CN102621334B - 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
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- CN102621334B CN102621334B CN201210099609.XA CN201210099609A CN102621334B CN 102621334 B CN102621334 B CN 102621334B CN 201210099609 A CN201210099609 A CN 201210099609A CN 102621334 B CN102621334 B CN 102621334B
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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 for follicle-stimulating hormone (FSH) (FSH) the fluorescent magnetic inspection-free test agent of particulate enzyme and preparation method specifically.
[background technology]
Follicular stimulating hormone claims that again follicle-stimulating hormone (FSH) (Follicle Stimulating Hormone, FSH) is a kind of glycoprotein hormones, and molecular weight is about 30KD, belongs to glycoprotein hormones family.The subunit that FSH can be dissociated by two non-covalent combinations is formed, be called α-subunit and β-subunit, in same, α-subunit is determined, 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 again specificity between kind, is hormone biologically active and immunocompetent determinative.α-subunit and β-subunit do not have separately biologically active, only have when both combine and cause the variation in spatial structure just to have biologically active.
FSH can promote granular cell hyperplasia, stimulation Steroidgenesis, regulate growth and the maturation of gametid, is one of major hormone in hypothalamic pituitary gonadal axis.There is very wide and tempting application prospect in fields such as the diagnoses and treatment of animal reproduction breeding, superfecundation, embryo transfer, oocyte maturation, in vitro fertilization and mankind's relevant disease.
For women, this hormone plays a role in HPO regulation loop, controls the menstrual cycle.FSH and LH paroxysmal from the gonadotroph of hypophysis discharges.Concentration in blood by steroids by hypothalamic negative feedback mechanism control.In ovary, FSH stimulates growth and the maturation of ovarian follicle together with LH, and then stimulates estrogenic biosynthesizing in ovarian follicle.FSH level presents a peak in the mid-term of menstrual cycle, although it is obvious to be not so good as LH.Due to the variation of ovarian function and the decline of estrogen level, climacteric, FSH reached high level.
For the male sex, FSH can regulate growth and the during spermatogenesis of seminiferous tubules.But different from estrogen, male sex hormone can not reduce the level of FSH, therefore also mean that FSH level in males is only subject to the adjusting of LH in serum.The patient of aspermia or oligospermia and azoospermatism there will be FSH to raise conventionally, but concrete mechanism it be unclear that.In orchioncus patients serum, the concentration of FSH generally can reduce.While there is high-caliber FSH in males, may be primary testicular failure or Klinefelter syndrome.
FSH detects has effect to the malfunction of finding out HPO system.FSH and LH detect for geneogenous disease, as the congenital disorders of chromosome abnormality, amenorrhoea (cause of disease), PCO (PCO) and climacteric syndrome etc.
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, develop again a new technology, particulate fluoroimmunoassay, utilize luminous chemical reaction chemiluminescence, and biological respinse (bioluminescence CL), BL) analyze ultramicron material, especially for checking ultramicron active substance in clinical immunoassay.This method, is transitioned into clinical medical conventional sense means from the rare technology in laboratory, has obtained nearly ten years very large development.It is highly sensitive with it that fluorescent particle enzyme is exempted from method, (in several seconds, produces luminous signal) fast, easy, do not use harmful reagent in test, therefore becomes one of up-and-coming method in on-radiation immunoassay.
Fluorescent magnetic particulate enzyme is exempted from method, no matter from the stability of sensitivity, detection time, the range of linearity, reagent, have the aspect contrasts such as non-environmental-pollution, its advantage is all very outstanding.And current domestic fluorescent magnetic particulate enzyme is exempted from carrying out of test item, mainly rely on external full-automatic determining instrument and matched reagent, expensive, thereby increased the burden of doctor and patient.
[summary of the invention]
The present invention, in order to overcome the deficiencies in the prior art, provides a kind of follicle-stimulating hormone (FSH) fluorescent magnetic particulate enzyme inspection-free test agent.
To achieve these goals, the present invention has designed the short inspection-free test agent of ripe hormone fluorescent magnetic particulate enzyme that soaks of a kind of ovum, it is characterized in that: comprise magnetic bead, and the enzyme conjugates of being combined with magnetic bead, described enzyme conjugates is the follicle-stimulating hormone (FSH) antibody that adopts 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 described magnetic bead is 1-1.5mm.
Described damping fluid is the one in Mes damping fluid, phosphate buffer or glycine buffer, and the pH value of damping fluid is 6-8; Described antiseptic is sodium azide.
The preparation method of the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescent magnetic particulate 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, both are mixed to rear vibration absorption 16 to 24 hours, add damping fluid sealing 2 to 3 hours, remove confining liquid.
The present invention compared with the existing technology, can be applied on the market and exempt from method on the instrument of detection means taking fluorescent magnetic particulate enzyme, and has easyly, and fast, stable detectability, can substitute the high reagent of foreign country.
[embodiment]
Below in conjunction with embodiment, the invention will be further described.
The present invention includes magnetic bead, and the enzyme conjugates of being combined with magnetic bead, enzyme conjugates is the follicle-stimulating hormone (FSH) antibody that adopts AP mark, magnetic bead is coated with follicle-stimulating hormone (FSH) antibody.
One, the preparation of FSH immune response reagent, adopts following processing step:
A. coated: to get 1mol/L Na
2hPO
477.4ml and 1mol/L NaH
2pO
422.6ml mixes, add deionized water be settled to 1000ml 10 times of coating buffers, dilute before use ten times, add appropriate follicle maturity hormone antibody to mix, then add in magnetic bead vortex mixer, every 12 magnetic bead 100ul coating buffers, 4 degree 20 hours.
B. sealing: discard coating buffer, add the phosphate buffer (PH7.4) that contains 1%BSA and 0.5% antiseptic (sodium azide), then add the confining liquid of every 12 magnetic bead 300ul to seal, 37 degree 3 hours.
C. encapsulation: discard confining liquid, enter oven for drying, temperature is 37 degree, and drying time is 4 hours, to put 12 magnetic beads in every group reagent, divides and installs 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.Follicle maturity hormone antibody is crosslinked by periodates oxidizing process and alkaline phosphatase.Being mixed with the concentration of 1: 1000 divides and installs in each reagent cup.
E. freeze-drying: pre-freeze;-35 DEG C of casing pre-freeze 2h-3h.A freeze-drying :-30 DEG C of constant temperature vacuumize, casing vacuum tightness, at 10-30pa, continues 12h-15h.Secondary freeze-drying: after a freeze-drying finishes, be warming up to 25 DEG C, then 25 DEG C of constant temperature keep 2h-3h.
F. sealing: each reagent rim of a cup is sealed with sealer.
G. packing: every 20 reagent cup are thrown in an agent plate, and be kept in the damp proof bag of aluminum.
Two, the using method of FSH reagent:
A) from aluminium flake protective seam, take out FSH immune response reagent cup.
B) in reagent cup, add the sample solution after 100 microlitre damping fluids and 50 microlitre standard items or dilution, on magnetite magnetic bead Vib., stir, under the room temperature of 37 degree, make antigen-antibody reaction 10 minutes.
C), with eluent wash reagent cup, remove wherein free enzyme connection label and sample composition.
D) add after matrix liquid, on magnetite magnetic bead Vib., stir, detect the fluorescence intensity of fluorescent material with fluorescence detector simultaneously, excitation wavelength 363mm, fluorescent absorption wavelength 447mm, and calculate the formation speed of fluorescent material.
E) detect according to titer the fluorescent material formation speed typical curve of drawing, calculate the concentration of sample F SH.
If when testing result exceedes 200mIU/ml, improve extension rate with sample diluting liquid and again dilute.
Three, the calculating of experimental result:
Graphing method: taking the lg value of standard items antigenic content as horizontal ordinate (x), draw out typical curve taking its corresponding signal value as ordinate (y), then the rate value recording according to detection sample is found corresponding antigenic content lg value on typical curve, thereby calculates the content of antigen.
Computing method: taking the lg value of standard items antigenic content value as independent variable (x), taking its corresponding signal value as dependent variable (y), obtain regression equation, bring the signal value of sample to be tested into regression equation, thereby calculate the content of corresponding antigen.
Claims (5)
1. the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescent magnetic particulate enzyme, it is characterized in that: comprise magnetic bead, and the enzyme conjugates of being combined with magnetic bead, described enzyme conjugates is the follicle-stimulating hormone (FSH) antibody that adopts AP mark, described magnetic bead is coated with follicle maturity hormone antibody; And the mean particle dia of described magnetic bead is 1-1.5mm; And described detection reagent is prepared as follows:
A. coated: to get 1mol/L Na
2hPO
477.4ml and 1mol/L NaH
2pO
422.6ml mixes, add deionized water be settled to 1000ml 10 times of coating buffers, dilute before use ten times, add appropriate follicle maturity hormone antibody to mix, then add in magnetic bead vortex mixer, every 12 magnetic bead 100ul coating buffers, 4 degree 20 hours;
B. sealing: discard coating buffer, add the phosphate buffer PH7.4 that contains 1%BSA and 0.5% antiseptic sodium azide, then add the confining liquid of every 12 magnetic bead 300ul to seal, 37 degree 3 hours;
C. encapsulation: discard confining liquid, enter oven for drying, temperature is 37 degree, and drying time is 4 hours, is magnetic particle to put 12 magnetic beads in every group reagent, divides and installs 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; Follicle maturity hormone antibody is cross-linked with periodates oxidizing process and alkaline phosphatase, and the concentration that is mixed with 1:1000 is divided and installed in each reagent cup;
E. freeze-drying: pre-freeze;-35 DEG C of casing pre-freeze 2h-3h; A freeze-drying :-30 DEG C of constant temperature vacuumize, casing vacuum tightness, at 10-30pa, continues 12h-15h; Secondary freeze-drying: after a freeze-drying finishes, be warming up to 25 DEG C, then 25 DEG C of constant temperature keep 2h-3h;
F. sealing: each reagent rim of a cup is sealed with sealer;
G. packing: every 20 reagent cup are thrown in an agent plate, and be kept in the damp proof bag of aluminum.
2. the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescent magnetic particulate 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) fluorescent magnetic particulate enzyme according to claim 1, is characterized in that: the using method of described detection reagent comprises step:
A) from aluminium flake protective seam, take out FSH immune response reagent cup;
B) in reagent cup, add the sample solution after 100 microlitre damping fluids and 50 microlitre standard items or dilution, on magnetite magnetic bead Vib., stir, under the room temperature of 37 degree, make antigen-antibody reaction 10 minutes;
C), with eluent wash reagent cup, remove wherein free enzyme connection label and sample composition;
D) add after matrix liquid, on magnetite magnetic bead Vib., stir, detect the fluorescence intensity of fluorescent material with fluorescence detector simultaneously, excitation wavelength 363mm, fluorescent absorption wavelength 447mm, and calculate the formation speed of fluorescent material;
E) detect according to titer the fluorescent material formation speed typical curve of drawing, calculate the concentration of sample F SH.
4. the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescent magnetic particulate enzyme according to claim 2, is characterized in that: described damping fluid is the one in Mes damping fluid, phosphate buffer or glycine buffer, the pH value of damping fluid is 6-8; Described antiseptic is sodium azide.
5. the preparation method of the inspection-free test agent of follicle-stimulating hormone (FSH) fluorescent magnetic particulate enzyme, adopt following processing step: damping fluid dilutes at normal temperatures follicle-stimulating hormone (FSH) antibody and magnetic particle ball is magnetic bead, the ratio of antibody and magnetic particle ball is 1 to 4:1, both are mixed to rear vibration absorption 16 to 24 hours, add damping fluid sealing 2 to 3 hours, removal confining liquid, wherein, the mean particle dia of described magnetic particle ball is 1-1.5mm;
And said method comprising the steps of
A. coated: to get 1mol/L Na
2hPO
477.4ml and 1mol/L NaH
2pO
422.6ml mixes, add deionized water be settled to 1000ml 10 times of coating buffers, dilute before use ten times, add appropriate follicle maturity hormone antibody to mix, then add in magnetic bead vortex mixer, every 12 magnetic bead 100ul coating buffers, 4 degree 20 hours;
B. sealing: discard coating buffer, add the phosphate buffer PH7.4 that contains 1%BSA and 0.5% antiseptic sodium azide, then add the confining liquid of every 12 magnetic bead 300ul to seal, 37 degree 3 hours;
C. encapsulation: discard confining liquid, enter oven for drying, temperature is 37 degree, and drying time is 4 hours, to put 12 magnetic beads in every group reagent, divides and installs 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; Follicle maturity hormone antibody is cross-linked with periodates oxidizing process and alkaline phosphatase, and the concentration that is mixed with 1:1000 is divided and installed in each reagent cup;
E. freeze-drying: pre-freeze;-35 DEG C of casing pre-freeze 2h-3h; A freeze-drying :-30 DEG C of constant temperature vacuumize, casing vacuum tightness, at 10-30pa, continues 12h-15h; Secondary freeze-drying: after a freeze-drying finishes, be warming up to 25 DEG C, then 25 DEG C of constant temperature keep 2h-3h;
F. sealing: each reagent rim of a cup is sealed with sealer;
G. packing: every 20 reagent cup are thrown in an agent plate, and be kept in the damp proof bag of aluminum.
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| 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 |
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Citations (5)
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|---|---|---|---|---|
| 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 |
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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. |
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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. |
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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. |