CN105319352A - Chemiluminescence immunodetection kit for detecting plasticiser - Google Patents
Chemiluminescence immunodetection kit for detecting plasticiser Download PDFInfo
- Publication number
- CN105319352A CN105319352A CN201410349858.9A CN201410349858A CN105319352A CN 105319352 A CN105319352 A CN 105319352A CN 201410349858 A CN201410349858 A CN 201410349858A CN 105319352 A CN105319352 A CN 105319352A
- Authority
- CN
- China
- Prior art keywords
- plasticiser
- liquid
- plasticizer
- detection reagent
- carrier protein
- 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.)
- Pending
Links
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a chemiluminescence immunodetection kit for detecting plasticizer, and belongs to the field of immunology detection. The kit is composed of an opaque white enzyme label plate of a plasticizer-carrier protein conjugate, a plasticizer standard sample, a plasticizer-peroxide enzymic-labelled antibody operating fluid, a luminescent substrate liquid, a concentrated-sample diluted liquid and a concentrated washing liquid. The plasticizer-carrier protein conjugate is obtained by coupling a plasticizer and a carrier protein through a mixed acid anhydride process or a carbodiimide process, and the concentrated washing liquid contains 0.05% of tween-20. Compared with a traditional ELISA adsorption analytical method, the kit possesses relatively high sensitivity, also is short in detection time and low in cost, and is applicable to residual quantity detection on a plasticizer in Chinese liquor and other samples.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune detection reagent kit detecting plasticiser, for detecting content of plasticizing agent in white wine or residual quantity.Belong to field of immunological detection.
Background technology
Plasticiser a kind ofly increases the flexibility of material or the adjuvant of materials liquefy.Phthalate plasticiser is classified as suspected environmental hormone, its bio-toxicity mainly belongs to estrogen and antiandrogen active, endocrinopathy can be caused to damage biosome Reproductive Performance, comprise the reduction of appreciation rate, miscarriage, congenital defect, abnormal sperm count, damage of testis, also can cause malignant tumour, cause lopsided baby.
Phthalate plasticiser is a kind of poisonous chemical industry plastics softening agent, belong to colourless, liquid with no taste, particulate molecular can be allowed after interpolation evenly scatter, therefore ductility, elasticity and pliability can be increased, the raw material of Chang Zuowei sofa, automotive seat, rubber tube, cosmetics and toy, belongs to industrial additive.
The present invention has the plasticiser antibody of high-affinity, high specific by distinctive immune animal preparation, and adopt enzymic-labelled antibody, set up a kind of chemiluminescence immunoassay kit that can detect plasticiser, detection will be stayed to provide new method for plasticiser medicine in Liquor Products, there is provided safely important theory and practice foundation to guarantee Liquor Products, safeguard that Spirits develops in a healthy way significant.
Summary of the invention
For problems of the prior art, the present invention mainly utilizes the ultimate principle of the specific immune response of antigen and antibody to realize.Chemiluminescence immune assay is the product that chemoluminescence method and immunoassay combine, and therefore has the high sensitivity of chemoluminescence method and the high specific of immunoassay simultaneously.In whole course of reaction, in sample, content of plasticizing agent is higher, and in reaction system, luminous intensity is more weak; Otherwise content of plasticizing agent is fewer in sample, luminous intensity is higher.
The present invention is a kind of chemiluminescence immune detection reagent kit detecting plasticiser, it is characterized in that containing following composition:
1, the opaque white color ELISA Plate of plasticiser-carrier protein couplet thing is coated with; Described plasticiser-carrier protein couplet thing is obtained by mixed anhydride method or carbodlimide method coupling plasticiser and carrier protein, and described carrier protein is human serum albumins, bovine serum albumin(BSA), egg albumin, mouse haemocyanin or rabbit serum proteins;
2, plasticiser standard items;
3, plasticiser-peroxidase labeled antibodies: this composition is the plasticiser antibody with peroxidase labelling, and described plasticiser antibody is monoclonal antibody or polyclonal antibody;
4, luminous substrate liquid: the Chemoluminescent substrate that this luminous substrate liquid is is luminous agent with the different luminol of luminol goods, is divided into A liquid and B liquid to preserve, presses 1:1 before use used in combination; 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 concentration and dilution liquid;
6,20 times of concentrated cleaning solutions.
In the present invention, described opaque white color ELISA Plate is the detachable of 96 holes or non-removable opaque white color ELISA Plate.
In the present invention, described plasticiser standard items, are made up of the plasticiser standard items of a series of variable concentrations, and concentration is the concentration ranges of 0.02 ~ 50 μ g/mL.
In the present invention, described plasticiser-peroxidase labeled antibodies is the plasticiser antibody with peroxidase labelling, as the plasticiser antibody that horseradish peroxidase (HRP) marks.
In the present invention, described luminous substrate liquid is any one Chemoluminescent substrate being luminous agent with the different luminol of luminol goods commercial.
In the present invention, described 2 times of concentration and dilution liquid, its composition is the phosphate buffer of 0.01mol/L, pH7.4, Glycine-HCl buffer or Tris-HCl damping fluid, please dilutes (1 part of concentrating sample dilution+1 part of deionized water) by 1:1 before using.
In the present invention, described 20 times of concentrated cleaning solutions, it comprises 0.05% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5, please dilutes (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using.
When the chemiluminescence immune detection reagent kit of plasticiser of the present invention is applied to the detection of plasticiser, detecting step is:
(1) pre-service testing sample is fluid sample by sample preparation to be tested, or uses Solvent Extract methods testing sample, and nitrogen dries up and redissolved in sample diluting liquid working fluid;
(2) required reagent is taken out from cold storage environment, be placed in room temperature (20 ~ 25 DEG C) balance more than 30min, notice that often kind of liquid reagent must shake up before using;
(3) get bag by the ELISA Plate of plasticiser antigen, add standard items/testing sample 50 μ L/ hole in the micropore of correspondence, standard items and each concentration of sample do two parallel laboratory tests;
(4) add plasticiser antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 45min with in the rearmounted 25 DEG C of light protected 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/ hole, fully washs 4 ~ 5 times, every minor tick 10s, pat dry (bubble be not 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/ hole, mixing of vibrating gently, detects luminous intensity (RLU) in chemiluminescence detector after mixing;
(7) calculating of testing result: calculate with the ratio of the standard solution obtained 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 (concentration is the standard solution of 0);
The natural logarithm of corresponding for the relative luminous intensity value calculated plasticiser (μ g/L) is made semilog coordinate system curve figure.The plasticiser concentration of each testing sample is found on typical curve according to its RLU value, or is calculated by the corresponding equation of typical curve.As having 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 plasticiser in sample.
Kit of the present invention can be used for the residues detection of plasticiser in the samples such as white wine.Detect the comparison of plasticiser residual quantity with existing other, kit of the present invention has following advantage:
(1) kit of the present invention of Chemiluminescence immunoassay is adopted, more more fast and convenient than chromatographic process (efficient 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) adopt the kit of the present invention of Chemiluminescence immunoassay, more sensitiveer than ELISA method, can detect that the plasticiser of lower concentration and content remains, the range of linearity is wider simultaneously;
(3) adopt the kit of the present invention of Chemiluminescence immunoassay, decrease two anti-use links, thus shorten detection time; Opaque white color ELISA Plate adds chemiluminescence detector sensitivity.In addition, with plasticiser-carrier protein couplet thing but not plasticiser antibody wrap by opaque white color ELISA Plate, decrease the instability of plasticiser antibody, ensure that the long-term effectiveness of kit.
Embodiment
Below by way of specific embodiment, the invention will be further described.These embodiments only for illustration of the present invention, and are not used for limiting the scope of the invention.
Embodiment
1, the preparation of each component of kit
(1) the haptenic preparation of plasticiser: by plasticiser acidifying, with sodium nitrite effect in 4 DEG C of unglazed low temperature environments, generates the intermediate containing diazo positive ion.Diazotizing plasticiser, as haptens, is used and is synthesized immunizing antigen and envelope antigen later;
(2) plasticiser-bovine serum albumin(BSA) (BSA) immunogenic preparation: adopt diazotising method to carry out coupling plasticiser and bovine serum albumin(BSA) (BSA) and obtain immunizing antigen;
The preparation of plasticiser-ovoserum albumin (OVA) envelope antigen: adopt diazotising method to carry out coupling plasticiser and ovoserum albumin (OVA) and obtain envelope antigen;
The preparation of plasticiser-peroxidase labeled antibodies: to the female BAl BIc/c mouse (body weight 18 ~ 20g) in 6 ~ 8 week age, heavy dose of immunization protocol is, first immunisation 160 μ g plasticiser-BSA mix with equivalent Freund's complete adjuvant, hypodermic injection.After 3 weeks, then mix with equivalent Freund's complete adjuvant with 80 μ g plasticiser-BSA, hypodermic injection.After this mixed with equivalent Freund's complete adjuvant with 80 μ g plasticiser-BSA every 3 weeks, lumbar injection.Last immunity in the spleen 80 μ g plasticiser-BSA is as booster immunization.Put to death mouse after three days, get its spleen, with myeloma cell fusion.Positive hybridoma cell is screened with indirect ELISA method.Prepare mouse ascites in a large number by mouse peritoneal injection hybridoma, ascites, after filtration, centrifugal preliminary purification, adopts sad method and affinity chromatography purifying ascites, then obtains the plasticiser monoclonal antibody of purifying through dialysis.Plasticiser monoclonal antibody and horseradish peroxidase, thus obtain plasticiser-peroxidase labeled antibodies;
Be coated with the opaque white color ELISA Plate preparation of plasticiser-OVA conjugate: be used for wrapping the detect aperture by opaque white color ELISA Plate after being diluted by plasticiser-OVA conjugate with damping fluid, 4 DEG C spend the night after use PBST buffer solution, then 180 μ L confining liquids (5% skim milk powder solution) are added, 37 DEG C of incubation 1.5h, incline liquid in hole, and thieving paper pats dry rear sealing and preserves.
2, the establishment of the chemiluminescence immune detection reagent kit of plasticiser is detected
The chemiluminescence immune detection reagent kit of the detection plasticiser set up, contains following ingredient:
(1) 96 hole opaque white color ELISA Plate (8 hole × 12) is coated with plasticiser-OVA conjugate, uses aluminium foil bag vacuum sealed package;
(2) plasticiser standard solution 6 bottles, concentration is respectively:
0μg/mL、0.02μg/mL、0.08μg/mL、0.5μg/mL、5μg/mL、50μg/mL
(3) plasticiser-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.Please dilute (1 part of concentration and dilution liquid+1 part of deionized water) by 1:1 before using and become working prototype dilution, the working prototype dilution after its dilution is the PBST damping fluid of 0.05mol/L, pH7.4;
(6) 20 times of concentrated cleaning solutions.Please dilute (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using and become work cleansing solution, the work cleansing solution after its dilution is between pH value range 7.0-7.5, containing 0.05% Tween-20, and the PBST damping fluid of 0.01mol/L.
3, the use of the chemiluminescence immune detection reagent kit of plasticiser
Get 5mL sample in clean glass test tube, add the chromatographically pure normal hexane of 2mL, vibrate after closing the lid 3min, then leaves standstill, and gets supernatant liquor 1mL after layering, dries up in clean glassware room temperature under nitrogen, and the methyl alcohol drying up rear 1mL35% redissolves rear to be measured.
The methanol solution of preparation 35%: get 35mL methyl alcohol and add in 65ml deionized water and mix
2) the chemiluminescence immune detection reagent kit operation steps of plasticiser
The hole bar of standard and sample requirement is inserted in microwell plate framework, the position of record standard and sample.50 μ L/ hole plasticiser standard solution and testing samples are added respectively in suitable micropore.Add 50 μ L/ hole plasticiser-Horseradish Peroxidase Conjugates in each micropore, fully after mixing, under room temperature, lucifuge leaves standstill incubation 45min.Liquid in hole is dried, fully washs 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/ hole.In chemiluminescence detector, luminous intensity (RLU) is detected immediately after mixing.
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 (concentration is the standard solution of 0).
The natural logarithm of corresponding for the relative luminous intensity value calculated plasticiser (μ g/L) is made semilog coordinate system curve figure.The plasticiser concentration of each testing sample is found on typical curve according to its RLU value, or is calculated by the corresponding equation of typical curve.As sample have 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 plasticiser, it is characterized in that the following composition in kit:
(1) the opaque white color ELISA Plate of plasticiser-carrier protein couplet thing is coated with; Described plasticiser-carrier protein couplet thing is obtained by mixed anhydride method or carbodlimide method coupling plasticiser and carrier protein, and described carrier protein is human serum albumins, bovine serum albumin(BSA), egg albumin, mouse haemocyanin or rabbit serum proteins;
(2) plasticiser standard items;
(3) plasticiser-peroxidase labeled antibodies: this composition is the plasticiser antibody with peroxidase labelling, and described plasticiser antibody is monoclonal antibody;
(4) luminous substrate liquid: the Chemoluminescent substrate that this luminous substrate liquid is is luminous agent with the different luminol of luminol goods, is divided into A liquid and B liquid to preserve, presses 1:1 before use used in combination; 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 concentration and dilution liquid;
(6) 20 times of concentrated cleaning solutions.
2. the chemiluminescence immune detection reagent kit of plasticiser according to claim 1, wherein, described opaque white color ELISA Plate is the detachable of 96 holes or non-removable opaque white color ELISA Plate.
3. the chemiluminescence immune detection reagent kit of plasticiser according to claim 1, wherein, the concentration of described plasticiser standard items is the concentration ranges of 0.02 ~ 50 μ g/mL.
4. the chemiluminescence immune detection reagent kit of plasticiser according to claim 1, wherein, described plasticiser-peroxidase labeled antibodies working fluid is diluted to 1:40000 ratio with antibody diluent.
5. the chemiluminescence immune detection reagent kit of plasticiser according to claim 1, wherein, described luminous substrate liquid is any one Chemoluminescent substrate being luminous agent with the different luminol of luminol goods commercial.
6. the chemiluminescence immune detection reagent kit of plasticiser according to claim 1, wherein, described 2 times of concentration and dilution liquid, its composition is 0.01mol/L, the phosphate buffer of pH7.4, Glycine-HCl buffer or Tris-HCl damping fluid, please dilute (1 part of concentrating sample dilution+1 part of deionized water) by 1:1 before using.
7. the chemiluminescence immune detection reagent kit of plasticiser according to 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 range 7.0-7.5, please dilutes (1 part of concentration and dilution liquid+19 parts of deionized waters) by 1:19 before using.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410349858.9A CN105319352A (en) | 2014-07-23 | 2014-07-23 | Chemiluminescence immunodetection kit for detecting plasticiser |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410349858.9A CN105319352A (en) | 2014-07-23 | 2014-07-23 | Chemiluminescence immunodetection kit for detecting plasticiser |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105319352A true CN105319352A (en) | 2016-02-10 |
Family
ID=55247200
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410349858.9A Pending CN105319352A (en) | 2014-07-23 | 2014-07-23 | Chemiluminescence immunodetection kit for detecting plasticiser |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105319352A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752525A (en) * | 2017-11-05 | 2019-05-14 | 江苏维赛科技生物发展有限公司 | The chemiluminescence immune detection reagent kit of o-phenyl phenol in a kind of detection fruits and vegetables |
-
2014
- 2014-07-23 CN CN201410349858.9A patent/CN105319352A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752525A (en) * | 2017-11-05 | 2019-05-14 | 江苏维赛科技生物发展有限公司 | The chemiluminescence immune detection reagent kit of o-phenyl phenol in a kind of detection fruits and vegetables |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104655846A (en) | Enzyme linked immunosorbent assay kit for detecting progesterone and detection method thereof | |
| CN105319354A (en) | Preparation method of chemiluminescence immunoassay detection kit used for detecting methyl parathion | |
| CN103869070B (en) | Detect enzyme linked immunological kit and the application thereof of dibutyl phthalate | |
| CN105067599A (en) | Chemiluminescence immune detection kit for detecting pro-gastrin-releasing peptide | |
| CN104655847A (en) | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof | |
| CN101413952A (en) | Chemiluminescence immune detection reagent kit for detecting ractopamine | |
| CN101413951A (en) | Chemiluminescence immune detection reagent kit for detecting Clenbuterol | |
| CN103421072A (en) | Dexamethasone semi-antigen, preparation method and applications thereof | |
| CN103698504B (en) | Phthalate total amount ELISA enzyme-linked immunologic detecting kit and using method thereof | |
| CN105319368A (en) | Enzyme linked immunosorbent assay kit used for detecting zearalenone, and detection method thereof | |
| CN103792374A (en) | Chemiluminesent immunoassay kit for detection of bisphenol A | |
| CN102331500A (en) | Method and enzyme linked immunosorbent assay kit for detecting lemon yellow | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| CN103792226A (en) | Chemiluminescence immunodetection kit used for detecting phenylethanolamine A | |
| CN102520158A (en) | Indirect-competition-law enzyme linked immunosorbent assay kit for detecting phenylethanolamine A | |
| CN105319352A (en) | Chemiluminescence immunodetection kit for detecting plasticiser | |
| CN103808921A (en) | Enzyme-linked immunosorbent assay kit for detecting residual zilpaterol and use method thereof | |
| CN103808933A (en) | Chemiluminiscence immunoassay kit for detecting zilpaterol | |
| CN103102319A (en) | Melamine semiantigen, preparation method and application thereof | |
| CN105319353A (en) | Preparation of enzyme linked immunosorbent assay kit used for detecting dihydropyridine drug residues | |
| CN102539762B (en) | Enzyme-linked immunosorbent assay kit for detecting Sudan red and paranitroaniline red medicaments and application thereof | |
| CN104655845A (en) | Preparation of chemiluminescence immunoassay kit for detecting chloroquine | |
| CN102980999A (en) | Chemiluminescence immunodetection kit for detecting ractopamine | |
| CN105301232A (en) | Chemiluminescence immunodetection kit for detecting cyproheptadine | |
| CN106610431A (en) | Enzyme-linked immunosorbent assay kit for detecting Benalaxyl and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160210 |
|
| WD01 | Invention patent application deemed withdrawn after publication |