CN109180519B - Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method - Google Patents
Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method Download PDFInfo
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- CN109180519B CN109180519B CN201810652749.2A CN201810652749A CN109180519B CN 109180519 B CN109180519 B CN 109180519B CN 201810652749 A CN201810652749 A CN 201810652749A CN 109180519 B CN109180519 B CN 109180519B
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- olaquindox metabolite
- olaquindox
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- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
Abstract
The invention discloses a olaquindox metabolite antigen, an antibody, an enzyme-linked immunosorbent assay kit and an assay method. The invention takes 4- (3-methyl-2-naphthamido) butyric acid as a hapten of a olaquindox metabolite, the hapten is coupled with carrier protein to obtain the antigen of the olaquindox metabolite, the antigen of the olaquindox metabolite can be applied to the preparation of a specific antibody of the olaquindox metabolite, and the specific antibody can be used for preparing an enzyme linked immunosorbent assay kit or a colloidal gold test paper card for detecting the olaquindox metabolite residue in food. The invention also discloses a corresponding kit detection method. The method is simple, convenient and rapid, the linear detection range is 0.2-16.2 ng/mL, the sensitivity is 0.54 ng/mL, the detection limit is 0.32 ng/mL, the recovery rate is 80-109.8%, the detection limit is low, the sensitivity is high, the specificity is strong, the stability is good, the cost is low, and the method is very suitable for detection of a large number of samples and rapid detection on site.
Description
Technical Field
The invention belongs to the technical field of drug residue detection. More particularly, relates to a olaquindox metabolite antigen, an antibody, an enzyme-linked immunoassay kit and a detection method.
Background
Olaquindox is an early product of quinoxaline medicines, has been widely applied worldwide since the 70 s in the 20 th century, but is forbidden to be used for food animal growth promotion all over the world at present. The european union banned the use of carbachol and quindox in 1998, and china banned the use of quindox and carbachol for growth promotion in food animals and specified the maximum residual limits of the indicators of the residual olaquindox in animal tissues. In recent years, olaquindox drugs have been mainly used in australia, brazil, japan, china, and the like.
The toxicity of olaquindox to animals mainly comprises acute toxicity and accumulative toxicity. Excessive intake of olaquindox can cause the animals to die due to stress massive hemorrhage, and when the olaquindox is accumulated in the animals to a certain degree, the olaquindox can also cause teratogenic, carcinogenic and mutagenic 'triple-effect' to animals or human beings. Therefore, the withdrawal period must be strictly controlled according to the application range and dosage concentration. In view of toxicity and potential hazard of olaquindox, people reevaluate safety of olaquindox and make new regulations on the use of olaquindox. Olaquindox itself is unstable, and it is metabolized in animals for a short time to more than ten kinds of metabolites, most of which do not have detection conditions, wherein 3-methylquinoxaline-2-carboxylic acid (MQCA) is the main metabolite, is relatively stable in vivo, and is a labeled residue recognized by the national committee of food code, so olaquindox metabolite MQCA is generally used as a target for residue analysis and monitoring. In 2003, the Ministry of agriculture in China stipulates that the maximum residual quantity of MQCA in muscle and liver tissues is 4 mug/kg and 50 mug/kg respectively.
However, olaquindox drugs are still illegally used or abused in production practice, and the residue of the drug poses a great potential threat to the health of consumers and the export of animal food, thereby driving the research of related detection methods. The detection methods include liquid chromatography-mass spectrometry, liquid chromatography, gas method, enzyme linked immunosorbent assay, colloidal gold adsorption detection, molecular imprinting, biosensor method, etc. Among them, the liquid chromatography-mass spectrometry and the enzyme-linked immunosorbent assay are mainstream analysis methods. Compared with the prior art, the immunoassay method is a screening method which is worthy of popularization and common use due to low cost, simple operation, high speed, large sample amount for one-time detection and low instrumentization degree.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art and provide a olaquindox metabolite antigen, an antibody, an enzyme-linked immunoassay kit and a detection method. The enzyme-linked immunoassay kit has the advantages of accuracy, sensitivity, rapidness and capability of detecting the olaquindox metabolite in a high-throughput manner, can efficiently detect the olaquindox metabolite, provides a certain theoretical guidance for the development of a rapid olaquindox metabolite detection product, and has important significance for monitoring the safety of medicines.
The first purpose of the invention is to provide a olaquindox metabolite hapten.
The second purpose of the invention is to provide the olaquindox metabolite antigen prepared by applying the olaquindox metabolite hapten.
The third purpose of the invention is to provide the application of the olaquindox metabolite antigen in preparing olaquindox metabolite antibodies.
The fourth purpose of the invention is to provide a olaquindox metabolite antibody prepared by applying the olaquindox metabolite antigen.
The fifth purpose of the invention is to provide the application of the olaquindox metabolite antibody in detection of olaquindox metabolites.
The sixth purpose of the invention is to provide the enzyme linked immunosorbent assay kit prepared by the olaquindox metabolite hapten, the olaquindox metabolite antigen and the olaquindox metabolite antibody.
The seventh purpose of the invention is to provide a method for detecting the olaquindox metabolite residues in the sample by using the enzyme linked immunosorbent assay kit.
The above purpose of the invention is realized by the following technical scheme:
the invention relates to a olaquindox metabolite hapten, wherein the hapten is 4- (3-methyl-2-naphthamido) butyric acid, and the molecular structural formula of the hapten is shown as a formula (1):
The preparation method of the hapten 4- (3-methyl-2-naphthamido) butyric acid comprises the following steps: dissolving 3-methyl-2-naphthoic acid in SOCl2Heating to 90-110 ℃ under the protection of nitrogen, and reacting for 1-3 h; removing solvent under reduced pressure, dissolving the residue in 1, 4-dioxane, and adding dropwise amino acid and Na under stirring2CO3The mixed aqueous solution of (1); stirring at room temperature overnight, adding a hydrochloric acid solution for acidification, extracting by using EtOAC, combining organic phases, washing by using a saturated NaCl solution, drying, and purifying by using silica gel column chromatography to obtain 4- (3-methyl-2-naphthamido) butyric acid, namely the hapten.
The invention also relates to a olaquindox metabolite antigen which is obtained by coupling the olaquindox metabolite hapten with carrier protein.
Wherein the carrier protein can be common carrier protein such as mouse serum albumin, thyroid protein (BCG), bovine serum albumin, rabbit serum albumin, human serum albumin, Ovalbumin (OVA), hemocyanin or fibrinogen.
Preferably, the carrier protein is ovalbumin.
The preparation method of the olaquindox metabolite antigen comprises the following steps: coupling the hapten (4- (3-methyl-2-naphthamido) butyric acid) with carrier protein by adopting an active ester method to obtain the olaquindox metabolite antigen.
The olaquindox metabolite antigen can be used as an immunogen to prepare an olaquindox metabolite antibody and can also be used as a coating antigen to prepare an ELISA plate.
The antibody can be a monoclonal antibody, a polyclonal antibody or a genetic engineering antibody, and is preferably a monoclonal antibody.
The preparation method of the olaquindox metabolite polyclonal antibody comprises the following steps:
immunizing rabbit or mouse with the above olaquindox metabolite antigen as immunogen to prepare olaquindox metabolite polyclonal antibody, collecting antiserum, purifying by plating and precipitating with caprylic acid and sulfuric acid, and purifying with affinity chromatography column.
The preparation method of the olaquindox metabolite monoclonal antibody comprises the following steps:
immunizing a mouse by taking the olaquindox metabolite antigen as an immunogen, fusing splenocytes of the mouse with SP2/0 myeloma cells to obtain hybridoma cells, culturing the hybridoma cells in a female Balb/c mouse to obtain ascites containing a high-concentration monoclonal antibody, and purifying the ascites to obtain the high-specificity anti-olaquindox metabolite monoclonal antibody.
Correspondingly, the antibody prepared by using the olaquindox metabolite antigen and the application of the antibody in detection of olaquindox metabolites are also within the protection scope of the invention.
The enzyme linked immunosorbent assay kit or the colloidal gold test paper card prepared by the olaquindox metabolite hapten, the olaquindox metabolite antigen and the olaquindox metabolite antibody is also within the protection scope of the invention.
Preferably, the enzyme linked immunosorbent assay kit comprises: the kit comprises an ELISA plate coated with a olaquindox metabolite antigen, an olaquindox metabolite antibody, an enzyme-labeled secondary antibody, an olaquindox metabolite series standard solution, a substrate buffer solution, a substrate working solution, a concentrated washing solution, a stop solution, a complex solution and a sulfuric acid solution.
Preferably, the coating solution for coating the olaquindox metabolite antigen is: dissolving 1.69 g of sodium carbonate and 2.95 g of sodium bicarbonate in 1L of double distilled water; the coating concentration of the olaquindox metabolite antigen is 0.125 mug/L.
Preferably, the ELISA plate can be a 96-hole detachable transparent ELISA plate made of polystyrene, is coated with olaquindox metabolite antigen capable of being specifically combined with the anti-olaquindox metabolite antibody, and seals sites on the surface of the micropores which do not adsorb the olaquindox metabolite antigen.
The preparation method of the ELISA plate coated with the olaquindox metabolite antigen comprises the following steps: diluting olaquindox metabolite antigen with coating solution as required, adding the coating solution into the pores, incubating overnight at 37 deg.C, removing the coating solution, washing with washing solution, adding blocking solution into each pore, incubating at 37 deg.C, removing the liquid in the pores, drying, and vacuum sealing with aluminum film for storage.
The overnight period herein means 8-16 h.
Wherein in the preparation process of the ELISA plate, the blocking solution is obtained by dissolving 0.1 g of BSA (bovine serum albumin), 5 g of glycine and 5 g of sucrose in 100 mL of PBS (0.01 mol/L, pH 7.4).
In addition, many substances that can be used as a solid phase carrier for immobilizing a olaquindox metabolite antigen include, for example, polystyrene, nitrocellulose, polyethylene, polypropylene, chitosan, cross-linked glucose, silicone rubber, agarose gel, and the like. The carrier may be in the form of wells, paper sheets, beads, etc.
Preferably, the enzyme-labeled secondary antibody is goat anti-rabbit IgG. The labeled enzyme of the enzyme labeled antibody is horseradish peroxidase or alkaline phosphatase, preferably horseradish peroxidase.
Preferably, the olaquindox metabolite series standard solution is diluted into a series of olaquindox metabolite standard solutions with the concentration of 0, 0.2, 0.6, 1.8, 5.4 and 16.2 ng/mL by using 0.2 mol/L, pH phosphate buffer solution with 5.4 at the time of use.
More preferably, the formulation of the phosphate buffer is: 2.9 g of NaH2PO4·12H2O, 8.5 g NaCl, 0.2 g KCl, 0.2 g KH2PO4And the volume is constant to 1L.
Preferably, the substrate working solution is a phosphoric acid-citric acid buffer solution with pH of 4.5-5.5 and containing hydrogen peroxide or carbamide peroxide; the substrate buffer solution is a phosphoric acid-citric acid buffer solution with pH of 4.5-5.5 and containing 3,3,5, 5-tetramethylbenzidine.
More preferably, the substrate working solution and the substrate buffer solution are mixed in a volume ratio of 1:1 to obtain a substrate solution.
Preferably, the concentrated washing solution has a pH of 7.0-7.8, contains 0.4-0.6% of Tween-20 and 0.3-0.5 mol/L of phosphate buffer solution, and the percentage is weight volume percentage.
More preferably, the concentrated wash is 20 times the concentrated wash; when in use, the detergent is diluted to 1 time by deionized water.
Preferably, the stop solution is 0.5M H2SO4。
Preferably, the sulfuric acid solution is a 3M sulfuric acid solution.
Preferably, the reconstituted solution is 0.2M PBS.
The invention also relates to a method for detecting the olaquindox metabolite residues in a sample by using the enzyme linked immunosorbent assay kit.
Particularly preferably, the method for detecting olaquindox metabolite residues in the sample comprises the following steps:
s1, taking the enzyme linked immunosorbent assay kit out of a refrigeration environment, and balancing for 30-45 min at 15-35 ℃;
s2, taking out the enzyme label plate, adding olaquindox metabolite series standard substance solutions with different concentrations into the standard holes, adding a sample to be detected into the sample holes, then respectively adding an enzyme-labeled secondary antibody and an olaquindox metabolite antibody working solution into each hole, and incubating; wherein the olaquindox metabolite standard solution is obtained by diluting olaquindox metabolite standard with phosphate buffer solution to different concentrations;
s3, absorbing the reaction solution in the plate holes, washing with concentrated washing solution, and patting the enzyme-labeled plate dry;
s4, adding the substrate working solution and the substrate buffer solution into each hole, patting and uniformly mixing, and covering a cover plate film;
s5.10-20 min later, adding a stop solution into each hole, and measuring the absorbance of each hole;
s6, analyzing the detection result: and determining the content of the olaquindox metabolite in the sample.
The results were calculated as the inhibition ratio (%) < B/B0X 100 (%); in the formula: b is the absorbance value of standard solution holes or sample holes to be detected of the olaquindox metabolites with different concentrations, B0The absorbance value of the 0-concentration olaquindox metabolite standard solution is shown; and drawing a standard curve by taking the inhibition rate as an ordinate and taking the logarithm of the concentration of the olaquindox metabolite standard solution as an abscissa, thereby determining the content of the olaquindox metabolite in the sample.
The sample to be tested includes, but is not limited to, animal tissues such as chicken, chicken liver, pork or pig liver.
Preferably, the sample to be tested in step S2 is chicken, duck, or pork.
Preferably, the kit is used for carrying out sample pretreatment before detection. The pretreatment of the sample is mainly to obtain a olaquindox metabolite solution from the sample so as to be used for subsequent detection.
The following is a common pretreatment method of several samples (chicken, duck and pork), comprising the following steps:
(1) taking out the sample, thawing, weighing the homogenized sample (2.00 +/-0.05) g to a 50 mL polystyrene centrifuge tube, adding 2 mL distilled water, and fully oscillating for 2 min;
(2) 8 mL of ethyl acetate, 1 mL of 1.5M H were added2SO4Shaking for 1 min, and centrifuging at room temperature (20-25 ℃) at a rotating speed of more than 4000 r/min for 5 min;
(3) taking out 4 mL of supernatant to a glass test tube, and drying the supernatant by nitrogen or air at 50 ℃;
(4) adding 0.5 mL of n-hexane, whirling for 30 s by using a vortex instrument, adding 0.5 mL of complex solution, whirling for 10 s by using the vortex instrument at more than 4000 r/min, and centrifuging for 5min at room temperature (20-25 ℃);
(5) removing the upper n-hexane phase, and taking 50 mu L of lower layer clear liquid to be tested.
Enzyme-linked immunoassay is a novel labeled immunoassay technology which combines enzyme catalysis reaction and immunoreaction and is used for detecting trace antigens or antibodies. The detection principle is as follows: (1) binding antigen or antibody to the surface of a solid phase carrier and keeping the immunological activity; (2) connecting the antigen or antibody with certain enzyme to form enzyme-labeled antigen or antibody, wherein the enzyme-labeled antigen or antibody retains the immunological activity and the enzyme activity; (3) and adding a substrate after washing, enzymatically enabling the substrate to generate a colored product, detecting the absorbance through a special instrument, and directly correlating the amount of the product with the amount of the detected substance in the sample so as to inversely calculate the concentration of the unknown antigen or antibody. The method has the outstanding advantages of high sensitivity, simple and quick analysis method, stable result, small error, good safety and long service life, and compared with an instrument analysis method, the method has the following advantages: can realize large-batch detection, and is more economical and time-saving.
Compared with the prior art, the invention has the beneficial effects that:
(1) the hapten plays an important role in the detection of the olaquindox metabolite residue, the hapten is used as a raw material to prepare an antigen system suitable for animal immunization to immunize animals, and the titer, the specificity and the affinity of the obtained antibody are good; the obtained antibody is used for an enzyme linked immunosorbent assay kit, is convenient to use, low in detection cost, efficient, accurate and rapid in detection method, can be used for simultaneously detecting a large number of samples, and is suitable for field monitoring of olaquindox metabolite residues in animal tissues and screening of a large number of samples.
(2) According to the enzyme linked immunosorbent assay kit and the method thereof provided by the invention, the maximum linear detection range is 0.2-16.2 ng/mL, the sensitivity is 0.54 ng/mL, the detection limit is 0.32 ng/mL, the recovery rate is 80-109.8%, the kit is rapid in detection, the detection time is greatly shortened, the influence of the operation proficiency of detection personnel is not considered, the whole detection process can be completed only by about 70 min, the detection limit is lower, and the sensitivity is higher; meanwhile, the stability and precision of the kit are improved by adopting the coated plate coated with the antigen, and the detection is more stable and accurate.
Drawings
FIG. 1 is a standard curve of olaquindox metabolite ELISA kit.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The reagents used in the examples of the invention were as follows:
coating liquid: 1.69 g of sodium carbonate and 2.95 g of sodium hydrogencarbonate were dissolved in 1L of double distilled water.
20 times of concentrated washing solution: 0.4 mol/L phosphate buffer solution with pH7.4 containing 0.5% Tween-20 was diluted 1-fold with deionized water at the time of use, and the percentage is weight volume percentage.
Sealing liquid: 0.1 g of BSA (bovine serum albumin), 5 g of glycine and 5 g of sucrose were dissolved in 100 mL of PBS (0.01 mol/L, pH 7.4).
The olaquindox metabolite is listed as standard solution: diluting the olaquindox metabolite standard solution to 1 mg/mL by using chromatographic grade methanol for later use; then diluted by PBS with the concentration of 0.2 mol/L, pH being 5.4 into a series of olaquindox metabolite standard solutions with the concentrations of 0 ng/mL, 0.2 ng/mL, 0.6 ng/mL, 1.8 ng/mL, 5.4 ng/mL and 16.2 ng/mL respectively, and stored at 4 ℃.
Substrate solution: the substrate solution consists of a solution A (substrate working solution) and a solution B (substrate buffer solution), wherein the solution A is a phosphoric acid-citric acid buffer solution with pH of 5.0 and containing carbamide peroxide; the solution B is a phosphoric acid-citric acid buffer solution with pH of 5.0 and containing 3,3,5, 5-tetramethylbenzidine; when in use, the liquid A and the liquid B are mixed according to the volume ratio of 1:1 to obtain a substrate solution.
Quinoethanol metabolite monoclonal antibody (2.5 mg/mL): the preparation in the early stage of the laboratory.
Horse radish peroxidase-labeled goat anti-rabbit IgG (10 mg/mL): purchased from biosgineers, Inc. of Dr. Wuhan.
EXAMPLE 1 preparation of Quinoethanol metabolite hapten, immunogen, coatingen and monoclonal antibody
1. Preparation of haptens, immunogens and coatinggens
Hapten 4- (3-methyl-2-naphthamido) butyric acid (abbreviated as MNBA) is respectively coupled with carrier protein Bovine Serum Albumin (BSA) and Ovalbumin (OVA) by adopting an active ester method to prepare immunogen MNBA-BSA and coating antigen MNBA-OVA. The specific method comprises the following steps:
(1) hapten synthesis:
3-methyl-2-naphthalenic acid (1.3 mmol) was dissolved in SOCl2(11 mmol), and the mixture is heated to 100 ℃ by microwave under the protection of nitrogen for reaction for 2 h; the solvent was removed under reduced pressure and the residue was dissolved in 1, 4-dioxane (5 mL); it was added dropwise to a mixture of amino acid (1.3 mmol/L) and Na with stirring2CO3(3.2 mmol/L) in 5 mL of an aqueous solution; stirring at room temperature overnight, adding 20 mL hydrochloric acid solution (1 mol/L), acidifying, and extracting with EtOAC (3X 15 mL); after the organic phases were combined, washed 3 times with saturated NaCl solution, dried over anhydrous sodium sulfate and the solvent was evaporated; purifying the residue by silica gel column chromatography to obtain 4- (3-methyl-2-naphthamido) butyric acid, namely the target hapten.
1H NMR (500 MHz, DMSO-d 6)：δ 8.29-8.22 (m, 4H), 7.97-7.90 (m, 2H), 7.88 (d, J = 0.6 Hz, 1H), 7.88-7.81 (m, 3H), 7.61-7.53 (m, 4H), 3.30 (q, J = 7.1 Hz, 4H), 2.57 (s, 6H), 2.37 (t, J = 7.1 Hz, 4H), 1.89 (p, J = 7.1 Hz, 4H)。
(2) Immunogen synthesis:
weighing 0.32 g of hapten 4- (3-methyl-2-naphthamido) butyric acid, weighing 0.131 g of NHS, adding the NHS into the hapten, dissolving the NHS into 400 mu L of DMF in a reaction device, and reacting for 10 h at room temperature under magnetic stirring; cooling the reaction final solution in a refrigerator at 4 ℃ for more than 2 h, centrifuging for 5min at 1.2 ten thousand rpm, taking the supernatant (namely the active ester solution), and slowly dripping the supernatant into 5 mL of BSA solution of 6 mg/mL for reaction; the reaction buffer was 0.2 mol/L Phosphate Buffer (PBS) pH7.4, and the reaction was carried out for 4 hours (from the end of the sample application) at room temperature under magnetic stirring; placing the final reaction solution into a dialysis bag, stirring and dialyzing the final reaction solution in 0.01 mol/L PBS (phosphate buffer solution) with pH of 7.4 at 4 ℃, and changing the dialysate every 4-8 h for 3 days; after dialysis, the protein solution (i.e. the immunogen MNBA-BSA of MQCA) in the dialysis bag is divided into a plurality of equal parts and is subpackaged in a 1 mL centrifuge tube, and the centrifuge tube is stored in a refrigerator at the temperature of-20 ℃ for later use.
(3) Coating original synthesis: the synthetic method of the coating antigen is the same as the process of the immunogen synthetic method, and the MNBA-OVA of the coating antigen can be obtained only by replacing the carrier protein BSA in the immunogen synthesis with OVA.
2. Preparation of olaquindox metabolite monoclonal antibody
(1) Animal immunization
Dissolving the immunogen prepared by the method in normal saline at a dose of 150 mu g/cell, uniformly mixing the dissolved immunogen with Freund's complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6 and 8 weeks old subcutaneously at the neck and back, uniformly mixing the immunogen and Freund's incomplete adjuvant in equal volume at 7, 14 and 28 days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by immune compound at a dose of 150 mu g/cell 3 days before fusion without adding Freund's adjuvant.
(2) Cell fusion
Mixing splenocytes of immunized mice with myeloma cells (SP 2/0) of mice in logarithmic growth phase, slowly adding preheated fusion agent (PEG 4000) within 45 s for fusion, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate, culturing at 37 deg.C and 5% CO2Culturing in an incubator, half-changing the culture medium with HT medium after 5 days, and completely changing the culture medium after 9 days.
(3) Screening for Positive hybridomas
After the cells are fused, when the cells grow to 1/4 of the area of the culture hole, screening the hybridoma cells by adopting a step screening method; the initial selection was carried out by indirect ELISA using the envelope antigen (the best envelope was titrated routinely by the square matrix method in advance)Diluted by concentration and positive serum), adding culture supernatant of a detected hole, incubating, washing, adding goat anti-mouse IgG-HRP and IgM-HRP, and carrying out color reaction on OPD; screening the screened positive hole by using an indirect competitive ELISA method, mixing cell supernatant and 100 mu g/mL olaquindox metabolite in equal volume, performing water bath at 37 ℃ for 30min, and adding the mixture into a coated enzyme label plate; meanwhile, PBS is used for replacing olaquindox metabolite as a control, and the rest steps are the same as the above; OD if blocked by Quinoethanol metabolite450The nm value is reduced to be less than 50 percent of the control hole, the control hole is judged to be positive, the control hole is detected to be positive for 2 times and 3 times, and the subcloning is immediately carried out by a limiting dilution method;
(4) expanded culture of hybridoma cells
Carrying out expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing and storing; injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of a Balb/c mouse aged 8-10 weeks, and injecting 1-2 × 10 hybridoma cells into the abdominal cavity after 7-10 days6And extracting ascites from the mice 7-10 days later, centrifuging to obtain supernatant, measuring titer, and freezing for later use.
EXAMPLE 2 establishment of enzyme-linked immunoassay of olaquindox metabolites
(1) The optimization of the concentration of the coating antigen and the concentration of the antibody comprises the following steps:
1) diluting the coating antigen with coating solution according to the concentration of 15.6, 8.9, 5.0, 2.5 and 1.25 ng/mL, longitudinally coating an enzyme label plate, incubating at 100 mu L/hole overnight at 37 ℃, washing twice with washing solution, and patting on absorbent paper to dry;
2) adding 170 mu L/hole of prepared sealing liquid for sealing, standing overnight at 37 ℃, spin-drying and putting into an oven;
3) adding a olaquindox metabolite standard solution diluted by 50 mu L/hole PBS solution;
4) adding 50 μ L/well of different gradients of olaquindox metabolite monoclonal antibodies (1: 32000, 1:64000, 1:96000, 1: 128000) diluted with PBS solution, incubating at 25 deg.C for 30min, washing the plate for 5 times, and drying on absorbent paper;
5) adding 100 μ L/hole enzyme-labeled secondary antibody (goat anti-mouse IgG) diluted 5000 times, incubating at 25 deg.C for 30min, washing the plate for 5 times, and drying on absorbent paper;
6) adding the substrate solution prepared in situ, reacting at 25 ℃ for 10 min with 100 mu L/hole;
7) adding stop solution, 50 μ L/well, reading with enzyme-labeling instrument, and reading absorbance value of each well at single wavelength of 450 nm or double waves of 450 nm/630 nm.
The experimental results show that the optimal working concentration of the coating antigen is 1.25 ng/mL, and the dilution factor of the antibody is 64000 times.
(2) Determination of antibody sensitivity
The antibody sensitivity was measured with the optimal concentration of coating antigen being 1.25 ng/mL and the antibody dilution factor being 64000 times. The method comprises the following steps:
1) taking a 96-well plate, diluting a coating source to 1.25 ng/mL by using a diluent, adding 100 mu L of the coating source into each well, incubating overnight at 37 ℃, washing twice by using a washing solution, and patting dry on absorbent paper;
2) adding 170 mu L/hole of prepared sealing liquid for sealing, standing overnight at 37 ℃, spin-drying and putting into an oven;
3) respectively adding olaquindox metabolite standard solutions with different gradients and antibodies diluted 64000 times into 50 mu L/hole PBS, incubating for 30min at 25 ℃, washing the plate for 5 times, and drying on absorbent paper;
4) adding 100 μ L/hole enzyme-labeled secondary antibody (goat anti-mouse IgG) diluted 5000 times, incubating at 25 deg.C for 30min, washing the plate for 5 times, and drying on absorbent paper;
5) adding the substrate solution prepared in situ, reacting at 25 ℃ for 10 min with 100 mu L/hole;
6) adding stop solution, reading by an enzyme-linked immunosorbent assay (ELISA) reader at 50 mu L/well, and reading the absorbance value of each well at a single wavelength of 450 nm or double waves of 450 nm/630 nm;
the results were calculated as the inhibition ratio (%) < B/B0X 100 (%), the lower the half inhibition, indicating the higher the sensitivity of the antibody; wherein B is the absorbance of solutions of different standard concentrations in competition, B0Absorbance values for 0 concentration olaquindox metabolite standard solutions at 50% inhibition (median inhibitory concentration, IC)50) Calculating quindoxyThe sensitivity of the metabolite monoclonal antibody is 0.54 ng/mL.
EXAMPLE 3 preparation of olaquindox metabolite ELISA kit
1. Preparation of reagents
(1) ELISA plate coated with olaquindox metabolite antigen
A 96-hole enzyme-detachable plate which is coated with olaquindox metabolite antigen and confining liquid, wherein the coating concentration is 1.25 mg/L; the olaquindox metabolite is a conjugate of olaquindox metabolite hapten 4- (3-methyl-2-naphthamido) butyric acid and Ovalbumin (OVA);
coating of microplate microplates: diluting the coating antigen to 1.25 mg/L with coating solution, adding 100 μ L of coating solution into each well, incubating overnight at 37 deg.C, removing the liquid in the well, washing with washing solution for 2 times, and patting to dry; then adding 170 mu L of confining liquid into each hole, incubating for 3 h at 37 ℃, pouring out the liquid in the hole, placing in an oven at 37 ℃ for drying, and then storing at 4 ℃ by using an aluminum foil bag in a vacuum sealing manner.
(2) Preparation of olaquindox metabolite standard solution
Accurately weighing olaquindox metabolite standard solution, diluting with chromatographic grade methanol to 1 mg/mL, and adding 0.2 mol/L PBS buffer solution (formulation is 2.9 g NaH)2PO4·12H2O, 8.5 g NaCl, 0.2 g KCl, 0.2 g KH2PO4And fixing the volume to 1L) to respectively prepare olaquindox metabolite solutions with the concentrations of 0, 0.2, 0.6, 1.8, 5.4 and 16.2 ng/mL, and storing at 4 ℃.
(3) Olaquindox metabolite antibody working solution
The concentration of the olaquindox metabolite monoclonal antibody is 1.25 mg/mL; the working concentration is 1:64000, and the solution is diluted by P solution.
(4) Substrate liquid
The liquid A (substrate working solution) and the liquid B (substrate buffer solution) are mixed according to a volume ratio of 1:1 to obtain substrate liquid; wherein the solution A is a phosphoric acid-citric acid buffer solution with pH of 5.0 and containing carbamide peroxide; the solution B is a phosphoric acid-citric acid buffer solution with pH of 5.0 and containing 3,3,5, 5-tetramethylbenzidine.
(5) 20 times concentrated washing liquid
0.4 mol/L phosphate buffer solution with pH7.4 containing 0.5% Tween-20 was diluted 1-fold with deionized water at the time of use, and the percentage is weight volume percentage.
2. Reagent split charging
After the determination of each reagent is qualified, aseptically packaging, and obtaining 7 mL/bottle of olaquindox metabolite antibody working solution; enzyme-labeled secondary antibody 7 mL/bottle; 1 mL/bottle of olaquindox metabolite standard solution; 7 mL of substrate solution per bottle; 7 mL of substrate buffer solution per bottle; 7 mL of stop solution per bottle; 50 mL/bottle of 3M sulfuric acid solution; the compound solution is 50 mL/bottle; the 20-fold concentrated washing solution is 50 mL/bottle. The transparent plastic bottles with covers of different colors are adopted for subpackaging and then are labeled, the batch number and the validity period are noted, and the product is stored at 4 ℃.
3. Assembly of the kit
Respectively placing 1 piece of the ELISA plate coated with the olaquindox metabolite antigen, 1 bottle of each of olaquindox metabolite antibody working solution, horseradish peroxidase-labeled secondary antibody, substrate solution, substrate buffer solution, 20-time concentrated washing solution, stop solution, 3M sulfuric acid solution and redissolution, 6 bottles of olaquindox metabolite standard substance working solution and 1 part of an instruction in a kit at a designated position, packaging the qualified kit after inspection, and storing the qualified kit at 4 ℃.
Example 4 application of olaquindox metabolite ELISA kit
1. The enzyme linked immunosorbent assay kit is used for detection
(1) Sample adding: fixing the required number of laths on a plate frame, adding 50 microliter of sample or standard substance into a microporous plate, adding 50 microliter of enzyme marker into the micropore, adding 50 microliter of antibody working solution into the microporous plate, covering with a cover plate membrane, fully oscillating and uniformly mixing;
(2) and (3) incubation: incubating at room temperature (25 ℃) for 30 min;
(3) washing: discarding liquid in the holes, filling the diluted washing liquid into each hole, standing for 30-60 s, and discarding the washing liquid in the holes; repeatedly washing for 4 times, and drying;
(4) color development: adding 50 mu L of substrate buffer solution into each hole, adding 50 mu L of substrate working solution, shaking and uniformly mixing (or mixing the substrate buffer solution and the substrate working solution in a ratio of 1:1, then adding 100 mu L of developing solution), and incubating at room temperature (25 ℃) for 10-15 min;
(5) and (4) terminating: adding 50 mu L of stop solution into each hole, and patting and uniformly mixing;
(6) reading value: reading the value by a microplate reader, and reading the absorbance value of each hole under the condition of single wavelength of 450 nm or double wavelength of 450 nm/630 nm.
2. Calculation and analysis of detection results
Inhibition ratio (%) ═ B/B0×100（%）
In the formula: b is the absorbance value of standard solution holes or sample holes to be detected of the olaquindox metabolites with different concentrations, B0The absorbance value of the 0-concentration olaquindox metabolite standard solution is shown.
And drawing a standard curve by taking the inhibition rate as an ordinate and taking the logarithm of the concentration of the olaquindox metabolite standard solution as an abscissa, and substituting the light absorption value of the olaquindox metabolite into the standard curve to calculate the content of the olaquindox metabolite in the sample to be detected.
The influence of the operation skill of the detection personnel is not considered, and the whole detection process can be completed only by about 70 min. The analysis of the detection result can also be calculated and analyzed by using computer professional software.
3. Standard curve
A olaquindox metabolite standard curve graph (shown in figure 1) is obtained by analyzing a detection result of a standard substance solution, and the results show that the kit has a linear detection range of 0.2-16.2 ng/mL for olaquindox metabolites, a sensitivity of 0.54 ng/mL and a detection limit of 0.32 ng/mL.
Example 5 evaluation of the application Effect of olaquindox metabolite ELISA kit
The detection method was the same as in example 4.
1. Pretreatment of sample to be tested
(1) Taking out the sample, thawing, weighing the homogenized sample (2.00 +/-0.05) g to a 50 mL polystyrene centrifuge tube, adding 2 mL distilled water, and fully oscillating for 2 min;
(2) 8 mL of ethyl acetate, 1 mL of 1.5M H were added2SO4Shaking for 1 min, and centrifuging for 5min at room temperature (20-25 ℃) at a rotating speed of more than 4000 r/min;
(3) taking out 4 mL of supernatant to a glass test tube, and drying the supernatant by nitrogen or air at 50 ℃;
(4) adding 0.5 mL of n-hexane, whirling for 30 s by using a vortex instrument, adding 0.5 mL of redissolution, whirling for 10 s by using the vortex instrument, and centrifuging for 5min at the room temperature (20-25 ℃) at the rotating speed of more than 4000 r/min;
(5) removing the upper n-hexane phase, and taking 50 mu L of lower layer clear liquid to be tested.
2. Repeatability test of olaquindox metabolite standard solution
From 3 batches of the microplate prepared in accordance with the method in example 3, 16 wells were randomly drawn out, the absorbance value of the olaquindox metabolite standard solution was measured according to the detection method of the kit in example 4, repeated 16 times, and the coefficient of variation (CV,%) was calculated, with the results shown in table 1.
TABLE 1 repeatability test for olaquindox metabolite standard solutions
Intra-batch coefficient of variation: coefficient of variation for each parallel sample in the same assay.
Inter-batch coefficient of variation: and (4) measuring the coefficient of variation of the results of different batches of the same sample, and taking the average value of the coefficients.
The result shows that the intra-batch variation coefficient range of the detection of the standard substance solution of the kit is between 2.45 and 10.77 percent, and the inter-batch variation coefficient is between 7.43 and 14.56 percent. The CV is less than 15%, which shows that the method has good accuracy, and the kit can be suitable for detecting the olaquindox metabolite in the actual sample.
3. Sample repeatability and accuracy test
The accuracy refers to the degree of coincidence between the measured value and the true value, and in enzyme-linked immunoassay, the accuracy is usually expressed by the recovery rate and the precision is usually expressed by the coefficient of variation. In the blank samples, olaquindox metabolites were added to final concentrations of 0.5, 1, 2. mu.g/L, 10 replicates of each concentration, and 3 batches were assayed. The average, the addition recovery and the intra-and inter-batch variation coefficients were calculated. The results are shown in Table 2.
TABLE 2 sample repeatability and accuracy test results
The result shows that the average adding recovery rate of the chicken sample is between 90.4 and 107.2 percent, the intra-batch variation coefficient is between 1.45 and 5.81 percent, and the inter-batch variation coefficient is between 1.93 and 10.93 percent, thereby meeting the national standard for each index of the kit.
4. Cross reaction
The specificity of an antibody is an important measure of the quality of an antibody. The higher the specificity of the antibody, the stronger the recognition capability of the antibody on a target antigen, and the smaller the cross reaction on a non-target antigen, so that the probability of false positive in detection is lower. The experiment selects the olaquindox metabolite structural analogue and the functional analogue to carry out antibody binding reaction. The smaller the cross reaction rate with other medicines is, the better the detection specificity of the olaquindox metabolite enzyme-linked immunoassay kit to the olaquindox metabolite is. The results are shown in Table 3.
TABLE 3 Cross-reaction results
Note: QCA is quinoxaline-2-carboxylic acid; ND stands for not detectable.
The result shows that the cross reaction rate of the olaquindox metabolite detection kit and other medicines is low, which indicates that the olaquindox metabolite enzyme-linked immunoassay kit has good detection specificity on olaquindox metabolites.
5. Shelf life test of kit
(1) The kit prepared in the embodiment 3 is placed at 2-8 ℃, the kits stored for 0, 2, 4, 6, 8, 9, 10, 11 and 12 months are respectively taken, and the absorbance value, 50% inhibition concentration, addition recovery rate and batch variation coefficient of the olaquindox metabolite standard solution are measured.
(2) The kit is placed for 12 days under the condition of being stored at 37 ℃, and the absorbance value, the 50% inhibition concentration, the addition recovery rate and the intra-batch variation coefficient of the olaquindox metabolite standard solution are measured every day.
(3) The kit is stored in a refrigerator at the temperature of 20 ℃ below zero for 12 days, and the absorbance value, the 50% inhibition concentration, the addition recovery rate and the batch variation coefficient of the olaquindox metabolite standard solution are measured every day.
The results show that through three condition storage tests, the absorbance value of the olaquindox metabolite standard solution is reduced by less than 10%, and all indexes meet the quality requirements, so that the kit can be stored at 2-8 ℃ for 12 months.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The olaquindox metabolite hapten is 4- (3-methyl-2-naphthamido) butyric acid, and the molecular structural formula of the olaquindox metabolite hapten is shown as a formula (1):
2. a olaquindox metabolite antigen obtained by coupling the olaquindox metabolite hapten of claim 1 to a carrier protein.
3. Use of a olaquindox metabolite antigen of claim 2 in the preparation of an olaquindox metabolite antibody.
4. An antibody against a olaquindox metabolite prepared using the olaquindox metabolite antigen of claim 2.
5. Use of a olaquindox metabolite antibody of claim 4 for the detection of olaquindox metabolites that is not aimed at the diagnosis or treatment of disease.
6. An enzyme linked immunosorbent assay kit or a colloidal gold test paper card prepared by using the olaquindox metabolite hapten as defined in claim 1, the olaquindox metabolite antigen as defined in claim 2 and the olaquindox metabolite antibody as defined in claim 4.
7. The ELISA kit of claim 6, comprising: an ELISA plate coated with the olaquindox metabolite antigen of claim 2, the olaquindox metabolite antibody of claim 4, an enzyme-labeled secondary antibody, an olaquindox metabolite series standard solution, a substrate buffer solution, a substrate working solution, a concentrated washing solution, a stop solution, a complex solution and a sulfuric acid solution.
8. The ELISA kit of claim 7, wherein the substrate working solution is a phosphate-citrate buffer solution with pH of 4.5-5.5 and containing hydrogen peroxide or carbamide peroxide; the substrate buffer solution is a phosphoric acid-citric acid buffer solution with pH of 4.5-5.5 and containing 3,3,5, 5-tetramethylbenzidine; the concentrated washing solution is 0.3-0.5 mol/L phosphate buffer solution with the pH value of 7.0-7.8 and containing 0.4-0.6% Tween-20 by weight volume.
9. A method for detecting olaquindox metabolite residues in a sample, characterized in that the enzyme linked immunosorbent kit of claim 7 or 8 is used for detection, and the method does not aim at diagnosis or treatment of diseases.
10. The method of detecting olaquindox metabolite residues in a sample of claim 9, comprising the steps of:
s1, taking the enzyme linked immunosorbent assay kit out of a refrigeration environment, and balancing for 30-45 min at 15-35 ℃;
s2, taking out the enzyme-labeled plate, adding olaquindox metabolite series standard substance solutions with different concentrations into the standard holes, adding a sample to be detected into the sample holes, then respectively adding an enzyme-labeled secondary antibody and an olaquindox metabolite antibody working solution into each hole, and incubating;
s3, absorbing the reaction liquid in the plate holes, washing with a concentrated washing liquid, and drying the enzyme-labeled plate;
s4, adding a substrate working solution and a substrate buffer solution into each hole, lightly beating and uniformly mixing, and covering a cover plate membrane;
s5.10-20 min later, adding a stop solution into each hole, and measuring the absorbance of each hole;
s6, analyzing a detection result: and determining the content of the olaquindox metabolite in the sample.
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| CN110927382A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof |
| CN114539171B (en) * | 2021-12-31 | 2023-05-05 | 华南农业大学 | MAC type multi-cluster tandem linear hapten and artificial antigen as well as preparation methods and applications thereof |
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