CN107677660B - Kitasamycin chemiluminescence enzyme-linked immunoassay kit - Google Patents
Kitasamycin chemiluminescence enzyme-linked immunoassay kit Download PDFInfo
- Publication number
- CN107677660B CN107677660B CN201610877176.4A CN201610877176A CN107677660B CN 107677660 B CN107677660 B CN 107677660B CN 201610877176 A CN201610877176 A CN 201610877176A CN 107677660 B CN107677660 B CN 107677660B
- Authority
- CN
- China
- Prior art keywords
- solution
- kitasamycin
- mug
- kit
- prepared
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention relates to a kitasamycin chemiluminescence enzyme-linked immunoassay kit with high sensitivity, high accuracy and high precision, which comprises a kit body, an ELISA plate arranged in the kit body and a reagent arranged in the kit body. Through screening monoclonal antibodies with excellent specificity by a large number of tests and determining the appropriate concentration of each component in the reaction solution, the kitasamycin chemiluminescence enzyme-linked immunoassay kit can quickly and accurately detect kitasamycin with lower content, and is suitable for detecting the requirement of kitasamycin drug residue in animal food of pigs or poultry.
Description
Technical Field
The invention relates to a chemiluminescence enzyme-linked immunoassay kit, in particular to a chemiluminescence enzyme-linked immunoassay kit for detecting kitasamycin.
Background
With the improvement of the quality of life of people, food safety problems are more and more concerned by consumers, and particularly, the problem of pesticide, antibiotic and other drug residues is more concerned by consumers. On the hot spot of global concern about food safety, how to detect food safety quickly and accurately becomes an important issue. To detect the antibiotic residue in food, a detection method with strong specificity, high sensitivity, high efficiency and high speed must be selected. Therefore, the method for detecting various antibiotic residues has very important significance in improving the sensitivity and selectivity of the method.
Antibiotics are widely used in the treatment of a variety of bacterial infectious diseases in humans and animals. Antibiotics used as clinical drugs are often taken in a short time and mainly enter animals by injection, oral administration, drinking water and the like. Excess antibiotic remains in the muscle and milk at the injection site before the drug holiday ends. When veterinarians use the antibiotics in a clinical way, the antibiotics are easy to remain in animals, and animal food pollution is caused. Some antibiotics are also used as pharmaceutical additives for preventing bacterial diseases and promoting the growth of animals for a long time. Such as oxytetracycline additives, aureomycin additives and the like enter animal bodies through oral cavities, cannot be completely discharged in a short time, and are easy to accumulate in the bodies, so that the antibiotic residues in animal food are caused.
The abuse of antibiotics caused by the rapid development of modern breeding industry, and the large amount of drug residues, have become a serious economic and social problem threatening the health of human beings. The problem of antibiotic contamination in animal derived foods has attracted considerable attention worldwide, and many countries have put forward a limited standard for antibiotic residues in various animal derived foods. The 'notice about the maximum residue limit of veterinary drugs in animal foods' announced by the Ministry of agriculture in 2002 in China defines the maximum residue limit of veterinary drugs in 92 animal foods, wherein the maximum residue limit of Kitasamycin (Kitasamycin) in the muscle, liver and kidney tissues of pigs/poultry is 200 mug/kg. Guitar mycin is also called columbamycin, is a multi-component macrolide antibiotic produced by streptomyces beilii, has stronger antibacterial activity on most gram-positive bacteria, part of gram-negative bacteria and mildews, is used for preventing and treating bacterial infection, mildews and the like of livestock and poultry and is used as a growth promoter, and in recent years, with the wide application of the kitasamycin, the problem of residue of the kitasamycin in animal food is increasingly prominent.
Therefore, it is highly desirable to establish a detection method capable of rapidly and sensitively detecting kitasamycin residues. There are several monitoring methods including traditional instrumentation, microbiological assays and emerging enzyme-linked immunoassays (ELISA). The traditional instrumental analysis methods for detecting drug residues include HPLC, LC-UV, LC-MS-MS and the like. The methods have the advantages of accuracy, stability and good specificity, can be used as standard methods, but have expensive instruments and equipment, are heavy, need a large amount of solvents, have high requirements on operators, have complex sample pretreatment, waste time and labor, are not easy to popularize, and are not suitable for carrying out real-time detection on large-scale samples. The microbiological assay method is capable of detecting a large number of samples in real time, but has poor specificity and cannot perform accurate qualitative and quantitative analysis. The enzyme linked immunosorbent assay (ELISA) overcomes the defects of the methods, is a rapid, sensitive and convenient detection method, and can be used for the instant detection of a large number of samples.
However, the market always has higher demands for improving the sensitivity and accuracy of the detection method. Compared with the common enzyme-linked immunoassay method, the chemiluminescence immunoassay (CLIA) is a detection method which has higher sensitivity and wider linear range and is more suitable for detecting the drug residue. It is a non-radioactive labeled immunoassay technology, which combines the luminescent immunoassay and the immune reaction to establish a novel labeled immunoassay technology for detecting trace antigens or antibodies.
There are related patent applications, such as CN201210049826.8, or cn201210049798. x. However, in order to meet the requirements of further lowering the detection limit, improving the sensitivity of the detection method, and improving the accuracy and precision of the detection kit, further improvement of the detection kit and the detection method is required.
Disclosure of Invention
In order to solve the technical problems, the inventor screens different monoclonal antibodies, obtains a monoclonal antibody with good effect after a large number of experiments, and prepares a chemical luminescence enzyme-linked immunoassay kit of kitasamycin with high sensitivity, high specificity, high reaction speed, high accuracy and high precision on the basis.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a chemical luminescence enzyme-linked immunoassay kit for kitasamycin comprises a kit body, an ELISA plate arranged in the kit body and a reagent arranged in the kit body;
the ELISA plate is a milky opaque polystyrene 96-hole chemiluminescence ELISA plate, each hole of the ELISA plate is coated with a coating antigen prepared by coupling kitasamycin and Ovalbumin (OVA), and the concentration of the coating antigen is preferably 1.0 mu g/mL. The coating solution is as follows: 1.75g of sodium carbonate and 2.72g of sodium bicarbonate were dissolved in 1L of water, and the pH was adjusted to 9.5. The enzyme label plate is prepared by putting the kitasamycin drug-OVA conjugate into a set coating solution, and carrying out reaction coating in a 37 ℃ thermostat, wherein the coating solution is sodium carbonate-sodium bicarbonate buffer solution with the pH value of-9.5. The micro-porous plate of the invention is wrapped inThe coated kitasamycin drug-OVA can be well combined on the plastic surface of a micropore plate in an alkaline environment and can be subjected to plate washing for many times, the concentration of the adopted coating antigen is 1.0 mu g/mL, the coated micropore plate can be sealed by a sealing solution, and the sealing solution is as follows: 10g OVA was dissolved in 1L of the washing solution, and 0.02% by weight of NaN was added3And then the product is obtained.
The reagent comprises: the kit comprises a kitasamycin monoclonal antibody, a horseradish peroxidase-labeled goat anti-mouse antibody, a kitasamycin series standard solution, a concentrated phosphate buffer solution, a concentrated washing solution and a chemiluminescent solution.
The sequence structures of the heavy chain variable region and the light chain variable region of the kitasamycin monoclonal antibody are respectively SEQ ID NO.1 and SEQ ID NO. 2. Specifically, SEQ ID NO.1 is EVKVEESGGGLVQPGGIVSPQGFKDEWCLLTLPMNWVRQSPEKGYYPMSRMRHCNGHSVTQLNTKAKGRFTMSRDDSKSSVYLQMSHMCMNMMNRQQCTSIYYDNLYYAMDYWGQGTSVTVSS, SEQ ID NO.2 is DILMTQSPASLSASVGHINWPCIGNLCSPSCYLAWYQQKQGKSPQLLVYYAKTLLFNNPITKYDMCLVERIAPSLKINSLRPCTGIWKMCWPLEVITFGGGTKLEIKR. The kitasamycin monoclonal antibody was diluted with the wash solution to 1: working concentration of 50000.
The goat anti-mouse antibody marked by horseradish peroxidase is horseradish peroxidase-goat anti-mouse IgG stock solution (purchased from Shanghai Langton bioscience, Ltd.), and is prepared into a washing solution for use, wherein the washing solution comprises the following components in percentage by weight: working concentration of 6000.
The kitasamycin series standard solution is obtained by diluting kitasamycin pure product with 0.05mmol/L, pH-7.5 PBS containing 10% methanol, and the concentrations of the kitasamycin standard product are 0 mug/L, 0.1 mug/L, 0.5 mug/L, 1.0 mug/L, 5 mug/L and 10 mug/L respectively, and the percentages are volume percentages.
The concentrated phosphate buffer solution is prepared from 5.24g NaH2PO4·2H2O and 33.6gNa2HPO4·12H2O is dissolved in 1L deionized water.
The concentrated washing solution is prepared by adding 0.05% of Tween-20 into 0.1mol/L salt buffer solution with the volume percentage of 0.05%, the pH value of 7.4 and the concentration.
The chemiluminescence solution comprises a solution A and a solution B, wherein the solution A is as follows: tris (hydroxymethyl) aminomethane solution with luminol 0.02M, p-cresol 0.002M, pH-8.8; the solution B is 100mL and contains 2.0g of citric acid and 2.35g of anhydrous Na2HPO4And 0.70mL of a 0.75% strength solution of carbamide peroxide.
The use method of the detection kit comprises the following steps:
1. sample pretreatment: removing muscle and kidney samples to be detected, and homogenizing the samples by using a homogenizer; weighing 1g of homogenized tissue sample into a 50mL polystyrene centrifuge tube, adding 5mL ethyl acetate, shaking for 15min by using an oscillator, and centrifuging for 10min at room temperature (about 25 ℃) at 4000 r/min; transferring 4mL of upper organic phase into a 10mL clean glass tube, drying by nitrogen in water bath at 50-55 ℃, adding 1mL of n-butane, whirling for 1min by using a vortex instrument, redissolving, whirling for 10S by using the vortex instrument, and centrifuging for 10min at the room temperature of 4000 r/min; the organic phase on the upper layer is removed, and the lower layer is taken out and placed in a 10mL centrifuge tube, water bath at 40 +/-5 ℃ and nitrogen blow-dried. Adding 2mL of PBS (pH 7.5), mixing uniformly in a vortex for 1min, and taking a clear water phase for detection.
2. A detection step:
1) sample adding: adding a kitasamycin series standard concentration solution or a sample solution with 50 mu L/hole into an enzyme label plate, then adding a kitasamycin antibody working solution with 50 mu L/hole, and incubating at room temperature (25 ℃) for 2.5 hours;
2) washing: pouring out the liquid in the hole, adding 280 mu L/hole of washing liquid into the ELISA plate, standing for 5min, then patting dry, and repeating for three times;
3) adding an enzyme-labeled secondary antibody: adding 100 mu L of enzyme-labeled secondary antibody working solution into each hole, and incubating at constant temperature for 1.5 h;
4) washing: pouring out the liquid in the hole, adding 280 mu L/hole of washing liquid into the ELISA plate, standing for 5min, then patting dry, and repeating for three times;
5) adding a luminescent liquid: adding 100 mu L of luminous liquid into each hole;
6) and (3) detection: the luminescence intensity of each well was measured with a chemiluminescence immunoassay analyzer.
The kitasamycin monoclonal antibody solution is an important factor for determining the measuring range and sensitivity of the kitasamycin enzyme-linked immunoassay kit. The kitasamycin monoclonal antibody solution provided by the invention can be diluted into 1: working concentration of 50000. The kit prepared according to the concentration of the kitasamycin monoclonal antibody solution can reach a good linear range.
The principle of the invention is to combine the high specificity of the antibody-antigen reaction with the high sensitivity of the enzyme catalysis, and to detect the product concentration by the chemiluminescence reaction of the enzyme catalysis substrate.
Has the advantages that: the chemiluminescence enzyme-linked immunoassay kit has the characteristics of high sensitivity, simplicity, rapidness and accuracy, compared with the traditional colorimetric ELISA method, the sensitivity can be improved by one order of magnitude, and the kit is expected to play an important role in kitasamycin drug residue detection in animal food.
Detailed Description
Example 1
Preparation of kitasamycin hapten, immunogen and monoclonal antibody
100mg of kitasamycin standard is weighed and dissolved in 50mL of methanol. The oxygen carboxymethyl light amine is weighed as 50mg, and 10mL of water is dissolved. The oxygen carboxymethyl light amine solution is slowly added into the kitasamycin methanol solution, and the reaction is stirred overnight. The reaction solution was rotary evaporated to dryness at 60 ℃. Accurately sucking 10mL of DMF to dissolve the evaporated material, adding 1g of dicyclohexylcarbodiimide, and stirring for reaction overnight to obtain the kitasamycin hapten.
Weighing 35mg carbonyl diimidazole, fully dissolving with 0.7ml acetone, adding 30mg kitasamycin hapten, oscillating at 37 ℃ for 2h, and drying in vacuum overnight; adding 10mg of BSA dissolved in 0.2mol/L pH8.0 boric acid buffer solution (1 ml), and shaking at 4 deg.C for 3.5 days; dialyzing with 0.2mol/L boric acid buffer solution with pH of 8.0 for 2d, adding thimerosal, and storing at 4 deg.C; and (3) identifying by adopting an ultraviolet scanning method, and confirming that the conjugate is successfully combined, thereby preparing the kitasamycin immunogen.
Animal immunization: dissolving the immunogen prepared by the method into normal saline according to 120 mu g/mouse, uniformly mixing the dissolved immunogen with Freund's complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection at the neck and back, uniformly mixing the immunogen with Freund's incomplete adjuvant in equal volume on 7 th, 14 th and 28 th days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using the immune compound 120 mu g/mouse 3 days before fusion without adding Freund's adjuvant.
Cell fusion: mixing splenocytes of immunized mice with myeloma cells (SP2/0) of mice in logarithmic growth phase, slowly adding preheated fusion agent (PEG4000) within 45 seconds for fusion, uniformly suspending with HAT culture medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate, culturing at 37 deg.C in 5% CO2 incubator, half-changing the culture medium with HT culture medium after 5 days, and completely changing the culture medium after 9 days.
Screening of hybridoma cells: after cell fusion, when the cells grow to 1/2 of the culture hole area, hybridoma cells are screened by a step screening method. And (3) primarily selecting by adopting an indirect ELISA method, coating an ELISA plate by coating antigen (the optimal coating concentration and the positive serum dilution are titrated by a square matrix method in advance), adding culture supernatant of a detected hole, incubating, washing, and then adding goat anti-mouse IgG-HRP, IgM-HRP and o-phenylenediamine (OPD) for color reaction. The screened positive hole is screened by an indirect competitive ELISA method, the cell supernatant is mixed with Sudan red with the volume equal to 100 mu g/mL, the mixture is subjected to water bath at 37 ℃ for 30min, and then the mixture is added into a coated enzyme label plate. The Sudan red control was also replaced with PBS and the rest was as above. If the OD450nm value after Sudan red blocking is reduced to below 50% of the control wells, the wells are judged to be positive, and the wells which are positive after 2-3 detections are subcloned immediately by a limiting dilution method.
Preparation of monoclonal antibody: performing expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernate, measuring titer by indirect ELISA, and freezing; injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of Balb/c mouse of 8-10 weeks old, injecting hybridoma cells 1-2 × 10 into the abdominal cavity after 7-10 days6Ascites from the mice are extracted 7 to 10 days later, the supernatant is centrifuged, the titer is determined, and the supernatant is frozen for later use.
Structural identification of the monoclonal antibody: the structure of the monoclonal antibody secreted by the hybridoma cells is sequenced by adopting a method which is conventional in the field, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively SEQ ID NO.1 and SEQ ID NO. 2. Specifically, SEQ ID NO.1 is EVKVEESGGGLVQPGGIVSPQGFKDEWCLLTLPMNWVRQSPEKGYYPMSRMRHCNGHSVTQLNTKAKGRFTMSRDDSKSSVYLQMSHMCMNMMNRQQCTSIYYDNLYYAMDYWGQGTSVTVSS, SEQ ID NO.2 is DILMTQSPASLSASVGHINWPCIGNLCSPSCYLAWYQQKQGKSPQLLVYYAKTLLFNNPITKYDMCLVERIAPSLKINSLRPCTGIWKMCWPLEVITFGGGTKLEIKR.
Example 2
Preparation of kitasamycin chemiluminescence enzyme-linked immunoassay kit
The chemiluminescent enzyme-linked immunoassay kit for preparing kitasamycin comprises a kit body, an ELISA plate arranged in the kit body and a reagent arranged in the kit body;
the ELISA plate is a milky opaque polystyrene 96-hole chemiluminescence ELISA plate, each hole of the ELISA plate is coated with a coating antigen prepared by coupling kitasamycin and Ovalbumin (OVA), and the concentration of the coating antigen is preferably 1.0 mu g/mL. The coating solution is as follows: 1.75g of sodium carbonate and 2.72g of sodium bicarbonate were dissolved in 1L of water, and the pH was adjusted to 9.5. The enzyme label plate is prepared by putting the kitasamycin drug-OVA conjugate into a set coating solution, and carrying out reaction coating in a 37 ℃ thermostat, wherein the coating solution is sodium carbonate-sodium bicarbonate buffer solution with the pH value of-9.5. The kitasamycin drug-OVA coated in the microporous plate can be well combined on the plastic surface of the microporous plate in an alkaline environment, and can be subjected to plate washing for many times, the concentration of the adopted coating antigen is 1.0 mu g/mL, the coated microporous plate can be sealed by a sealing solution, and the sealing solution is as follows: 10g OVA was dissolved in 1L of the washing solution, and 0.02% by weight of NaN was added3And then the product is obtained.
The reagent comprises: the kit comprises a kitasamycin monoclonal antibody, a horseradish peroxidase-labeled goat anti-mouse antibody, a kitasamycin series standard solution, a concentrated phosphate buffer solution, a concentrated washing solution and a chemiluminescent solution.
The sequence structures of the heavy chain variable region and the light chain variable region of the kitasamycin monoclonal antibody are SEQ ID NO.1 and SEQ ID NO. 2. Specifically, SEQ ID NO.1 is EVKVEESGGGLVQPGGIVSPQGFKDEWCLLTLPMNWVRQSPEKGYYPMSRMRHCNGHSVTQLNTKAKGRFTMSRDDSKSSVYLQMSHMCMNMMNRQQCTSIYYDNLYYAMDYWGQGTSVTVSS, SEQ ID NO.2 is DILMTQSPASLSASVGHINWPCIGNLCSPSCYLAWYQQKQGKSPQLLVYYAKTLLFNNPITKYDMCLVERIAPSLKINSLRPCTGIWKMCWPLEVITFGGGTKLEIKR. The kitasamycin monoclonal antibody was diluted with the wash solution to 1: working concentration of 50000.
The goat anti-mouse antibody marked by horseradish peroxidase is horseradish peroxidase-goat anti-mouse IgG stock solution (purchased from Shanghai Langton bioscience, Ltd.), and is prepared into a washing solution for use, wherein the washing solution comprises the following components in percentage by weight: working concentration of 6000.
The kitasamycin series standard solution is obtained by diluting kitasamycin pure product with 0.05mmol/L, pH-7.5 PBS containing 10% methanol, and the concentrations of the kitasamycin standard product are 0 mug/L, 0.1 mug/L, 0.5 mug/L, 1.0 mug/L, 5 mug/L and 10 mug/L respectively, and the percentages are volume percentages.
The concentrated phosphate buffer solution is prepared from 5.24g NaH2PO4·2H2O and 33.6gNa2HPO4·12H2O is dissolved in 1L deionized water.
The concentrated washing solution is prepared by adding 0.05% of Tween-20 into 0.1mol/L salt buffer solution with the volume percentage of 0.05%, the pH value of 7.4 and the concentration.
The chemiluminescence solution comprises a solution A and a solution B, wherein the solution A is as follows: tris (hydroxymethyl) aminomethane solution with luminol 0.02M, p-cresol 0.002M, pH-8.8; the solution B is 100mL and contains 2.0g of citric acid and 2.35g of anhydrous Na2HPO4And 0.70mL of a 0.75% strength solution of carbamide peroxide.
The use method of the detection kit comprises the following steps:
1. sample pretreatment: removing muscle and kidney samples to be detected, and homogenizing the samples by using a homogenizer; weighing 1g of homogenized tissue sample into a 50mL polystyrene centrifuge tube, adding 5mL ethyl acetate, shaking for 15min by using an oscillator, and centrifuging for 10min at room temperature (about 25 ℃) at 4000 r/min; transferring 4mL of upper organic phase into a 10mL clean glass tube, drying by nitrogen in water bath at 50-55 ℃, adding 1mL of n-butane, whirling for 1min by using a vortex instrument, redissolving, whirling for 10S by using the vortex instrument, and centrifuging for 10min at the room temperature of 4000 r/min; the organic phase on the upper layer is removed, and the lower layer is taken out and placed in a 10mL centrifuge tube, water bath at 40 +/-5 ℃ and nitrogen blow-dried. Adding PBS 2mL, mixing for 1min by vortex, taking the clear water phase for detection.
2. A detection step:
1) sample adding: adding a kitasamycin series standard concentration solution or a sample solution with 50 mu L/hole into an enzyme label plate, then adding a kitasamycin antibody working solution with 50 mu L/hole, and incubating at room temperature (25 ℃) for 2.5 hours;
2) washing: pouring out the liquid in the hole, adding 280 mu L/hole of washing liquid into the ELISA plate, standing for 5min, then patting dry, and repeating for three times;
3) adding an enzyme-labeled secondary antibody: adding 100 mu L of enzyme-labeled secondary antibody working solution into each hole, and incubating at constant temperature for 1.5 h;
4) washing: pouring out the liquid in the hole, adding 280 mu L/hole of washing liquid into the ELISA plate, standing for 5min, then patting dry, and repeating for three times;
5) adding a luminescent liquid: adding 00 μ L of luminescent solution into each well;
6) and (3) detection: the luminescence intensity of each well was measured with a chemiluminescence immunoassay analyzer.
Example 3
Guitar mycin residue detection by using kitasamycin chemiluminescence enzyme-linked immunoassay kit
The use method of the detection kit comprises the following steps:
1. sample pretreatment: taking a sample to be detected, homogenizing the sample (a muscle sample, a liver sample or a kidney sample of a pig or poultry) by using a homogenizer, weighing 1g of the homogenized tissue sample into a 50mL polystyrene centrifuge tube, adding 5mL ethyl acetate, oscillating for 15min by using an oscillator, and centrifuging for 10min at the room temperature (about 25 ℃) of 4000 r/min; transferring 4mL of upper organic phase into a 10mL clean glass tube, drying by nitrogen in water bath at 50-55 ℃, adding 1mL of n-butane, whirling for 1min by using a vortex instrument, redissolving, whirling for 10S by using the vortex instrument, and centrifuging for 10min at the room temperature of 4000 r/min; the organic phase on the upper layer is removed, and the lower layer is taken out and placed in a 10mL centrifuge tube, water bath at 40 +/-5 ℃ and nitrogen blow-dried. Adding 2mL of PBS (pH 7.5), mixing uniformly in a vortex for 1min, and taking a clear water phase for detection.
2. A detection step:
1) sample adding: adding a kitasamycin series standard concentration solution or a sample solution with 50 mu L/hole into an enzyme label plate, then adding a kitasamycin antibody working solution with 50 mu L/hole, and incubating at room temperature (25 ℃) for 2.5 hours;
2) washing: pouring out the liquid in the hole, adding 280 mu L/hole of washing liquid into the ELISA plate, standing for 5min, then patting dry, and repeating for three times;
3) adding an enzyme-labeled secondary antibody: adding 100 mu L of enzyme-labeled secondary antibody working solution into each hole, and incubating at constant temperature for 1.5 h;
4) washing: pouring out the liquid in the hole, adding 280 mu L/hole of washing liquid into the ELISA plate, standing for 5min, then patting dry, and repeating for three times;
5) adding a luminescent liquid: adding 100 mu L of luminous liquid into each hole;
6) and (3) detection: the luminescence intensity of each well was measured with a chemiluminescence immunoassay analyzer.
The measured luminescence value of the standard was divided by the luminescence value of the first standard (0 standard) and multiplied by 100 to obtain the inhibition ratio (B/B0), and the inhibition ratio was plotted as ordinate and the logarithm of the kitasamycin concentration was plotted as abscissa, and the concentration of each sample was read from the standard curve.
Example 4
Quality test of kitasamycin chemiluminescence enzyme-linked immunoassay kit
Weighing homogenized pig muscle, pig liver, chicken muscle and chicken liver samples, adding kitasamycin standard solution, and making the concentration of the added medicine in the tissue be 0.05, 0.1, 0.5 and 1 time of the maximum residual limit. The samples were treated according to the pretreatment method of example 3 and tested according to the test method of example 3.5 replicates were assayed for each sample concentration and repeated 3 times at different times. The average recovery, standard deviation, and intra-and inter-batch coefficient of variation were calculated. The results are shown in Table 1. Therefore, the kit has the advantages that the addition recovery rate of kitasamycin in tissues is 90-110%, and the intra-batch and inter-batch variation coefficients are less than 15%.
TABLE 1 Guitar mycin addition recovery and coefficient of variation
The results show that the kitasamycin chemiluminescence enzyme-linked immunoassay kit is superior to the existing detection kit in terms of detection limit, precision and accuracy. Such superior results result from the optimal selection of the monoclonal antibody used in the present invention, as well as the other components and amounts in the kit. Therefore, the kitasamycin chemiluminescence enzyme-linked immunoassay kit has good application prospect.
Claims (1)
1. The kit is characterized by comprising a kit body, an ELISA plate arranged in the kit body and a reagent arranged in the kit body;
the ELISA plate is a milky opaque polystyrene 96-hole chemiluminescent ELISA plate, each hole of the ELISA plate is coated with a coating antigen prepared by coupling kitasamycin and ovalbumin, and the concentration of the coating antigen is preferably 1.0 mu g/mL; the coating solution is as follows: 1.75g of sodium carbonate and 2.72g of sodium bicarbonate are dissolved in 1L of water, and the pH value is adjusted to 9.5; the enzyme label plate is prepared by putting the kitasamycin drug-ovalbumin conjugate into a set coating solution, and carrying out reaction coating in a 37 ℃ thermostat, wherein the coating solution is sodium carbonate-sodium bicarbonate buffer solution with the pH value of-9.5; the concentration of the adopted coating antigen is 1.0 mug/mL, and the coated microporous plate can be sealed by a sealing solution which is: dissolving 10g egg albumin in 1L washing solution, and adding 0.02 wt% NaN3Then the product is prepared;
the reagent arranged in the box body comprises: the kit comprises a kitasamycin monoclonal antibody, a horse radish peroxidase-labeled goat anti-mouse antibody, a kitasamycin series standard solution, a concentrated phosphate buffer solution, a concentrated washing solution and a chemiluminescent solution;
the sequence structures of the heavy chain variable region and the light chain variable region of the kitasamycin monoclonal antibody are respectively SEQ ID NO.1 and SEQ ID NO. 2; the kitasamycin monoclonal antibody was diluted with the wash solution to 1: a working concentration of 50000; specifically, SEQ ID NO.1 is EVKVEESGGGLVQPGGIVSPQGFKDEWCLLTLPMNWVRQSPEKGYYPMSRMRHCNGHSVTQLNTKAKGRFTMSRDDSKSSVYLQMSHMCMNMMNRQQCTSIYYDNLYYAMDYWGQGTSVTVSS, SEQ ID NO.2 is DILMTQSPASLSASVGHINWPCIGNLCSPSCYLAWYQQKQGKSPQLLVYYAKTLLFNNPITKYDMCLVERIAPSLKINSLRPCTGIWKMCWPLEVITFGGGTKLEIKR;
the goat anti-mouse antibody marked by the horseradish peroxidase is horseradish peroxidase-goat anti-mouse IgG stock solution, and when the goat anti-mouse IgG stock solution is used, a washing solution is prepared into a solution with the weight ratio of 1: a working concentration of 6000;
the kitasamycin series standard solution is obtained by diluting kitasamycin pure product with 0.05mmol/L, pH-7.5 PBS containing 10% methanol, the concentrations of the kitasamycin standard product are respectively 0 mug/L, 0.1 mug/L, 0.5 mug/L, 1.0 mug/L, 5 mug/L and 10 mug/L, and the percentages are volume percentages;
the concentrated phosphate buffer solution is prepared from 5.24g NaH2PO4·2H2O and 33.6g Na2HPO4·12H2Dissolving O in 1L deionized water to obtain the product;
the concentrated washing solution is prepared by adding 0.05 percent of tween-20 into 0.1mol/L salt buffer solution according to the volume percentage, the pH value of 7.4 and the concentration of 0.1 mol/L;
the chemiluminescence solution comprises a solution A and a solution B, wherein the solution A is as follows: tris (hydroxymethyl) aminomethane solution with luminol 0.02M, p-cresol 0.002M, pH-8.8; the solution B is 100mL and contains 2.0g of citric acid and 2.35g of anhydrous Na2HPO4And 0.70mL of a 0.75% strength solution of carbamide peroxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610877176.4A CN107677660B (en) | 2016-09-30 | 2016-09-30 | Kitasamycin chemiluminescence enzyme-linked immunoassay kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610877176.4A CN107677660B (en) | 2016-09-30 | 2016-09-30 | Kitasamycin chemiluminescence enzyme-linked immunoassay kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107677660A CN107677660A (en) | 2018-02-09 |
| CN107677660B true CN107677660B (en) | 2020-11-24 |
Family
ID=61133271
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610877176.4A Active CN107677660B (en) | 2016-09-30 | 2016-09-30 | Kitasamycin chemiluminescence enzyme-linked immunoassay kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107677660B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102042964A (en) * | 2010-11-12 | 2011-05-04 | 武汉人福药业有限责任公司 | Method for detecting dissolution rates of acetylkitasamycin capsules |
| CN103288963A (en) * | 2012-02-29 | 2013-09-11 | 华中农业大学 | Monoclonal antibody for kitasamycin residual detection and preparation method and application thereof |
| CN103645322A (en) * | 2013-12-12 | 2014-03-19 | 洛阳莱普生信息科技有限公司 | Chemiluminescent enzyme-linked immunosorbent assay (ELISA) kit for streptomycin |
| CN104988206A (en) * | 2015-06-09 | 2015-10-21 | 姜洪波 | Ureaplasma urealyticum/mycoplasma hominis combined rapid culture and drug sensitivity detection kit |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9175086B2 (en) * | 2010-02-10 | 2015-11-03 | Immunogen, Inc. | CD20 antibodies and uses thereof |
| CN103288960B (en) * | 2012-02-29 | 2015-01-07 | 华中农业大学 | Gene engineering antibody for kitasamycin residue detection, and preparation method and application thereof |
| CN106405088A (en) * | 2015-08-03 | 2017-02-15 | 镇江亿特生物科技发展有限公司 | Immunocolloidal-gold detection card for kitasamycin and preparation method thereof |
-
2016
- 2016-09-30 CN CN201610877176.4A patent/CN107677660B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102042964A (en) * | 2010-11-12 | 2011-05-04 | 武汉人福药业有限责任公司 | Method for detecting dissolution rates of acetylkitasamycin capsules |
| CN103288963A (en) * | 2012-02-29 | 2013-09-11 | 华中农业大学 | Monoclonal antibody for kitasamycin residual detection and preparation method and application thereof |
| CN103645322A (en) * | 2013-12-12 | 2014-03-19 | 洛阳莱普生信息科技有限公司 | Chemiluminescent enzyme-linked immunosorbent assay (ELISA) kit for streptomycin |
| CN104988206A (en) * | 2015-06-09 | 2015-10-21 | 姜洪波 | Ureaplasma urealyticum/mycoplasma hominis combined rapid culture and drug sensitivity detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107677660A (en) | 2018-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2008098438A1 (en) | Elisa test kit of detecting a nitrofurazone metabolite | |
| CN1811436B (en) | Method for detecting Ennoxacin and its special enzyme-linked immune reagent kit | |
| CN101639477B (en) | ELISA detection method for doxycycline remnant, kit and application | |
| CN102967709A (en) | Enzyme linked immunosorbent assay kit for detecting zearalenone drug and application thereof | |
| CN105131121B (en) | Detect monoclonal antibody, ELISA method and the kit of Furaxone metabolite | |
| CN101299045B (en) | Method for detecting florfenicol and florfenicol amine and special-purpose enzyme-linked immunologic reagent kit thereof | |
| CN106831498B (en) | Furacilin metabolite SEM derivatizations haptens, artificial antigen preparation method and applications | |
| CN105566493B (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting florfenicol | |
| CN108178777B (en) | Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof | |
| CN101358967A (en) | Method for detecting chlorpromazine and special ELISA kit thereof | |
| Samsonova et al. | Enzyme-linked immunosorbent assay of ampicillin in milk | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| CN105273021B (en) | Erythromycin haptens, artificial antigen and antibody and its preparation method and application | |
| CN103645322B (en) | A kind of chemical luminescence ELISA detection kit of streptomysin | |
| CN106645764B (en) | Detect enzyme linked immunological kit and its application of diazepam | |
| CN107677807A (en) | A kind of kitasamycin magnetic immunochemiluminescence detection kit | |
| CN107677660B (en) | Kitasamycin chemiluminescence enzyme-linked immunoassay kit | |
| CN105524174B (en) | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting thiamphenicol and florfenicol | |
| JP2009031024A (en) | Measuring method of antigen or antibody | |
| CN103509068B (en) | Amikacin haptens and its preparation method and application | |
| CN104558187A (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cephalosporin antibiotics | |
| CN109734675B (en) | Method and product suitable for detecting olaquindox content in veterinary drug preparation | |
| CN111377888B (en) | Rhododendrin mollis toxin III hapten as well as preparation method and application thereof | |
| CN103420879B (en) | Dapsone hapten and its preparation method and application | |
| CN109061168B (en) | Enzyme linked immunosorbent assay kit for detecting diclazuril and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20201105 Address after: No. 170, Meihe jiulu, Tongan District, Xiamen City, Fujian Province 361000 Applicant after: XIAMEN ZHONGJI TESTING TECHNOLOGY Co.,Ltd. Address before: 266071 Shandong city of Qingdao province Ningxia City Road No. 308 Applicant before: QINGDAO University |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |