CN115469105A - One-step method rapid detection kit for African swine fever ELISA antibody - Google Patents
One-step method rapid detection kit for African swine fever ELISA antibody Download PDFInfo
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Abstract
The invention discloses a one-step method African swine fever ELISA antibody rapid detection kit, the kit adds a detection sample and enzyme-labeled antigen-HRP in a pre-coated micropore, captures antigen through a sample antibody, forms an antigen-antibody-enzyme-labeled antigen compound with the enzyme-labeled antigen, the substrate reacts with the compound to amplify the catalytic action of enzyme for color development, and reads a test value through an enzyme-labeling instrument; meanwhile, PEG6000 is added into the sample diluent, so that the combination chance of the solid-phase antigen, the antibody and the enzyme-labeled antigen compound is increased, the immune compound formation is accelerated, the critical concentration of the liquid-phase and solid-phase reaction is increased, and the hook effect is reduced; compared with the traditional ELISA, the kit combines the primary antibody incubation and the enzyme labeling into one step, reduces one-step washing and one-step incubation, and shortens the detection time by nearly 50 minutes; the kit has good sensitivity, specificity and stability, and is superior to a commercial kit.
Description
Technical Field
The invention relates to a one-step enzyme-linked immunosorbent assay detection kit, in particular to African swine fever P30 protein antibody detection, and relates to an immunoassay method for detecting antibodies in a sample based on antigen-antibody reaction and amplification of enzyme on a substrate.
Background
Enzyme linked immunosorbent assay (ELISA), which is a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid phase carrier such as polystyrene and immunoreaction is carried out by utilizing specific binding of antigen and antibody. It is an immunoenzyme technology developed following immunofluorescence and radioimmunoassay technologies. Since the early 70 s, this technology has developed very rapidly and is now widely used in many areas of biology and medical science. In practice, the specific method steps may vary depending on the design. Namely: indirect methods for detecting antibodies, double antibody sandwich methods for detecting antigens, and antigen competition methods for detecting small molecule antigens or haptens, etc. ELISA double antibody sandwich method and ELISA indirect method are commonly used.
African Swine Fever Virus (ASFV) can cause hyperpyrexia, generalized bleeding of organs of the whole body and acute enlargement of spleen of pigs of different ages, and the death rate of the disease reaches 100%. Until now, no effective vaccine is available for the disease, and the disease is still prevented and controlled by biological safety prevention and control and clinical detection and accurate elimination. At present, the infected pigs are screened by parallel detection of antigen and antibody by adopting a fluorescent quantitative PCR method and an enzyme-linked immunosorbent assay method respectively, and the sensitivity and convenience of an antibody detection kit become important application indexes.
The detection of ASFV antibody by African swine fever antigen proteins P72, P30, P62 and P54 is proved to be effective, but the existing ELISA method has long detection time, complex operation and higher requirements for personnel, and is not beneficial to the prevention and control of African swine fever. Therefore, the existing diagnostic technology is improved, is more efficient and convenient, is necessary for serological detection of African swine fever, and is an urgent need for relieving the control pressure of ASFV.
Disclosure of Invention
Aiming at the defects in the prior art, the embodiment of the invention aims to provide a one-step method rapid detection kit for African swine fever ELISA antibody, and the detection is carried out by the principle of a double-antigen sandwich method; the recombinant protein P30 of the African swine fever is combined on a polystyrene solid phase carrier, and simultaneously a serum sample and an enzyme-labeled antigen are added, wherein both the recombinant protein P30 and the enzyme-labeled antigen can be combined with a sample antibody and are provided with a plurality of combinable sites; meanwhile, a reaction system is optimized, PEG6000 is added into a sample diluent, the combination chance of the solid-phase antigen, the antibody and the enzyme-labeled antigen compound is increased, the immune compound formation can be accelerated, the critical concentration of the liquid-phase and solid-phase reaction is increased, the hook effect is reduced, and the sensitivity and the specificity of the method are ensured.
In order to achieve the purpose, the invention provides the following technical scheme:
a one-step method African swine fever ELISA antibody rapid detection kit comprises a reaction plate, wherein the reaction plate is a coating plate pre-coated with an African swine fever recombinant antigen; and sample diluent, enzyme-labeled antigen, substrate solution, stop solution, washing solution, negative control and positive control.
As a further scheme of the invention, a sample and an enzyme-labeled antigen are added into the reaction plate, an antibody in the sample is combined with a detection antigen coupled with HRP, and an antigen-antibody-enzyme-labeled antigen complex is formed after incubation.
As a further scheme of the invention, the enzyme-labeled antigen is a P30 recombinant protein marked by HRP and can be specifically combined with an African swine fever antibody, the coating concentration of the P30 recombinant protein is 50-120 ng/hole, the concentration of the enzyme-labeled antigen is 50-120 ng/hole, and the dilution of a serum sample is 1.
As a further aspect of the present invention, the sample diluent comprises: BSA, 1.5% Tween 20 and 3% PEG6000 were added to the PBS buffer at a final concentration of 1%, and the mixture was sufficiently dissolved and mixed. The method comprises the steps of coating a P30 recombinant protein on a coating plate in advance to prepare a stationary phase, adding a diluted sample into micropores coated with an antigen, adding an HRP-labeled ASFV antigen, combining an enzyme-labeled antigen with other sites of an antibody to form an antigen-antibody-enzyme-labeled antigen complex, washing, adding a substrate TMB for color development, adding serum into the precoated micropores simultaneously to combine the sample and the HRP-labeled ASFV antigen, and combining primary antibody incubation and enzyme-labeled binding into one step.
As a further embodiment of the present invention, the optimal coating conditions for the P30 recombinant protein are 4 ℃ overnight and the optimal blocking conditions are 1% casein at 37 ℃ for 2 hours.
As a further aspect of the present invention, the washing solution is a PBS buffer solution containing 0.5% Tween-20, the substrate solution is a TMB color developing solution, and the stop solution is 2mol/L sulfuric acid.
As a further scheme of the invention, the negative control is serum without a target antibody, the positive control is serum containing the target antibody, the ASFV P30 recombinant protein is coated in advance to prepare a stationary phase, a sample is added into micropores coated with an antigen, an ASFV antigen marked by HRP is added to form an antigen-antibody-enzyme-labeled antigen complex, and the antigen-antibody-enzyme-labeled antigen complex is washed and then added with a substrate TMB for color development.
The invention has the following beneficial effects:
1. the invention uses a double-antigen sandwich method to determine whether a sample contains ASFV antibody. The coating plate is coated with recombinant ASFV antigen in advance to prepare a stationary phase, a sample and the ASFV antigen marked by HRP are added into micropores of the coating antigen to form an antigen-antibody-enzyme-labeled antigen compound, and the antigen-antibody-enzyme-labeled antigen compound is washed and then added with substrate TMB for color development. TMB is converted to blue by the catalysis of HRP enzyme and yellow by acid termination. The darker the color, the more ASFV antibody content in the sample, and the lighter the color is positively correlated with the ASFV antibody content in the sample. And (4) measuring the absorbance (OD value) at the wavelength of 450nm by using a microplate reader, and calculating an S/P value through the OD value to judge whether the sample contains the ASFV antibody.
2. According to the detection kit, the detection sample and the enzyme-labeled antigen-HRP are added simultaneously, so that the operation steps are reduced, and primary antibody incubation and enzyme-labeled binding are combined into one step; the operation time is shortened, one-step washing and one-step incubation are reduced, and at least 50 minutes are shortened; compared with the traditional ELISA, the one-step method African swine fever antibody ELISA rapid detection kit envelope plate comprises capture antigen, a sample and enzyme-labeled antigen can be added simultaneously during detection, the enzyme-labeled antigen is specific, only a target antibody in the sample is identified, an antigen-antibody-enzyme-labeled antigen compound is formed by capturing the antigen, a substrate reacts with the antigen-antibody-enzyme-labeled antigen compound to amplify the catalytic action of enzyme for color development, and a test value is read by an enzyme-labeling instrument. The whole test time only needs 1 hour, the manual operation time is within 20 minutes, and meanwhile, the artificial dilution operation error can be avoided, so that the kit is an ideal choice for the African swine fever ELISA antibody detection kit which is high in accuracy, high in repeatability, time-saving and efficient.
3. The detection kit of the invention reduces the operation time: primary antibody incubation time; diluting the standard substance for a certain time; preparing time of secondary antibody; repeated washing times, etc. The probability of human misoperation is reduced. The difference between batches is reduced, and the accuracy and the repeatability are improved.
To more clearly illustrate the structural features and effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 is a flow chart of the operation of a conventional ELISA test kit.
FIG. 2 is a flow chart of the ELISA detection kit according to the present invention.
FIG. 3 is a schematic SDS-PAGE diagram according to the present invention.
Detailed Description
The invention will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.
Referring to fig. 1-3, the kit for rapidly detecting the African swine fever ELISA antibody by one-step method provided by the invention comprises a reaction plate, a sample diluent, an enzyme-labeled antigen, a substrate solution, a stop solution, a washing solution, a negative control and a positive control; the invention is based on a double-antigen sandwich method, and the reaction plate is a coating plate which pre-coats the African swine fever recombinant antigen. Preferably, the sample and the enzyme-labeled antigen are added into the pre-coated reaction plate, so that the antibody in the sample can be combined with the detection antigen coupled with the HRP, and an antigen-antibody-enzyme-labeled antigen complex can be formed after incubation.
The enzyme-labeled antigen is an HRP-labeled African swine fever antigen which is specifically combined with an African swine fever antibody and can be specifically combined with a target antibody in a sample.
In the present invention, the sample diluent is: phosphate buffer containing BSA, tween 20 and PEG 6000; the washing liquid is: PBS buffer containing Tween-20; the substrate solution is: TMB color development liquid; the stop solution is: 2mol/L sulfuric acid; the negative control is serum without the antibody of interest; the positive control was serum containing the antibody of interest.
The invention adopts a double-antigen sandwich method to detect the African swine fever antibody; the enzyme-labeled antigen and the fixed antigen are the same antigen and can be combined with a sample antibody simultaneously.
The following provides specific embodiments of the invention
Example 1
The ELISA detection kit of the invention comprises the components shown in Table 1
TABLE 1
The specific process comprises the following steps:
1. preparation and purification of African swine fever virus P30 recombinant protein
1.1. Construction and identification of recombinant expression vectors
At the same time useXhoI andBam Hi, carrying out double enzyme digestion on the recovered P30 target gene segment and the P ET-57 carrier, recovering the corresponding segment, and connecting, wherein the connecting reaction system is as follows: 1 mul of T4 DNA Ligase Buffer, 0.5 mul of T4 DNA Ligase, 6.5 mul of target DNA fragment and 2 mul of vector fragment; after ligation at 16 ℃ for 2h, transformation intoE.coating the competent cells of coli BL21 (DE 3) on a kanamycin-containing plate, culturing at 37 ℃ for 12-16 h, selecting a single white colony, inoculating the single white colony in a liquid culture medium containing kanamycin, performing shake culture at 37 ℃ and 220 r/min, extracting plasmid DNA, performing PCR (polymerase chain reaction) identification on bacterial liquid, and extracting recombinant plasmid at 16 DEG CXho I / Bam HI, double enzyme digestion is carried out overnight, and the enzyme digestion product is subjected to nucleic acid gel electrophoresis detection. The recombinant plasmid identified as positive was designated pET-57-P30 and sequenced.
1.2 Expression and purification of P30 recombinant protein
Transforming the plasmid P ET-57-P30 with correct sequencing into escherichia coli BL21 (DE 3) competent cells, plating for culture, selecting a single colony, inoculating into a kanamycin-resistant LB culture medium, performing shake culture at 37 ℃ and 220 r/min, and culturing to OD 600 When the concentration reached 0.6, IPTG was added to a final concentration of 1 mmol/L and induced at 37 ℃ for 6 hours at 220 rpm. 2000 Centrifuging at rpm/min for 10 min, collecting thallus, ultrasonicating, centrifuging at 10000 r/min for 10 min, collecting supernatant and precipitate, and performing SDS-PAGE analysis. Washing the precipitate obtained by ultrasonic centrifugation with washing solution I (2 mol/L urea, 0.1% Triton 100, 50 mmol/L NaCl, 0.2 mmol/L EDTA) and washing solution II (2 mol/L urea, 0.1% Triton 100), centrifuging, and finally using 8 mol/L urea (8 mol/L urea, 0.5 mol/L NaCl, 20 mmol/LNa) 2 HPO 4 、20 mmol/L Na H 2 PO 4 ) Dissolving the purified P30 protein, determining the concentration of the purified protein by using a BCA kit, analyzing and identifying by SDS-PAGE and Western Blot, and subpackaging and storing for later use.
2. Preparation of enzyme-labeled antigen:
preparing an enzyme-labeled antigen by using a carbodiimide (EDC) method, comprising the following steps of (1) diazotizing African swine fever: weighing 25 mg of African swine fever, and dissolving in 4 mL of precooled 1mol/L hydrochloric acid; adding 0.3 mol/L NaNO dropwise 2 Detecting the solution by using a starch potassium iodide test paper, and stopping dripping NaNO when the solution is dark purple 2 A solution; the reaction was carried out at 4 ℃ in ice bath and in dark for 45 min. (2) Weighing 15 mg of tyrosine, dissolving in 0.5 ml of 1mol/L sodium hydroxide solution, and diluting to 2ml by using a carbonate buffer solution with the pH value of 9.6; slowly dropwise adding the diazotized African swine fever recombinant protein in the step (1) into a tyrosine solution, continuously stirring, and simultaneously adjusting the pH value with 1mol/L NaOH solution to keep the pH value between 9.0 and 9.6; the reaction solution is placed in a refrigerator at 4 ℃ and slowly stirred for 12 h, and then is filtered by a 0.22 mu m microporous filter membrane to obtain the diazotized derivative of the African swine fever recombinant protein. (3) Coupling enzyme-labeled antigen: taking 2mL African swine fever diazotization derivative solution, sequentially adding 14.3 mg carbodiimide and 2.4 mg succinimide, and stirring at room temperature for reaction for 2h; then 10mg of horse radish peroxidase is added, and the mixed reaction solution is put into a refrigerator at 4 ℃ to be slowly stirred for 12 hours; 4. dialyzing at the temperature of DEG C to remove free micromolecules such as carbodiimide, succinimide, african swine fever, diazotized derivatives of African swine fever and the like; after the free active groups are sealed, the coupling solution is used after being qualified by adopting a direct ELISA method.
3. The preparation method of each solution related by the invention comprises the following steps:
coating buffer solution: carbonate buffer solution (0.05M, pH = 9.6), and 1.59g of Na was weighed 2 CO 3 ，2.94gNaHCO 3 And dissolving the antigen in distilled water, and diluting to 1000mL, wherein the solution is mainly used for diluting the coating antigen.
Sealing liquid: 1wt% casein solution: 100mg of casein was weighed and dissolved in 10mL of 0.01M PBS with pH = 7.4.
PBS solution (0.01m, ph = 7.4): 8.0g of NaCl,0.1g of KCl,0.29g of NaCl were weighed out NaH 2 PO 4 ·2H 2 O，2.96g Na 2 HPO 4 ·12H 2 The O is dissolved in distilled water and the volume is up to 1000mL.
20 × concentrated washing solution: PBST solution (0.01m, ph = 7.4) was added with 500 μ L tween-20 to 1000mL of PBS buffer.
Substrate color development solution: commercial TMB solutions.
Stopping liquid: 2M sulfuric acid solution, 21.7mL of concentrated sulfuric acid was weighed and added to 200mL of purified water, and the mixture was stirred while adding the solution to mix well.
Example 1
A one-step method African swine fever ELISA antibody rapid detection kit, the detection process comprises the following steps:
preparing a coating plate, namely pre-coating the African swine fever P30 antigen, and specifically comprising the following steps of: coating: diluting the African swine fever P30 coating antigen into a 96-well enzyme label plate coated with 100 ng/well by using a carbonate buffer solution, and standing overnight in a refrigerator at 4 ℃; then washing the enzyme label plate 3 times by using PBST washing liquid, and finally drying by beating for the last time; and (3) sealing: adding casein blocking solution with the mass fraction of 1%, performing incubation at 37 ℃ for 2h at 200 mu L/hole; then washing the enzyme label plate 3 times by using PBST washing liquid, and finally drying by beating for the last time;
adding a sample and enzyme labeling: 50 μ L of 1;
washing the plate: preparing 1 Xwashing liquid to wash the ELISA plate, wherein each hole is 300 mu L for 4 times, and the last time is carried out for drying;
color development: adding 100 μ L substrate TMB into each well, and developing at room temperature for 15min;
and (4) terminating: add 50. Mu.L of H per well 2 SO 4 Terminating the reaction;
reading: the absorbance (OD) at 450nm of each well was measured on a microplate reader.
Example 2
A one-step method African swine fever ELISA antibody rapid detection kit, the detection process comprises the following steps:
preparing a coating plate for pre-coating the African swine fever P30 antigen, and specifically comprising the following steps of: coating: diluting the African swine fever P30 coating antigen into a 1 mu g/mL coating 96-hole enzyme label plate by using a carbonate buffer solution, and standing overnight in a refrigerator at 4 ℃; then washing the enzyme label plate 3 times by using PBST washing liquid, and finally drying by beating for the last time; and (3) sealing: adding casein blocking solution with the mass fraction of 1%, performing incubation at 37 ℃ for 2h at 200 mu L/hole; then washing the enzyme label plate 3 times by using PBST washing liquid, and finally drying by beating for the last time;
adding a sample and enzyme labeling: 50 mu L of 1:40 diluted serum sample and 50 mu L of enzyme-labeled antigen are added into each sample hole; adding 50 mu L of sample diluent and 50 mu L of enzyme-labeled antigen into each hole of the standard hole site, and incubating for 30min at 37 ℃;
washing the plate: preparing 1 Xwashing liquid to wash the ELISA plate, wherein each hole is 300 mu L for 4 times, and the last time is carried out for drying;
color development: adding 100 μ L substrate TMB into each well, and developing at room temperature for 15min;
and (4) terminating: add 50. Mu.L of H per well 2 SO 4 Terminating the reaction;
reading: the absorbance (OD) at 450nm of each well was measured on a microplate reader.
And establishing a standard curve by taking the logarithm of the concentration of the ASFV standard solution as an abscissa and the light absorption value as an ordinate to obtain a linear equation of the standard curve. Substituting the linear relation into the light absorption value to calculate the concentration of the ASFV antibody to be detected.
The detection kit of the invention reduces the operation time: diluting the standard substance for a certain time; primary antibody incubation time; the preparation time of the secondary antibody is complicated; repeated washing times, etc. The probability of human misoperation is reduced. The difference between batches is reduced, and the accuracy and the repeatability are improved. The performance indexes of the evaluation kit mainly comprise sensitivity, specificity and repeatability, the kit and the commercialized kit are evaluated and compared according to the three indexes, and the judgment basis and the comparison result are as follows.
1. Basis of judgment
1) And judging results of the commercialized kit: positive control OD 450nm Average value is more than 0.85; negative control OD 450nm The average value is less than 0.15; the test result is judged to be valid.
S/P = (sample OD) 450nm Value-negative control OD 450nm Mean)/(positive control OD 450nm Mean-negative control OD 450nm Mean value)
And (4) judging a result: S/P is more than or equal to 0.4, and is positive; S/P is more than 0.3 and less than 0.4, and suspicious; S/P is less than or equal to 0.3 negative.
2) The kit has the following judgment results: positive control OD 450nm Average value is more than 1.0; negative control OD 450nm Average value is less than 0.25; the test result is judged to be valid.
S/P = (sample OD) 450nm Value-negative control OD 450nm Mean value)/(positive control OD 450nm Mean-negative control OD 450nm Mean value)
And (5) judging a result: S/P is more than or equal to 0.3, and is positive; S/P is less than 0.3, and the result is negative.
2. Comparison results
1) Sensitivity test
The diagnostic sensitivity studies (tables 2-4) were performed on 10 positive sera (positive sample plate) with known background and 20 weak positive sera (clinical immune sera) with the kit of the invention and the commercial kit.
TABLE 2 different dilution times of the test kit of the present invention on known positive serum
Table 3 detection results of different dilution times of known positive serum by commercial kit
TABLE 4 detection results of clinical samples by the kit of the present invention and the commercial kit
After 10 parts of positive serum with known background are respectively diluted by 100 times, 200 times and 500 times, the 10 parts of serum diluted by 500 times of the kit are all positive, the 10 parts of serum diluted by 200 times of the commercialized kit are all positive, 3 parts of serum are suspicious and 1 part of serum is negative; as for the detection results of 20 parts of clinical serum, the kit and the commercialized kit are positive in detection, so that the diagnostic sensitivity of the kit is higher than that of the commercialized kit.
) Experiment of specificity
The kit and the commercial African swine fever virus antibody detection kit are selected to respectively detect the special nature control serum and 10 parts of background negative serum (tables 5-6).
TABLE 5 results of specificity test of two batches of kits
TABLE 6 negative serum test results for African swine fever virus
The result shows that the kit does not have a false positive detection result, the negative rate of the clinical negative sample commercialized kit is 90%, and one sample is detected to be suspicious, which indicates that the specificity of the kit is superior to that of the commercialized kit.
) Repeatability test
3 kits prepared in a laboratory are selected for 3 kits per batch, 5 African swine fever virus negative sera (1-5), 5 African swine fever weak positive sera (6-10) and 5 African swine fever strong positive sera (11-15) are repeatedly determined for 4 times, and batch repeatability research is carried out. The known negative, weak positive and strong positive samples were tested 4 times repeatedly using 3 batches of laboratory prepared kits for batch-to-batch reproducibility studies.
TABLE 7 in-batch repeatability test results
TABLE 8 test results of repeatability between batches
The result shows that the kit has better repeatability. The intra-batch variation coefficient is between 2.96 and 11.73 percent, and the inter-batch variation coefficient is between 4.01 and 12.53 percent. The kit has higher coefficient of variation when the S/P value is smaller, and has lower coefficient of variation when the S/P value is larger.
The performance of the kit is better than that of a commercial kit in terms of the performance index of the kit.
The kit has the advantages of few steps, short time and convenient operation in the aspect of operation process, the commercial kit needs 122 minutes for completing the experiment, the kit only needs 75 minutes, and 47 minutes are saved. (Table 9).
TABLE 9 operational procedures and times for the kits of the present invention and commercial kits
The invention uses a double-antigen sandwich method to determine whether a sample contains ASFV antibody. The preparation method comprises the steps of coating a recombinant ASFV antigen on a coating plate in advance to prepare a stationary phase, adding a sample into micropores of the coating antigen, adding an ASFV antigen marked by HRP (horse radish peroxidase), forming an antigen-antibody-enzyme-labeled antigen complex, washing, and adding a substrate TMB (Tetramethylbenzidine) for color development. TMB is converted to blue by the catalysis of HRP enzyme and yellow by the termination of sulfuric acid. The darker the color, the more ASFV antibody content in the sample, and the lighter the color is positively correlated with the ASFV antibody content in the sample. And (4) measuring the absorbance (OD value) at the wavelength of 450nm by using a microplate reader, and calculating an S/P value through the OD value to judge whether the sample contains the ASFV antibody.
According to the detection kit, the detection sample and the enzyme-labeled antigen-HRP are added simultaneously, so that the operation steps are reduced, and primary antibody incubation and enzyme-labeled antigen combination are combined into one step; the operation time is shortened, one-step washing and one-step incubation are reduced, and about 50 minutes is shortened; compared with the traditional ELISA, the coated plate of the one-step African swine fever antibody ELISA rapid detection kit comprises capture antigen, a sample and enzyme-labeled antigen can be added simultaneously during detection, the enzyme-labeled antigen is specific, only a target antibody in the sample is identified, an antigen-antibody-enzyme-labeled antigen complex is formed by capturing the antigen, a substrate reacts with the complex to amplify the catalytic action of enzyme for color development, and a test value is read by an ultraviolet spectrophotometer.
The technical principle of the present invention has been described above with reference to specific embodiments, which are merely preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. Other embodiments of the invention will occur to those skilled in the art without the exercise of inventive faculty, and such will fall within the scope of the invention.
Claims (6)
1. A one-step method African swine fever ELISA antibody rapid detection kit is characterized by comprising a reaction plate, wherein the reaction plate is a coating plate pre-coated with African swine fever P30 recombinant protein; and sample diluent, enzyme-labeled antigen, substrate solution, stop solution, washing solution, negative control and positive control.
2. The kit for rapidly detecting the African swine fever ELISA antibody by the one-step method of claim 1, wherein the enzyme-labeled antigen is HRP-labeled P30 recombinant protein, and the P30 recombinant protein and the antibody have multiple binding sites.
3. The kit for rapidly detecting the African swine fever ELISA antibody according to the claim 1, wherein the P30 recombinant protein coat concentration is 50-120 ng/well, the concentration of the enzyme-labeled antigen is 50-120 ng/well, and the dilution of the serum sample is 1.
4. The kit for rapidly detecting the African swine fever ELISA antibody according to the claim 1, wherein the components of the sample diluent are as follows: BSA, 1.5% Tween 20 and 3% PEG6000 were added to the PBS buffer at a final concentration of 1%, and the mixture was sufficiently dissolved and mixed.
5. The kit for rapidly detecting the African swine fever ELISA antibody by the one-step method of claim 1, wherein a coating plate is coated with P30 recombinant protein in advance to prepare a stationary phase, a diluted sample is added into micropores coated with an antigen, an ASFV antigen marked by HRP is added, an enzyme-labeled antigen is combined with other sites of the antibody to form an antigen-antibody-enzyme-labeled antigen complex, and a substrate TMB is added for color development after washing.
6. The kit for rapidly detecting the African swine fever ELISA antibody by the one-step method as claimed in claim 1, wherein the sample and the ASFV antigen marked by HRP are added into the pre-coated micropores simultaneously, and the primary antibody incubation and enzyme labeling combination are combined into one step.
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Denomination of invention: A one-step ELISA kit for rapid detection of African swine fever antibodies Granted publication date: 20230324 Pledgee: Jiangsu Taicang Rural Commercial Bank Co.,Ltd. Yuewang Branch Pledgor: LONGKUO (SUZHOU) BIOLOGICAL ENGINEERING Co.,Ltd. Registration number: Y2024980021654 |