CN101793894A - Direct competitive enzyme-linked immunoassay kit for detecting medroxyprogesterone acetate - Google Patents
Direct competitive enzyme-linked immunoassay kit for detecting medroxyprogesterone acetate Download PDFInfo
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- CN101793894A CN101793894A CN200910045733A CN200910045733A CN101793894A CN 101793894 A CN101793894 A CN 101793894A CN 200910045733 A CN200910045733 A CN 200910045733A CN 200910045733 A CN200910045733 A CN 200910045733A CN 101793894 A CN101793894 A CN 101793894A
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Abstract
The invention belongs to the biomedicine field and discloses a direct competitive enzyme-linked immunoassay kit for detecting medroxyprogesterone acetate. The invention provides the synthesis scheme of the medroxyprogesterone acetate antigen as follows: firstly synthesizing medroxyprogesterone acetate-3-carboxymethyloxime with medroxyprogesterone acetate, activating medroxyprogesterone acetate-3-carboxymethyloxime with N-hydroxysuccinimide under catalysis of carbodiimide and binding the activated substance with bovine serum albumin to synthesize the immunogen; and binding the immunogen with horseradish peroxidase to synthesize the enzyme-labeled antigen. The immunogen is used for immunizing the New Zealand white rabbits to obtain the medroxyprogesterone acetate polyclonal antibody. During assay, the goat anti-rabbit polyclonal antibody (second antibody) is used for coating the enzyme label plate, the second antibody is bound with the medroxyprogesterone acetate polyclonal antibody, and samples or medroxyprogesterone acetate standard and the enzyme-labeled antigen are added after washing for developing and assay, thus obtaining the result. The reagents prepared in the kit are convenient to use. The invention provides a rapid, peculiar, sensitive and accurate method for detecting medroxyprogesterone acetate during food security and medicine detection.
Description
Technical field the invention belongs to biological medicine technology field, drug test, food safety detection reagents.Particularly, the present invention utilizes immunoreactive high sensitivity and specificity, and the enlarge-effect of enzymatic reaction and easily detection property, a kind of direct competitive ELISA measuring reagent kit that detects Medroxyprogesterone of setting up
(Medroxyprogesterone acetate MPA) is a kind of derivatives of progesterone of synthetic to the background technology medroxyprogesterone acetate, claims medroxyproges-terone acetate again, and its progestin is 10～30 times of progesterone.Similarly derivatives of progesterone still has megestrol acetate (Megestrol acetate) with it, chlormadinone (Chlormadinineacetate) etc., and this compounds is referred to as acetylizad progestational agents.Medically be used for practising contraception treatment dysmenorrhoea, functional amenorrhea, threatened abortion etc.On aquaculture, this compounds has the acceleration growth of animal, increases the effect of efficiency of feed utilization, so the Ceng Zuowei feed addictive.But Medroxyprogesterone etc. easily store in animal body, and the people is edible the residual food of this compounds, and the sexual dysfunction of causing is arranged, liver and kidney dysfunction, potentiality harm such as induced tumor even.So each state of the world all expressly forbids adding such material in feed.China also with Medroxyprogesterone as " veterinary drug of forbidding in the food animal feed and compound ".
The detection method of relevant Medroxyprogesterone adopts gas chromatography-mass spectrography (GC-MS) more now, liquid chromatograph mass spectrography (LC-MS) or multistage coupling technique such as LC-MS-MS, and its equipment requirements height, technical difficulty is big, requires also high to the technician.The present invention utilizes the enzyme-linked immunoassay principle, detection by quantitative animal tissue, ight soil, bile. and the Medroxyprogesterone in the urine is residual, is a kind of expensive instrument input that do not need, and the personnel of general medium educational level are through short-term cultivation, the just detection method that can grasp.This kit detects the Medroxyprogesterone in several samples, for Medroxyprogesterone in the food and in the clinical medicine detects, provides a kind of efficient, fast, and special detection method.
Summary of the invention the invention provides a kind of enzyme linked immunological kit of fast detecting Medroxyprogesterone.The Medroxyprogesterone kit of its preparation, the range of linearity are 0.1ng/ml～20ng/ml, IC
50For about 1.9ng/ml, sensitivity is 0.08ng/ml.Stationary phase is (4 ℃ of refrigerators are preserved) more than 6 months.
The present invention discloses immunogenic synthetic, the immunity of animal, the preparation of specific antibody, the debugging of kit and assembling, the pre-treatment and the assay method of determining to reach sample of the various parameters of kit.
Medroxyprogesterone, chemical name: 17 β-hydroxyl-19-demethyl-androstane-4-alkene-3-ketone.Be abbreviated as MPA, molecular weight 274.4 is small-molecule substance, does not have immunogenicity and only has an immunoreactivity, produces the ELISA measuring reagent kit of Medroxyprogesterone, must prepare the antibody that Medroxyprogesterone is had high degree of specificity.
Obtain Medroxyprogesterone is had the antibody of high degree of specificity, the most important condition is that the antigen that can excite animal to produce antibody will be arranged.Therefore, immunogenic preparation is the most important.The scheme of its solution is the covalently bound last macromolecular substances of Medroxyprogesterone, normally with a kind of protein cross.Commonly used have a bovine serum albumin(BSA), ovalbumin, and thyroglobulin, big keyhole maple hemocyanin etc., the present invention discloses the immunogenic method of a kind of preparation Medroxyprogesterone.At first use a hydroxylamine compound, the ethyloic azanol is modified MPA, generates Medroxyprogesterone-3-ethyloic oxime (MPA-3-CMO), the latter and N-maloyl imine reaction are with two hexamethylene carbodiimides (DCC) or water-soluble carbodiimide { 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; 1-hexamethylene-3-(2-agate quinoline-4-ethyl) carbodiimide is to the methylbenzene sulfinate } catalysis, make it activation, link to each other with free amido in the carrier protein molecule then, generate the Medroxyprogesterone immunogene.
Immunogene has been arranged, and second step was exactly to use the immunogen immune animal, and the present invention is New Zealand's Female rabbits of immunity 1.5～2kg size, obtained Medroxyprogesterone is had the polyclonal antibody of specific reaction.The invention provides a kind of immunization protocol that can produce high titre antibody.
For detection specificity antibody, set up the direct competitive enzyme-linked immunoassay, must synthesize Medroxyprogesterone and the crosslinked antigen-enzyme conjugate (enzyme-labelled antigen) of a kind of enzyme, crosslinked enzyme comprises: horseradish peroxidase, alkaline phosphatase, beta galactosidase etc.The present invention discloses a kind of synthetic Medroxyprogesterone and the crosslinked method of enzyme, with the preparation enzyme-labelled antigen.
On above basis, the assembling detection kit.With polystyrene 96 holes or 48 hole ELISA Plate, bag is anti-by two.(select goat anti-rabbit antibody for use, commodity supply is arranged.Generally select the height of tiring for use, tire 1: 1000～more than 2000, good stability two anti-.)。
Optimize bag by concentration, antibody concentration, enzyme-labelled antigen concentration makes it to reach the detection requirement.
The invention provides preparation and the assembling flow path of forming the kit related reagent:
(1) with the two anti-coated elisa plate of choosing.What (2) hatch that back washing goes not adsorb is unnecessary two anti-.(3) with not adsorbing two anti-unnecessary sites on the another kind of protein blocking plate, the non-specific adsorption when measuring to reduce.(4) provide Medroxyprogesterone antibody and Medroxyprogesterone-enzyme conjugate (enzyme-labelled antigen) concentrate.(5) provide antibody and enzyme-labelled antigen to use dilution.(6) provide concentrated cleaning solution (7) that the titer of Medroxyprogesterone is provided, with 7 standard: 0ng/ml of specimen preparation liquid preparation, 0.1ng/ml, 0.5ng/ml, 1.0ng/ml, 5.0ng/ml, 10.0ng/ml, 20.0ng/ml (8) provide enzyme colour developing buffer solution and zymolyte liquid, the used zymolyte of this kit is H
2O
2And tetramethyl benzidine (TMP), H
2O
2Be assigned in the colour developing buffer solution, tetramethyl benzidine (TMP) is preparation separately.(8) provide the enzyme reaction stop buffer, stop buffer is a 2M sulfuric acid.(9) sampling prepares liquid.The preparation of sample during for preparation Medroxyprogesterone standard and mensuration.
Above reagent is all put 4 ℃ of refrigerators preservations.
Use the detection step that this kit detects Medroxyprogesterone:
(1) according to working sample number and the required hole count of production standard curve (every sample and standard are all done two holes), take out the hole bar of required usefulness from refrigerator, placement makes temperature balance to room temperature (25 ℃).
(2) with the Medroxyprogesterone antibody concentrated solution with antibody diluent by dilution in 1: 100 after, every hole adds 100 μ l, adds a cover, room temperature was placed 30 minutes.
(3) with concentrated cleaning solution with DDW by dilution in 1: 9 after, as cleansing solution, remove antibody liquid in the hole, with cleansing solution washing 3 times, every hole, each 250 μ l, add cleansing solution at every turn after, left standstill 3 minutes, outwell, dry, wash again next time.Last washing is got rid of liquid as far as possible.
(4) each hole adds standard or sample, and each standard and sample respectively add 2 holes, and every hole adds 50 μ l.With the enzyme-labelled antigen concentrate, with antibody diluent by 1: 100 the dilution after, every hole adds 50 μ l.Other gets 2 holes, adds 0 standard, 50 μ l and antibody diluent 50 μ l as blank well, parallel oscillation ELISA Plate slightly, liquid in each hole of mixing.Add a cover, room temperature was placed 30 minutes.
(5) wash each hole with (3), dry at last.
(6) each hole adds colour developing liquid 100 μ l, and colour developing liquid is made up of colour developing damping fluid and TMB liquid, faces the time spent with preparation in 10: 1 (i.e. 1000 μ l colour developing damping fluid adds 100 μ l TMB liquid).After the adding, add a cover, room temperature dark place lucifuge was placed 15 minutes.
(7) every hole adds stop buffer 50 μ l, and mixing in 30 minutes, is used microplate reader, under wavelength 450nm, measures its optical density.
OD with each hole
450Deduct the OD of blank well
450, get the practical measurement value in each hole.Obtain the mean value of two parallel holes, get the measured value of each standard or sample.Obtain the inhibiting rate of each standard and sample according to following formula:
The inhibiting rate %=of each standard or sample
(measured value of the measured value of each standard or sample/0 standard) * 100%
Logarithm value with each normal concentration is X, and its corresponding inhibition ratio is Y, maps on coordinate axis, get the semilog coordinate typical curve of Medroxyprogesterone concentration, on typical curve, find out the point on the corresponding X-axis of sample inhibiting rate, just can calculate the concentration of Medroxyprogesterone in the sample.The related software that now existing ELISA calculates utilizes these software, as long as the input measured value just can be obtained a result immediately.
The detection method that this kit is used belongs to direct competitive enzyme-linked immunoassay method.96 hole ELISA Plate are wrapped in advance by two anti-, add the specific antibody of Medroxyprogesterone, after this antibody and two resistive connections close, are fixed on the ELISA Plate.Then, add standard antigen or contain the sample and the enzyme mark-Medroxyprogesterone of Medroxyprogesterone, Medroxyprogesterone and enzyme mark-Medroxyprogesterone in standard or the sample, common competition and Medroxyprogesterone antibodies are after hatching, unconjugated enzyme mark-Medroxyprogesterone, chromogenic assay are removed in washing.The logarithm value of Medroxyprogesterone amount is inverse ratio in measured value and standard or the sample, reaches the purpose of quantitative measurement Medroxyprogesterone with this.
Description of drawings
The Medroxyprogesterone typical curve of Fig. 1 for doing with this kit measurement.
Following embodiment can further be understood the present invention.A kind of method for optimizing only is provided herein, but content of the present invention and claim are not constituted any limitation.
Synthesizing of embodiment 1 Medroxyprogesterone immunogene and enzyme-labelled antigen
1.1 Medroxyprogesterone-3-ethyloic oxime (MPA-3-CMO) is synthetic
Take by weighing Medroxyprogesterone 83mg (0.3mmmol) and be dissolved in the 1ml anhydrous pyridine, stir down, add 45mg (0.45mmol) ethyloic azanol, about 24 hours of stirring at room (sampling midway, silica gel thin-layer chromatography is identified, stopped when oximate is complete .), the decompressing and extracting pyridine gets yellow oil.Add the 2ml acetic acid ethyl dissolution, it is inferior to give a baby a bath on the third day after its birth with distilled water, isolates organic phase.The heating decompression goes ethyl acetate closely dried, adds the 3ml ether, puts refrigerator, gets white precipitate.Centrifugal, will precipitate drying, this is Medroxyprogesterone-3-ethyloic oxime, must about 40mg.Be dissolved in 1ml DMF, identify with silica gel thin-layer chromatography (TLC).Among the TLC figure, MPA-3-CMO stays initial point, and MPA then leaves initial point, moves forward with chromatographic solution.
1.2 the activation of Medroxyprogesterone-3-ethyloic oxime
The Medroxyprogesterone of last preparation-3-ethyloic oxime DMF liquid, add 40mg DCC (about 0.2mmol), under 4 ℃ of stirrings, add NHS 23mg (about 0.2mmol), 4 ℃ of stirrings of lucifuge are spent the night. next day, the centrifugal precipitation of going. and supernatant is the Medroxyprogesterone-3-ethyloic oxime (MPA-3-CMO) of activation.
1.3 immunogene: the synthetic BSA of the taking by weighing 50mg of Medroxyprogesterone-bovine serum albumin(BSA) (MPA-BSA) adds the distilled water and the 2000 μ l DMF dissolving of 2000 μ l PH8～9, under 4 ℃ of stirrings, MPA-3-CMO 500 μ of an adding activation l.4 ℃ stirring spend the night. next day, take out with 0.01M PBS (pH7.5) dialysis 3 days. change dislysate every day 2 times. after the dialysis, decide protein concentration, and, calculating crosslinking rate with uv scanning evaluation. this method crosslinking rate is generally about 15～20.
1.4 enzyme-labelled antigen: MPA-horseradish peroxidase (MPA-HRP) synthetic
Take by weighing HRP 10mg and add the distilled water of 500 μ l PH8～9 and 500 μ l DMF dissolving, under 4 ℃ of stirrings, MPA-3-CMO 100 μ of an adding activation l.4 ℃ stirring spend the night. next day, take out with 0.01MPBS (pH7.5) dialysis 3 days. change dislysate every day 2 times. after the dialysis, cross Sephadex G 25 posts, collect coloured peak. be synthetic enzyme-labelled antigen: MPA-horseradish peroxidase (MPA-HRP).
The production of embodiment 2 Medroxyprogesterone polyclonal antibodies
With 3 New Zealand screech owl does, every rabbit immunity 4 times. be made into 1mg/ml concentration with immunogene MPA-BSA first, get 3ml, add the 3ml Freund's complete adjuvant, after fully emulsified. every rabbit with 2ml (being the 1mg immunogene) in rabbit back, the multiple spot hypodermic injection is a first immunisation. later on respectively at the 21st day, 51 days, 91 days immune 3 times again. the 21st day, 51 days, immunogene with 3ml 1mg/ml concentration, add the emulsification of 3ml incomplete Freund, every rabbit is used 1ml, in buttocks muscles two side injections. back 10 days of immunity in the 51st day, the antibody production is checked in blood sampling. the 91st day, immunogene is made into 0.5mg/ml solution with 0.9% sodium chloride liquid, exempts from the immunogene from ear vein injection 1ml for every. this time, and the 10th day, just can be from rabbit ear vein or heart results blood. to collect blood and condense naturally by it. centrifugal collection serum, this is the polyclonal antibody of anti-Medroxyprogesterone..
According to this immunization protocol, generally can both gather in the crops the polyclonal antibody of high titre.
With two anti-coated elisa plates, utilize synthetic enzyme-labelled antigen (MPA-HRP), to measure polyclonal antibody and tire. the antiserum titre of production can reach more than 100,000. with using by dilution in 1: 10000～1: 20000.
Embodiment 3 optimizes kit and forms, and determines each reagent ultimate density
Determine that with the square formation method bag is anti-by two, how anti-MPA is, MPA-HRP three's optium concentration.
3.1 coated elisa plate: the two anti-1mg/ml concentration that are made into, after diluting by 1: 1000 (1000 μ l damping fluids add 1 μ l two and resist) with 0.01M carbonate buffer solution (pH9.6), every hole adds 100 μ l, adds a cover, and puts into 4 ℃ of refrigerator overnight.Inferior daily cleansing solution is washed 3 times, and after last the drying, every hole adds 200 μ l confining liquids, and confining liquid is the 0.01M carbonate buffer solution (pH9.6) that contains 2% ovalbumin.After the adding, add a cover, put into 4 ℃ of refrigerator overnight.Inferior daily cleansing solution is washed 3 times, after last the drying, ELISA Plate is put into 37 ℃ of baking ovens 30 minutes, oven dry.Take out back aluminium foil vacuum packaging.
3.2 join antibody concentrated solution: how anti-with preparation MPA, dilution (preserving liquid) in 1: 1500 with antibody.Use by dilution in 1: 100 with antibody diluent during mensuration.
3.3 join the MPA-HRP concentrate: synthetic MPA-HRP, preserve liquid with enzyme, by dilution in 1: 10.
Use by dilution in 1: 100 with antibody diluent during mensuration.
3.4 join concentrated cleaning solution: the Tween-20 of 0.5M PBS liquid (pH7.2)+0.5% volume is a cleansing solution.
Time spent is used by dilution in 1: 9 with distilled water or deionized water.
3.5 join antibody diluent: for 0.1M PBS (pH7.2) adds 0.1% gelatin.
3.6 join sample preparation liquid: add 0.05% Tween-20 for 0.1M PBS (pH7.2) adds 0.1% sodium salicylate.
3.7 preparation MPA standard: join with sample preparation liquid.Prepare 7 standard: 0ng/ml altogether, 0.1ng/ml, 0.5ng/ml, 1.0ng/ml, 5.0ng/ml, 10.0ng/ml, 20.0ng/ml.
3.8 preparation colour developing liquid: colour developing liquid is partly formed by two, and the one, " colour developing damping fluid ", another is " TMB " liquid.Colour developing damping fluid: Na
2HPO
41.84g+ citric acid 0.47g+H
2O 100ml+30%H
2O
250 μ l,
TMB liquid: the ethanol with 95% or be made into 1mg/ml concentration with dimethyl sulfoxide.
3.9 join stop buffer: 2M sulfuric acid.The concentrated sulphuric acid then can by 1: 8 dilute with water.
3.10 kit is formed:
(1) wrapped by two anti-96 holes or 48 hole ELISA Plate (vacuum In Aluminium Foil Packing)
(2) MPA standard: 7 standard: 0ng/ml, 0.1ng/ml, 0.5ng/ml, 1.0ng/ml, 5.0ng/ml, 10.0ng/ml, 20.0ng/ml
(3) MPA antibody concentrated solution (time spent is used by dilution in 1: 100)
(4) MPA-HRP concentrate (time spent is used by dilution in 1: 100)
(5) antibody diluent
(6) colour developing damping fluid
(7) TMB liquid
(8) stop buffer.
(9) 10X cleansing solution
(10) specimen preparation liquid.
Implement 4 sample preparation and mensuration
4.1 the processing of working sample:
Urine sample: the 2ml freshly voided urine adds 2ml 0.1M acetate buffer solution (pH4.8), adds 40 μ l glusulase liquid, 6ml distilled water, and 37 ℃ are incubated 4 hours, enzymolysis.Then, the boiling water bath heating is 5 minutes.Centrifugal, to get supernatant 1ml and add the 4ml normal hexane, rotation mixes extracting 10 minutes.Centrifugal, get organic phase 2ml, N
2Air-blowing is done, and residue adds 200 μ l specimen preparation liquid, and rotation mixes.Equilibrium at room temperature 30 minutes is made sample determination liquid.
Tissue sample: the 1g tissue sample adds 5ml homogenate (0.9% sodium chloride+0.1% sodium salicylate), homogenate.Then, the boiling water bath heating is 5 minutes.The homogenate constant volume is an original volume.Centrifugal, to get supernatant 1ml and add the 4ml normal hexane, rotation mixes extracting 10 minutes.Centrifugal, get organic phase 2ml, N
2Air-blowing is done, and residue adds 200 μ l specimen preparation liquid, and rotation mixes.Equilibrium at room temperature 30 minutes is made sample determination liquid.
The dilution factor of urine samples and tissue sample dilution factor are 2.
4.2 the mensuration of sample and titer: the 4th, 5 page of described detection step to specifications, measure with the kit that assembles.
It is as follows that mensuration gained standard items are counted a tree name:
The typical curve of being painted as shown in Figure 3.IC
50＝1.9ng/ml
Implement each parameter of 5 kits
The specificity of kit and cross reaction:
Medroxyprogesterone 100%
Megestrol acetate 60%
Chlormadinone 60%
Progesterone＜2%
Hydrocortisone＜2%
Accuracy: do 24 holes, replication with the 0.5ng/ml standard.Calculate " variation in the plate " CV=4.6% " to make a variation between plate "＜15%.Sensitivity is 0.08ng/ml.
Degree of accuracy: do recovery test with urine, add 0.1ng/ml respectively, 1ng/ml, its recovery of 10ng/ml is respectively: 73%, 86%, and 83%
Kit stability:
Heat stabilization test is put kit in 37 degree, puts for 3 weeks, is equivalent to 4 degree and preserves 7.4 months.The optical density of its 0 standard and each standard descends about 25%. but its trend is constant. and the stationary phase of visible kit is more than half a year.
Claims (6)
1. the present invention discloses a kind of direct competitive enzyme linked immunological kit of measuring Medroxyprogesterone.
Its feature comprises: immunogenic preparation, the preparation of enzyme-labelled antigen, the preparation of Antibody Preparation and kit each component and the bag quilt of ELISA Plate.
2. according to right 1 described enzyme linked immunological kit, it is characterized in that the synthetic immunogenic method of Medroxyprogesterone.Be dissolved in anhydrous pyridine with Medroxyprogesterone, stir down, add the ethyloic azanol, the mol ratio of Medroxyprogesterone and ethyloic azanol is about 1: 1.5, and stirring at room is after 24 hours, and the decompressing and extracting pyridine gets yellow oil.Add acetic acid ethyl dissolution, it is inferior to give a baby a bath on the third day after its birth with distilled water, isolates organic phase.The heating decompression goes ethyl acetate closely dried, adds diethyl ether, and puts refrigerator, gets white precipitate.Be Medroxyprogesterone-3-ethyloic oxime, again with N-maloyl imine reaction, with two hexamethylene carbodiimides (DCC) or water-soluble carbodiimide { 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; 1-hexamethylene-3-(2-agate quinoline-4-ethyl) carbodiimide-to the methylbenzene sulfinate } catalysis, getting Medroxyprogesterone-3-ethyloic oxime activator, this activator and carrier protein link with amino on the carrier protein under alkali condition, are the immunogene of Medroxyprogesterone.Carrier protein is as bovine serum albumin(BSA), ovalbumin, thyroglobulin, big keyhole maple hemocyanin.
3. according to right 1-2, its spy is, the method for synthetic a kind of enzyme-labelled antigen: with Medroxyprogesterone-3-ethyloic oxime activator, link with enzyme under alkali condition, its enzyme can be: horseradish peroxidase, alkaline phosphatase, beta galactosidase.
4. according to right 1 described enzyme linked immunological kit, it is characterized in that the optimization preparation of kit solution.The preparation of standard solution and sample is the phosphate buffered solution with the 0.1～0.01M that contains 0.1%～0.5% sodium salicylate and 0.05%～0.1% Tween-20.
Antibody diluent 0.1～0.01M the phosphate buffered solution that contains 0.1～0.5% gelatin.
Colour developing liquid is two components, and wherein, a component is made up of sodium hydrogen phosphate and citric acid for the colour developing damping fluid, and every milliliter contains 0.2～0.5 μ l30%H
2O
2Another component is tetramethyl benzidine (TMB) liquid.TMB liquid is mixed with 1mg/ml concentration with 95% ethanol or dimethyl sulfoxide.
5. according to right 1 described enzyme linked immunological kit, it is characterized in that, with adopting goat anti-rabbit antibody (two is anti-) bag by 96 or 48 hole ELISA Plate.With not adsorbing two anti-positions in the 2% ovalbumin fluid-tight closed pore.
6. one kind with direct competitive enzyme-linked immunoassay method, measures the method for Medroxyprogesterone in the various samples.It is characterized in that:
(1) pre-treating method of sample.Use the solvent extraction method, simplified the mensuration process greatly.
(2) with the kit measurement of the described technology assembling of right 1-5, testing process is:
With the ELISA Plate that is coated with goat anti-rabbit antibody, add Medroxyprogesterone antibody, to hatch the back washing and dry, adding standard or sample solution and enzyme-labelled antigen (Medroxyprogesterone-horseradish peroxidase) are hatched the back washing and are dried, and add chromogenic reagent, stop, and measure.The logarithm value and the OD of standard or sample Medroxyprogesterone concentration
450Value is inverse ratio.Reach the purpose of quantitative measurement Medroxyprogesterone with this.This direct competitive ELISA measuring reagent kit, sensing range are 0.1～20ng/ml, and sensitivity can reach 0.08ng/ml.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102504002A (en) * | 2011-11-29 | 2012-06-20 | 南京农业大学 | Progesterone artificial antigen, preparation method for same and application thereof |
| CN103134715A (en) * | 2011-11-30 | 2013-06-05 | 内蒙古蒙牛乳业（集团）股份有限公司 | Pretreatment method and detection method for detecting prolactin content in lactobacillis beverage |
| CN103134716A (en) * | 2011-11-30 | 2013-06-05 | 内蒙古蒙牛乳业（集团）股份有限公司 | Pretreatment method and detection method for detecting progesterone content in lactobacillis beverage |
| CN103175960A (en) * | 2013-03-19 | 2013-06-26 | 南京农业大学 | Enzyme immunoassay method for progestational hormone in excrement of sows and method for detecting oestrous cycle of sow |
| CN103235143A (en) * | 2012-12-25 | 2013-08-07 | 中山大学达安基因股份有限公司 | Kit for quantitatively measuring neonatal 17alpha-OHP with time-resolved fluoroimmunoassay method |
| CN103712982A (en) * | 2013-12-13 | 2014-04-09 | 山东博科生物产业有限公司 | High-sensitivity TMB (tetramethylbenzidine) color developing liquid and preparation method thereof |
| CN104086647A (en) * | 2014-07-15 | 2014-10-08 | 江南大学 | Method for synthesizing artificial antigen of progesterone |
| CN104215780A (en) * | 2013-05-31 | 2014-12-17 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting medroxyprogesterone and application thereof |
| CN104655846A (en) * | 2013-11-20 | 2015-05-27 | 镇江先创生物科技有限公司 | Enzyme linked immunosorbent assay kit for detecting progesterone and detection method thereof |
| CN107607702A (en) * | 2017-07-21 | 2018-01-19 | 上海科华生物工程股份有限公司 | The preparation method and applications of sterols haptens alkaline phosphatase enzyme conjugate |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472533A (en) * | 2002-07-29 | 2004-02-04 | 中国农业科学院饲料研究所 | Reagent box and method for determining content of medroxyprogesterone acetate |
| CN101066998A (en) * | 2007-04-27 | 2007-11-07 | 江南大学 | Prepn of specific antibody of provera acetate and method of using the antibody in homogenous or heterogenous enzyme-linked immune analysis |
-
2009
- 2009-02-03 CN CN200910045733A patent/CN101793894A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472533A (en) * | 2002-07-29 | 2004-02-04 | 中国农业科学院饲料研究所 | Reagent box and method for determining content of medroxyprogesterone acetate |
| CN101066998A (en) * | 2007-04-27 | 2007-11-07 | 江南大学 | Prepn of specific antibody of provera acetate and method of using the antibody in homogenous or heterogenous enzyme-linked immune analysis |
Non-Patent Citations (5)
| Title |
|---|
| 刘 等: "醋酸甲羟孕酮人工抗原的合成及间接ELISA的建立", 《化学试剂》 * |
| 刘烜 等: "肠衣中醋酸甲羟孕酮残留ELISA检测方法的应用", 《中国国境卫生检疫杂志》 * |
| 杨泽晓 等: "醋酸甲羟孕酮人工抗原的合成与鉴定", 《西北农林科技大学学报( 自然科学版)》 * |
| 郭金花 等: "抗醋酸甲羟孕酮抗体的制备及初步鉴定", 《细胞与分子免疫学杂志》 * |
| 郭金花: "食品中残留醋酸甲羟孕酮酶免疫检测方法研究", 《中国优秀博硕士学位论文数据库（硕士） 农业科技辑》 * |
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