzyme-linked immunosorbent assay kit for clostridium perfringens toxin antibody of cattle A.
Background
Clostridium perfringens (c.perfringens), known as clostridium welchii (c.welchii), is an important pathogen of zoonotic bacterial infectious diseases, which is itself nonpathogenic, and the pathogenic factor is exotoxin produced by thallus, and is classified into A, B, C, D, E five types according to the main exotoxin produced.
The main pathogenic toxin of clostridium perfringens type a is alpha toxin, but more and more studies have found that beta 2 toxin is also potentially pathogenic. Alpha toxin is a multifunctional metalloenzyme dependent on zinc ions, and can destroy phospholipids on the surface of cell membranes of host cells, thereby destroying the functions of the cell membranes. The mycotoxin can cause necrotic enteritis, enterotoxemia and other diseases, the disease presents sporadic or endemic diseases, the disease is urgent, the disease death rate is too high, serious economic loss is brought to the breeding industry, and immunization is an effective method for preventing and controlling the disease.
Clostridium perfringens toxin antibody levels are an index for evaluating the resistance of animals to clostridium perfringens disease, and a mouse toxin neutralization test is a classical method for detecting clostridium perfringens toxin antibodies of type a, which is accurate and reliable, but time-consuming, labor-consuming and complex to operate, requires a large number of experimental animals, and individual differences can affect test results; the lecithin hydrolysis inhibition test is insensitive, has poor repeatability and complex operation, and is difficult to popularize and apply; the indirect hemagglutination experiment needs to prepare hemagglutination antigen, is relatively complex and is difficult to popularize in production practice. The ELISA can better solve the problems, and the ELISA kit for detecting the clostridium perfringens toxin antibody of the type A bovine is urgently needed to be developed for detecting the level of clostridium perfringens toxin antibody of the type A bovine for matched vaccines and also for detecting epidemiology conditions of clostridium perfringens of the type A bovine.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for detecting bovine clostridium perfringens type A toxin, which utilizes an antigen epitope polypeptide composition of bovine clostridium perfringens type A alpha toxin as a coating antigen, establishes an indirect ELISA method with good specificity, sensitivity and repeatability, and is used for detecting whether bovine serum contains bovine clostridium perfringens type A toxin antibodies.
In order to achieve the purpose, the bovine clostridium perfringens type A alpha toxin antigen epitope polypeptide composition with excellent performance is firstly screened and obtained, and the bovine clostridium perfringens type A alpha toxin antigen epitope polypeptide composition provided by the invention consists of a polypeptide shown in a sequence 1 in a sequence table, a polypeptide shown in a sequence 2 in the sequence table or a polypeptide shown in a sequence 3 in the sequence table. In a preferred embodiment of the present invention, the polypeptide shown in sequence 1, the polypeptide shown in sequence 2 and the polypeptide shown in sequence 3 have a mass ratio of any three polypeptides of (0.5 to 1.5): (0.5-1.5): (0.5-1.5); more preferably, they have a mass ratio of 1:1:1, a step of;
the invention relates to a beef clostridium antibody enzyme-linked immunosorbent assay kit, which comprises an enzyme-linked reaction plate, positive control serum, negative control serum, enzyme-labeled secondary antibodies, sample diluent, 20-time concentrated washing liquid, substrate liquid A, substrate liquid B and stop solution, wherein the enzyme-linked reaction plate is coated with a bovine type A clostridium perfringens alpha toxin antigen epitope polypeptide composition.
The ELISA plate is a detachable 96-hole ELISA plate; the alpha toxin epitope polypeptide composition of the clostridium perfringens A is obtained by chemical and artificial synthesis.
The optimal preparation method and the condition of the ELISA plate are that the clostridium perfringens type A alpha toxin epitope polypeptide composition is dissolved in carbonate solution with pH of 9.6, then the solution is added into a 96-hole polystyrene ELISA plate, 200ng polypeptide per hole is placed for 8 to 12 hours at the temperature of 2 to 8 ℃ so that polypeptide antigen and the ELISA plate are fully combined, then PBS buffer solution containing 1 percent (g/ml) Bovine Serum Albumin (BSA) with pH of 7.4 is added according to 300 mu l/hole, sealing treatment is carried out for 2 to 3 hours at the temperature of 37 ℃, and after spin-drying, the ELISA plate is dried and then sealed and stored at the temperature of 4 ℃.
The positive control serum is bovine serum collected after the inactivated clostridium perfringens type a toxin is immunized; the negative control serum is bovine clostridium perfringens type a toxin antibody negative bovine serum.
The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-bovine IgG antibody.
The substrate solution A is a citric acid phosphate buffer solution containing 0.06% (g/ml) of urea hydrogen peroxide, the substrate solution B is a tetramethyl benzidine solution with the concentration of 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when the substrate solution A is used. The stop solution is 2mol/L sulfuric acid solution.
The kit also comprises a sample diluent and a 20-time concentrated washing liquid; the sample dilution was a phosphate buffer with a value of 7.4 of 0.01M, pH containing 0.5% (g/100 ml) casein; the concentrated washing solution is phosphate buffer solution with pH value of 7.4 and 0.01M containing Tween-20 with concentration of 0.8% -1.2% (ml/ml).
The detection program of the kit provided by the invention is as follows:
1. balance: taking the kit out of the refrigeration environment, and standing at room temperature for 30min for standby; the liquid reagent is mixed evenly before use.
2. Preparing liquid: diluting the concentrated washing solution with distilled water or deionized water for 20 times to obtain a washing buffer solution;
3. setting: 2 negative control wells and 2 positive control wells, the remainder being wells for samples to be tested.
4. Pre-diluting a sample to be tested: sample serum to be detected, negative control serum and positive control serum are subjected to a sample dilution according to a ratio of 1: 20.
5. Sample adding: each well was pre-set with 100. Mu.l of diluted test sample. The time span of the sample adding process should be as short as possible.
6. Incubation: shaking and mixing uniformly, and placing in a 37 ℃ incubator for reaction for 30min.
7. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, the mixture was immersed for 15 seconds, the washing solution was discarded, and the plate was washed continuously for 4 times and then dried by pipetting.
8. Adding enzyme: 100 μl horseradish peroxidase was added to each well to label the goat anti-bovine IgG antibody.
9. Incubation: placing in a 37 ℃ incubator for reaction for 30min.
10. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, immersed for 15 seconds, the washing solution was thrown away, and the plate was washed continuously for 4 times and then dried by shaking.
11. Adding 100 μl of substrate working solution (substrate working solution A and substrate working solution B are mixed in equal amounts to obtain substrate working solution, and mixing immediately before use), shaking, mixing, placing in a 37 deg.C incubator, and reacting for 15min in dark place.
12. 50 μl of the color development stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well 450nm Value (the reaction plate with stop solution should read OD within 15 min) 450nm Values).
And (3) judging a detection result:
1. negative control OD 450nm The average value should be less than or equal to 0.15, otherwise, the method is ineffective.
2. The positive control should have a value between 1.0 and 2.5 per test, otherwise it is ineffective.
3. Calculating a critical value: critical value = 0.17 x positive control OD 450nm Average value of values.
Determination of OD by serum to be examined 450nm If the value is more than or equal to the critical value, judging the value as positive; determination of OD by serum to be examined 450nm Value of<The threshold value is judged as negative.
The kit provided by the invention can be used for detecting the clostridium perfringens type A toxin antibody of the cattle so as to judge whether the clostridium perfringens type A toxin antibody of the cattle exists in the detected animal.
The invention has the positive effects that: the invention adopts bioinformatics method to accurately analyze the epitope of the clostridium perfringens type A alpha toxin of the cattle, and screens out peptide segments suitable for ELISA detection from the main epitope of clostridium perfringens type A alpha toxin. The peptide segment integrates antigen epitope, has the advantages of high sensitivity and strong specificity, and can be used for detecting clostridium perfringens type A antibody of cattle.
Meanwhile, an advanced solid-phase peptide synthesis technology is adopted to synthesize the polypeptide antigen for preparing the coated enzyme-labeled reaction plate.
In addition, the coating antigen used in the kit is a chemically synthesized polypeptide, so that the kit does not contain mixed proteins, has high purity, and further improves the sensitivity and detection efficiency of detecting the bovine type-A clostridium perfringens toxin antibody.
In a word, the kit adopts the enzyme-linked reaction plate coated by the chemically synthesized clostridium perfringens type A alpha toxin epitope polypeptide composition, has less antigen consumption, high sensitivity and strong specificity, and can effectively detect clostridium perfringens type A toxin antibodies of cattle. Experimental results show that the kit provided by the invention has the advantages of good repeatability, strong specificity and high sensitivity. Can meet the needs of people of different levels, and has wide market prospect and good economic and social benefits.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of coated antigen of bovine clostridium perfringens toxin A antibody ELISA kit
The experiment adopts a bioinformatics method to accurately analyze main epitope of clostridium perfringens type-a alpha toxin of cattle, combines biological functional verification to screen out proper peptide segments, synthesizes 3 peptides respectively by a full-automatic polypeptide synthesizer, and prepares coating antigen with purity of about 85% as shown in sequence 1, sequence 2 and sequence 3 in a sequence table so as to improve the detection rate of positive antibody. The polypeptide synthesis method may be a conventional method, and the present invention synthesizes 3 polypeptides of the present invention as coating precursors of the kit of the present invention using the following method.
The coating antigen of the invention can be prepared by using a Applied Biosystem full-automatic polypeptide synthesizer. Fmoc (9-fluoroethylene carbonyl, 9-fluorenylmethoxycarbonyl) modified amino acid was used by Merrifield solid phase synthesis, using Rink Amide MBHA resin as solid phase carrier. The production process includes the steps of polypeptide antigen solid phase synthesis, polypeptide cleavage and identification, antigen purification, freeze drying and preservation. The following description will be given respectively:
1. solid phase synthesis of coated antigen
1. Preparation of synthetic reagents
The amino acid sequences of the synthetic coating antigens are shown as sequence 1, sequence 2 and sequence 3.
Sequence 1:
FVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNA
sequence 2:
LNLVIIGPSADIIQFECKSFGHEVLNLKK
sequence 3:
EVYKYIVTNWLAKVNTQIDLIKYINVVVKN
appropriate Fmoc modified amino acids (available from NOVA) were prepared according to the coating antigen sequence and the scale of synthesis and added to the corresponding Carridge. The resin 5g was weighed according to the required synthesis scale, placed in a reaction chamber, and the upper and lower lids were screwed down and labeled, and the name, lot number, TARE of the reaction chamber and the weight of the resin were recorded. The reaction chamber was loaded into the synthesizer. The appropriate amounts of synthetic reagents were formulated and placed into corresponding reagent bottles, including 100% NMP, 3% AIM (caproyl imidazole), 35% PIP (piperidine), 100% MeOH (methanol), etc.
2. Detection of synthesizer state
And (3) checking whether the polypeptide synthesis instrument runs normally, and running a Run Self Test program after starting up, wherein the instrument automatically checks whether various indexes are normal. Additionally, it was checked whether nitrogen was sufficient and the system gauge pressure was normal (normal gauge pressure 10.2 psi). The performance of the instrument should be known prior to synthesis, so the flow rate of each synthetic reagent is measured. The synthesizer comprises: sending Flow Rate1-18 to synthesizer, selecting Main Menu-Module Test-find Module A, moduleD, moduleI, moduleI, module A according to Prer or next) -measure or observe according to Start-according to more, if the Flow is not proper, regulating the lower valve pressure until the requirement is met (specific detection requirement see Table 1 below).
TABLE 1 flow rate detection Standard Meter for polypeptide synthesizer
| Reagent(s) | Bottle number | Module | Standard range |
| 35％Piperidine | 1 | A | 1.0～1.2ml |
| 3％AIM | 4 | D | 1.0～1.2ml |
| 100％MeOH | 9 | I | 3.5～4.0ml |
| DIC | 8 | I | 0.45～0.55g |
| 100％NMP | 10 | A | 2.6～2.8ml |
3. Coated antigen synthesis begins
The amino acid sequence to be synthesized was sent Std Fmoc 1.0sol dic90 to the synthesizer during the synthesizer procedure. File-New-Sequence-edit Sequence of synthetic peptide, save. File-New-Run, check Chemistry for Std Fmoc 1.0Sol DIC 90; whether Sequence is a stored name; setting the Cycles; and (5) preserving. And finally, sending the obtained product to a synthesizer.
Main Menu-Cycle Monitor-begin, start running.
4. Coated antigen synthesis
The Fmoc group is removed, the electron-withdrawing effect of the fluorene ring system of the Fmoc group enables the 9-H to be acidic and easy to be removed by weak base, piperidine (PIP) is used for attacking the 9-H during reaction, beta is eliminated to form dibenzofluorenene, and the dibenzofluorenene is easy to be attacked by secondary cyclic amine to form a stable addition product. After removal of the Fnov group, the "-NH2" group is exposed for the synthesis reaction. The activated next Fmoc group protected amino acid and 1-Hydroxybenzotriazole (HOBT) were then added to the reactor.
The above polypeptide sequence is synthesized by sequentially repeating the synthesis steps from the C terminal to the N terminal according to a specific sequence (the synthesizer automatically completes the synthesis according to the program, and the specific circulation steps are shown in the following table 2). And (5) observing and recording the dosage and the running condition of the reagent during the process.
TABLE 2 coating antigen Synthesis cycle procedure
5. End of coating antigen synthesis
The synthesizer will automatically stop after the coated antigen synthesis is completed and the peptide resin (peptide now also attached to the resin) is essentially washed clean. Then the reactor is taken down from the polypeptide synthesizer, the peptide resin is washed 3 times by using 100 percent methanol, and then the peptide resin is dried in a fume hood, and then the peptide resin is completely transferred into a brown polyethylene bottle and is put into a refrigerator with the temperature of minus 20 ℃ and sealed by a sealing film for standby.
2. Cleavage and identification of coating antigen
1. Cleavage of polypeptide antigen
The polypeptide obtained by the above reaction is bound to the solid phase carrier by chemical bond, and the polypeptide must be separated from the solid phase carrier by acidolysis with a specific organic strong acid. The acid hydrolysis also removes the protecting groups on the functional groups of each amino acid. The method comprises the following steps:
the synthesized polypeptide resin (referring to the peptide also attached to the resin) was removed from the freezer and placed in a 2L round bottom flask, 90ml trifluoroacetic acid (Trifluoroacetic acid, TFA), 10ml Tripropylsilane (TIS) and magnetic stirrer were added to the flask in a fume hood, and the flask was then stably placed on a magnetic stirrer and stirred at room temperature for 1h until the reaction was complete. After the reaction is finished, the TFA in the crude product is removed by continuous evaporation for 30-120 min by using a rotary evaporator with a cold trap. And then, repeatedly cleaning the crude product of the polypeptide antigen by using Dimethylformamide (DMF), and finally, filtering the mixed resin by using a sand core funnel to obtain the coating antigen.
2. Identification of coating antigen
After the synthesis of the polypeptide antigen, qualitative and quantitative analysis is carried out by using matrix-assisted laser desorption/flight time mass spectrometry (MODAL-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC), and the synthesized peptide is identified by using common amino acid analysis.
3. Coated antigen purification
The cyclized polypeptide antigen is ultrafiltered by using a circulating tangential filter membrane bag, the polypeptide antigen can not pass through a filter membrane with a certain aperture as a macromolecule, and small molecular impurities formed or introduced by reaction in the early synthesis process can pass through the filter membrane. Then sterilizing by a filter with the aperture of 0.2 mu m, subpackaging the obtained solution into sterile plastic bottles, and labeling. The label is marked with the name, number, production lot number, concentration, production date, shelf life and preservation condition of the polypeptide, and the polypeptide is packaged and stored at-20 ℃ or-40 ℃ for standby.
4. Freeze drying of coated antigen
In order to facilitate long-term storage and transport, the coated antigen needs to be freeze-dried to obtain the polypeptide in a solid state. And (3) placing the pre-frozen coated antigen on a freeze dryer for drying to obtain the coated antigen in a solid state. And (5) labeling after packaging. The label is marked with the name, number, production lot number, concentration, date of production, shelf life and storage conditions of the polypeptide.
Example 2 preparation of a bovine clostridium perfringens toxin antibody enzyme-linked immunosorbent assay kit
The clostridium perfringens toxin antibody enzyme-linked immunosorbent assay kit for cattle comprises:
(1) 96-hole removable polystyrene enzyme-linked reaction plate coated with clostridium perfringens toxin antigen of cattle A; 2X 96 wells.
(2) Positive control serum: bovine serum collected after immunization with inactivated clostridium perfringens type a toxin was used as positive control serum for the kit (1 tube, 1.5 ml/tube).
(3) Negative control serum: bovine clostridium perfringens type a toxin antibody negative bovine serum was used as a negative control serum for the kit (1 tube, 1.5 ml/tube).
(4) Enzyme-labeled secondary antibody: is prepared by taking horseradish peroxidase labeled goat anti-bovine IgG (purchased from Earth Ox company) as stock solution and diluting the stock solution by 1:10000, and 2 bottles (12 ml/bottle).
(5) Sample dilution: 1 bottle (24 ml/bottle) of phosphate buffer with a value of 7.4 of 0.01M, pH containing 0.5% (g/100 ml) casein.
(6) Substrate solution A: phosphate buffer (1 bottle, 12 ml/bottle) of citric acid containing 0.6mg/ml urea hydrogen peroxide
(7) Substrate solution B: a solution of 0.2mg/ml Tetramethylbenzidine (TMB) (1 bottle, 12 ml/bottle).
(8) Stop solution: 2mol/L sulfuric acid solution (1 bottle, 12 ml/bottle).
(9) 20-fold concentrated washing solution: phosphate buffer (50 ml/bottle, 2 bottles) containing Tween-20 at a concentration of 0.8% -1.2% (ml/ml) and having a pH of 7.4.
Serum dilution plates (2, 96 wells/block) can also be included in the kit as needed for dilution of serum samples.
The preparation method of the 96-hole removable polystyrene enzyme-linked reaction plate coated with the clostridium perfringens toxin antigen of cattle A comprises the following steps: 1. the polypeptide antigen prepared in example 1 was dissolved in a carbonate solution at pH9.6, then added to a 96-well polystyrene enzyme-linked reaction plate, 200ng of the polypeptide per well (7 sets of treatment were set, wherein XJ001 per well of the polypeptide shown in sequence 1, XJ002 per well of the polypeptide shown in sequence 2, XJ003 per well of the polypeptide shown in sequence 3, XJ004 per well of the polypeptide shown in sequence 1 and 100ng of the polypeptide shown in sequence 2, XJ005 per well of the polypeptide shown in sequence 1 and 100ng of the polypeptide shown in sequence 3, XJ006 per well of the polypeptide shown in sequence 2 and 100ng of the polypeptide shown in sequence 3, XJ007 per well of the polypeptide shown in sequence 1, 66.7ng of the polypeptide shown in sequence 2 and 66.7ng of the polypeptide shown in sequence 3) were left at 2 to 8℃for sufficient binding with the enzyme-linked reaction plate, then buffer containing 1% (g/ml) Bovine Serum Albumin (BSA) at pH7.4℃was added per well, PBS was blocked at 37℃for 4℃for 4 hours, and after sealing, the enzyme-linked reaction plate was dried, and dried.
Example 3 sensitivity test of bovine clostridium perfringens A toxin antibody ELISA kit
1. Application method of clostridium perfringens toxin antibody enzyme-linked immunosorbent assay kit for cattle A
1. Balance: taking the kit out of the refrigeration environment, and standing at room temperature for 30min for standby; the liquid reagent is mixed evenly before use.
2. Preparing liquid: diluting the concentrated washing solution with distilled water or deionized water for 20 times to obtain a washing buffer solution;
3. setting: 2 negative control wells and 2 positive control wells, the remainder being wells for samples to be tested.
4. Pre-diluting a sample to be tested: sample serum to be detected, negative control serum and positive control serum are subjected to a sample dilution according to a ratio of 1: 20.
5. Sample adding: each well was pre-set with 100. Mu.l of diluted test sample. The time span of the sample adding process should be as short as possible.
6. Incubation: shaking and mixing uniformly, and placing in a 37 ℃ incubator for reaction for 30min.
7. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, the mixture was immersed for 15 seconds, the washing solution was discarded, and the plate was washed continuously for 4 times and then dried by pipetting.
8. Adding enzyme: 100 μl horseradish peroxidase was added to each well to label the goat anti-bovine IgG antibody.
9. Incubation: placing in a 37 ℃ incubator for reaction for 30min.
10. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, immersed for 15 seconds, the washing solution was thrown away, and the plate was washed continuously for 4 times and then dried by shaking.
11. Adding 100 μl of substrate working solution (substrate working solution A and substrate working solution B are mixed in equal amounts to obtain substrate working solution, and mixing immediately before use), shaking, mixing, placing in a 37 deg.C incubator, and reacting for 15min in dark place.
12. 50 μl of the color development stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well 450nm Value (the reaction plate with stop solution should read OD within 15 min) 450nm Values).
And (3) judging a detection result:
1. negative control OD 450nm The average value should be less than or equal to 0.15, otherwise, the method is ineffective.
2. The positive control should have a value between 1.0 and 2.5 per test, otherwise it is ineffective.
3. Calculating a critical value: critical value = 0.17 x positive control OD 450nm Average value of values.
Determination of OD by serum to be examined 450nm If the value is more than or equal to the critical value, judging the value as positive; determination of OD by serum to be examined 450nm Value of<The threshold value is judged as negative.
2. Sensitivity test to known positive serum
The sensitivity test was conducted on 230 parts of bovine clostridium perfringens toxin-inactivated vaccine against bovine type a clostridium perfringens toxin antibody enzyme-linked immunosorbent assay kit prepared according to the method of example 2 (XJ 001, XJ002, XJ003, XJ004, XJ005, XJ006, XJ 007) according to the method of using the above-mentioned clostridium perfringens toxin-producing enzyme-linked immunosorbent assay kit, respectively, with a sensitivity of 50% for the kit of XJ001 to 230 parts of known positive serum, a sensitivity of 57% for the kit of XJ002 to 230 parts of known positive serum, a sensitivity of 43% for the kit of XJ003 to 230 parts of known positive serum, a sensitivity of 70% for the kit of XJ004 to 230 parts of known positive serum, a sensitivity of 67% for the kit of XJ005 to 230 parts of known positive serum, and a sensitivity of 67% for the kit of XJ006 to 230 parts of known positive serum, and a sensitivity of 93% for the kit of known positive serum to 93% were carried out.
TABLE 3 sensitivity detection results of clostridium perfringens toxin A antibody ELISA kit for cattle
| Kit lot number | Detection rate of | Sensitivity to |
| XJ001 (sequence 1) | 115/230 | 50％ |
| XJ002 (sequence 2) | 131/230 | 57％ |
| XJ003 (sequence 3) | 99/230 | 43％ |
| XJ004 (sequencer 1+ sequencer 2) | 161/230 | 70％ |
| XJ005 (SEQ ID NO: 1+SEQ ID NO: 3) | 154/230 | 67％ |
| XJ006 (SEQ ID NO: 2+ 3) | 154/230 | 67％ |
| XJ007 (SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3) | 214/230 | 93％ |
3. Minimum detection limit test
3 parts of bovine serum positive for clostridium perfringens type a antibodies are selected and diluted 1:20-1:320 times, 3 batches of clostridium perfringens type a antibodies enzyme-linked immunosorbent assay (batches XJ007-1, XJ007-2 and XJ 007-3) are prepared by using the example 2, and the diluted bovine serum is detected by the method for using the clostridium perfringens type a antibodies enzyme-linked immunosorbent assay according to the method, wherein the result shows that the kit can detect positive serum diluted 1:160 times, and the table 4 shows the experimental result of one batch. In Table 4, the positive control was bovine serum collected after immunization with clostridium perfringens type A toxin-inactivating antigen as a positive control serum for the kit (1 tube, 1.5 ml/tube). Negative control: bovine serum negative for clostridium perfringens type a toxin antibodies served as negative control serum for the kit (1 tube, 1.5 ml/tube). Critical value (Cut-off value) =0.17×positive control OD 450nm Average value of values.
TABLE 4 detection results of minimum detection limit test of clostridium perfringens toxin A antibody ELISA kit
Example 4 specificity test of bovine clostridium perfringens A toxin antibody ELISA kit
140 healthy bovine serum (supplied by the middle-animal industry, inc. Lanzhou biological pharmaceutical factory), 2B clostridium perfringens toxin antibody positive serum (supplied by the middle-animal industry, inc. Lanzhou biological pharmaceutical factory), 2C clostridium perfringens toxin antibody positive serum (supplied by the middle-animal industry, inc. Lanzhou biological pharmaceutical factory) and 2C bovine clostridium perfringens toxin antibody positive serum (supplied by the middle-animal industry, inc. Lanzhou biological pharmaceutical factory) were each tested according to the method of using the clostridium perfringens toxin antibody ELISA kit of type A of bovine type described in example 3.
The specific detection results of the kit are shown in the following table (table 5), and the detection results of 140 healthy bovine serum show that the specificity of the XJ001 kit is 100.0%, and the kit is negative; the specificity of the XJ002 kit is 100.0%, and both are negative; the specificity of the XJ003 kit is 100.0%, and the XJ003 kit is negative; the specificity of the XJ004 kit is 100.0%, and the kit is negative; the specificity of the XJ005 kit is 100.0%, and the XJ005 kit is negative; the specificity of the XJ006 kit is 100.0%, and the specificity is negative; the specificity of the XJ007 kit was 100.0% and was negative. The detection results of 2 parts of clostridium perfringens toxin antibody positive serum, 2 parts of clostridium perfringens toxin antibody positive serum and 2 parts of clostridium perfringens toxin antibody positive serum are all negative. Therefore, the specificity of the 7 batches of kit for the positive serum detection of the related pathogenic antibodies is 100%.
TABLE 5 specific detection results of clostridium perfringens toxin antibody ELISA kit for bovine A-type
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Claims (9)
1. The clostridium perfringens type a alpha toxin antigen epitope polypeptide composition of cattle consists of polypeptide shown in a sequence 1 in a sequence table, polypeptide shown in a sequence 2 in the sequence table and polypeptide shown in a sequence 3 in the sequence table.
2. The clostridium perfringens a toxin alpha epitope polypeptide composition of claim 1 wherein: the mass ratio of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 and the polypeptide shown in the sequence 3 is (0.5-1.5): (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1:1.
3. an enzyme-linked immunosorbent assay kit for clostridium perfringens type a toxin antibodies of cattle, comprising an enzyme-linked reaction plate and an enzyme-labeled secondary antibody, wherein the enzyme-linked reaction plate is coated with the clostridium perfringens type a toxin antigen epitope polypeptide composition of cattle of claim 1 or 2.
4. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the ELISA plate is a detachable 96-hole ELISA plate; the clostridium perfringens alpha toxin polypeptide composition of the cattle A type is obtained by chemical artificial synthesis.
5. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the method for obtaining the ELISA plate comprises the steps of dissolving the clostridium perfringens type A alpha toxin epitope polypeptide composition in the method in claim 1 or 2 in 100 mu l of carbonate solution with the pH of 9.6, adding the solution into a 96-hole polystyrene ELISA plate, placing 200ng of the clostridium perfringens type A alpha toxin epitope polypeptide composition in each hole at the temperature of 2-8 ℃ for 8-12 hours, fully combining the clostridium perfringens type A alpha toxin epitope polypeptide composition with the ELISA plate, adding PBS buffer solution with the bovine serum albumin of 0.01g/ml with the pH of 7.4 according to 300 mu l/hole, sealing at the temperature of 37 ℃ for 2-3 hours, drying the ELISA plate, and sealing at the temperature of 4 ℃ for storage.
6. The enzyme-linked immunosorbent assay kit according to claim 3, wherein:
the kit also comprises positive control serum and negative control serum; the positive control serum is serum obtained by immunizing cattle with clostridium perfringens toxin A (inactivated) serving as an immunogen; the negative control serum is bovine clostridium perfringens type a toxin antibody negative bovine serum.
7. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-bovine IgG antibody.
8. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the kit also comprises a substrate solution A, a substrate solution B and a stop solution; the substrate solution A is a citric acid phosphate buffer solution containing 0.0006g/ml of urea hydrogen peroxide, the substrate solution B is a tetramethyl benzidine solution of 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when in use; the stop solution is 2mol/L sulfuric acid solution.
9. A kit according to claim 3, wherein: the kit also comprises a sample diluent and a 20-time concentrated washing liquid; the sample diluent is phosphate buffer solution with 0.01mol/L, pH value of 7.4 and containing 0.00005g/ml casein; the concentrated washing liquid is phosphate buffer solution with the pH value of 7.4 and 0.01mol/L of Tween-20 with the volume percentage concentration of 0.8-1.2%.
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