CN112462071A - Special enzyme-linked immunoassay kit for ribavirin - Google Patents
Special enzyme-linked immunoassay kit for ribavirin Download PDFInfo
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Abstract
The invention provides an enzyme-linked immunoassay kit special for ribavirin, which comprises a horseradish peroxidase-labeled ribavirin antibody working solution, a ribavirin standard, a 20 Xconcentrated washing solution, a substrate solution A, a substrate solution B and a stop solution; 1% of PEG4000, 3% of trehalose, 0.5% of calf serum, 0.1% of EDTA and 0.01% of Proclin300 are added into the horseradish peroxidase-labeled ribavirin antibody working solution to serve as antibody stabilizers; the micropore ELISA plate is coated with ribavirin hapten-HSA coupling antigen, and a step of sealing by adding PBS buffer solution containing 5% of sucrose, 0.05% of PEG4000, 6% of glycerol, 0.1% of gelatin, 0.01% of Proclin300 and 0.1mol/L in the preparation process. The enzyme-linked immunoassay kit special for ribavirin has high detection sensitivity and specificity, good accuracy and precision parameters of detection results and excellent stability performance index, and is an ideal ribavirin detection product.
Description
Technical Field
The invention belongs to the field of biotechnology detection, and particularly relates to an enzyme-linked immunoassay kit special for ribavirin.
Background
Ribavirin, chemical name is 1-beta-D-ribofuranosyl-1H-1, 2, 4-triazole-3-carboxamide, molecular formula is C8H12N4O, is a white or quasi-white knotThe crystal powder is odorless and tasteless, and belongs to the synthetic nucleoside drugs. Ribavirin is a broad-spectrum antiviral drug that inhibits a wide variety of DNA and RNA viruses and is commonly used to treat viral infectious diseases in humans. Research shows that the long-term use of ribavirin can damage the reproductive system and has reproductive toxicity.
The 560 document issued by the Ministry of agriculture at 10.2005 shows that the antiviral drug ribavirin for human use is prohibited from being used for the treatment of diseases of animals such as pigs and chickens. Although the use of ribavirin as a veterinary drug has been clearly banned by the country, there is sometimes a phenomenon in which farmers illegally use ribavirin for the treatment of animal diseases. Once the ribavirin residual medicine is accumulated in animal tissues, corresponding harm is generated after the ribavirin residual medicine is eaten by human, and the ribavirin residual medicine has higher safety risk.
At present, the detection methods of ribavirin mainly comprise liquid chromatography, liquid chromatography-tandem mass spectrometry, electrophoresis, immunoassay and the like. Although the instrumental analysis method has the advantages of high detection accuracy, good repeatability and the like, the method also has the defects of complicated pretreatment procedures, long detection time, high instrument cost, high requirement on the professional degree of detection personnel and the like, is not beneficial to the screening work of large-batch samples, and is also not beneficial to the effective supervision of the use of ribavirin medicines. The enzyme-linked immunosorbent assay is developed based on the specific binding reaction between antigen and antibody, takes enzyme as a rapid screening method of reaction medium, and has the characteristics of simple and rapid detection, large detection sample amount, low requirement on detection personnel and the like.
Disclosure of Invention
The invention aims to provide the special ribavirin enzyme-linked immunosorbent assay kit which has the advantages of high detection stability, high detection sensitivity, strong specificity and short detection time.
The technical scheme of the invention is as follows:
a special enzyme-linked immunoassay kit for ribavirin comprises a micropore elisa plate coated with ribavirin hapten-HSA coupling antigen, a horseradish peroxidase-labeled ribavirin antibody working solution, a ribavirin standard, 20 x concentrated washing liquid, a substrate liquid A, a substrate liquid B and stop solution, wherein 1% of PEG4000, 3% of trehalose, 0.5% of calf serum, 0.1% of EDTA and 0.01% of Proclin300 are added into the horseradish peroxidase-labeled ribavirin antibody working solution to serve as antibody stabilizers, and the micropore elisa plate is sealed by buffer solution containing 5% of sucrose, 0.05% of PEG4000, 6% of glycerol, 0.1% of gelatin, 0.01% of Proclin300 and 0.1mol/L in the preparation process.
Further, the ribavirin hapten is prepared by the following steps: weighing 1.0g of raw material ribavirin, dissolving the ribavirin with 20mL of acetone and 20mL of pyridine, uniformly stirring, weighing 0.8g of glutaric anhydride, adding the glutaric anhydride, stirring and reacting at 80 ℃ for 6 hours, stopping, drying by distillation, feeding the mixture to a silica gel column, eluting by using an eluent of petroleum ether and ethyl acetate (1: 2), and separating and purifying to obtain the ribavirin hapten.
Wherein the ribavirin antibody is prepared by immunizing a mouse or a rabbit with a ribavirin hapten-KLH conjugate as immunogen.
Furthermore, the concentrated washing solution contains 1% Tween-20 and 0.2mol/LPBS buffer solution.
Further, the substrate A solution is hydrogen peroxide, the substrate B solution is TMB, and the mixture is uniformly mixed within 10min before use.
Further, the terminating solution is 1mol/L H2SO4。
When a specific sample is detected, the enzyme-linked immunoassay kit special for ribavirin can also comprise a sample extractant and a sample diluent, wherein the sample extractant contains 50% of methanol and 0.01mol/L citric acid-sodium citrate buffer solution, the pH value is 6.0, and the sample diluent is 0.05mol/L citric acid-sodium citrate buffer solution, and the pH value is 6.0.
The invention has the beneficial effects that:
the special enzyme-linked immunoassay kit for ribavirin coats the coupling antigen in a microporous plate, uses enzyme-labeled antibody working solution to react with a sample to compete for the coated object, performs analysis and determination on ribavirin residual quantity by substrate color development, and establishes a direct competition ELISA method. When the enzyme-linked immunoassay kit special for ribavirin is prepared, a stabilizer buffer solution is used for sealing in the process of preparing an enzyme label plate, and an antibody stabilizer is added into an enzyme-labeled antibody working solution, so that the stability of the detection kit is effectively improved, the storage time of the kit is greatly prolonged, and the phenomena of inaccurate detection result and reagent waste caused by unstable reagents are avoided. The detection sensitivity of the special enzyme-linked immunoassay kit for ribavirin reaches 1ng/L, the detection recovery rate is 89-111%, the intra-plate variation coefficient is 4.8-5.3%, and the inter-plate variation coefficient is 6.1%. The kit disclosed by the invention is simple in structure, high in detection sensitivity, good in accuracy and precision detection data, stable in reagent, long in storage time, low in detection cost, low in requirement on detection personnel, good in portability of detection products, and suitable for rapid screening of ribavirin in large-batch samples.
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FIG. 1 is a standard graph of the kit.
Detailed Description
For a further understanding of the present invention, reference will now be made to the preferred embodiments of the present invention by way of examples, but it is to be understood that these descriptions are intended only to further illustrate the features and advantages of the present invention, and not to limit the scope of the claims, and that the reagents of the present invention, unless otherwise specified, are conventional and commercially available.
Example one
1. The special enzyme-linked immunoassay kit for ribavirin comprises the following components:
(1) the enzyme label plate with 96 holes is coated with ribavirin hapten-HSA coupling antigen.
(2) An enzyme-labeled antibody working solution, specifically a horseradish peroxide ribavirin monoclonal antibody, 7 mL/bottle. 1% PEG4000, 3% trehalose, 0.5% calf serum, 0.1% EDTA, 0.01% Proclin300 as antibody stabilizer are added into the enzyme-labeled antibody working solution. The percentage is mass percentage.
(3) Ribavirin standard 0ng/L, 1ng/L, 3ng/L, 9ng/L, 27ng/L, 81ng/L, 1 mL/bottle.
(4) The washing solution is concentrated by 20 times, the ingredients comprise 1% Tween-20, 0.2mol/LPBS buffer solution, pH 7.2, 30 mL/bottle, and when in use, the solution is diluted by deionized water according to the proportion of 1: 19.
(5) The sample extractant comprises 50% methanol, 0.01mol/L citric acid-sodium citrate buffer solution, pH6.0, and 4 mL/bottle.
(6) The sample diluent comprises 0.05mol/L citric acid-sodium citrate buffer solution, pH6.0 and 8 mL/bottle.
(7) The substrate A solution is hydrogen peroxide, and the substrate B solution is TMB, which are all 7 mL/bottle.
(8) The stop solution is 1mol/L H2SO47 mL/bottle.
2. Preparation of main components:
2.1 Synthesis of ribavirin haptens
Weighing 1.0g of raw material ribavirin, dissolving the ribavirin with 20mL of acetone and 20mL of pyridine, uniformly stirring, weighing 0.8g of glutaric anhydride, adding the glutaric anhydride, stirring and reacting for 6 hours at 80 ℃, stopping, drying by distillation, applying to a silica gel column, eluting by using an eluent of petroleum ether and ethyl acetate (1: 2), separating and purifying to finally obtain the ribavirin hapten.
2.2 Synthesis of immunogen and coatingen
The immunogen is ribavirin hapten-KLH, the coating antigen is ribavirin hapten-HSA, and the preparation process comprises the following steps:
weighing ribavirin hapten 12mg and dissolving in 1mL of DMF to obtain hapten diluent; weighing 15mgEDC and 15mgNHS, dissolving in 0.2mL deionized water, adding into hapten diluent, and stirring at room temperature for 30min to obtain activated hapten diluent; weighing carrier protein KLH or HSA50mg, and dissolving in 4ml PBS; dropwise and slowly adding the activated hapten diluent into the prepared carrier protein solution, stirring at room temperature for 8 hours for reaction, dialyzing with 0.01mol/L PBS (phosphate buffer solution), intercepting the molecular mass of 8000-14000 u, dialyzing at 4 ℃ for 3 days, changing the dialysate 3 times per day, subpackaging after the preparation and storing at-20 ℃.
2.3 preparation of ribavirin monoclonal antibody
2.3.1 animal immunization
Healthy Balb/c mice, 6-8 weeks old, were selected and injected subcutaneously with ribavirin hapten-KLH conjugate containing Freund's complete adjuvant at an immunization dose of 200 μ g/mouse, boosted at 2,4 and 8 weeks, and finally immunized at 12 weeks to generate multiple antisera.
2.3.2 preparation of myeloma and splenocytes
Culturing SP2/0 myeloma cells in RPMI-1640 culture medium to make the cells in logarithmic growth phase to obtain SP2/0 myeloma cell suspension; taking the spleen of the immunized mouse, crushing, filtering by a 200-mesh stainless steel gauze to prepare spleen cell suspension, and counting for later use.
2.3.3 cell fusion and Mono-cloning
SP2/0 myeloma cells and spleen cells were fused and positive wells were screened using an indirect competition ELISA assay. And (3) adopting a limiting dilution method to perform monoclonality on the positive hole until obtaining a hybridoma cell strain which stably secretes the ribavirin monoclonal antibody.
2.3.4 cell cryopreservation and Resuscitation
The ribavirin monoclonal hybridoma cell strain is 1 multiplied by 106one/mL cell suspension, 500. mu.L each in cryotubes, was stored in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting into 37 ℃ water bath for fast melting, transferring into a conical flask, adding into 15mL of complete culture solution, and using CO2The incubator is used for culture.
2.3.5 Mass production and purification of monoclonal antibodies
Taking a healthy Balb/c female mouse, injecting 0.5mL of paraffin oil into the abdominal cavity, and injecting ribavirin monoclonal hybridoma cell strain 10 into the abdominal cavity after 10 days6Every other day, and ascites were collected 7-10 days later. Ascites was centrifuged at 4000rpm for 10min and the supernatant was collected. The purified antibody was stored at-20 ℃.
2.3.6 specificity of ribavirin monoclonal antibodies
Antibody specificity refers to the ability of an antibody to bind to an epitope of a different structure, expressed as the rate of cross-reactivity. The cross reaction rate is low, and the specificity of the antibody can be proved to be high. The 50% inhibitory concentrations (i.e., IC) were obtained from the standard curves for each drug50). The cross-reactivity of the kit to other drugs was calculated using the following formula:
cross reversePercent stress [ (%) ] (ribavirin IC)50Ribavirin analog IC50)×100％
The cross reaction rate of the special enzyme-linked immunoassay kit for ribavirin is 100 percent, the kit has no cross reaction to amantadine, zanamivir, ofloxacin, ciprofloxacin and other medicaments, and the content of ribavirin in a sample can be specifically detected.
2.4 preparation of enzyme-labeled antibodies
The enzyme-labeled antibody is a ribavirin monoclonal antibody labeled by horseradish peroxidase. The horseradish peroxidase and ribavirin monoclonal antibody are reacted by a sodium periodate method to obtain the horseradish peroxidase-ribavirin monoclonal antibody.
2.5 Elisa plate
The ribavirin hapten-HSA coupling coating antigen is diluted to 2 mu g/mL by 0.05mol/L, pH9.6 carbonate buffer solution, 100 mu L of the ribavirin hapten-HSA coupling coating antigen is added into each hole, the ribavirin hapten-HSA coupling coating antigen is incubated for 2h at 37 ℃, washed for 2 times and dried by spinning. Then adding 250 mu L of coating stabilizer into each hole for sealing, incubating for 2h at 37 ℃, pouring out liquid in the hole, patting the hole dry by absorbent paper, and then sealing and storing by an aluminum film in vacuum. The coating stabilizer is 0.1mol/L PBS buffer solution containing 5% of sucrose, 0.05% of PEG4000, 6% of glycerol, 0.1% of gelatin and 0.01% of Proclin300, the pH value is 7.4, and the percentage is mass percentage. The coating stabilizer not only has the functions of ensuring the activity of macromolecular protein and preventing degradation, but also has the functions of improving the stability of the ELISA plate and sealing, and reduces the influence of the background of reagent holes.
The enzyme label plate after being coated and other reagents of the invention are put in a constant temperature box with 37 ℃ for aging test, and the absorbance value is measured after the 25 th day, and the A value is reduced by less than 10%.
The stability of the ELISA plate and the reagent is a key index for measuring the product quality, is an important guarantee for the safety and effectiveness of the kit, and aims to obviously improve the stability of the kit. In the development process of the kit, 1% of PEG4000, 3% of trehalose, 0.5% of calf serum, 0.1% of EDTA and 0.01% of Proclin300 are added into the working solution of the enzyme-labeled antibody as stabilizers, and a stabilizer coating step is introduced into an enzyme-labeled plate for coating the coupled antigen in the coating process, so that the stability of the kit is obviously improved.
The assembled kit can also comprise a specification, a cover plate film, a self-sealing bag, a certificate and the like, and the shelf life of the kit can reach more than 3 years at the temperature of 2-8 ℃.
3. The detection principle of the special enzyme-linked immunoassay kit for ribavirin is as follows:
by utilizing the property that the ribavirin antibody and the ribavirin antigen can generate specific binding, the antigen is pre-coated on the enzyme label plate, then the standard substance or the sample and the ribavirin antibody are added, the ribavirin and the antigen fixed on the enzyme label plate compete for the ribavirin antibody, finally the substrate is added for catalytic color development, and at the moment, the color development depth is in inverse proportion to the content of the ribavirin in the standard substance or the sample.
4. Specific detection steps of kit
(1) And taking out the enzyme label plates with required quantity, inserting the enzyme label plates into the enzyme label plate frame, recording the positions of each standard product and each sample, and performing a double-hole parallel experiment for reducing the fluctuation of detection values.
(2) Adding 50 mu L of standard substance/sample into corresponding micropores, adding 50 mu L of enzyme-labeled antibody working solution into the micropores, lightly oscillating for 5s, mixing, and placing the cover plate at the rear part in a dark environment at 25 ℃ for reaction for 20 min.
(3) Uncovering the cover plate film, drying liquid in the holes, adding 250 mu L of washing liquid into the holes, washing for 2 times, pouring the washing liquid, and patting the washing liquid dry by absorbent paper.
(4) Adding 100 mul/hole substrate color developing solution, mixing, covering, and reacting in a dark environment at 25 deg.C for 10 min.
(5) Adding stop solution 50 μ L/hole, mixing, setting enzyme-labeling instrument at 450nm after 5min, and determining OD value.
5. Analysis of detection results
Let the average absorbance of each standard (or sample) be B, zero standard, i.e. the standard with a concentration of 0. mu.g/L, and the average absorbance B0With (B/B)0) X 100% is the percentage of absorbance corresponding to each standard (or sample): and (5) substituting a semi-logarithmic system into the percentage of absorbance corresponding to the standard substance, and fitting a standard curve with the concentration of the standard substance. The absorbance of a sample to be detected is measuredAnd substituting the ratio into a fitted standard curve equation to obtain the corresponding concentration of the sample, and multiplying the concentration by the corresponding dilution multiple of the sample to obtain the content of the detected substance in the sample.
Example 2 detection of ribavirin in pork samples
1. Sample pretreatment method
Weighing 1.0g of pork homogeneous sample in a 10mL polystyrene centrifuge tube, adding 4mL deionized water, adding 40 μ L of sample extractant, and oscillating for 1 min; centrifuging at 4000rpm for 5min at room temperature; taking 400 mu L of supernatant into a 2mL centrifuge tube, adding 80 mu L of sample diluent, and oscillating for 30 s; 50 μ L was taken for analysis.
The sample extract is a buffer solution containing 50% of methanol and 0.01mol/L of citric acid-sodium citrate, and the pH value is 6.0; the sample diluent is 0.05mol/L citric acid-sodium citrate buffer solution, and the pH value is 6.0. The percentage is mass percentage.
2. Fitting of standard curve
The standard curve consisted of 6 concentrations (0ng/L, 1ng/L, 3ng/L, 9ng/L, 27ng/L, 81ng/L) and was measured 2 times for each of the 6 standard points to determine the working concentration range and IC50. Taking the logarithm value of the concentration of the ribavirin standard substance as an abscissa, and the inhibition rate (B/B) of each corresponding concentration0%) is the ordinate and a standard curve is made, see fig. 1. Kit IC50The concentration was 3.6 ng/L.
3. Sample precision and accuracy testing
Accuracy is expressed as recovery and precision as coefficient of variation. Pork samples were recovered by addition with ribavirin to a final concentration of 10 ng/kg. And extracting different kits for determination, determining the same sample for 3 times, and respectively calculating the recovery rate and the coefficient of variation of the determined sample. The results are shown in Table 1.
TABLE 1 recovery and coefficient of variation determination
The results show that the pork samples added with 10ng/kg of ribavirin have the measured recovery rate of 89-111%, the intra-plate coefficient of variation of 4.8-5.3% and the inter-plate coefficient of variation of 6.1%.
EXAMPLE III
(1) A 96-hole enzyme label plate, wherein the enzyme label plate is coated with ribavirin hapten-HSA conjugate.
(2) An enzyme-labeled antibody working solution, specifically a horseradish peroxidase-labeled ribavirin polyclonal antibody, which is 7 mL/bottle.
(3) Ribavirin standard 0ng/L, 1ng/L, 3ng/L, 9ng/L, 27ng/L, 81ng/L, 1 mL/bottle.
(4) The washing solution is concentrated by 20 times, the ingredients comprise 1% Tween-20, 0.2mol/LPBS buffer solution, pH 7.2, 30 mL/bottle, and when in use, the solution is diluted by deionized water according to the proportion of 1: 19.
(5) The sample extractant comprises 50% methanol, 0.01mol/L citric acid-sodium citrate buffer solution, pH6.0, and 4 mL/bottle.
(6) The sample diluent comprises 0.05mol/L citric acid-sodium citrate buffer solution, pH6.0 and 8 mL/bottle.
(7) The substrate A solution is hydrogen peroxide, and the substrate B solution is TMB, which are all 7 mL/bottle.
(8) The stop solution is 1mol/L H2SO47 mL/bottle.
The above description is only a preferred embodiment of the present invention, and 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. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (7)
1. An enzyme-linked immunoassay kit special for ribavirin comprises a micropore enzyme label plate, a horseradish peroxidase-labeled ribavirin antibody working solution, a ribavirin standard, a 20 Xconcentrated washing solution, a substrate solution A, a substrate solution B and a stop solution; 1% of PEG4000, 3% of trehalose, 0.5% of calf serum, 0.1% of EDTA and 0.01% of Proclin300 are added into the horseradish peroxidase-labeled ribavirin antibody working solution to serve as antibody stabilizers; the micropore ELISA plate is coated with ribavirin hapten-HSA coupling antigen, and a step of sealing by adding PBS buffer solution containing 5% of sucrose, 0.05% of PEG4000, 6% of glycerol, 0.1% of gelatin, 0.01% of Proclin300 and 0.1mol/L in the preparation process.
2. The enzyme-linked immunoassay kit special for ribavirin according to claim 1, wherein the ribavirin hapten is prepared by the following steps: weighing 1.0g of raw material ribavirin, dissolving the ribavirin with 20mL of acetone and 20mL of pyridine, uniformly stirring, weighing 0.8g of glutaric anhydride, adding the glutaric anhydride, stirring and reacting at 80 ℃ for 6 hours, stopping, drying by distillation, feeding the mixture to a silica gel column, eluting by using an eluent of petroleum ether and ethyl acetate (1: 2), and separating and purifying to obtain the ribavirin hapten.
3. The special ribavirin enzyme-linked immunoassay kit according to claim 1 or 2, wherein the concentrated washing solution is a buffer solution containing 1% tween-20 and 0.2mol/LPBS, and has a pH of 7.2.
4. The ribavirin-specific enzyme-linked immunoassay kit according to claim 1 or 2, wherein the substrate a solution a is hydrogen peroxide and the substrate B solution is TMB.
5. The special ribavirin enzyme-linked immunoassay kit according to claim 1 or 2, wherein the stop solution is 1mol/L H2SO4。
6. The ribavirin-specific enzyme-linked immunoassay kit according to claim 1 or 2, wherein: the kit also comprises a sample extractant and a sample diluent, wherein the sample extractant is a buffer solution containing 50% of methanol and 0.01mol/L of citric acid-sodium citrate and has pH of 6.0, and the sample diluent is a buffer solution containing 0.05mol/L of citric acid-sodium citrate and has pH of 6.0.
7. The ribavirin-specific enzyme-linked immunoassay kit according to claim 1 or 2, wherein: the ribavirin antibody is prepared by taking a ribavirin hapten-KLH conjugate as an immunogen.
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| CN109438424A (en) * | 2018-11-16 | 2019-03-08 | 中国农业大学 | Ribavirin haptens and artificial antigen and the preparation method and application thereof |
| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunoassay kit and detection method thereof |
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