CN106053861A - Follicle-stimulating hormonebiological activity intensity detection method - Google Patents
Follicle-stimulating hormonebiological activity intensity detection method Download PDFInfo
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- CN106053861A CN106053861A CN201610608726.2A CN201610608726A CN106053861A CN 106053861 A CN106053861 A CN 106053861A CN 201610608726 A CN201610608726 A CN 201610608726A CN 106053861 A CN106053861 A CN 106053861A
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- fsh
- follicule
- stimulating hormone
- estradiol
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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
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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Abstract
The invention relates to a follicle-stimulating hormonebiological activity intensity detection method, which comprises the following steps of (1) preparing follicle-stimulating-hormone-free serum; (2) preparing support cells; (3) analyzing the follicle-stimulating hormonebiological activity intensity in a serum sample of a subject: performing back calculation on the detection result of estradiol on a follicle-stimulating hormone standard curve, and finding out the follicle-stimulating hormonebiological activity intensity in the sample to be detected. The method provided by the invention (i.e., a testis cell method) can be used for detecting the follicle-stimulating hormonebiological activity intensity in the sample to be detected, and the sensitivity is high.
Description
Technical field
The present invention relates to a kind of follicule-stimulating hormone (FSH) biological activity strength detecting method.
Background technology
Follicule-stimulating hormone (FSH) (FSH) is a kind of proteohormone of gene expression secretion.Male, act on testis bent
Seminiferous tubule, can promote spermiogenesis tail；Act on support (Sertoli) emiocytosis estradiol.In women, promote stratum granulosum of ovarian follicle
Hyperplasia is broken up, and promotes that ovary is grown up.The detection of follicule-stimulating hormone (FSH) at present is mainly by euzymelinked immunosorbent assay (ELISA) and chemiluminescence
Method.These methods are all direct or indirect detection follicule-stimulating hormone (FSH) itself, do not detect its biological activity.And have biological living only
Property is relevant to Clinical symptom and sign.Simultaneously as euzymelinked immunosorbent assay (ELISA) and chemoluminescence method are all to be carried out by immunoreation principle
Detection, the sensitivity of its detection is extremely restricted.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes the deficiencies in the prior art, it is provided that a kind of follicule-stimulating hormone (FSH) biology is lived
Property strength detecting method.
The present invention solves its technical problem and employed technical scheme comprise that, a kind of follicule-stimulating hormone (FSH) biological activity intensity detection side
Method, follicule-stimulating hormone (FSH) (FSH) stimulating animal support (Sertoli) the cell generation estradiol by person under inspection's blood serum sample:
The biological activity intensity of follicule-stimulating hormone (FSH) (FSH) is gone out by measuring the concentration inverse of estradiol.
The present invention specifically includes following steps:
(1) without the preparation of follicule-stimulating hormone (FSH) serum:
Silicone rubber tube is filled up estradiol crystallization, is subcutaneously implanted back part of animal 4-6 week in order to suppress dividing of animal follicule-stimulating hormone (FSH)
Secrete；Collecting 40-60 milliliter animal serum, add the activated carbon of 10-20mg, at room temperature absorption more than 12 hours, use filter paper mistake
Filter steroid hormone, filtrate be stored in-40～-80 DEG C of refrigerators in preserve；
(2) preparation of support cell (Sertoli cell):
Take animal testis 2, remove peplos, by the consumption of 4～6.5ml 199 culture fluid/2 testis, testis is put in 199 cultivations
In liquid, adding collagenase III and separate enzyme II, making the ultimate density of collagenase III in culture fluid is 0.25-0.29mg/
Ml, the ultimate density separating enzyme II is 0.11-0.15 μ g/ml；Hatching 15～20 minutes at 34～37 DEG C, gained suspension is used
Equivalent 199 culture fluid dilutes, and adds ficoll, make ficoll final concentration of 11～14g/100mL, by it after filtration in filtrate
It is centrifuged 3～20 minutes under 1000～1500g；By centrifugal gained containing supporting that the precipitation of (Sertoli) cell is with 4～8mL 199
Culture fluid is resuspended；Then, add 3-isobutyl-methyl xanthine, bovine serum albumin and hyclone, its final concentration respectively: 3-is different
Fourth methylxanthine is 0.125-0.150mmol/L；Bovine serum albumin is 0.5-0.6g/100mL；Hyclone is cumulative volume
0.5-2%；With trypanblue exclusion method labor cell calculate, (Sertoli) cell suspension must be supported；
(3) follicule-stimulating hormone (FSH) biological activity intensity analysis in person under inspection's blood serum sample:
(A) stimulation of the follicule-stimulating hormone (FSH) in blood serum sample support (Sertoli) cell generation estradiol:
Take 70-100 μ l step (two) gained and support cell suspension, add person under inspection serum 30-50 μ l；Take 70-100 μ l step simultaneously
(2) gained support (Sertoli) cell suspension, adds the follicule-stimulating hormone (FSH) of each concentration that 30-50 μ l phosphate buffer diluted
Standard substance (wherein contain with person under inspection's specimen equivalent but without the serum of follicule-stimulating hormone (FSH))；By the reagent that configured at 37 DEG C, use
5% CO2And 95%O2After hatching 4-6 hour, add the 1.5-1.6ml phosphorus containing 0.01-0.012g/100mL thimerosal gelatin
Phthalate buffer is to terminate reaction；
(B) detection of estradiol: the person under inspection's blood serum sample that will process through step (A) and standard substance are according to existing estradiol
Detection kit or other method carry out the detection of estradiol concentration, draw follicule-stimulating hormone (FSH) biological activity intensity-estradiol dense
Scale directrix curve；
The follicule-stimulating hormone (FSH) standard substance of each concentration of configuration in step (A), its concentration and follicule-stimulating hormone (FSH) biological activity intensity in
Linear positive proportionate relationship, i.e. follicule-stimulating hormone (FSH) standard concentration is the highest, and follicule-stimulating hormone (FSH) biological activity intensity is the highest.But research is sent out
Existing, in person under inspection's serum, follicule-stimulating hormone (FSH) concentration and activity intensity are not the most linearly proportionate relationships, and therefore, we utilize this
Principle carries out the detection of activity intensity.
The reckoning of (c) follicule-stimulating hormone (FSH) (FSH) concentration:
By the testing result of estradiol in person under inspection's blood serum sample at follicule-stimulating hormone (FSH) biological activity intensity-estradiol concentration mark
Inverse on directrix curve, finds follicule-stimulating hormone (FSH) biological activity intensity in sample to be tested.
The advantage of the present invention (i.e. testicular cell method): by using the support cell of person under inspection serum stimulation animal to produce female
Glycol, goes out the biologically active level of follicule-stimulating hormone (FSH) (FSH) by measuring the concentration inverse of estradiol.Detection is that follicle stimulates
The biological activity of element rather than the content of follicule-stimulating hormone (FSH) itself.Testing result is closely related with Clinical symptom and sign.And it is clever
Sensitivity is high.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment: this experiment is supported as a example by cell detection people FSH by Cavia porcellus:
(1) without the preparation of follicule-stimulating hormone (FSH) serum:
The silicone rubber tube of 0.25cm length (internal diameter 0.062 inch, external diameter 0.095 inch) fills up estradiol crystallization, and (estradiol is tied
Crystalline substance, purchased from Sigma, St. Louis, the Missouri State, the U.S.), it is subcutaneously implanted guinea pig back 6 weeks in order to suppress Cavia porcellus follicle
The secretion of stimulin；Collecting 50 milliliters of guinea pig serum, add the activated carbon of 10mg, at room temperature absorption 12 hours, use filter paper mistake
Filter, removes steroid hormone (i.e. estradiol etc.), and filtrate is stored in-80 DEG C of refrigerators preservation；
(2) preparation of Cavia porcellus support cell:
Take 2, the testis of the Cavia porcellus that body weight is 550-700g, remove peplos；By the consumption of 6.2ml 199 culture fluid/2 testis, will
Testis is put in 199 culture fluid (purchased from Sigma, St. Louis, the Missouri State, the U.S.), add collagenase III and
Separating enzyme II, making the ultimate density of collagenase III in culture fluid is 0.28mg/ml；The ultimate density separating enzyme II is 0.14 μ g/
ml；Hatching 20 minutes at 37 DEG C, gained suspension equivalent 199 culture fluid dilutes, and adds ficoll, make after filtration in filtrate
The final concentration of 13g/100mL of ficoll, is centrifuged it 10 minutes under 1500g；Centrifugal gained is used containing the precipitation supporting cell
8mL 199 culture fluid is resuspended；Then, 3-isobutyl-methyl xanthine, bovine serum albumin and hyclone are added.Its final concentration is respectively
It is: 3-isobutyl-methyl xanthine is 0.125 mmol/L；Bovine serum albumin is 0.5g/100mL；Hyclone is cumulative volume
2%；With trypanblue exclusion method labor cell calculate, (Sertoli) cell suspension must be supported；
(3) follicule-stimulating hormone (FSH) biological activity intensity analysis in person under inspection's blood serum sample
(A) stimulation of the follicule-stimulating hormone (FSH) in blood serum sample Sertoli cell generation estradiol:
Take 100 μ l step (two) gained support (Sertoli) cell suspension, add person under inspection's serum 50 μ l；Take 100 μ l steps simultaneously
(2) gained support (Sertoli) cell suspension, adds the follicule-stimulating hormone (FSH) mark of each concentration that 50 μ l phosphate buffers diluted
Quasi-product (wherein contain with person under inspection's specimen equivalent but without the serum of follicule-stimulating hormone (FSH))；By the reagent that configured at 37 DEG C, with 5%
CO2And 95%O2After hatching 4 hours, add the 1.5ml phosphate buffer containing 0.01g/100mL thimerosal gelatin anti-to terminate
Should；
(B) detection of estradiol:
The person under inspection's blood serum sample processed through step (A) and standard substance are entered according to existing estradiol detectable cassette method
The detection of row estradiol concentration, draws follicule-stimulating hormone (FSH) biological activity intensity-estradiol concentration standard curve；
The present embodiment is to detect with Mei Lian bio tech ltd, Shanghai estradiol detection kit.It is below illustratively
The method of book operation:
1. before experiment, all of reagent, sample and micropore lath reach room temperature (18～25 DEG C)；
2. the washing liquid (washing liquid of dilution is stored in 2～8 DEG C can preserve 2 weeks) that distilled water 1:40 dilution concentrates；
3. every hole adds 25 μ l standard substance, quality-control product, sample (96 orifice plates requirements were loaded complete in 3 minutes).
4. every hole adds the enzyme conjugates of 200 μ l, is sufficiently mixed 10 seconds, incubated at room 120 minutes.
5. wash plate: discard mixtures incubated, absorbent paper pats dry.Every hole adds washing liquid 400 μ L, washes soak time between plate
It is 10 seconds or vibration 5 seconds, discards, repeat aforesaid operations 3 times.Pat dry in absorbent paper until there is no moisture after washing.
6. the substrate solution of 100 μ l, mixing, incubated at room 15 minutes are added in every hole.
7. every hole adds the stop buffer of 50 μ l, mixing, terminates reaction.
8. reading OD value in 10 minutes, the wavelength (OD value) of microplate reader uses 450 ± 10nm.
9. result calculates: 1. calculate the mean OD value of standard, quality-control product and sample.All of OD value all deducts " 0 " standard
The mean OD value of product；2. Criterion curve, uses the OD value of each standard substance concentration value plotted versus corresponding thereto.
OD value is the longitudinal axis (Y-axis), and concentration is transverse axis (X-axis).3. the estradiol concentration of sample, first searches OD value in Y-axis, laterally prolongs
Extend standard curve, draw vertical line to intersect with X-axis with the point on standard curve, draw the concentration value of estradiol.
(C) reckoning of follicule-stimulating hormone (FSH) (FSH) concentration:
By the testing result of estradiol in person under inspection's blood serum sample at follicule-stimulating hormone (FSH) biological activity intensity-estradiol concentration mark
Inverse on directrix curve, finds follicule-stimulating hormone (FSH) biological activity intensity in sample to be tested.
Evaluation of result:
From 1.00 mIU/ml, FSH standard substance (Pharmacia S.P.A., the U.S.) are carried out serial dilution is: 1.00 mIU/
Ml, 0.100 mIU/ml, 0.010 mIU/ml, 0.001 mIU/ml, 0.0001 mIU/ml, the most respectively with putting the method for exempting from, chemistry
The testicular cell method of luminescence method and the present invention detects.Each diluted concentration carries out three detections.Experiment is repeated 5 times.Knot
Fruit is listed in table 1:
From result, testicular cell method detection people FSH ratio puts the method for exempting from and sensitive 100 times of chemoluminescence method.
Claims (3)
1. a follicule-stimulating hormone (FSH) biological activity strength detecting method, it is characterised in that comprise the following steps:
(1) without the preparation of follicule-stimulating hormone (FSH) serum:
Silicone rubber tube is filled up estradiol crystallization, is subcutaneously implanted back part of animal 4-6 week in order to suppress dividing of animal follicule-stimulating hormone (FSH)
Secrete；Collecting 40-60 milliliter animal serum, add the activated carbon of 10-20mg, at room temperature absorption more than 12 hours, use filter paper mistake
Filter steroid hormone, filtrate be stored in-40～-80 DEG C of refrigerators in preserve；
(2) preparation of support cell:
Take animal testis 2, remove peplos, by the consumption of 4～6.5ml 199 culture fluid/2 testis, testis is put in 199 cultivations
In liquid, adding collagenase III and separate enzyme II, making the ultimate density of collagenase III in culture fluid is 0.25-0.29mg/
Ml, the ultimate density separating enzyme II is 0.11-0.15 μ g/ml；Hatching 15～20 minutes at 34～37 DEG C, gained suspension is used
Equivalent 199 culture fluid dilutes, and adds ficoll, make ficoll final concentration of 11～14g/100mL, by it after filtration in filtrate
It is centrifuged 3～20 minutes under 1000～1500g；By centrifugal gained containing the precipitation 4～8mL 199 culture fluid weights supporting cell
Outstanding；Then, 3-isobutyl-methyl xanthine, bovine serum albumin and hyclone are added；With trypanblue exclusion method labor cell calculate,
Cell suspension must be supported；
(3) follicule-stimulating hormone (FSH) biological activity intensity analysis in person under inspection's blood serum sample:
(A) stimulation of the follicule-stimulating hormone (FSH) in blood serum sample support cell generation estradiol:
Take 70-100 μ l step (two) gained and support cell suspension, add person under inspection serum 30-50 μ l；Take 70-100 μ l step simultaneously
(2) gained supports cell suspension, adds the follicule-stimulating hormone (FSH) standard substance of each concentration that 30-50 μ l phosphate buffer diluted；Will
The reagent configured, at 37 DEG C, uses 5% CO2And 95%O2After hatching 4-6 hour, add 1.5-1.6ml containing 0.01-
The phosphate buffer of 0.012g/100mL thimerosal gelatin is to terminate reaction；
(B) detection of estradiol: the person under inspection's blood serum sample that will process through step (A) and standard substance are according to existing estradiol
Detection kit or other method carry out the detection of estradiol concentration, draw follicule-stimulating hormone (FSH) biological activity intensity-estradiol dense
Scale directrix curve；
The reckoning of (c) follicule-stimulating hormone (FSH) concentration:
By the testing result of estradiol in person under inspection's blood serum sample at follicule-stimulating hormone (FSH) biological activity intensity-estradiol concentration mark
Inverse on directrix curve, finds follicule-stimulating hormone (FSH) biological activity intensity in sample to be tested.
Follicule-stimulating hormone (FSH) biological activity strength detecting method the most according to claim 1, it is characterised in that step (two)
In, detect is the sustentacular cell of testis of animal.
Follicule-stimulating hormone (FSH) biological activity strength detecting method the most according to claim 1, it is characterised in that step (two)
In, 3-isobutyl-methyl xanthine, bovine serum albumin and hyclone final concentration respectively: 3-isobutyl-methyl xanthine is
0.125-0.150mmol/L；Bovine serum albumin is 0.5-0.6g/100mL；Hyclone is the 0.5-2% of cumulative volume.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002102996A1 (en) * | 2001-06-19 | 2002-12-27 | The Population Council, Inc. | Male contraceptives |
| CN101937003A (en) * | 2010-09-14 | 2011-01-05 | 高常青 | Method for detecting trace luteinizing hormone |
| CN103068839A (en) * | 2010-08-04 | 2013-04-24 | 葛莱高托普有限公司 | Improved recombinant human follicle-stimulating hormone |
-
2016
- 2016-07-28 CN CN201610608726.2A patent/CN106053861A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002102996A1 (en) * | 2001-06-19 | 2002-12-27 | The Population Council, Inc. | Male contraceptives |
| CN103068839A (en) * | 2010-08-04 | 2013-04-24 | 葛莱高托普有限公司 | Improved recombinant human follicle-stimulating hormone |
| CN101937003A (en) * | 2010-09-14 | 2011-01-05 | 高常青 | Method for detecting trace luteinizing hormone |
Non-Patent Citations (2)
| Title |
|---|
| GAUTAM,M等: "Hormone responsiveness of cultured Sertoli cells obtained from adult rats after their rapid isolation underless harsh conditions", 《ANDROLOGY》 * |
| 高菊兴等: "人精浆中端粒酶活性测定及其临床意义", 《山东医学高等专科学校学报》 * |
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Application publication date: 20161026 |