CN101710122A - Method for quickly immunologically detecting beta-lactamase in milk - Google Patents
Method for quickly immunologically detecting beta-lactamase in milk Download PDFInfo
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- CN101710122A CN101710122A CN200910175355A CN200910175355A CN101710122A CN 101710122 A CN101710122 A CN 101710122A CN 200910175355 A CN200910175355 A CN 200910175355A CN 200910175355 A CN200910175355 A CN 200910175355A CN 101710122 A CN101710122 A CN 101710122A
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Abstract
The invention provides a method for quickly immunologically detecting a beta-lactamase in milk, and belongs to the technical field of immunological detection. The method is characterized in that: a three-antibody sandwich method reagent consists of a rat anti-beta-lactamase monoclonal antibody coated on an elisa plate, a rabbit anti-beta-lactamase polyclonal antibody and a horseradish peroxidase labelled sheep and rabbit antibody; and immunological detection is performed to detect the beta-lactamase in a raw milk by a three-antibody sandwich method. Compared with the prior art, the method can accurately, specially and quickly immunologically detect the beta-lactamase illegally added into the milk so as to timely monitor and control the quality of the raw milk from the source and guarantee the safety of the milk and the health of human, and is good in social and economic efficiency.
Description
Technical field
The invention belongs to technical field of immunoassay, specifically is the ELISA detection method of the beta-lactamase that adds in a kind of animal food safety or the animal husbandry production field raw milk.
Background technology
At present, penicillin is the choice drug of treatment bovine mastitis as Beta-lactam medicine, is modal residual antibiotic in the milk.Because the cow's milk that domestic most dairy products enterprise exceeds standard to antibiotic residue is taked the principle refusing to purchase, driving for economic interests, some illegal milking stations are in order to seek the economic interests of oneself, use some biopreparates residual microbiotic in the cow's milk that goes to degrade artificially, produce so-called artificial " antibiotic-free milk ".2005 so far, and the existing company of counting avows the anti-agent of separating of selling break down bovine Ruzhong residual antibiotic.This principal ingredient of separating anti-agent is a beta-lactamase, and it is produced by gram-positive bacterium and secretion, residual beta-lactam antibiotic in the alternative decomposing milk.Beta-lactamase is that China does not allow the food additives that use, and the actual microbiotic that contains in the milk has been covered in the use of this enzyme.The beta-lactam endonuclease capable makes the structural failure of penicillin lactams and loses activity, and causes antibiotics drug resistances such as penicillin, cynnematin to increase, thereby greatly reduces the ability that people resist infectious disease, and that gives the consumer healthyly brings harm.
Current, the inspection technology of beta-lactamase in the milk has several methods such as cylinder plate method, iodimetric titration and acidity method, but all lacks specificity.And beta-lactamase has endogenous and exogenous difference.Exogenous artificial the interpolation endangers huge; Endogenic amount seldom but can be to the result who utilizes above-mentioned several method to detect, and produces and disturbs, and measuring error is big.Still do not have at present a kind of can be accurate, special and the immune detection method of illegally adding the beta-lactamase in the milk to apace.
Summary of the invention
Purpose of the present invention provides a kind of tachysynthesis to detect the method for beta-lactamase in the milk, utilize this method, can be accurate, special and apace immune detection illegally add beta-lactamase in the milk to, so that from the source, in time monitor and a former milk quality of control, guarantee the security and the human health of milk, society and business efficiency are good.
The present invention seeks to realize like this: a kind of tachysynthesis detects the method for beta-lactamase in the milk, it is characterized in that utilizing bag by the mouse-anti beta-lactamase monoclonal antibody on ELISA Plate, form tri-antibody sandwich (Tas-ELISA) method reagent with rabbit anti-beta-lactamase polyclonal antibody and horseradish peroxidase-labeled goat anti-rabbit antibody, carry out tri-antibody sandwich (Tas-ELISA) method immunology detection, detect the beta-lactamase in the raw milk.
Described mouse-anti beta-lactamase monoclonal antibody is to adopt the preparation of beta-lactamase immunity BALB/c mouse.
Tiring of described mouse-anti beta-lactamase monoclonal antibody is 1: 655 ten thousand～1,310 ten thousand, and hypotype is IgG1, and affinity is 1.000 * 10
9L/mol～1.046 * 10
10L/mol, preferable.
Described rabbit anti-beta-lactamase polyclonal antibody is to adopt the preparation of beta-lactamase immunity new zealand white rabbit, or preparation such as Japan large ear rabbit.
Described rabbit anti-beta-lactamase polyclonal antibody is tired and is 1: 320 ten thousand～6,550,000, and is preferable.
After all adopting the albumin A purifying, described mouse-anti beta-lactamase monoclonal antibody and rabbit anti-beta-lactamase polyclonal antibody use, and preferable.
The present invention compared with prior art has following advantage:
One, high specificity: because mouse-anti monoclonal antibody and the anti-polyclonal antibody of rabbit all are special, only the beta-lactamase that adds in the raw milk is had positive reaction, the interference of its endogenic beta-lactamase can be excluded, the accuracy height.
Two, detection is quick, cost is low: the detection of raw milk sample can once can detect 96 samples about 3 hours, and because agents useful for same is conventional reagent, does not need specific installation, and general basic unit all can carry out in the inspection laboratory, and cost is low.
Three, social benefit is good: realize inspection, monitoring to raw milk from the source of food security, guarantee numerous consuming public's safe diet, help people health.
Description of drawings
Accompanying drawing is a detection principle schematic of the present invention.
1. ELISA Plate among the figure, 2. mouse-anti beta-lactamase monoclonal antibody, 3. beta-lactamase, 4. rabbit anti-beta-lactamase polyclonal antibody, 5. horseradish peroxidase-labeled goat anti-rabbit antibody.
Embodiment
For better implement the present invention, as follows especially exemplified by preferred embodiment.
Adopt beta-lactamase immune BALB/c mouse and new zealand white rabbit respectively, prepared mouse-anti beta-lactamase monoclonal antibody and rabbit anti-beta-lactamase polyclonal antibody.Tiring of mouse-anti beta-lactamase monoclonal antibody is 1: 1,310 ten thousand, and hypotype is that IgG1 affinity is 1.046 * 10
10L/mol with other related substances reaction negatives, only has positive reaction with the antigen beta-lactamase; It is 1: 655 ten thousand that the rabbit anti-beta-lactamase is tired, only with antigen beta-lactamase reacting positive.Monoclonal antibody and polyclonal antibody are used after all adopting the albumin A purifying.The mouse-anti beta-lactamase monoclonal antibody that is used for wrapping quilt can realize the tri-antibody sandwich method of raw milk beta-lactamase is detected with rabbit anti-beta-lactamase polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit antibody.Detection sensitivity is 40U/mL.The detection step of this invention is:
1, with 0.01mol/L pH9.6 carbonate buffer solution monoclonal antibody is diluted to 5 μ g/mL coated elisa plates, every hole 100 μ L, 4 ℃ are spent the night;
2, wash plate 3 times with PBS/T20, every hole 200 μ L, each 3 minutes;
3, with the PBS/T20 sealing that contains 3% calf serum, every hole 200 μ L were hatched 1 hour for 37 ℃;
4, wash plate, with step 2; Add milk sample, every hole 100 μ L were hatched 30 minutes for 37 ℃;
5, wash plate, with step 2; The polyclonal antibody that adds 0.75mg/L, every hole 100 μ L, 37 ℃, 30 minutes;
6, wash plate, with step 2; The horseradish peroxidase-labeled goat anti-rabbit antibody that adds 0.08mg/L, every hole 100 μ L, 37 ℃, 30 minutes;
7, wash plate, with step 2; Add tetramethyl benzidine (TMB) colour developing liquid, every hole 100 μ L, 37 ℃ of lucifuge reactions added 50 μ L 2mol/L H after 15 minutes
2SO
4Solution; O.D. value when reading wavelength and being 450nm.
8, criterion: positive threshold value=2.1 * blank, blank＜0.2.More than or equal to positive threshold value, then contain beta-lactamase in this sample as sample O.D. value.
9, result: the blank average equals 0.106, and positive threshold value is 0.223.
Sample one: the O.D. value is 0.248, then contains the beta-lactamase of interpolation in this sample;
Sample two: the O.D. value is 0.101, does not then contain the beta-lactamase of interpolation in this sample.
Claims (6)
1. a tachysynthesis detects the method for beta-lactamase in the milk, it is characterized in that utilizing bag by the mouse-anti beta-lactamase monoclonal antibody on ELISA Plate, form tri-antibody sandwich method reagent with rabbit anti-beta-lactamase polyclonal antibody and horseradish peroxidase-labeled goat anti-rabbit antibody, carry out tri-antibody sandwich method immunology detection, detect the beta-lactamase in the raw milk.
2. a kind of tachysynthesis according to claim 1 detects the method for beta-lactamase in the milk, it is characterized in that described mouse-anti beta-lactamase monoclonal antibody is to adopt the preparation of beta-lactamase immunity BALB/c mouse.
3. a kind of tachysynthesis according to claim 2 detects the method for beta-lactamase in the milk, it is characterized in that tiring of described mouse-anti beta-lactamase monoclonal antibody is 1: 655 ten thousand～1,310 ten thousand, and hypotype is IgG1, and affinity is 1.000 * 10
9L/mol～1.046 * 10
10L/mol.
4. a kind of tachysynthesis according to claim 1 detects the method for beta-lactamase in the milk, it is characterized in that described rabbit anti-beta-lactamase polyclonal antibody is to adopt the preparation of beta-lactamase immunity new zealand white rabbit.
5. a kind of tachysynthesis according to claim 4 detects the method for beta-lactamase in the milk, it is characterized in that it is 1: 320 ten thousand～6,550,000 that described rabbit anti-beta-lactamase polyclonal antibody is tired.
6. a kind of tachysynthesis according to claim 1 detects the method for beta-lactamase in the milk, it is characterized in that using after described mouse-anti beta-lactamase monoclonal antibody and rabbit anti-beta-lactamase polyclonal antibody all adopt the albumin A purifying.
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| CN2009101753553A CN101710122B (en) | 2009-12-16 | 2009-12-16 | Method for quickly immunologically detecting beta-lactamase in milk |
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| CN2009101753553A CN101710122B (en) | 2009-12-16 | 2009-12-16 | Method for quickly immunologically detecting beta-lactamase in milk |
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| CN101710122B CN101710122B (en) | 2013-08-28 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893631A (en) * | 2010-06-28 | 2010-11-24 | 中国检验检疫科学研究院 | Detection method for arabis mosaic virus and special kit |
| CN101923093A (en) * | 2010-07-13 | 2010-12-22 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN101936998A (en) * | 2010-08-19 | 2011-01-05 | 武汉中博生物股份有限公司 | Antirabies virus IgG (Immunoglobulin G) antibody ELISA (Enzyme Linked Immunosorbent Assay) detection kit for dogs |
| CN101936997A (en) * | 2010-08-19 | 2011-01-05 | 武汉中博生物股份有限公司 | Human anti-rabies virus IgG antibody ELISA test kit |
| CN102495211A (en) * | 2011-12-07 | 2012-06-13 | 河北省科学院生物研究所 | Beta-fructofuranosidase enzyme-linked immunoassay detection kit |
| CN103134937A (en) * | 2013-01-05 | 2013-06-05 | 江南大学 | Double antibody sandwich method of detecting beta-lactamase of milk |
| CN106093403A (en) * | 2016-05-26 | 2016-11-09 | 东北农业大学 | A kind of method with double antibody sandwich ELISA detection beta lactamase of optimization |
| CN108445217A (en) * | 2018-03-08 | 2018-08-24 | 山东绿都生物科技有限公司 | A kind of fluorescent microsphere detection card quantitatively detecting beta-lactamase residues in milk |
-
2009
- 2009-12-16 CN CN2009101753553A patent/CN101710122B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 韩奕奕等: "牛乳中三聚氰胺和β-内酰胺酶快速检测方法的评估——双流向酶联免疫法", 《中国奶牛》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893631A (en) * | 2010-06-28 | 2010-11-24 | 中国检验检疫科学研究院 | Detection method for arabis mosaic virus and special kit |
| CN101923093A (en) * | 2010-07-13 | 2010-12-22 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN101923093B (en) * | 2010-07-13 | 2013-12-18 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN101936998A (en) * | 2010-08-19 | 2011-01-05 | 武汉中博生物股份有限公司 | Antirabies virus IgG (Immunoglobulin G) antibody ELISA (Enzyme Linked Immunosorbent Assay) detection kit for dogs |
| CN101936997A (en) * | 2010-08-19 | 2011-01-05 | 武汉中博生物股份有限公司 | Human anti-rabies virus IgG antibody ELISA test kit |
| CN102495211A (en) * | 2011-12-07 | 2012-06-13 | 河北省科学院生物研究所 | Beta-fructofuranosidase enzyme-linked immunoassay detection kit |
| CN102495211B (en) * | 2011-12-07 | 2015-05-20 | 河北省科学院生物研究所 | Beta-fructofuranosidase enzyme-linked immunoassay detection kit |
| CN103134937A (en) * | 2013-01-05 | 2013-06-05 | 江南大学 | Double antibody sandwich method of detecting beta-lactamase of milk |
| CN106093403A (en) * | 2016-05-26 | 2016-11-09 | 东北农业大学 | A kind of method with double antibody sandwich ELISA detection beta lactamase of optimization |
| CN108445217A (en) * | 2018-03-08 | 2018-08-24 | 山东绿都生物科技有限公司 | A kind of fluorescent microsphere detection card quantitatively detecting beta-lactamase residues in milk |
| CN108445217B (en) * | 2018-03-08 | 2021-12-10 | 山东绿都生物科技有限公司 | Fluorescent microsphere detection card for quantitatively detecting beta-lactamase residue in milk |
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