CN104865391B - The anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone - Google Patents
The anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone Download PDFInfo
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- CN104865391B CN104865391B CN201510226042.1A CN201510226042A CN104865391B CN 104865391 B CN104865391 B CN 104865391B CN 201510226042 A CN201510226042 A CN 201510226042A CN 104865391 B CN104865391 B CN 104865391B
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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 the anti-Seedling of people and strangle the enzyme linked immunosorbent detection reagent of hormone, including solid phase carrier and be coated on the antibody on solid phase carrier, antibody is that antigen connects with hole relative hemocyanin Rhizoma Nelumbinis, after immunity new zealand rabbit, purification obtains, this antibody anti-Seedling of behaviour strangles hormone antibody, this antigen is 6 polypeptide of synthetic, shown in the aminoacid sequence of 6 polypeptide such as SEQ ID NO1～SEQ ID NO6；This enzyme linked immunosorbent detection reagent can effectively detect the anti-Seedling of people and strangle hormone, and with 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。
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection reagent, be specifically related to the anti-Seedling of people and strangle the enzyme linked immunosorbent detection reagent of hormone。
Background technology
People AMH (anti-Mulerianhormone, AMH) the biglycan albumen being made up of two identical 70kb subunits, relative molecular mass is 140000, constitutes transforming growth factor β superfamily with inhibin, activin, bone morphogenetic protein and growth and differentiation factor etc.。Mankind's AMH gene is positioned at No. 19 the short arm of a chromosome (19p13.3), long 2.4～2.8kb, has 5 exons, encodes 560 amino acid whose amyloid protein precursors。
Summary of the invention
The technical problem to be solved is to provide the anti-Seedling of a kind of people and strangles the enzyme linked immunosorbent detection reagent of hormone, and this enzyme linked immunosorbent detection reagent can effectively detect the anti-Seedling of people and strangle hormone, and detection sensitivity reaches 0.1ng/ml。
This invention address that the technical scheme that above-mentioned technical problem adopts is: the anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone, including solid phase carrier and be coated on the antibody on solid phase carrier, antibody is that antigen connects with hole relative hemocyanin Rhizoma Nelumbinis, after immunity new zealand rabbit, purification obtains, the described antibody anti-Seedling of behaviour strangles hormone antibody, described antigen is 6 polypeptide of synthetic, and the aminoacid sequence of 6 polypeptide is such as shown in SEQIDNO1～SEQIDNO6。
Compared with prior art, it is an advantage of the current invention that the anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone, including solid phase carrier and be coated on the antibody on solid phase carrier, antibody is that antigen connects with hole relative hemocyanin Rhizoma Nelumbinis, after immunity new zealand rabbit, purification obtains, this antibody anti-Seedling of behaviour strangles hormone antibody, and this antigen is 6 polypeptide of synthetic, and the aminoacid sequence of 6 polypeptide is such as shown in SEQIDNO1～SEQIDNO6；This enzyme linked immunosorbent detection reagent can effectively detect the anti-Seedling of people and strangle hormone, and with 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。
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail。
Embodiment
One, the design of antigen: AMH albumen is carried out epitope analysis from hydrophilic, accessibility, antigenicity, plasticity, CHARGE DISTRIBUTION, secondary structure prediction, design, filter out the polypeptide antigen (the raw work synthesis in Shanghai) of 6 synthetic, article 6, the aminoacid sequence of polypeptide antigen is such as shown in SEQIDNO1～SEQIDNO6, respectively with hole relative hemocyanin (KLH) Rhizoma Nelumbinis even。
Two, the preparation of specific antibody: immune programme for children: select the healthy new zealand white rabbit of 13 2 monthly ages, body weight 1.5～2.0kg, takes 1 and is only used as negative control group, and remaining 12 is test group；Being fed 2 weeks by 13 new zealand white rabbit elder generation adaptabilities, edge of picking up the ears prior to before every animal immune extracts 2mL venous blood, and 4 DEG C of standings overnight, take serum and make negative control reagent；The antigen of above-mentioned 6 kinds of KLH couplings is additionally added 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, emulsifying in vortex oscillator, water presents spherical until an Emulsion is instilled and does not disperse, obtain immunizing antigen reagent；Immunizing antigen reagent (antigen+complete Freund's complete adjuvant) at White Rabbit back multi-point injection, is injected 1mL for every by initial immunity。The 28th day immunizing antigen reagent White Rabbit intramuscular injection 1mL 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, if titer does not reach requirement, continue to raise, repeat booster immunization, until obtaining satisfied titer；After titer reaches requirement, carotid artery takes blood results antiserum。
Three, the purification of antibody: take the above-mentioned antiserum of 12mL, adds equal-volume 12mLPBS(concentration 0.01M, pH7.8, lower same), mixing；Add ammonium sulfate saturated solution 24mL while stirring, make into 50% saturation, put the centrifugal 10min of 60min, 10000rpm, abandon supernatant, with the mixed solution washing and precipitating secondary of saturated ammonium sulfate 12mL and PBS12mL, remove supernatant for 4 DEG C；Precipitation is dissolved in 12mLPBS, drips saturated ammonium sulfate solution 6mL, put 30min for 4 DEG C, be centrifuged and remove supernatant, repeat this step once；Adding 2.4mL normal saline in precipitation, move in bag filter, dialysis does not have sulfate ion to lye, with the BaCl of mass percentage concentration 1%2Solution titration detects, then changes a normal saline dialysis, prepares antibody purification 2.5mL。
Four, the labelling 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。Adding normal saline to 1mL, add 5mg antibody purification, add 0.1mL1M carbonate buffer solution (pH9.6), 4 ° of C put 24 hours；Adding 0.1mL lysine (concentration 0.2M), room temperature effect 2 hours, in PBS(concentration 0.15M, pH7.2) in, fully to dialyse, centrifugation, supernatant is the antibody purification of horseradish peroxidase-labeled, is namely that the anti-Seedling of people strangles hormone ELISA detectable。
Five, the anti-Seedling of people strangles foundation and the detection of hormone ELISA detectable: be coated: strangle hormone ELISA detectable (volume ratio 800:1) with the anti-Seedling of people of pH9.5 carbonate buffer solution dilution, it is coated the ELISA Plate in 96 holes, 100 μ L/ holes, totally 88 detection hole, 4 DEG C are coated overnight, negative control hole (is coated with carbonate buffer solution dilution with negative control reagent) and sets 4/plate, and blank control wells (carbonate buffer solution) sets 4 holes/plate；Wash plate, wash plate three times with automatic washer, 5 minutes/time, cleaning mixture TBS；Close: with the site being not bound with upper antibody on the BSA sealase target of mass percentage concentration 1%, 200 μ L/ holes, room temperature effect 1 hour, wash plate；Adding sample or standard protein diluent, 100 μ L/ holes, room temperature effect 1 hour, detection hole adds the anti-Seedling of people of pH9.5 carbonate buffer solution dilution and strangles hormone ELISA detectable (volume ratio 600:1), 100 μ L/ holes；Detection: sample (can be human serum, urine, cultivation cell and cultivation cell conditioned medium etc., include 3,3 ', 5,5 '-tetramethyl benzidine) is added dropwise in detection hole, negative control hole, blank control wells, and dark place is reacted 20 minutes, adds 2NH2SO4, every hole 50 μ L, terminate reaction, after 10 minutes, read OD490, blank tube returns to zero, and OD measured by sample490After look into standard curve and show that the anti-Seedling of people in sample strangles the content of hormone, with the antibody combined effect of 6 kinds of antigens, sensitivity is higher, and detection sensitivity reaches 0.1ng/ml。
<110>Xue Jinfeng, Zeng Qiao
<120>the anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone
<160>6
<170>PatentInversion3.5
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<211>15
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CysTrpArgGlyArgAspProArgGlyProGlyArgAlaGlnArg
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CysProProArgProSerAlaGluLeuGluGluSerProProSer
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ProSerLeuArgPheGlnGluProProProGlyGlyAlaGlyCys
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CysLeuGlnProArgGlyGluAspSerArgLeuSerThrAlaArg
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CysLeuSerAspProAlaAlaLeuGluArgLeuLeuAspGlyGlu
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ValCysAsnThrGlyAspArgGlnAlaAlaLeuProSerLeuCys
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Claims (1)
1. the anti-Seedling of people strangles the enzyme linked immunosorbent detection reagent of hormone, including solid phase carrier and be coated on the antibody on solid phase carrier, antibody is by antigen and hole relative hemocyanin coupling, after immunity new zealand rabbit, purification obtains, it is characterized in that the described antibody anti-Seedling of behaviour strangles hormone antibody, described antigen is 6 polypeptide of synthetic, the aminoacid sequence of 6 polypeptide such as SEQIDNO:1～SEQIDNO:6。
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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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| 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 |
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| US20160274130A1 (en) * | 2012-11-09 | 2016-09-22 | Ansh Labs Llc | Antibody Compositions and Immunoassay Methods to Detect Isoforms of Anti-Müllerian Hormone |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
Non-Patent Citations (1)
| Title |
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| The journey from the old to the new AMH assay:how to avoid getting lost in the values;SM Nelson et al.;《Reproductive BioMedicine Online》;20111031;第23卷(第4期);第411-420页 * |
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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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