CN1472533A - Reagent box and method for determining content of medroxyprogesterone acetate - Google Patents

Reagent box and method for determining content of medroxyprogesterone acetate Download PDF

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Publication number
CN1472533A
CN1472533A CNA021253706A CN02125370A CN1472533A CN 1472533 A CN1472533 A CN 1472533A CN A021253706 A CNA021253706 A CN A021253706A CN 02125370 A CN02125370 A CN 02125370A CN 1472533 A CN1472533 A CN 1472533A
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China
Prior art keywords
reagent bottle
reagent
enzyme
medroxyprogesterone acetate
housed
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CNA021253706A
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Chinese (zh)
Inventor
丁宏标
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Feed Research Institute of Chinese Academy of Agricultural Sciences
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Feed Research Institute of Chinese Academy of Agricultural Sciences
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Priority to CNA021253706A priority Critical patent/CN1472533A/en
Publication of CN1472533A publication Critical patent/CN1472533A/en
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Abstract

The kit for the checking method includes kit body, enzyme saleplate, reagent bottle A packed with standard methocorporin acetate reagent, reagent bottle B packed with methocorporin acetate antibody diluted solution, reagent bottle C packed with enzyme scale antigen reagent, reagent bottle D packed with enzyme seale antigen diluted solution, reagent bottle E packed with phosphate solid of tween-20, reagent bottle F packed with O-phenylenediamine, reagent bottle G packed with sodium acetate-citrin buffer solution, reagent bottle H packed with H2O2 solution and reagent bottle I packed with sulhpuric acid solution.

Description

A kind of kit and detection method thereof that detects medroxyprogesterone acetate content
Technical field
The present invention relates to a kind of kit and detection method thereof that detects medroxyprogesterone acetate content, particularly relate to a kind of immunity detection reagent and detection method thereof that detects medroxyprogesterone acetate in animal food and the feed.
Background technology
(Medroxyprogesterone MPA), has another name called DMPA to medroxyprogesterone acetate, and Amen is a kind steroid hormone medicine, has been widely used in the replacement therapy of menopausal women syndrome disease.But the medroxyprogesterone acetate in the food has serious adverse to the people, and the sperm that can suppress the people generates, and causes sterilely, so the medroxyprogesterone acetate in the animal food is residual to the mankind, and particularly children's health has constituted potential serious harm.The U.S. and the European Community are to the residual qualification that strictness is arranged of medroxyprogesterone acetate in animal body and the animal food, and China does not formulate the food hygienic standard of medroxyprogesterone acetate residual quantity as yet.Because medroxyprogesterone acetate has significant assimilation, can obviously promote growth of animal, and is effective and inexpensive, is used as feed addictive at the U.S., Australia, New Zealand's approved, but strict withdrawal time is arranged.China and European Union forbid making feed addictive with it, but still have the people illegally to use.In order to ensure animal food eater's health, the detection that enlarges animal product and feed is very important.
Measure the method for the residual amount of medroxyprogesterone acetate, mainly contain the active method of mouse bioassay, chromatography, radioimmunology, enzyme immunoassay (EIA) etc., domestic and foreign literature has many reports, but it is more easy to seek operation, degree of accuracy is higher, and the detection method that specificity is stronger is the direction that vast researcher is endeavoured to study always.
Summary of the invention
The purpose of this invention is to provide a kind of simple in structure, easy to operate, low-cost medroxyprogesterone acetate enzyme immunoassay kit.
A kind of medroxyprogesterone acetate enzyme immunoassay kit, it comprises box body; ELISA Plate; The reagent bottle A of standard medroxyprogesterone acetate reagent is housed; The reagent bottle B of medroxyprogesterone acetate antibody diluent is housed; The reagent bottle C of enzyme-labelled antigen reagent is housed; The reagent bottle D of enzyme-labelled antigen dilution is housed; The reagent bottle E of the phosphate solid that contains Tween-20 is housed; The reagent bottle F of o-phenylenediamine is housed; The reagent bottle G of sodium acetate-citrate buffer solution is housed; H is housed 2O 2The reagent bottle H of solution; The reagent bottle I of sulfuric acid solution is housed.
Dilution among the described reagent bottle B is for containing 10g/L BAS, 0.01mol/L, the PBST solution of pH7.4; Enzyme-labelled antigen reagent among the described reagent bottle C is medroxyprogesterone acetate-horseradish peroxidase or enzyme mark goat anti-rabbit antibody or the anti-rabbit antibody of enzyme mark pig; Enzyme-labelled antigen dilution among the described reagent bottle D is for containing 1% gelatin, 0.01mol/L, the PBS solution of pH7.4.
In order to make the reagent bottle in the kit place firm more also difficult brokenly, also be provided with the sponge carriage in the described kit.Be shaped on 9 hole and grooves of laying reagent bottle on the described sponge carriage, described reagent bottle A, B, C, D, E, F, G, H, I all are contained in interior corresponding hole of sponge carriage and the groove.
Described ELISA Plate is made up of the plastic strip in plastic stent and some caves with holes that separate separately.
Kit should be placed under the 2-8 ℃ of condition and store.
Second purpose of the present invention provides the method that the above-mentioned medroxyprogesterone acetate enzyme immunoassay kit of a kind of the present invention of utilization detects medroxyprogesterone acetate.
For realizing this purpose, the present invention adopts the medroxyprogesterone acetate in enzyme immunoadsorption (ELISA) the competition ratio juris test sample, and its technical scheme may further comprise the steps:
1) handles detected sample;
2) with polyclone or monoclonal medroxyprogesterone acetate antibody sandwich on ELISA Plate, ELISA Plate is placed on 2 ℃ of-8 ℃ of refrigerator overnight;
3) the dilution mixing in the adding 20-30ml B reagent bottle in the A reagent bottle;
4) the enzyme-labelled antigen dilution in the adding 10-20ml D reagent bottle in the C reagent bottle, the dissolving mixing is preserved down at 2-8 ℃;
5) in the E reagent bottle, add deionized water and be mixed with cleansing solution, wash ELISA Plate 2-4 time, each 3-4 minute at interval, wash the back and pat dry with thieving paper with this cleansing solution;
6) in the ELISA Plate aperture, add A reagent bottle and reagent in the B reagent bottle and the sample diluting liquid of handling for preparing respectively, reagent in A reagent bottle and the B reagent bottle is as the positive and negative control, add the immune response that is at war with of reagent in the C reagent bottle prepare then, incubation is after 1.5 hours under 37-38 ℃ of condition, wash plate 4-6 time with the E cleansing solution again, each 3-4 minute at interval, pat dry;
7) in the ELISA Plate aperture, add the zymolyte mixed liquor respectively, mixing, 37-38 ℃ of lucifuge effect 15-20 minute, colour developing;
8) in the ELISA Plate aperture, add stop buffer stopped reaction in the I reagent bottle respectively, range estimation or measure A with microplate reader 490nmValue.
Described zymolyte mixed liquor is to prepare with the reagent among reagent bottle F, G, the H in half an hour before use, the method of preparation is, the reagent of G reagent bottle and F reagent bottle by volume portion rate is the ratio mixing of 7-9: 3-1, and by the H that adds in every milliliter of this mixed liquor in the 14 μ l H reagent bottles 2O 2Formulated.
Utilize kit provided by the present invention to detect easy, quick, sensitive, exactly and whether contain medroxyprogesterone acetate in animal food or the feed, cheap, sample pre-treatments is simple, extracts the back and just can directly measure, and once can measure great amount of samples.Detect medroxyprogesterone acetate with method of the present invention, lowest detectable limit can reach the MPA residue detection that 100ng/kg is particularly useful for meat milk egg products.
Embodiment
The preparation of embodiment 1, specific antibody (MPA monomer):
1, antigen preparation
With the medroxyprogesterone acetate-bovine serum albumin(BSA) (MPA-BSA) of synthetic as immunizing antigen.
2, immune animal
Adopt BALB/C small white mouse or rabbit as immune animal, immunizing antigen dosage is 50-100ng, hypodermic injection, and 3-4 is after week, 2-4 extracting spleen cell of lumbar injection booster immunization.
3, Fusion of Cells
SP2/0 myeloma cell and splenocyte are carried out Fusion of Cells in the solvent of polyvinyl alcohol (PVA), cultivation elects in the HAT nutrient solution.
4, hybridoma cell cloneization
Adopt limiting dilution assay screening hybridoma, up to monoclonal antibody that obtains complete homogeneity and stable monoclonal hybridoma strain.
5, the preservation of monoclonal antibody (McAb)
At liquid nitrogen or-20 ℃ of preservations, 37 ℃ of water-bath quick-thawings during use.
6, the production of McAb and purification
Light mouse peritoneal injecting fluid paraffin, 0.5ml/, 7-10 days pneumoretroperitoneum injection hybridomas 5 * 10 5-10 6/ only, gather ascites after 7-10 days.Carry out purifying through ammonium sulfate salting-out process, the bottle packing is stand-by.
The processing of embodiment 2, test sample milk
1, centrifugal 10 minutes degreases of milk sample 3500rpm;
2, draw and discard the upper strata fat deposit;
3, get 5ml degrease milk and add 150 μ l leather Rui Shi liquid I, shake up the back vibration and add 150 μ l leather Rui Shi liquid II again, shake up rapidly;
4,3500rpm is centrifugal 10 minutes;
5, remove supernatant, again with damping fluid with its 1: 1 the dilution (1 portion of supernatant+1 portion damping fluid);
6, getting 50 μ l is used for detecting.
The processing of embodiment 3, tested meat sample
1, with homogenizer or bruiser meat sample or kidney are carried out homogeneous;
2, get sample 5g behind the homogeneous, add 20ml acetonitrile solution (84 parts of acetonitrile+16 part water) and extracted 10 minutes;
3,3000rpm is centrifugal 10 minutes;
4, with 3ml deionized water dilution 3ml supernatant, use the 4.5ml ethyl acetate extraction again;
5,3000rpm is centrifugal 10 minutes;
6, the ethyl acetate layer is dried up;
7, with the 1.5ml damping fluid residue is dissolved, add 1.5ml hexane or normal heptane degrease again;
8, thoroughly remove the normal hexane or the normal heptane layer on upper strata;
9, getting 50 μ l subnatants is used for detecting.
Embodiment 4, usefulness kit of the present invention detect medroxyprogesterone acetate
1, handles detected sample;
2, with polyclone or monoclonal medroxyprogesterone acetate antibody sandwich on ELISA Plate, ELISA Plate is 12 * 8 holes, every hole 50-100 μ l, 37 ℃, incubation 2hr is with the sealing of 10g/L gelatin.Every ELISA Plate behind the bag quilt is sealed with aluminium foil bag, is placed on 4 ℃ of refrigerator overnight;
3, the dilution mixing in the adding 25ml B reagent bottle in the A reagent bottle; Be standard medroxyprogesterone acetate reagent in the A reagent bottle, in the B reagent bottle for containing 10g/L BAS, 0.01mol/L, the PBST solution of pH7.4;
4, the enzyme-labelled antigen dilution in the adding 15ml D reagent bottle in the medroxyprogesterone acetate-horseradish peroxidase in the C reagent bottle, the dissolving mixing is preserved down at 4 ℃; Enzyme-labelled antigen dilution in the D reagent bottle is for containing 1% gelatin, 0.01mol/L, the PBS solution of pH7.4;
5, in the E reagent bottle of the phosphate that contains 0.1% Tween-20 (Tween-20) (PBS), add 200ml distilled water and be mixed with the E cleansing solution, be used for the ELISA Plate washing.Wash ELISA Plate 3 times with this cleansing solution, washing lotion must not be overflowed, and each 3 minutes at interval, washes the back and pats dry with thieving paper;
6, substrate mixed liquor preparation: face with in preceding half an hour, according to each institute expense, with G: the F=8 by volume of the sodium acetate-citrate buffer solution in o-phenylenediamine in the F reagent bottle and the G reagent bottle: 1 ratio is mixed, and the ratio that adds the hydrogen peroxide in the 14 μ l H reagent bottles in every milliliter of this mixed liquor is mixed with the substrate mixed liquor again;
7, with the kit balance to room temperature, in the ELISA Plate aperture, add A reagent bottle and reagent in the B reagent bottle and the sample diluting liquid of handling for preparing respectively, reagent in A reagent bottle and the B reagent bottle is as the positive and negative control, add the immune response that is at war with of reagent in the C reagent bottle prepare then, concrete listed as table 1, add reagent and the testing sample dilution for preparing successively, the 1-3 hole is the standard control hole, the 4-12 hole is the sample determination hole, during every batch of mensuration, can put a testing sample in every hole, also can put same testing sample in several holes, carry out replicate determination, also available another piece ELISA Plate detects simultaneously, its 1-12 hole expansion is sample well, and the number of visual concrete testing sample is decision how much and flexibly.
Table 1:
Order Addition Hole number
????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 ????9 ????10 ????11 ????12
????1 ???25μl ????A ????B ????A Dilution back sample liquid
????2 Shake all
????3 ???25μl ????C ????D ????C ????C ????C ????C ????C ????C ????C ????C ????C
????4 Shake all
, in 4-12 sample determination hole, successively add determined antigen and enzyme-labelled antigen, and in 1-3 standard control hole, only add or not enzyme-added mark antigen by last table.Incubation is washed plate 4-6 time with the E cleansing solution after 1.5 hours again under 37 ℃ of conditions, each 3 minutes at interval, pats dry;
8, in each ELISA Plate aperture, add 50 μ l zymolyte mixed liquors respectively, mixing, 37 ℃ of lucifuge effects 15 minutes, colour developing;
9, in the ELISA Plate aperture, add sulfuric acid stop buffer stopped reaction (2mol/L in the I reagent bottle respectively; 50 μ l/ holes);
10, measure: compare 1-3 hole color earlier, No. 1 Kongzui is dark, and No. 2 take second place in the hole, and No. 3 holes are near colourless, and description standard is correct.
Visual method: comparative sample hole and No. 2 hole colors, if more of light color than No. 2 holes, then positive, on the contrary negative.If color and No. 2 holes are approaching, then survey A with microplate reader 490nmValue is to reach the purpose of accurate detection.

Claims (8)

1, a kind of medroxyprogesterone acetate enzyme immunoassay kit is characterized in that it comprises box body; ELISA Plate; The reagent bottle A of standard medroxyprogesterone acetate reagent is housed; The reagent bottle B of medroxyprogesterone acetate antibody diluent is housed; The reagent bottle C of enzyme-labelled antigen reagent is housed; The reagent bottle D of enzyme-labelled antigen dilution is housed; The reagent bottle E of the phosphate solid that contains Tween-20 is housed; The reagent bottle F of o-phenylenediamine is housed; The reagent bottle G of sodium acetate-citrate buffer solution is housed; H is housed 2O 2The reagent bottle H of solution; The reagent bottle I of sulfuric acid solution is housed.
2, kit according to claim 1 is characterized in that: the dilution among the described reagent bottle B is for containing 10g/L BAS, 0.01mol/L, the PBST solution of pH7.4; Enzyme-labelled antigen reagent among the described reagent bottle C is medroxyprogesterone acetate-horseradish peroxidase or enzyme mark goat anti-rabbit antibody or the anti-rabbit antibody of enzyme mark pig; Enzyme-labelled antigen dilution among the described reagent bottle D is for containing 1% gelatin, 0.01mol/L, the PBS solution of pH7.4.
3, kit according to claim 1 and 2 is characterized in that: also be provided with the sponge carriage in the described kit.
4, kit according to claim 3 is characterized in that: be shaped on 9 hole and grooves of laying reagent bottle on the described sponge carriage, described reagent bottle A, B, C, D, E, F, G, H, I all are contained in interior corresponding hole of sponge carriage and the groove.
5, kit according to claim 1 is characterized in that: described ELISA Plate is made up of the plastic strip in plastic stent and some caves with holes that separate separately.
6, a kind of method of utilizing the described kit of claim 1 to detect medroxyprogesterone acetate may further comprise the steps:
1) handles detected sample;
2) with polyclone or monoclonal medroxyprogesterone acetate antibody sandwich on ELISA Plate, ELISA Plate is placed on 2 ℃ of-8 ℃ of refrigerator overnight;
3) the dilution mixing in the adding 20-30ml B reagent bottle in the A reagent bottle;
4) the enzyme-labelled antigen dilution in the adding 10-20ml D reagent bottle in the C reagent bottle, the dissolving mixing is preserved down at 2-8 ℃;
5) in the E reagent bottle, add deionized water and be mixed with cleansing solution, wash ELISA Plate 2-4 time, each 3-4 minute at interval, wash the back and pat dry with thieving paper with this cleansing solution;
6) in the ELISA Plate aperture, add A reagent bottle and reagent in the B reagent bottle and the sample diluting liquid of handling for preparing respectively, reagent in A reagent bottle and the B reagent bottle is as the positive and negative control, add the immune response that is at war with of reagent in the C reagent bottle prepare then, incubation is after 1.5 hours under 37-38 ℃ of condition, wash plate 4-6 time with the E cleansing solution again, each 3-4 minute at interval, pat dry;
7) in the ELISA Plate aperture, add the zymolyte mixed liquor respectively, mixing, 37-38 ℃ of lucifuge effect 15-20 minute, colour developing;
8) in the ELISA Plate aperture, add stop buffer stopped reaction in the I reagent bottle respectively, range estimation or measure A with microplate reader 490nmValue.
7, the method for detection medroxyprogesterone acetate according to claim 6, it is characterized in that: described zymolyte mixed liquor is to prepare with the reagent among reagent bottle F, G, the H in half an hour before use, the method of preparation is, the reagent of G reagent bottle and F reagent bottle by volume portion rate is the ratio mixing of 7-9: 3-1, and by the H that adds in every milliliter of this mixed liquor in the 14 μ l H reagent bottles 2O 2Formulated.
8, the method for detection medroxyprogesterone acetate according to claim 6 is characterized in that: the dilution among the described reagent bottle B is for containing 10g/L BAS, 0.01mol/L, the PBST solution of pH7.4; Enzyme-labelled antigen reagent among the described reagent bottle C is medroxyprogesterone acetate-horseradish peroxidase or enzyme mark goat anti-rabbit antibody or the anti-rabbit antibody of enzyme mark pig; Enzyme-labelled antigen dilution among the described reagent bottle D is for containing 1% gelatin, 0.01mol/L, the PBS solution of pH7.4.
CNA021253706A 2002-07-29 2002-07-29 Reagent box and method for determining content of medroxyprogesterone acetate Pending CN1472533A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101793894A (en) * 2009-02-03 2010-08-04 王武康 Direct competitive enzyme-linked immunoassay kit for detecting medroxyprogesterone acetate
CN101957381A (en) * 2010-10-15 2011-01-26 无锡安迪生物工程有限公司 Double-channel test card for simultaneously testing estradiol and medroxyprogesterone
CN102128931A (en) * 2010-12-21 2011-07-20 江南大学 Method for detecting medroxyprogesterone (MPA) based on silver enhancement
CN101066998B (en) * 2007-04-27 2011-09-07 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
CN101598735B (en) * 2009-07-28 2012-10-24 南京农业大学 Method for measuring paper milk sample reproductive hormones concentration by enzyme-immunoassay method
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
CN108303553A (en) * 2017-12-05 2018-07-20 广东农工商职业技术学院(农业部华南农垦干部培训中心) Method and kit based on magnetic microsphere chemiluminescence determination medroxyprogesterone acetate content and application

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101066998B (en) * 2007-04-27 2011-09-07 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
CN101793894A (en) * 2009-02-03 2010-08-04 王武康 Direct competitive enzyme-linked immunoassay kit for detecting medroxyprogesterone acetate
CN101598735B (en) * 2009-07-28 2012-10-24 南京农业大学 Method for measuring paper milk sample reproductive hormones concentration by enzyme-immunoassay method
CN101957381A (en) * 2010-10-15 2011-01-26 无锡安迪生物工程有限公司 Double-channel test card for simultaneously testing estradiol and medroxyprogesterone
CN102128931A (en) * 2010-12-21 2011-07-20 江南大学 Method for detecting medroxyprogesterone (MPA) based on silver enhancement
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
CN104215780B (en) * 2013-05-31 2016-06-22 北京勤邦生物技术有限公司 The enzyme linked immunological kit of detection medroxyprogesterone and application thereof
CN108303553A (en) * 2017-12-05 2018-07-20 广东农工商职业技术学院(农业部华南农垦干部培训中心) Method and kit based on magnetic microsphere chemiluminescence determination medroxyprogesterone acetate content and application
CN108303553B (en) * 2017-12-05 2023-09-08 广东农工商职业技术学院(农业部华南农垦干部培训中心) Method for determining medroxyprogesterone acetate content based on magnetic microsphere chemiluminescence method, kit and application

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