CN104865391A - Anti-Mulerian hormone (AMH) enzyme-linked immunosorbent assay reagent - Google Patents
Anti-Mulerian hormone (AMH) enzyme-linked immunosorbent assay reagent Download PDFInfo
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- CN104865391A CN104865391A CN201510226042.1A CN201510226042A CN104865391A CN 104865391 A CN104865391 A CN 104865391A CN 201510226042 A CN201510226042 A CN 201510226042A CN 104865391 A CN104865391 A CN 104865391A
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- amh
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- linked immunosorbent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Abstract
The invention discloses an AMH enzyme-linked immunosorbent assay reagent. The reagent comprises solid-phase carriers and antibodies coated on the solid-phase carriers. The antibodies are formed by coupling of antigens with keyhole limpet hemocyanin and are obtained through purification after immunizing New Zealand rabbits. The antibodies are AMH antibodies. The antigens are 6 artificially synthesized polypeptides, the amino acid sequence of the 6 polypeptides are shown as SEQ ID NO1-SEQ ID NO6. By means of the reagent, AMH can be detected effectively, the antibodies with 6 kinds of antigens, so that the sensitivity is high, the detection sensitivity reaches 0.1ng/ml, and an effective detection mean is provided for AMH detection.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection reagent, be specifically related to the enzyme linked immunosorbent detection reagent that the anti-seedling of people strangles hormone.
Background technology
People's AMH (anti-Mulerian hormone, AMH) the biglycan albumen be made up of two identical 70kb subunits, relative molecular mass is 140000, forms transforming growth factor β superfamily with inhibin, activin, bone morphogenetic protein and growth and differentiation factor etc.Mankind AMH gene is positioned at No. 19 the short arm of a chromosome (19p13.3), and long 2.4 ~ 2.8 kb, have 5 extrons, 560 amino acid whose amyloid protein precursors of encoding.
Summary of the invention
Technical matters to be solved by this invention is to provide the enzyme linked immunosorbent detection reagent that the anti-seedling of a kind of people strangles hormone, and this enzyme linked immunosorbent detection reagent effectively can detect the anti-seedling of people and strangle hormone, and detection sensitivity reaches 0.1ng/ml.
The present invention solves the problems of the technologies described above adopted technical scheme: the anti-seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone, comprise solid phase carrier and be coated on the antibody on solid phase carrier, antibody is that antigen and hole relative hemocyanin lotus root connect, after immunize New Zealand rabbit, purifying obtains, described antibody anti-seedling of behaving strangles hormone antibody, described antigen is 6 polypeptide of Prof. Du Yucang, and the amino acid sequence of 6 polypeptide is as shown in SEQ ID NO1 ~ SEQ ID NO6.
Compared with prior art, the invention has the advantages that the anti-seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone, comprise solid phase carrier and be coated on the antibody on solid phase carrier, antibody is that antigen and hole relative hemocyanin lotus root connect, after immunize New Zealand rabbit, purifying obtains, this antibody anti-seedling of behaving strangles hormone antibody, and this antigen is 6 polypeptide of Prof. Du Yucang, and the amino acid sequence of 6 polypeptide is as shown in SEQ ID NO1 ~ SEQ ID NO6; This enzyme linked immunosorbent detection reagent effectively can detect the anti-seedling of people and strangle hormone, and owing to there being the antibody of 6 kinds of antigen, sensitivity is higher, and detection sensitivity reaches 0.1ng/ml, and the detection for people AMH provides effective detection means.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
One, the design of antigen: epitope analysis is carried out to AMH albumen from water wettability, accessibility, antigenicity, plasticity, CHARGE DISTRIBUTION, secondary structure prediction, design, filter out the polypeptide antigen (the raw work synthesis in Shanghai) of 6 Prof. Du Yucang, article 6, the amino acid sequence of polypeptide antigen is as shown in SEQ ID NO1 ~ SEQ ID NO6, connects respectively with hole relative hemocyanin (KLH) lotus root.
Two, the preparation of specific antibody: immune programme for children: the healthy new zealand white rabbit selecting 13 2 monthly ages, body weight 1.5 ~ 2.0kg, gets 1 as negative control group, and remaining 12 is test group; 13 first adaptability of new zealand white rabbit fed 2 weeks, edge of picking up the ears prior to before every animal immune extracts 2mL venous blood, and 4 DEG C of hold over night, get serum and make negative control reagent; The antigen of above-mentioned 6 kinds of KLH couplings is added in addition at Freund's complete adjuvant or Freund's incomplete adjuvant, wherein Freund's complete adjuvant or Freund's incomplete adjuvant V: antigenic solution (6 kinds of antigen equivalent, total concentration 500 μ g/ mL) V=1:1, emulsification in vortex oscillator, until do not disperse presenting spherical in an emulsion instillation water, obtain immunizing antigen reagent; Immunizing antigen reagent (antigen+Freund's complete adjuvant completely) at White Rabbit back multi-point injection, is injected 1 mL for every by initial immunity.The 28th day immunizing antigen reagent White Rabbit intramuscular injection 1 mL booster immunization with antigen+complete incomplete Freund's adjuvant, every 14d booster immunization 1 time later, 7d after 3rd booster immunization, from White Rabbit ear edge vein exploitating blood, adopt Dot-ELISA to measure antiserum titre, do not reach requirement if tire, continue to raise, repeat booster immunization, until obtain satisfied tiring; Tire after reaching requirement, arteria carotis gets blood results antiserum.
Three, the purifying of antibody: get the above-mentioned antiserum of 12mL, adds equal-volume 12mL PBS(concentration 0.01M, pH7.8, lower same), mixing; Add ammonium sulfate saturated solution 24mL while stirring, make into 50% saturation degree, put 60min for 4 DEG C, the centrifugal 10min of 10000rpm, abandons supernatant, with saturated ammonium sulfate 12 mL and PBS 12 mL mixed solution washing and precipitating secondary, removes supernatant; Precipitation is dissolved in 12mL PBS, drips saturated ammonium sulfate solution 6mL, put 30min for 4 DEG C, centrifugally remove supernatant, repeat this step once; Add 2.4mL physiological saline in precipitation, move in bag filter, dialyse does not have sulfate ion to lye, with the BaCl of mass percentage concentration 1%
2solution titration detects, then changes a normal saline dialysis, obtained antibody purification 2.5mL.
Four, the mark of antibody purification: 10mg horseradish peroxidase is dissolved in 0.2mL glutaraldehyde (whole mass percentage concentration 1.25%)+PBS(final concentration 0.1M, pH6.8) in solution, room temperature effect 18 hours, glutaraldehyde is removed in dialysis.Add physiological saline to 1mL, add 5mg antibody purification, add 0.1mL 1M carbonate buffer solution (pH9.6), 4 ° of C put 24 hours; Add 0.1mL lysine (concentration 0.2M), room temperature effect 2 hours, in PBS(concentration 0.15M, pH7.2) in, enough hemodialysis, centrifugation, supernatant is the antibody purification of horseradish peroxidase-labeled, namely strangles hormone ELISA for the anti-seedling of people and detects reagent.
Five, the anti-seedling of people strangles foundation and the detection that hormone ELISA detects reagent: bag quilt: strangle hormone ELISA with the anti-seedling of people of pH 9.5 carbonate buffer solution dilution and detect reagent (volume ratio 800:1), bag is by the ELISA Plate in 96 holes, 100 μ L/ holes, totally 88 detect aperture, 4 DEG C of bags are spent the night, negative control hole (diluting bag quilt with negative control reagent and carbonate buffer solution) establishes 4/plate, and blank control wells (carbonate buffer solution) establishes 4 holes/plate; Wash plate, wash plate three times with automatic washer, 5 minutes/time, cleansing solution TBS; Close: with the site BSA sealase target of mass percentage concentration 1% not having combine upper antibody, 200 μ L/ holes, room temperature effect 1 hour, washes plate; Add sample or standard protein dilution, 100 μ L/ holes, room temperature effect 1 hour, the anti-seedling of people that detect aperture adds the dilution of pH 9.5 carbonate buffer solution again strangles hormone ELISA detection reagent (volume ratio 600:1), 100 μ L/ holes; Detect: sample (can be human serum, urine, cultured cell and cultured cell supernatant etc., include 3,3 ', 5,5 '-tetramethyl benzidine) is added dropwise in detect aperture, negative control hole, blank control wells, and dark place is reacted 20 minutes, adds 2 N H
2sO
4, every hole 50 μ L, cessation reaction, read OD after 10 minutes
490, blank tube returns to zero, and OD measured by sample
490after look into typical curve and show that in sample, the anti-seedling of people strangles the content of hormone, owing to there being the antibody acting in conjunction of 6 kinds of antigens, sensitivity is higher, and detection sensitivity reaches 0.1ng/ml.
<110> Xue Jin cutting edge of a knife or a sword, Zeng Qiao
The anti-seedling of <120> people strangles the enzyme linked immunosorbent detection reagent of hormone
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> protein
<213> Prof. Du Yucang
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Cys Trp Arg Gly Arg Asp Pro Arg Gly Pro Gly Arg Ala Gln Arg
5 10 15
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<211> 15
<212> protein
<213> Prof. Du Yucang
<400> 2
Cys Pro Pro Arg Pro Ser Ala Glu Leu Glu Glu Ser Pro Pro Ser
5 10 15
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<212> protein
<213> Prof. Du Yucang
<400> 3
Pro Ser Leu Arg Phe Gln Glu Pro Pro Pro Gly Gly Ala Gly Cys
5 10 15
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<212> protein
<213> Prof. Du Yucang
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Cys Leu Gln Pro Arg Gly Glu Asp Ser Arg Leu Ser Thr Ala Arg
5 10 15
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<211> 15
<212> protein
<213> Prof. Du Yucang
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Cys Leu Ser Asp Pro Ala Ala Leu Glu Arg Leu Leu Asp Gly Glu
5 10 15
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<211> 15
<212> protein
<213> Prof. Du Yucang
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Val Cys Asn Thr Gly Asp Arg Gln Ala Ala Leu Pro Ser Leu Cys
5 10 15
Claims (1)
1. the anti-seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone, comprise solid phase carrier and be coated on the antibody on solid phase carrier, antibody is that antigen and hole relative hemocyanin lotus root connect, after immunize New Zealand rabbit, purifying obtains, it is characterized in that described antibody anti-seedling of behaving strangles hormone antibody, described antigen is 6 polypeptide of Prof. Du Yucang, and the amino acid sequence of 6 polypeptide is as shown in SEQ ID NO1 ~ SEQ ID NO6.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510226042.1A CN104865391B (en) | 2015-05-06 | 2015-05-06 | The anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone |
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| CN201510226042.1A CN104865391B (en) | 2015-05-06 | 2015-05-06 | The anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone |
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| CN104865391A true CN104865391A (en) | 2015-08-26 |
| CN104865391B CN104865391B (en) | 2016-06-22 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105527448A (en) * | 2015-12-31 | 2016-04-27 | 苏州市博纳泰科生物技术有限公司 | A fluorescence immunochromatographic detecting method for anti-mullerian hormone and a kit |
| CN106674348A (en) * | 2017-01-06 | 2017-05-17 | 刘玲 | Anti-mullerian hormone (AMH) antibody and preparation method thereof |
| CN107014991A (en) * | 2017-03-29 | 2017-08-04 | 广东工业大学 | A kind of peptide polyclonal antibody of growth hormone release inhibiting hormone 28 and preparation method thereof |
| CN108303554A (en) * | 2018-02-08 | 2018-07-20 | 江苏麦得科生物科技有限公司 | A kind of magnetic microparticle chemiluminescence immune assay kit of anti-gyneduct hormone |
| CN111273042A (en) * | 2020-01-21 | 2020-06-12 | 迈克生物股份有限公司 | Anti mullerian tube hormone detect reagent box |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101573382B (en) * | 2006-11-02 | 2013-08-21 | 法国国家健康医学研究院 | Monoclonal antibodies and directed against the human anti-mullerian hormone type ii receptor (amhr-ii) |
| CN103728459A (en) * | 2013-12-25 | 2014-04-16 | 李志荣 | Preparation method and application of kit for double-sandwich immunofluorescence quantitative detection of human anti-Mullerian hormone (AMH) on basis of quantum dots |
| WO2014074835A2 (en) * | 2012-11-09 | 2014-05-15 | Ansh Labs Llc | Antibody compositions and immunoassay methods to detect isoforms of anti-mullerian hormone |
-
2015
- 2015-05-06 CN CN201510226042.1A patent/CN104865391B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101573382B (en) * | 2006-11-02 | 2013-08-21 | 法国国家健康医学研究院 | Monoclonal antibodies and directed against the human anti-mullerian hormone type ii receptor (amhr-ii) |
| WO2014074835A2 (en) * | 2012-11-09 | 2014-05-15 | Ansh Labs Llc | Antibody compositions and immunoassay methods to detect isoforms of anti-mullerian hormone |
| CN103728459A (en) * | 2013-12-25 | 2014-04-16 | 李志荣 | Preparation method and application of kit for double-sandwich immunofluorescence quantitative detection of human anti-Mullerian hormone (AMH) on basis of quantum dots |
Non-Patent Citations (1)
| Title |
|---|
| SM NELSON ET AL.: "The journey from the old to the new AMH assay:how to avoid getting lost in the values", 《REPRODUCTIVE BIOMEDICINE ONLINE》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105527448A (en) * | 2015-12-31 | 2016-04-27 | 苏州市博纳泰科生物技术有限公司 | A fluorescence immunochromatographic detecting method for anti-mullerian hormone and a kit |
| CN106674348A (en) * | 2017-01-06 | 2017-05-17 | 刘玲 | Anti-mullerian hormone (AMH) antibody and preparation method thereof |
| CN107014991A (en) * | 2017-03-29 | 2017-08-04 | 广东工业大学 | A kind of peptide polyclonal antibody of growth hormone release inhibiting hormone 28 and preparation method thereof |
| CN108303554A (en) * | 2018-02-08 | 2018-07-20 | 江苏麦得科生物科技有限公司 | A kind of magnetic microparticle chemiluminescence immune assay kit of anti-gyneduct hormone |
| CN111273042A (en) * | 2020-01-21 | 2020-06-12 | 迈克生物股份有限公司 | Anti mullerian tube hormone detect reagent box |
| CN111273042B (en) * | 2020-01-21 | 2023-09-01 | 迈克生物股份有限公司 | Anti-mullerian hormone detection kit |
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| CN104865391B (en) | 2016-06-22 |
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Effective date of registration: 20180118 Address after: 315040 Ningbo province high tech Zone, Hui Hui Road, No. 139, No. Patentee after: Chen Binglai Address before: 315040 Zhejiang high tech District of Ningbo City Xinhui Road No. 139 Building No. 2 Co-patentee before: Zeng Qiao Patentee before: Xue Jinfeng |
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