CN102621334A - Fluorescent magnetic microparticle enzyme-linked immunosorbent assay reagent for follicle-stimulating hormone and preparation method thereof - Google Patents
Fluorescent magnetic microparticle enzyme-linked immunosorbent assay reagent for follicle-stimulating hormone and preparation method thereof Download PDFInfo
- Publication number
- CN102621334A CN102621334A CN201210099609XA CN201210099609A CN102621334A CN 102621334 A CN102621334 A CN 102621334A CN 201210099609X A CN201210099609X A CN 201210099609XA CN 201210099609 A CN201210099609 A CN 201210099609A CN 102621334 A CN102621334 A CN 102621334A
- Authority
- CN
- China
- Prior art keywords
- follicle
- stimulating hormone
- fsh
- enzyme
- magnetic particle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a diagnosis reagent, specifically a fluorescent magnetic microparticle enzyme-linked immunosorbent assay reagent for follicle-stimulating hormone, which is characterized by comprising magnetic beads and an enzyme conjugate conjugated with the magnetic beads, wherein the enzyme conjugate is AP (alkaline phosphatase)-labeled follicle-stimulating hormone antibody, and the magnetic beads are coated by a follicle-stimulating hormone antibody. Compared with the prior art, the reagent can be applied to commercial instruments taking the enzyme-linked immunosorbent assay as a detection means, is simple, convenient, rapid and stable in detection capability, and can replace imported expensive reagents.
Description
[technical field]
The present invention relates to a kind of diagnostic reagent, a kind of specifically follicle-stimulating hormone (FSH) (FSH) fluorescence inspection-free test agent of magnetic particle enzyme and preparation method of being used for.
[background technology]
Follicular stimulating hormone claims that again (Follicle Stimulating Hormone is a kind of glycoprotein hormones FSH) to follicle-stimulating hormone (FSH), and molecular weight is about 30KD, belongs to glycoprotein hormones family.FSH is made up of the subunit that two non-covalent combinations can be dissociated, and is called α-subunit and β-subunit, is confirming with α-subunit in a kind of; Even the amino acid sequence of the α-subunit in all mammals is all guarded; Their α-subunit can exchange, and β-subunit can not exchange, and β-subunit has hormone specificity separately; Having specificity between kind again, is hormone biologically active and immunocompetent determinative.α-subunit and β-subunit be biologically actives not separately, have only when both combine to cause the variation on the spatial structure, just biologically active.
FSH can promote granular cell hyperplasia, the growth and the maturation that stimulate steroids to generate, regulate gametid, is one of major hormone in the hypothalamic pituitary gonadal axis.Has very wide and tempting application prospect in animal reproduction breeding, superfecundation, embryo transfer, oocyte maturation, fields such as diagnoses and treatment in vitro fertilization and human relevant disease.
For the women, this hormone plays a role in the HPO regulation loop, the control menstrual cycle.FSH and LH paroxysmal from the gonadotroph of hypophysis discharges.Concentration in the blood is controlled through hypothalamic negative feedback mechanism by the steroid hormone.FSH and LH stimulate the growth and the maturation of ovarian follicle together in ovary, and then stimulate estrogenic biosynthesizing in the ovarian follicle.The FSH level presents a peak in the mid-term of menstrual cycle, although it is obvious to be not so good as LH.Because the variation of ovarian function and the decline of estrogen level, climacteric, FSH reached high level.
For the male sex, FSH can regulate the growth and the spermatogenesis process of seminiferous tubules.Yet different with estrogen, male sex hormone can not reduce the level of FSH, means also that therefore the interior FSH level of males only receives the adjusting of LH in the serum.The patient of aspermia or oligospermia and azoospermatism can occur FSH usually and raise, but concrete mechanism it be unclear that.The concentration of FSH generally can reduce among the orchioncus patients serum.When occurring high-caliber FSH in the males, possibly be primary testicular failure or Klinefelter syndrome.
FSH detects has effect to the malfunction of finding out the HPO system.FSH and LH detect and are used for geneogenous disease, like congenital disorders, amenorrhoea (cause of disease), PCO (PCO) and the climacteric syndrome etc. of chromosome abnormality.
The fluoroimmunoassay technology just has application as far back as the forties in 20th century, is with fluorescein-labelled antigen or antibody at that time, but because the sensitivity of high background and detecting instrument is low, fails to realize the clinical practice of fluoroimmunoassay always.After this, developed a new technology again, the particulate fluoroimmunoassay; Promptly utilize luminous chemical reaction chemiluminescence; CL) and biological respinse (bioluminescence BL) analyzes the ultramicron material, especially for check ultramicron active substance in the clinical immunoassay.This method carries out the transition to clinical medical conventional sense means from breadboard rare technology, has obtained very big development over past ten years.It is highly sensitive with it that the fluorescent particle enzyme is exempted from method, (produces luminous signal in several seconds) fast, easy, do not use harmful reagent in the test, therefore becomes one of up-and-coming method in the on-radiation immunoassay.
Fluorescence magnetic particle enzyme is exempted from method, no matter from the stability of sensitivity, detection time, the range of linearity, reagent, aspect contrast such as non-environmental-pollution is arranged, its advantage is all very outstanding.And carrying out of the inspection-free survey project of present domestic fluorescence magnetic particle enzyme mainly relies on external full-automatic determining instrument and matched reagent, costs an arm and a leg, thereby increased the burden of doctor and patient.
[summary of the invention]
The present invention provides a kind of follicle-stimulating hormone (FSH) fluorescence magnetic particle enzyme inspection-free test agent in order to overcome the deficiency of prior art.
To achieve these goals; The present invention has designed the short inspection-free test agent of ripe hormone fluorescence magnetic particle enzyme that soaks of a kind of ovum; It is characterized in that: comprise magnetic bead; And the enzyme conjugates that combines with magnetic bead, described enzyme conjugates is for adopting the follicle-stimulating hormone (FSH) antibody of AP mark, and described magnetic bead is coated with follicle-stimulating hormone (FSH) antibody.
Described enzyme conjugates comprises damping fluid 25-250mmol/L, stabilizing agent 1-20mmol/L, electrolyte 50-300mmol/L, promoter 4-5mmol/L, antiseptic 2-5mmol/L; All the other are deionized water.
The mean particle dia of said magnetic bead is 1-1.5mm.
Said damping fluid is a kind of in Mes damping fluid, phosphate buffer or the glycine buffer, and the pH value of damping fluid is 6-8; Described antiseptic is a sodium azide.
The preparation method of the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescence magnetic particle enzyme; Adopt following processing step: damping fluid dilutes follicle-stimulating hormone (FSH) antibody and magnetic particle ball at normal temperatures; The ratio of antibody and magnetic particle ball is 1 to 4: 1; With vibration absorption behind both mixings 16 to 24 hours, add damping fluid sealing 2 to 3 hours, remove confining liquid and get final product.
The present invention compares with prior art, can be applied on the market with fluorescence magnetic particle enzyme to exempt from the instrument that method is a detection means, and have easyly, and fast, the stable detection ability can substitute the high reagent of foreign country.
[embodiment]
Below in conjunction with embodiment the present invention is further described.
The present invention includes magnetic bead, and the enzyme conjugates that combines with magnetic bead, enzyme conjugates is for adopting the follicle-stimulating hormone (FSH) antibody of AP mark, and magnetic bead is coated with follicle-stimulating hormone (FSH) antibody.
One, the preparation of FSH immune response reagent, adopt following processing step:
A. encapsulate: get 1mol/L Na
2HPO
477.4ml with 1mol/L NaH
2PO
422.6ml mixing, add deionized water be settled to 1000ml 10 times of coating buffers, face with ten times of preceding dilutions, add an amount of follicle maturity hormone antibody mixing, add then in the magnetic bead vortex mixer, per 12 magnetic bead 100ul coating buffers, 4 spend 20 hours.
B. sealing: discard coating buffer, add the phosphate buffer (PH7.4) that contains 1%BSA and 0.5% antiseptic (sodium azide), the confining liquid that adds per 12 magnetic bead 300ul again seals, 37 degree 3 hours.
C. encapsulation: discard confining liquid, get into oven for drying, temperature is 37 degree, and drying time is 4 hours, to put 12 magnetic beads in every group reagent, divides to install in each reagent cup.
D. the preparation of enzyme mark: with containing 2.5%BSA, 1.5% skimmed milk power and 0.05% antiseptic (Proclin
TM300) phosphate buffer (PH7.4) preparation buffer system.The follicle maturity hormone antibody is crosslinked with periodates oxidizing process and alkaline phosphatase.The concentration branch that was mixed with 1: 1000 installs in each reagent cup.
E. freeze-drying: pre-freeze;-35 ℃ of casing pre-freeze 2h-3h.A freeze-drying :-30 ℃ of constant temperature vacuumize, and casing vacuum tightness continues 12h-15h at 10-30pa.The secondary freeze-drying: a freeze-drying is warming up to 25 ℃ after finishing, and 25 ℃ of constant temperature keep 2h-3h then.
F. seal: each reagent rim of a cup is sealed with sealer.
G. packing: per 20 reagent cup are thrown on the agent plate, and are kept in the damp proof bag of aluminum.
Two, the method for application of FSH reagent:
A) from the aluminium flake protective seam, take out FSH immune response reagent cup.
B) sample solution behind adding 100 microlitre damping fluids and 50 microlitre standard items or the dilution in reagent cup stirs on magnetite magnetic bead Vib., under the room temperature of 37 degree, makes antigen-antibody reaction 10 minutes.
C), remove wherein free enzyme and join label and sample composition with eluent wash reagent cup.
D) add matrix liquid after, on magnetite magnetic bead Vib., stir, detect the fluorescence intensity of fluorescent material simultaneously with fluorescence detector, excitation wavelength 363mm, fluorescent absorption wavelength 447mm, and calculate the formation speed of fluorescent material.
E) detect the fluorescent material formation speed typical curve of drawing according to titer, calculate the concentration of sample F SH.
If when testing result surpasses 200mIU/ml, improve extension rate with sample diluting liquid and dilute again.
Three, the calculating of experimental result:
Graphing method: the lg value with the standard items antigenic content is horizontal ordinate (x); With its corresponding signal value is that ordinate (y) is drawn out typical curve; The rate value that records according to the detection sample is then found corresponding antigen content lg value on typical curve, thereby calculates the content of antigen.
Computing method: the lg value with standard items antigenic content value is independent variable (x), is dependent variable (y) with its corresponding signal value, obtains regression equation, brings the signal value of sample to be tested into regression equation, thereby calculates the content of corresponding antigen.
Claims (5)
1. inspection-free test agent of follicle-stimulating hormone (FSH) fluorescence magnetic particle enzyme; It is characterized in that: comprise magnetic bead; And the enzyme conjugates that combines with magnetic bead, described enzyme conjugates is for adopting the follicle-stimulating hormone (FSH) antibody of AP mark, and described magnetic bead is coated with the follicle maturity hormone antibody.
2. the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescence magnetic particle enzyme according to claim 1; It is characterized in that: described enzyme conjugates comprises damping fluid 25-250mmol/L, stabilizing agent 1-20mmol/L, electrolyte 50-300mmol/L; Promoter 4-5mmol/L, antiseptic 2-5mmol/L; All the other are deionized water.
3. the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescence magnetic particle enzyme according to claim 1, it is characterized in that: the mean particle dia of said magnetic bead is 1-1.5mm.
4. the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescence magnetic particle enzyme according to claim 2 is characterized in that: said damping fluid is a kind of in Mes damping fluid, phosphate buffer or the glycine buffer, and the pH value of damping fluid is 6-8; Described antiseptic is a sodium azide.
5. the preparation method of the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescence magnetic particle enzyme; Adopt following processing step: damping fluid dilutes follicle-stimulating hormone (FSH) antibody and magnetic particle ball at normal temperatures; The ratio of antibody and magnetic particle ball is 1 to 4: 1; With vibration absorption behind both mixings 16 to 24 hours, add damping fluid sealing 2 to 3 hours, remove confining liquid and get final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210099609.XA CN102621334B (en) | 2012-04-06 | 2012-04-06 | Fluorescent magnetic microparticle enzyme-linked immunosorbent assay reagent for follicle-stimulating hormone and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210099609.XA CN102621334B (en) | 2012-04-06 | 2012-04-06 | Fluorescent magnetic microparticle enzyme-linked immunosorbent assay reagent for follicle-stimulating hormone and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102621334A true CN102621334A (en) | 2012-08-01 |
| CN102621334B CN102621334B (en) | 2014-11-12 |
Family
ID=46561372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210099609.XA Active CN102621334B (en) | 2012-04-06 | 2012-04-06 | Fluorescent magnetic microparticle enzyme-linked immunosorbent assay reagent for follicle-stimulating hormone and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102621334B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5238815A (en) * | 1985-08-30 | 1993-08-24 | Toyo Soda Manufacturing Co., Ltd. | Enzymatic immunoassay involving detecting fluorescence while oscillating magnetic beads |
| CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
| CN101463342A (en) * | 2007-12-21 | 2009-06-24 | 上海市肿瘤研究所 | Method for isolating and identifying human lung adenocarcinoma stem cell |
| CN101750487A (en) * | 2008-12-02 | 2010-06-23 | 博阳生物科技（上海）有限公司 | Dry method photic stimulation chemiluminescence immunoassay reagent kit and preparation and application thereof |
| CN102095878A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for follicle-stimulating hormone |
-
2012
- 2012-04-06 CN CN201210099609.XA patent/CN102621334B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5238815A (en) * | 1985-08-30 | 1993-08-24 | Toyo Soda Manufacturing Co., Ltd. | Enzymatic immunoassay involving detecting fluorescence while oscillating magnetic beads |
| CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
| CN101463342A (en) * | 2007-12-21 | 2009-06-24 | 上海市肿瘤研究所 | Method for isolating and identifying human lung adenocarcinoma stem cell |
| CN101750487A (en) * | 2008-12-02 | 2010-06-23 | 博阳生物科技（上海）有限公司 | Dry method photic stimulation chemiluminescence immunoassay reagent kit and preparation and application thereof |
| CN102095878A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for follicle-stimulating hormone |
Non-Patent Citations (3)
| Title |
|---|
| TIMOTHY S. LAWRUK ET AL.: "Quantification of Alachlor in Water by a Novel Magnetic Particle-Based ELISA", 《ENVIRON. CONTAM. TOXICOL.》 * |
| 王雅杰等: "改良荧光磁微粒酶免分析法检测三种肿瘤标志物及应用", 《标记免疫分析与临床》 * |
| 章尧等: "血清FSH、LH 磁微粒分离酶联免疫法检测评价", 《蚌埠医学院学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102621334B (en) | 2014-11-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hu et al. | Advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold as label in lateral flow assays for detection of ractopamine | |
| TWI536019B (en) | Immunochromatographic assay | |
| EP2523001A1 (en) | Reagent composition for immunochromatography | |
| CN104698184B (en) | The test kit of detection sugar antigen | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN103048446B (en) | Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof | |
| CN102901812A (en) | Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb) | |
| CN108333360A (en) | Gastrin-releasing peptide precursor dilution and its application and kit | |
| CN103048452A (en) | Nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, as well as preparation method and determining method of same | |
| CN105067599A (en) | Chemiluminescence immune detection kit for detecting pro-gastrin-releasing peptide | |
| CN101614747A (en) | Prolactin time-resolved fluoroimmunoassay method and kit | |
| CN108918848B (en) | Vitamin D releasing agent and preparation method and application thereof | |
| CN105891463A (en) | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence | |
| CN101614742A (en) | The magnetic microparticle chemiluminescence immune assay determination kit of luteinizing principle | |
| CN102156121B (en) | Chemiluminescent substrate liquid applied to detection system taking horseradish peroxidase (HRP) as enzymatic reaction | |
| CN103048476A (en) | Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof | |
| CN103048453B (en) | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof | |
| CN103048477B (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same | |
| CN103344774A (en) | Chemiluminescence immune assay kit and method for magnetic particle of human estradiol (E2) | |
| CN102103142B (en) | In-vitro assay method for thrombin-activatable fibrinolysis inhibitor (TAFI) content | |
| CN103063852B (en) | Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof | |
| CN102341707B (en) | Test reagent, and method for measuring analyte in test sample using same | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| CN103048473B (en) | Nano magnetic particle chemiluminiscence determining kit for promoting hormone generation by follicles and preparation and detection method thereof | |
| CN103267866B (en) | A kind of FSH nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 201100 Shanghai East Road, Minhang District, No. 85 Patentee after: SHANGHAI AILEX TECHNOLOGY Co.,Ltd. Address before: 201100 Shanghai East Road, Minhang District, No. 85 Patentee before: SHANGHAI AILEX TECHNOLOGY Co.,Ltd. |
|
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 201108 Lane 152, 468, Beihengsha River Road, Minhang District, Shanghai Patentee after: AILEX TECHNOLOGY GROUP Co.,Ltd. Address before: No. 85 Youdong Road, Minhang District, Shanghai 201100 Patentee before: SHANGHAI AILEX TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20181212 Address after: 201108 Lane 152, 468, Beihengsha River Road, Minhang District, Shanghai Co-patentee after: ZHEJIANG AILEX MEDICAL Co.,Ltd. Patentee after: AILEX TECHNOLOGY GROUP Co.,Ltd. Address before: 201108 Lane 152, 468, Beihengsha River Road, Minhang District, Shanghai Patentee before: AILEX TECHNOLOGY GROUP Co.,Ltd. |