CN105301263A - Enzyme linked immunosorbent assay kit for detecting progesterone residues in chicken tissues and application of enzyme linked immunosorbent assay kit - Google Patents
Enzyme linked immunosorbent assay kit for detecting progesterone residues in chicken tissues and application of enzyme linked immunosorbent assay kit Download PDFInfo
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- CN105301263A CN105301263A CN201410355991.5A CN201410355991A CN105301263A CN 105301263 A CN105301263 A CN 105301263A CN 201410355991 A CN201410355991 A CN 201410355991A CN 105301263 A CN105301263 A CN 105301263A
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- progesterone
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- linked immunosorbent
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Abstract
The invention relates to an enzyme linked immunosorbent assay kit for detecting progesterone residues in chicken tissues. The enzyme linked immunosorbent assay kit comprises the following components at proportion: a coating board, an enzyme marker working solution, a substrate solution A, a substrate solution B, a stop solution, a concentrated washing liquid and a concentrated extracting solution, and has the sensitivity of 0.1 ppb, the precision that the in-assay variation coefficient CV% is smaller than 5% and the interassay variation coefficient CV% is smaller than 10%, the accuracy shown as the recovery rate ranging from 80% to 110%, the IC50 of 0.3-0.5 ppb, and the linearly dependent coefficient |r| larger than or equal to 0.9900. The enzyme linked immunosorbent assay kit has the advantages of being wide in application range, convenient to use and accurate in detection.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to the progestomimetic enzyme linked immunological kit of a kind of detection hormone, it is specially adapted to the detection that in chicken, progesterone is residual.
Technical background
Progesterone is also called progesterone, progesterone, corpus luteum sterone, corpus luteum hormone, progesterone, gestogen or helps progesterone to be a kind ofly relate to menstrual cycle of female, gestation and to the mankind's also influential steroids of the embryo of other animals again.Progesterone is a kind of estrogen, participates in the female menstrual cycle of the mankind and other animals, supports conceived and embry ogenesis.Progesterone belongs to the hormone that a class is called progestational hormone, is also the most important progestational hormone of the mankind.Hormone medicine can improve the feed conversion rate of poultry food, and then reaches the object of weightening finish, but can cause series of problems because progesterone is residual in vivo, as sex character change, precocious etc., more obvious to Children and teenager effect.At present, hormonal substance is forbidden using in aquaculture by most countries, and must not detect in food.But the domestic detection residual to forbidden drugs such as progesterone in livestock products is almost blank, and remain monitoring regulation and the requirement about importer according to China to Animal by-product, studying various detection method becomes the task of top priority.
At present, detect the method mainly high performance liquid chromatography (HPLC) of progesterone residual quantity, due to the instrument and equipment of complexity and loaded down with trivial details process and the high professional qualification requirement to reviewer, be not suitable for the examination of on-site supervision and great amount of samples.
Summary of the invention
The object of the invention is to provide a kind of for progesterone residue detection enzyme linked immunological kit in chicken, the screening of its applicable on-the-spot batch samples simple to operate for above-mentioned deficiency.
Kit of the present invention, it contains:
(1) ELISA Plate (coating antigen is antigen, antibody or antiantibody) of coating antigen is coated with;
(2) enzyme marker (for ENR-HRP, enzymic-labelled antibody or enzyme labeling antiantibody);
(3) progesterone antibody working fluid (when envelope antigen in ELISA Plate and enzyme marker be enzyme labeling antiantibody or ELISA Plate are wrapped by antiantibody and enzyme marker is enzyme-labelled antigen time contain);
(4) progesterone standard solution;
(5) substrate nitrite ion;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) the concentrated liquid that redissolves.
The U.S. linked immunoassay reagent kit of detection progesterone residual quantity provided by the invention, comprise ELISA Plate and the enzyme marker working fluid of progesterone specific antibody working fluid and pre-coated coating antigen, described enzyme marker is the antiantibody of enzyme labeling, enzyme labeling P-3-O-CMO or enzyme labeling progesterone specific antibody; Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody.
The marker enzyme of described enzyme marker is horseradish peroxidase, and the sheep anti mouse antiantibody of enzyme labeling adopts mixed acid anhydride or carbodiimide method that marker enzyme and antiantibody are carried out coupling and obtains.
Described progesterone specific antibody is progesterone monoclonal antibody, and they obtain as immunogene with the conjugate that P-3-O-CMO and carrier protein adopt mixed anhydride method to obtain, the preferred progesterone mouse monoclonal antibody of described progesterone monoclonal antibody.
In order to more convenient on-site supervision and great amount of samples examination, described kit also comprises progesterone standard solution, developer, stop buffer, concentrated cleaning solution, the concentrated liquid that redissolves.
The preferred concentrated cleaning solution of described cleansing solution is pH7.1--7.7, and containing 0.8% ~ 1.2% Tween 80 and 0.03--0.05% thiomersal preservative, 0.02--0.04mol/L phosphate buffer, described number percent is percent weight in volume.
When the marker enzyme of enzyme marker is horseradish peroxidase, substrate nitrite ion A is hydrogen peroxide or urea peroxide, and substrate nitrite ion B is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1 ~ 2mol/L sulfuric acid or hydrochloride buffer;
Described redissolution liquid is for containing 0.08--0.12% polysorbas20,3--8% ovalbumin, 0.2 ~ 0.5mol/L phosphate buffer, and described number percent is percent weight in volume.
In the present invention, the preparation process of ELISA Plate is for be diluted to 0.2 ~ 0.3 μ g/ml with damping fluid by coating antigen, every hole adds 100 μ L, 37 DEG C of incubation 2h or 4 DEG C spend the night, and incline coating buffer, wash twice with the cleansing solution after dilution, each 30s, pat dry, in every hole, then add 150 ~ 200 μ L confining liquids, 37 DEG C of incubation 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
The building-up process of antigen in the present invention:
1. haptenic synthesis
P-3-O-CMO by progesterone and succinic anhydride by being obtained by reacting;
2. the preparation of progesterone antibody
Adopt carbodiimide method to carry out coupling P-3-O-CMO and carrier protein and obtain immunogene.
Progesterone standard solution in kit of the present invention; Mark product solution 6 bottles is 0 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L.
Cleaning Principle of the present invention is:
When on capillary strip during pre-coated progesterone antigen, after adding sample solution or standard solution, add ELIAS secondary antibody immediately, add progesterone specific antibody solution again, progesterone residual in sample and ELISA Plate are wrapped the progesterone antigenic competition progesterone specific antibody of quilt, enzyme labeling antiantibody carries out amplification, develops the color with nitrite ion, sample light absorption value becomes negative correlation with the content of progesterone, and comparing with typical curve can the concentration range of progesterone residual quantity in judgement sample roughly.
Present invention also offers and a kind ofly apply above-mentioned enzyme linked immunological kit and detect the method that progesterone is residual in chicken, it comprises step:
(1) sample pre-treatments;
(2) detect with kit;
(3) testing result is analyzed.
Sample pretreatment mainly in order to obtain progesterone solution from sample, thus for follow-up detection.The method of Sample pretreatment: take 2.0 ± 0.05 chicken in 50mL polystyrene centrifuge tube, add 5 ~ 8mL ethyl acetate, oscillator vibrates 10min, the centrifugal 5min of more than 3000r, gets supernatant 1 ~ 3mL, to the glass test tube that 10mL is clean, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up, adding 1 ~ 3mL sample redissolution liquid, with vortex instrument vortex 1min, getting 50 μ L for analyzing.
When detecting with kit in the present invention, when coating antigen is progesterone antigen, in ELISA Plate micropore, add standard solution or sample solution, add ELIAS secondary antibody immediately, then add antibody working fluid, after incubation, washing pats dry, colour developing, stops, measures absorbance by microplate reader.Interpretation of result process in the present invention for: with the absorbance values (B) of the standard solution of each concentration the obtained absorbance (B divided by first standard solution (0 standard)
0) being multiplied by 100% again, i.e. percentage absorbance, computing formula is:
Percentage absorbance (%)=(B/B
0) × 100%
With the semilog value of the concentration of progesterone standard items (μ g/L) for X-axis, percentage absorbance is Y-axis, drawing standard curve map, with the percentage absorbance in same way calculation sample, the concentration of corresponding each sample then can read the residual quantity of progesterone in sample from typical curve.
In the present invention, the analysis of testing result also can adopt regression equation method, calculates the concentration of sample solution.
In the present invention, the analysis of testing result can also utilize computer software, and this method is more convenient for the express-analysis of a large amount of sample, and whole testing process only needs can complete less than 1h.
The present invention detects enzyme linked immunological kit that progesterone in Chicken Tissues remains and mainly adopts indirect competitive ELISA method qualitative or quantitatively detect the residual quantity of the progesterone in sample, require low to sample pre-treatments, sample pretreatment process is simple, can detect batch samples fast, main agents provides with the form of working fluid, and the method for inspection is convenient and easy simultaneously, there is specificity high, highly sensitive, degree of accuracy is high, accuracy high.Enzyme linked immunological kit structure of the present invention is simple, and easy to use, carrying convenience, detection method efficiently and accurately, is suitable for batch samples selective mechanisms.Kit of the present invention plays a significant role in the detection that progesterone is residual.
Accompanying drawing explanation
Accompanying drawing 1 progesterone chemical structural formula.
The typical curve of accompanying drawing 2 progesterone enzyme linked immunological kit.
Embodiment
The present invention is set forth further below in conjunction with concrete enforcement step example:
1, the preparation of ELISA Plate
Be buffered liquid with bag and progesterone antigen diluent is become 0.20 μ g/mL, every hole adds 100 μ L, 37 DEG C of incubations two hours, incline coating buffer, washes twice with the concentrated cleaning solution of dilution, each 30s, pat dry, and then every hole adds 200 μ L confining liquids, 37 DEG C of incubation 2h, incline liquid in hole, preserves after dry with the vacuum seal of aluminium film.
2, set up the enzyme linked immunological kit detecting progesterone and remain, make it comprise following component:
(1) bag is by the ELISA Plate of progesterone antigen;
(2) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled;
(3) progesterone antibody working fluid;
(4) progesterone standard items 6 bottles, concentration is respectively 0 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L;
(5) substrate nitrite ion is made up of A liquid and B liquid, and substrate colour developing A liquid is urea peroxide, and substrate colour developing B liquid is tetramethyl benzidine;
(6) stop buffer is 2mol/L hydrochloric acid;
(7) concentrated cleaning solution is for being pH7.1 ~ 7.7, containing 0.8% ~ 1.2% Tween 80 and 0.03--0.05% thiomersal preservative, and 0.02--0.04mol/L phosphate buffer, described number percent is percent weight in volume;
(8) the concentrated liquid that redissolves is for containing 0.08--0.12% polysorbas20,3--8% ovalbumin, 0.2 ~ 0.5mol/L phosphate buffer, and described number percent is percent weight in volume.
3, the detection that in sample, progesterone is residual:
(1) 2.0+ (-) 0.5 chicken is taken in 50mL polystyrene centrifuge tube, add 5 ~ 8mL ethyl acetate, oscillator vibrates 10min, the centrifugal 5min of more than 3000r, gets supernatant 1 ~ 3mL, to the glass test tube that 10mL is clean, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up, adding 1 ~ 3mL sample redissolution liquid, with vortex instrument vortex 1min, getting 50 μ L for analyzing.
(2) detect with kit:
Progesterone standard solution or sample solution 50 μ L is added in the ELISA Plate micropore being coated with progesterone antigen, add ELIAS secondary antibody 50 μ L immediately, add progesterone antibody working fluid 50 μ L again, with cover plate film shrouding, 30min is reacted in 37 DEG C of constant temperature ovens, pour out liquid in hole, every hole adds 250 μ L cleansing solutions, liquid in hole is poured out after 30s, repeatable operation like this washes plate 5 times, pat dry with thieving paper, every hole adds substrate colour developing A liquid urea peroxide, substrate colour developing B liquid tetramethyl benzidine, to vibrate gently mixing, 37 DEG C of constant temperature oven colour developing 15min, every hole adds 2mol/L stop buffer hydrochloric acid 50 μ L, to vibrate gently mixing, 450nm place is set in by microplate reader, measure every hole absorbance (OD value).The typical curve painted as shown in Figure 2, IC
50=1.9ng/mL.
4, standard items precision test:
Respectively the ELISA Plate prepared from three different time periods respectively extract a collection of ELISA Plate out, often criticize each extraction 10 kits, 20 micropores extracted out by every plate, measure the absorbance of 2.7 μ g/L standard solution, calculate the coefficient of variation.
Can be found out by above-mentioned test findings, often criticize each 10 the standard items coefficient of variation of kit between 4.6% ~ 11.2%, meet the regulation that precision is less than or equal to 25%.
5, sample accuracy experiment:
The progesterone standard items getting two concentration are respectively 10 μ g/kg, and 20 μ g/kg, carry out interpolation recovery experiment to sample respectively, each concentration do 4 parallel, respectively accuracy in computation.
Result shows that the TIANZHU XINGNAO Capsul of chicken sample is between 82.0% ~ 89.4%.
6, cross reacting rate experiment
Select the drug monitoring cross reacting rate having similar structures and similar functions with progesterone, obtain its inhibition concentration of 50% respectively by the typical curve of various medicine, calculate kit to the cross reacting rate of other drug with lower examination.Cross reacting rate is larger, and so this kit is better to the detection specificity of progesterone.
Cross reacting rate (%)=(causing the analog concentration of the concentration of 50% suppression progesterone/cause 50% suppression) × 100%
7, kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 6 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, progesterone added practical measurement value all in normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 6 days under 37 DEG C of preservation conditions by kit, carry out accelerated aging test, result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 5 days, the bright kit indices of measurement result page table is completely normal, and can draw from above result, kit can preserve 6 months at 2 ~ 8 DEG C.
Claims (5)
1. detect the enzyme linked immunological kit that progesterone in Chicken Tissues is residual, it is characterized in that: its constituent and ratio as follows:
Comprise by plate, standard items, enzyme marker working fluid, substrate solution A, substrate solution B, stop buffer, concentrated cleaning solution, concentrated extracting solution: described bag is be coated with progesterone antigen at the bottom of hole by plate, and described enzyme marker is enzyme labeling progesterone antibody.
2. kit as described in right 1, is characterized in that described progesterone antibody is monoclonal antibody.
3. kit as claimed in claim 1 or 2 is characterized in that the marker enzyme of enzyme marker is horseradish peroxidase, substrate nitrite ion A is hydrogen peroxide or urea peroxide, substrate nitrite ion B is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1 ~ 2mol/L sulfuric acid or hydrochloride buffer.
4. kit as claimed in claim 1 or 2, it is characterized in that: concentrated cleaning solution is pH7.1-7.7, containing 0.8% ~ 1.2% Tween 80 and 0.03--0.05% thiomersal preservative, 0.02-0.04mol/L phosphate buffer: the concentrated liquid that redissolves is for containing 0.08-0.12% polysorbas20,3-8% ovalbumin, 0.2 ~ 0.5mol/L phosphate buffer, the concentration of progesterone standard items is respectively 0 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L, described number percent is percent weight in volume.
5. detect the method that in Chicken Tissues, progesterone is residual, comprise step:
(1) sample pre-treatments;
(2) detect with enzyme linked immunological kit in claim 1;
(3) testing result is analyzed.
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| CN201410355991.5A CN105301263A (en) | 2014-07-25 | 2014-07-25 | Enzyme linked immunosorbent assay kit for detecting progesterone residues in chicken tissues and application of enzyme linked immunosorbent assay kit |
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| CN201410355991.5A CN105301263A (en) | 2014-07-25 | 2014-07-25 | Enzyme linked immunosorbent assay kit for detecting progesterone residues in chicken tissues and application of enzyme linked immunosorbent assay kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116333115A (en) * | 2023-05-12 | 2023-06-27 | 北京纳百生物科技有限公司 | Anti-progesterone monoclonal antibody, kit and application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62272156A (en) * | 1986-05-02 | 1987-11-26 | Teikoku Hormone Mfg Co Ltd | Quantitative determination of progesterone in bovine nonfat milk |
| EP0325449A2 (en) * | 1988-01-19 | 1989-07-26 | Idexx Laboratories, Inc. | Immunoassays |
| JP2010032283A (en) * | 2008-07-28 | 2010-02-12 | Fujifilm Corp | Immunological measuring method |
| CN101923094A (en) * | 2009-12-28 | 2010-12-22 | 张洪友 | Progesterone detection kit of dairy cow milk |
| CN101949940A (en) * | 2010-08-11 | 2011-01-19 | 吉林大学 | Detection kit of cow milk progesterone content |
| CN203365441U (en) * | 2013-05-31 | 2013-12-25 | 北京勤邦生物技术有限公司 | Medroxyprogesterone ELISA detection kit |
-
2014
- 2014-07-25 CN CN201410355991.5A patent/CN105301263A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62272156A (en) * | 1986-05-02 | 1987-11-26 | Teikoku Hormone Mfg Co Ltd | Quantitative determination of progesterone in bovine nonfat milk |
| EP0325449A2 (en) * | 1988-01-19 | 1989-07-26 | Idexx Laboratories, Inc. | Immunoassays |
| JP2010032283A (en) * | 2008-07-28 | 2010-02-12 | Fujifilm Corp | Immunological measuring method |
| CN101923094A (en) * | 2009-12-28 | 2010-12-22 | 张洪友 | Progesterone detection kit of dairy cow milk |
| CN101949940A (en) * | 2010-08-11 | 2011-01-19 | 吉林大学 | Detection kit of cow milk progesterone content |
| CN203365441U (en) * | 2013-05-31 | 2013-12-25 | 北京勤邦生物技术有限公司 | Medroxyprogesterone ELISA detection kit |
Non-Patent Citations (2)
| Title |
|---|
| R. SIMERSKY ET AL.: "Development of an ELISA-based kit for the on-farm determination of progesterone in milk", 《VETERINARNI MEDICINA》 * |
| 庞观龙等: "奶牛孕酮—卵清蛋白交联物、孕酮—牛血清白蛋白交联物的制备", 《广西农学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116333115A (en) * | 2023-05-12 | 2023-06-27 | 北京纳百生物科技有限公司 | Anti-progesterone monoclonal antibody, kit and application |
| CN116333115B (en) * | 2023-05-12 | 2023-07-28 | 北京纳百生物科技有限公司 | Anti-progesterone monoclonal antibody, kit and application |
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Application publication date: 20160203 |