CN111505294A - Application of artificial antigen of variolothricin in enzyme linked immunosorbent assay kit - Google Patents
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Abstract
The invention provides an enzyme linked immunosorbent assay kit for detecting variolothricin, which comprises: the kit comprises an ELISA plate coated with a coating antigen, a variegated aspergillon standard solution, a variegated aspergillon antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a washing solution, wherein the coating antigen is a variegated aspergillon conjugate antigen, the enzyme conjugate is an enzyme-labeled variegated aspergillon antibody, and the variegated aspergillon antibody is obtained by immunizing animals with immunogen. The invention also discloses a method for detecting the variolothricin by applying the enzyme linked immunosorbent assay kit, which comprises the following steps: firstly, preprocessing a sample, then detecting by using a kit, and finally analyzing a detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of the variolothricin in grain and feed samples, is simple and convenient to operate, low in cost and high in sensitivity, can be monitored on site, and is suitable for screening a large number of samples.
Description
Technical Field
The invention relates to an application technology of a variegated aspergillomycin artificial antigen in an enzyme-linked immunosorbent assay kit, in particular to a variegated aspergillomycin artificial antigen molecular structure and an enzyme-linked immunosorbent assay kit for detecting variegated aspergillomycin, which can qualitatively and quantitatively detect the residual quantity of variegated aspergillomycin medicines in grains and feeds.
Background
Variegated aspergillin (ST for short) can induce experimental animals to generate a plurality of cancers. High incidence of liver cancer in certain regions of south africa may be associated with ST. ST is associated with gastric cancer in humans. ST can also be converted to the more potent carcinogen aflatoxin. ST can cause some animal diseases. ST can be produced by metabolism of more than 10 fungi, and the yield on artificial culture medium reaches 10 g/kg. Foods known to be contaminated with ST include: rice, wheat, corn, peanut, soybean, ham, cheese, coffee, etc.
Since the damages of the ST are large and the distribution is wide, the effective and rapid detection of the ST in food and feed is very important to reduce the influence of the ST on human and livestock. Compared with the traditional biological detection method, the thin layer chromatography, the high performance liquid chromatography and the like, the immunochemical detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like when detecting the mycotoxin. The prerequisite for immunochemical detection of ST is the requirement for antibodies against ST. The invention is a rapid detection method, has short time, simple operation and lower cost, and is suitable for detecting samples in large batch by a basic unit.
Disclosure of Invention
The invention aims to provide an enzyme linked immunosorbent assay kit capable of detecting the residual quantity of the variolox aspergillin in grains and feeds, and provides a qualitative and quantitative detection method which is efficient, accurate, simple and convenient and is suitable for screening large-batch samples.
The kit of the invention comprises: the kit comprises an ELISA plate coated with a coating antigen, a variegated aspergillon standard solution, a variegated aspergillon antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a washing solution, wherein the coating antigen is a variegated aspergillon coupling antigen, and the enzyme conjugate is an enzyme-labeled variegated aspergillon antibody.
The variegated aspergillomycin specific antibody is prepared by taking a variegated aspergillomycin artificial antigen as an immunogen, and can be a variegated aspergillomycin monoclonal antibody or a variegated aspergillomycin polyclonal antibody, wherein the variegated aspergillomycin monoclonal antibody is preferred.
The labeled enzyme of the enzyme conjugate is horseradish peroxidase or alkaline phosphatase extracted from bacteria, wherein horseradish peroxidase is preferred; the enzyme conjugate is obtained by coupling enzyme and a variolomycin antibody.
In order to facilitate field monitoring and large-scale sample screening, the kit further comprises a variegated aspergillin standard solution, a substrate color developing solution, a stop solution and a washing solution.
The concentration of the variegated aspergillon standard solution in 6 bottles is respectively 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L.
When the marker enzyme is horseradish peroxidase, the substrate color developing solution consists of a substrate solution A and a substrate solution B, wherein the A is hydrogen peroxide or carbamide peroxide, the B is o-phenylenediamine or tetramethylbenzidine, the stop solution is a sulfuric acid solution or a hydrochloric acid buffer solution with the concentration of 1-2 mol/L, when the marker enzyme is bacteria extracted alkaline phosphatase, the substrate color developing solution is a para-nitrophosphate buffer solution, and the stop solution is a sodium hydroxide solution with the concentration of 1-2 mol/L.
The washing liquid preferably has a pH value of 7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
The coating buffer solution used in the preparation process of the enzyme label plate is carbonate buffer solution with the pH value of 9.6 and 0.05 mol/L, and the confining solution is phosphate buffer solution with the pH value of 7.1-7.5 and containing 1-3% of casein and 0.1-0.3 mol/L, wherein the percentages are weight volume percentages.
The preparation process of the ELISA plate comprises the steps of diluting a coating source into 20 mu g/m L by using a coating buffer solution, adding 100 mu l of the coating source into each hole, incubating for 2 hours at 25 ℃ in the dark or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting the liquid dry, then adding 150-200 mu l of a sealing solution into each hole, incubating for 1-2 hours at 25 ℃ in the dark, pouring off liquid in the holes, patting the liquid dry, drying and then sealing and storing in vacuum by using an aluminum film.
The detection principle of the invention is as follows:
the kit adopts a direct competition E L ISA method to pre-coat the coupling antigen on the enzyme label plate microporous strip, the residual variolox in the sample and the coupling antigen pre-coated on the enzyme label plate microporous strip compete for the enzyme conjugate resisting the variolox, the TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the residual variolox in the sample, the sample is compared with a standard curve, and the sample is multiplied by the corresponding dilution multiple, so that the residual quantity of the variolox in the sample can be obtained.
The invention also provides a method for detecting the variolothricin by using the enzyme linked immunosorbent assay kit, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using the kit;
(3) and analyzing the detection result.
The enzyme linked immunosorbent assay kit for detecting the variegated aspergillomycin mainly adopts an E L ISA method to qualitatively or quantitatively detect the content of the variegated aspergillomycin in a sample, has low requirements on the pretreatment of the sample, simple pretreatment process of the sample, and can simultaneously and rapidly detect a large number of samples, the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement, and has the characteristics of high specificity, high sensitivity, high accuracy and the like.
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FIG. 1: molecular structural formula of variegated aspergillin hapten
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of kit Components
1. Preparation of variolothricin hapten
Taking 16mg of variegated triptycene, adding 20ml of methanol for dissolution, adding 18mg of 3-maleimide hydrazine propionate, fully adding the triptycene, adding 1.7g of sodium acetate, stirring for dissolution, heating, carrying out reflux reaction for 12 hours, stopping the reaction, carrying out rotary evaporation, removing the methanol, adding 50ml of water, adding 60ml of chloroform, fully shaking, standing, separating an aqueous phase, adding 70ml of water, shaking, standing, separating an aqueous phase, drying and evaporating an organic phase by anhydrous sodium sulfate, adding 3ml of diethyl ether, fully washing, removing a supernatant, carrying out solid precipitation, and carrying out vacuum drying to obtain 13mg of a maleimido propionic acid variegated triptycene hapten product, wherein the yield is 53%.
2. Preparation of antigens
Immunogen preparation-conjugation of variegated aspergillose hapten and hemocyanin (K L H) gives the immunogen.
Taking 25mg of Bovine Serum Albumin (BSA), adding 0.1M PB8.0 for dissolving, adding 1.9mg of dithiothreitol, and reacting at room temperature for 12g to obtain solution A; taking 9mg of maleimide propionic acid sterigmatocystin hapten, adding DMF1ml for dissolving to obtain solution B, slowly dripping into solution A, continuously reacting for 9h at room temperature, stopping reaction, dialyzing and purifying for three days by 0.02M PBS, changing solution 3 times a day to obtain BSA-sterigmatocystin conjugate which is immunogen, subpackaging, and storing at-20 ℃ for later use.
Preparation of coating antigen-coupling of variolothricin hapten and Ovalbumin (OVA) to obtain immunogen.
Dissolving Ovalbumin (OVA)20mg in PB8.0 0 0.1M, adding dithiothreitol 0.94mg, and reacting at room temperature for 12g to obtain solution A; dissolving 4mg of maleimidopropylated variegated aspergillin hapten in DMF1ml to obtain solution B, slowly dripping into solution A, continuously reacting for 9h at room temperature, stopping reaction, dialyzing and purifying with 0.02M PBS for three days, changing solution 3 times per day to obtain OVA-variegated aspergillin conjugate, namely coating antigen, subpackaging, and storing at-20 ℃ for later use.
3. Preparation of variolothricin monoclonal antibody
Animal immunization: injecting the immunogen obtained in the above steps into Balb/c mice, wherein the immunization dose is 150 mug/mouse, so that antiserum is generated.
Cell fusion and cloning, namely, after the determination result of mouse serum is higher, taking splenocytes, fusing the splenocytes with SP2/0 myeloma cells according to the ratio of 8:1 (quantitative ratio), adopting indirect competition E L ISA to determine cell supernatant, screening positive holes, and cloning the positive holes by using a limiting dilution method until obtaining a hybridoma cell strain secreting a variolomycin monoclonal antibody.
Freezing and thawing the cell, namely preparing the frozen solution of the monoclonal hybridoma cell strain into 1 × 106The cell suspension of L per m is preserved in liquid nitrogen for a long time, the frozen tube is taken out during recovery, the frozen tube is immediately put into a water bath at 37 ℃ for fast melting, and after the frozen solution is removed by centrifugation, the frozen tube is transferred into a culture bottle for culture.
Production and purification of monoclonal antibody, injecting sterilized paraffin oil 0.5m L/mouse into Balb/c mouse abdominal cavity, injecting stable monoclonal hybridoma cell strain 5 × 10 into abdominal cavity 7 days later5Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.
4. Preparation of enzyme conjugates
Taking a goat as an immune animal and taking a variegated aspergillon monoclonal antibody as an immunogen to immunize a goat without a pathogen to obtain the variegated aspergillon antibody. The enzyme conjugate is obtained by coupling the variolothricin antibody and horseradish peroxidase (HRP).
5. Preparation of ELISA plates
Diluting the coating source to 20 μ g/m L with a coating buffer solution, adding 100 μ l into each well, incubating at 25 deg.C in the dark for 2h, decanting off the liquid in the well, washing with a washing solution for 2 times, each time for 30s, patting to dryness, adding 200 μ l of blocking solution into each well, incubating at 25 deg.C in the dark for 2h, decanting off the liquid in the well, patting to dryness, drying, and vacuum sealing with aluminum film for storage.
Example 2 construction of enzyme-linked immunosorbent assay kit for detecting variolothricin
An enzyme linked immunosorbent assay kit for detecting the variolothricin is constructed, and comprises the following components:
(1) an ELISA plate coated with a variolothricin coupling antigen;
(2) the concentration of the variolothricin standard solution in 6 bottles is respectively 0 mug/L, 0.02 mug/L, 0.06 mug/L, 0.18 mug/L, 0.54 mug/L and 1.62 mug/L;
(3) a variolomycin antibody labeled with horseradish peroxidase;
(4) the substrate color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(5) the stop solution is 2 mol/L sulfuric acid;
(6) the washing liquid is phosphate buffer solution with the pH value of 7.4, containing 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L, and the percentage is weight volume percentage;
example 3 detection of variolothricins in cereals and feeds
1. Detection with a kit
And numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes in parallel for each sample and standard product, and recording the positions of the standard holes and the sample holes. Adding 50 mul of standard substance/sample into corresponding micropores, then adding 50 mul/pore of the working solution of the enzyme conjugate, and reacting for 30min at 25 ℃ in a dark environment. And (5) spin-drying the liquid in the holes, washing for 4-5 times, and patting dry. Adding 50 mul/hole of the substrate solution A, adding 50 mul/hole of the substrate solution B, mixing uniformly, and reacting for 15min at 25 ℃ in a dark environment. Adding 50 mul/well of stop solution, mixing, setting an enzyme-labeling instrument at 450nm, and measuring the OD value of each well.
2. Analysis of detection results
And the percent absorbance of the standard or the sample is equal to the average value (double holes) of the absorbance values of the standard or the sample divided by the average value of the absorbance values of the first standard (0 standard), and then multiplied by 100 percent to obtain the percent absorbance value of the standard or the sample, the percent absorbance of the standard is taken as the ordinate, the logarithm of the concentration (mu g/L) of the variolomycin standard is taken as the abscissa, a standard curve graph is drawn, the percent absorbance of the sample is substituted into the standard curve, the concentration corresponding to the sample is read from the standard curve, and the dilution multiple corresponding to the percent absorbance is multiplied to obtain the actual concentration of the variolomycin in the sample.
3. Pretreatment method
The pretreatment method of the grain sample (soybean meal, rice meal, corn meal, flour and the like) comprises the following steps:
homogenizing the grain sample with a homogenizer; weighing the homogenized grain sample into a 50ml polystyrene centrifuge tube; adding organic solvent and deionized water, respectively, shaking with oscillator for 5min, and centrifuging for 5 min; transferring supernatant into a 50ml polystyrene centrifuge tube, adding organic solvent, shaking for 5min with an oscillator, and centrifuging for 5 min; removing the upper liquid layer, taking the lower organic phase layer into a clean and dry glass tube, and drying by blowing under water bath nitrogen flow; adding organic solvent, whirling with vortex instrument, adding redissolution working solution, and centrifuging for 5 min; the upper organic solvent phase was removed and 50. mu.l of the lower aqueous phase was taken for analysis.
A feed (raw material, batch and concentrate) sample pretreatment method comprises the following steps:
weighing feed samples into a 50ml polystyrene centrifuge tube, adding an organic solvent, oscillating for 5min by using an oscillator, and centrifuging for 5 min; transferring supernatant, adding redissolution working solution, shaking with oscillator for 1min, and mixing; 50 μ l was taken for analysis.
Example 4 determination of technical parameters of variolothricins
1. Sensitivity and detection limit of kit
The sensitivity of the kit is determined according to a conventional method, and the range of a standard curve is 0.02-1.62 mu g/L50The (50% inhibition concentration) floating range is 0.09-0.15 mu g/L, 20 samples are detected, and the standard curve is obtainedThe concentration corresponding to each percent absorbance value is checked, the detection limit is represented by adding 3 times of standard deviation to the average value of the concentration of 20 samples, and the result shows that the detection limit of the method for the heterochromomycin in the grains and the feed is respectively 0.05 mu g/kg and 2 mu g/kg.
2. Sample precision and accuracy testing
The calculation formula is that the recovery ratio (%) is × 100% of actual measured value/theoretical value, wherein the theoretical value is the added concentration of the sample, and the relative standard deviation RSD% is SD/X × 100%, wherein SD is the standard deviation, and X is the average value of the measured data.
Grain samples are subjected to addition recovery measurement according to two concentrations of the variegated aspergillin of 0.05 mug/kg and 0.1 mug/kg, feed samples are subjected to addition recovery measurement according to two concentrations of the variegated aspergillin of 2 mug/kg and 4 mug/kg, 4 samples are subjected to parallel measurement by three different reagents, and the average recovery rate and precision result of the calculated samples are shown in the following table.
TABLE 1 grain and feed sample precision and accuracy tests
The grain sample is subjected to addition recovery measurement according to the variegated aspergillomycin with two concentrations of 0.05 mug/kg and 0.1 mug/kg, the feed sample is subjected to addition recovery measurement according to the variegated aspergillomycin with two concentrations of 2 mug/kg and 4 mug/kg, and the average recovery rates are respectively 96.2% -101.5% and 91.4% -113.7%; the relative standard deviation in each batch and between batches is less than 15 percent.
3. Stability test of kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), the 50% inhibition concentration and the actual measurement value of the addition of the variolothricin of the kit are all within the normal range after the measurement for 12 months. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 7 days under the storage condition of 37 ℃ for accelerated aging experiments, and the results show that all indexes of the kit completely meet the requirements. And in consideration of the occurrence of the freezing condition of the kit, the kit is frozen for 7 days in a refrigerator at the temperature of-20 ℃, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it can be obtained that the kit can be stored at 2 to 8 ℃ for at least 12 months.
Claims (4)
1. An enzyme linked immunosorbent assay kit for detecting variolothricin is characterized by comprising: the kit comprises an ELISA plate coated with a coating antigen, a variegated aspergillon standard solution, a variegated aspergillon antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a washing solution, wherein the coating antigen is a variegated aspergillon conjugate antigen, the enzyme conjugate is an enzyme-labeled variegated aspergillon antibody, the variegated aspergillon antibody is obtained by immunizing animals with immunogen, the variegated aspergillon conjugate antigen is obtained by coupling a variegated aspergillon hapten and carrier protein, and the variegated aspergillon hapten is obtained by carrying out a series of chemical reactions on the variegated aspergillon and 3-maleimide hydrazine and other substances.
2. The kit of claim 1, wherein the molecular structural formula of the variolothricin hapten is:
3. the kit according to claim 1, characterized in that the immunogen is prepared as follows:
taking 25mg of Bovine Serum Albumin (BSA), adding 0.1M PB8.0 for dissolving, adding 1.9mg of dithiothreitol, and reacting at room temperature for 12g to obtain solution A; taking 9mg of maleimide propionic acid sterigmatocystin hapten, adding DMF1ml for dissolving to obtain solution B, slowly dripping into solution A, continuously reacting for 9h at room temperature, stopping reaction, dialyzing and purifying for three days by 0.02M PBS, changing solution 3 times a day to obtain BSA-sterigmatocystin conjugate which is immunogen, subpackaging, and storing at-20 ℃ for later use.
4. A method for detecting the content of the variolothricin in a sample comprises the following steps:
(1) pretreating a sample;
(2) detecting with the kit according to any one of claims 1 to 3;
(3) and analyzing the detection result.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112904008A (en) * | 2021-02-04 | 2021-06-04 | 浙江省食品药品检验研究院 | Enzyme linked immunosorbent assay kit for detecting protein A and other impurities in biological products and application thereof |
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| CN111505294B (en) | 2022-11-18 |
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