CN103792374A - Chemiluminesent immunoassay kit for detection of bisphenol A - Google Patents
Chemiluminesent immunoassay kit for detection of bisphenol A Download PDFInfo
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- CN103792374A CN103792374A CN201210432991.1A CN201210432991A CN103792374A CN 103792374 A CN103792374 A CN 103792374A CN 201210432991 A CN201210432991 A CN 201210432991A CN 103792374 A CN103792374 A CN 103792374A
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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Abstract
Belonging to the technical field of industrial compound environmental hormone detection, the invention provides a chemiluminesent immunoassay kit for detection of bisphenol A. The kit provided by the invention is composed of a non-transparent white enzyme-labeled plate coated with a bisphenol A-carrier protein conjugate, a bisphenol A standard substance, a bisphenol A-peroxidase labeled antibody working solution, a luminescent substrate solution, a concentrated sample dilution solution, and a concentrated washing solution. The bisphenol A-carrier protein conjugate is obtained by coupling bisphenol A and carrier protein through a mixed anhydride method or a carbodiimide method, and the concentrated washing solution contains 0.05% Tween-20. Compared with the traditional enzyme linked immunosorbent assay method, the kit provided by the invention has the advantages of higher sensitivity, short detection time and low cost, and can be used for detection of residual bisphenol A in an environmental water sample.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune detection reagent kit that detects bisphenol-A, for detection of the content of bisphenol A in environmental water sample or residual quantity.Belong to industrial compound class environmental hormone detection technique field.
Background technology
Bisphenol-A (BPA) is the starting material of polycarbonate, epoxy resin and phenolics, is to use in the world one of industrial compound widely.In packaging for foodstuff and container inner wall application, be widely used, in the manufacture process of fluid sealant, lens and other hundreds of commodity as used in the packing of tinned food and beverage, feeding bottle, water bottle, tooth filling.BPA enters water environment by atmosphere or sewage in production and use procedure, then under the effect of microorganism, decomposes gradually, and its half life period is approximately 2. 5～4. 0 d.BPA belongs to incretion interferent.Recent study shows, BPA has estrogen active, and the trace even concentration of trace also may cause harmful effect to animal physiological situation, reproductive system and development of fetus.Under some contaminated environment, BPA can disturb the endocrine function of multiple biology, affects wildlife safety, even threatens human life's health.There is data to show that bisphenol-A has certain embryotoxicity and teratogenesis, can obviously increase the generation of the cancers such as animal ovary cancer, prostate cancer, leukaemia.Therefore BPA has been listed in the blacklist of priority pollutant by some countries of European Union.Along with production and the use of industry and expanding economy BPA roll up, whole world BPA use amount has exceeded 2,000,000 tons, its final home to return to is to enter water environment by various approach, and is acted in aquatic ecosystem and transmitted by food chain, and hydrobiont is produced to endocrine.Therefore, in water sample, the detection technique research tool of bisphenol-A is of great significance.
Bisphenol-A has multiple analysis test method, mainly contain at present spectrophotometric method and fluorometry, capillary electrophoresis, vapor-phase chromatography gentle-matter coupling method, liquid phase chromatography and liquid-matter coupling method etc., it is high that these methods detect degree of accuracy, the features such as inspection positive rate is low, but detecting instrument is expensive, operation is more loaded down with trivial details, length consuming time, testing cost is high.Enzyme linked immunosorbent assay analysis method is one of detection technique that current application is the widest, and major advantage is that detection speed is fast, and sample pre-treatments is simple, simple to operate, and testing cost is low, is convenient to the detection for batch samples simultaneously.Wei Kongji etc. have set up fast, the biotin-avidin amplification enzyme linked immunosorbent assay of Sensitive Detection bisphenol-A, envelope antigen concentration 13. 8 mg/L, antibody dilution 2. 4 × 10
5doubly, biotinylation two optimum diluting multiple anti-and enzyme mark Avidin is respectively 2000 times and 500 times, the range of linearity 0. 2 g/L ~ 1000 g/L of method; Minimum detectability 0. 05 g/L; Method is for the mensuration of food and saliva content of bisphenol A, and sample preparation is simple, and result is satisfied.
Chemiluminescence immunoassay detection technique is the product of chemoluminescence method and immunoassay combination, therefore has the high sensitivity of chemiluminescence detection technology and the high specific of immuno analytical method simultaneously.The present invention has the bisphenol-A antibody of high-affinity, high specific by distinctive immune animal preparation, and adopts enzymic-labelled antibody, sets up a kind of chemiluminescence immunoassay kit that can detect bisphenol-A in water sample.The feature such as that this method has is easy and simple to handle, quick, highly sensitive, specificity is good.
Summary of the invention
For problems of the prior art, the present invention utilizes the ultimate principle of the specific immune response of antigen and antibody to realize.Chemiluminescence immune assay is the product of chemoluminescence method and immunoassay combination, therefore has the high sensitivity of chemoluminescence method and the high specific of immunoassay simultaneously.In whole course of reaction, in sample, content of bisphenol A is higher, and in reaction system, luminous intensity is more weak; Otherwise content of bisphenol A is fewer in sample, luminous intensity is higher.
The present invention is a kind of chemiluminescence immune detection reagent kit that detects bisphenol-A, it is characterized in that containing following composition:
1, be coated with the opaque white color ELISA Plate of bisphenol-A-carrier protein couplet thing; Described bisphenol-A-carrier protein couplet thing is that bisphenol-A and carrier protein are obtained by mixed anhydride method or carbodlimide method coupling, and described carrier protein is ovoserum albumin, bovine serum albumin(BSA);
2, bisphenol-A standard items;
3, bisphenol-A-peroxidase labeled antibodies: this composition is the bisphenol-A antibody with peroxidase labelling, and described bisphenol-A antibody is monoclonal antibody or polyclonal antibody;
4, luminous substrate liquid: this luminous substrate liquid is the Chemoluminescent substrate take the different luminol of luminol goods as luminous agent, is divided into A liquid and B liquid and preserves, and presses before use 1:1 and mixes use; Wherein A liquid level luminescence enhancer adds luminol goods luminescence enhancer and adds different luminol, and B liquid is superoxol or urea hydrogen peroxide solution;
5,2 times of concentrating sample dilutions;
6,20 times of concentrated cleaning solutions.
In the present invention, described opaque white color ELISA Plate is the detachable or non-removable opaque white color ELISA Plate in 96 holes.
In the present invention, described bisphenol-A standard items, are made up of the bisphenol-A standard items of a series of variable concentrations, and concentration is 0 ng/mL, 0.05 ng/mL, 0.15 ng/mL, 0.45 ng/mL, 1.35 ng/mL, 4.05 ng/mL.
In the present invention, described bisphenol-A-peroxidase labeled antibodies is the bisphenol-A antibody with peroxidase labelling, as the bisphenol-A antibody of horseradish peroxidase (HRP) mark.
In the present invention, described luminous substrate liquid is commercial any Chemoluminescent substrate take the different luminol of luminol goods as luminous agent.
In the present invention, described 2 times of concentration and dilution liquid, its composition is 0.01mol/L, phosphate buffer, glycocoll-HCl damping fluid or the Tris-HCl damping fluid of pH7.4 please dilute (1 part of concentrating sample dilution+1 part deionized water) by 1:1 before use.
In the present invention, described 20 times of concentrated cleaning solutions, it comprises 0.05% Tween-20, and the PBST of 0.01mol/L, between pH value scope 7.0-7.5, please dilutes (1 part of concentration and dilution liquid+19 part deionized water) by 1:19 before use.
When the chemiluminescence immune detection reagent kit of bisphenol-A of the present invention is applied to the detection of bisphenol-A, detecting step is:
(1) pre-service testing sample is fluid sample by sample preparation to be tested;
(2) required reagent is taken out from cold storage environment, more than being placed in room temperature (20～25 ℃) balance 30 min, note must shaking up before every kind of liquid reagent uses;
(3) get the ELISA Plate that is coated with bisphenol-A antigen, add standard items/testing sample 50 μ L/ holes in corresponding micropore, the each concentration of standard items and sample is done two parallel laboratory tests;
(4) add bisphenol-A antibody working fluid, 50 μ L/ holes, vibration mixes gently, with reacting 45 min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate;
(5) carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 μ L/ holes, fully wash 4 ~ 5 times, every minor tick 10 s, pat dry (bubble not being eliminated after patting dry can be poked with original rifle head) with thieving paper;
(6) add luminous substrate liquid mixed liquor (A liquid mixes by 1:1 before use with B liquid) 100 μ L/ holes, vibration mixes gently, detects luminous intensity (RLU) after mixing in chemiluminescence detector;
(7) calculating of testing result: use the ratio of obtained standard solution and sample solution luminous value and blank solution to calculate.See following formula:
Relative luminous intensity=RLU/RULmax
In formula:
The luminous intensity values of RLU=standard (or sample) solution;
The luminous intensity values of RLUmax=blank (standard solution that concentration is 0);
The natural logarithm of the corresponding bisphenol-A of the relative luminous intensity value of calculating (μ g/L) is made to semilog coordinate system curve map.The bisphenol A concentration of each testing sample is found on typical curve according to its RLU value, or calculates by the corresponding equation of typical curve.As there being dilution in sample preparation, the sample concentration that should draw according to typical curve will be multiplied by its extension rate again.Be the actual concentrations of bisphenol-A in sample.
Kit of the present invention can be used for the residues detection of bisphenol-A in the samples such as animals urine, blood sample, tissue and internal organ.With the comparison of existing other detection bisphenol A residues amount, kit of the present invention has following advantage:
(1) kit of the present invention of employing chemiluminescence immunoassay method, more more fast and convenient than chromatographic process (high efficiency liquid phase, LC-MS, gas chromatography mass spectrometry), capillary electrophoresis method, required instrument is more simple, and testing cost is more cheap, has high-throughout feature simultaneously;
(2) kit of the present invention of employing chemiluminescence immunoassay method, more sensitiveer than ELISA method, can detect the bisphenol A residues of lower concentration and content, and the range of linearity is wider simultaneously;
(3) adopt the kit of the present invention of chemiluminescence immunoassay method, reduced by two anti-use links, thereby shortened detection time; Opaque white color ELISA Plate has increased chemiluminescence detector sensitivity.In addition, with bisphenol-A-carrier protein couplet thing but not bisphenol-A antibody is coated with opaque white color ELISA Plate, reduce the instability of bisphenol-A antibody, guaranteed the long-term effectiveness of kit.
Embodiment
Below by specific embodiment, the invention will be further described.These embodiment are only for the present invention is described, and are not used for limiting the scope of the invention.
Embodiment 1
1, the preparation of the each component of kit
(1) the haptenic preparation of bisphenol-A: by bisphenol-A acidifying, with sodium nitrite effect, generate the intermediate containing diazo positive ion in 4 ℃ of unglazed low temperature environments.Diazotizing bisphenol-A is as haptens, for synthesizing immunizing antigen and envelope antigen below;
(2) the immunogenic preparation of bisphenol-A-bovine serum albumin(BSA) (BSA): adopt diazotising method to carry out coupling bisphenol-A and bovine serum albumin(BSA) (BSA) and obtain immunizing antigen;
(3) preparation of bisphenol-A-ovoserum albumin (OVA) envelope antigen: adopt diazotising method to carry out coupling bisphenol-A and ovoserum albumin (OVA) and obtain envelope antigen;
(4) preparation of bisphenol-A-peroxidase labeled antibodies: (g), heavy dose of immunization protocol is that first immunisation mixes with 160 μ g bisphenol-A-BSA and equivalent Freund's complete adjuvant, hypodermic injection to body weight 18 ~ 20 to the female BALB/c mouse to 6 ~ 8 week age.After 3 weeks, then mix hypodermic injection with 80 μ g bisphenol-A-BSA and equivalent Freund's complete adjuvant.After this mixed lumbar injection with 80 μ g bisphenol-A-BSA and equivalent Freund's complete adjuvant every 3 weeks.Last immunity in the spleen 80 μ g bisphenol-A-BSA are as booster immunization.After three days, put to death mouse, get its spleen, merge with myeloma cell.Screen positive hybridoma cell with indirect ELISA method.Inject hybridoma by mouse peritoneal and prepare in a large number mouse ascites, ascites, after filtration, centrifugal preliminary purification, adopts sad method and affinity chromatography purifying ascites, then obtains the bisphenol A monoclonal antibody of purifying through dialysis.Bisphenol A monoclonal antibody and horseradish peroxidase, thus bisphenol-A-peroxidase labeled antibodies obtained;
(5) be coated with the opaque white color ELISA Plate preparation of bisphenol-A-OVA conjugate: with damping fluid by the detection hole for coated opaque white color ELISA Plate after the dilution of bisphenol-A-OVA conjugate, 4 ℃ spend the night after with the washing of PBST damping fluid, then add 180 μ L confining liquids (5% skimmed milk power solution), 37 ℃ of incubation 1.5 h, the liquid in hole that inclines, thieving paper pats dry rear sealing and preserves;
(6) luminous substrate liquid preparation: the Chemoluminescent substrate take the different luminol of luminol goods as luminous agent, is divided into A liquid and B liquid and preserves; Wherein A liquid level luminescence enhancer adds luminol goods luminescence enhancer and adds different luminol, and B liquid is superoxol or urea hydrogen peroxide solution;
(7) 2 times of concentrating sample dilutions: its composition is 0.01mol/L, the phosphate buffer of pH7.4;
(8) 20 times of concentrated cleaning solutions: its composition is for containing 0.05% Tween-20, and the PBST of 0.01mol/L, between pH value scope 7.0-7.5.
2, detect the establishment of the chemiluminescence immune detection reagent kit of bisphenol-A
The chemiluminescence immune detection reagent kit of the detection bisphenol-A of setting up, has comprised following ingredient:
(1) 96 hole opaque white color ELISA Plate (8 hole × 12) is coated with bisphenol-A-OVA conjugate, uses aluminium foil bag vacuum sealed package;
(2) 6 bottles of bisphenol-A standard solution, concentration is respectively:
0?ng/mL、0.05?ng/mL、0.15?ng/mL、0.45?ng/mL、1.35?ng/mL、4.05?ng/mL；
(3) bisphenol-A-Horseradish Peroxidase Conjugates solution;
(4) luminous substrate A liquid (luminol and reinforcing agent), luminous substrate B liquid (urea hydrogen peroxide);
(5) 2 times of concentrating sample dilutions.Before use, please diluting (1 part of concentration and dilution liquid+1 part deionized water) by 1:1 becomes working prototype dilution, and the working prototype dilution after its dilution is 0.05 mol/L, the PBST damping fluid of pH 7.4;
(6) 20 times of concentrated cleaning solutions.Before use, please diluting (1 part of concentration and dilution liquid+19 part deionized water) by 1:19 becomes work cleansing solution, and the work cleansing solution after its dilution is between pH value scope 7.0-7.5, containing 0.05% Tween-20, and the PBST damping fluid of 0.01mol/L.
3, the chemiluminescence immune detection reagent kit of bisphenol-A is on probation
(1) pre-treatment of sample
Get water sample to be checked, if when sample is more muddy, can be more than 5000 r/min, centrifugal 5 min or filtration;
(2) chemiluminescence immune detection reagent kit operation steps
The hole bar of standard and sample requirement is inserted in microwell plate framework to the position of record standard and sample.In suitable micropore, add respectively 50 μ L/ hole bisphenol-A standard solution and testing samples.Add 50 μ L/ hole bisphenol-A-Horseradish Peroxidase Conjugates in each micropore, after fully mixing, under room temperature, lucifuge leaves standstill incubation 45 min.Liquid in hole is dried, fully wash 4 ~ 5 times with wash operating solution.Remove the liquid in hole completely, pat dry with thieving paper, add luminous substrate liquid mixed liquor (A liquid mixes by 1:1 before use with B liquid) 100 μ L/ holes.After mixing, in chemiluminescence detector, detect immediately luminous intensity (RLU);
(3) computational analysis of testing result
Calculate with the ratio of obtained standard solution and sample solution luminous value and blank solution.See following formula:
Relative luminous intensity=RLU/RULmax
In formula:
The luminous intensity values of RLU=standard (or sample) solution;
The luminous intensity values of RLUmax=blank (standard solution that concentration is 0);
The natural logarithm of the corresponding bisphenol-A of the relative luminous intensity value of calculating (μ g/L) is made to semilog coordinate system curve map.The bisphenol A concentration of each testing sample is found on typical curve according to its RLU value, or calculates by the corresponding equation of typical curve.As sample has passed through beforehand dilution, the sample concentration that should draw according to typical curve will be multiplied by its extension rate again.
Claims (7)
1. detect a chemiluminescence immune detection reagent kit for bisphenol-A, it is characterized in that the following composition in kit:
(1) be coated with the opaque white color ELISA Plate of bisphenol-A-carrier protein couplet thing; Described bisphenol-A-carrier protein couplet thing is that bisphenol-A and carrier protein are obtained by mixed anhydride method or carbodlimide method coupling, and described carrier protein is ovoserum albumin, bovine serum albumin(BSA);
(2) bisphenol-A standard items;
(3) bisphenol-A-peroxidase labeled antibodies: this composition is the bisphenol-A antibody with peroxidase labelling, and described bisphenol-A antibody is monoclonal antibody;
(4) luminous substrate liquid: this luminous substrate liquid is the Chemoluminescent substrate take the different luminol of luminol goods as luminous agent, is divided into A liquid and B liquid and preserves, and presses before use 1:1 and mixes use; Wherein A liquid is that luminescence enhancer adds luminol goods luminescence enhancer and adds different luminol, and B liquid is superoxol or urea hydrogen peroxide solution;
(5) 2 times of concentrating sample dilutions;
(6) 20 times of concentrated cleaning solutions.
2. according to the chemiluminescence immune detection reagent kit of the bisphenol-A of claim 1, wherein, described opaque white color ELISA Plate is the detachable or non-removable opaque white color ELISA Plate in 96 holes.
3. according to the chemiluminescence immune detection reagent kit of the bisphenol-A of claim 1, wherein, the concentration of described bisphenol-A standard items is 0 ng/mL, 0.05 ng/mL, 0.15 ng/mL, 0.45 ng/mL, 1.35 ng/mL, 4.05 ng/mL.
4. according to the chemiluminescence immune detection reagent kit of the bisphenol-A of claim 1, wherein, described bisphenol-A-peroxidase labeled antibodies working fluid is to be diluted to 1:20000 ratio with antibody diluent.
5. according to the chemiluminescence immune detection reagent kit of the bisphenol-A of claim 1, wherein, described luminous substrate liquid is commercial any Chemoluminescent substrate take the different luminol of luminol goods as luminous agent.
6. according to the chemiluminescence immune detection reagent kit of the bisphenol-A of claim 1, wherein, described 2 times of concentrating sample dilutions, its composition is 0.01mol/L, phosphate buffer, glycocoll-HCl damping fluid or the Tris-HCl damping fluid of pH7.4, please dilute (1 part of concentrating sample dilution+1 part deionized water) by 1:1 before use.
7. according to the chemiluminescence immune detection reagent kit of the bisphenol-A of claim 1, wherein, described 20 times of concentrated cleaning solutions, it comprises 0.05% Tween-20, the PBST of 0.01mol/L, between pH value scope 7.0-7.5, please dilutes (1 part of concentration and dilution liquid+19 part deionized water) by 1:19 before use
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Cited By (4)
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| CN105277701A (en) * | 2014-07-22 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Chemiluminiscence immunodetection kit for detecting adprin |
| CN105440136A (en) * | 2014-08-25 | 2016-03-30 | 上海近岸科技有限公司 | A recombinant anti-bisphenol A monoclonal antibody, a preparing method thereof and uses of the antibody |
| CN105973874A (en) * | 2016-03-24 | 2016-09-28 | 中国农业科学院棉花研究所 | Detection method of 2,2-bis(4-hydroxyphenyl)propane in water |
| CN112213481A (en) * | 2020-08-13 | 2021-01-12 | 茅台学院 | Hapten directly coated bisphenol A ABC enzyme-linked immunosorbent assay method based on monoclonal antibody |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105277701A (en) * | 2014-07-22 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Chemiluminiscence immunodetection kit for detecting adprin |
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| CN105973874A (en) * | 2016-03-24 | 2016-09-28 | 中国农业科学院棉花研究所 | Detection method of 2,2-bis(4-hydroxyphenyl)propane in water |
| CN112213481A (en) * | 2020-08-13 | 2021-01-12 | 茅台学院 | Hapten directly coated bisphenol A ABC enzyme-linked immunosorbent assay method based on monoclonal antibody |
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Application publication date: 20140514 |