CN109932516A - A kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity - Google Patents
A kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity Download PDFInfo
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- CN109932516A CN109932516A CN201910208247.5A CN201910208247A CN109932516A CN 109932516 A CN109932516 A CN 109932516A CN 201910208247 A CN201910208247 A CN 201910208247A CN 109932516 A CN109932516 A CN 109932516A
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Abstract
The invention discloses a kind of analysis methods using attached cell-Human ovarian granulosa cell (KGN cell) evaluation Human Fallicle-Stimulating Hormone (hFSH) receptor affinity.This method has selected a kind of specific anti-human Follicle Stimulating Hormone Receptors (hFSHR) antibody, by the hFSHR of hFSH KGN cell surface in conjunction with anti-hFSHR antibody competition, so that progesterone content generates variation, thus may compare hFSH to the relative affinity of hFSHR.Present method avoids radiological hazard caused by isotope marked price method and the cumbersome operating process of flow cytometry is used, effective measurement hFSH can be stablized for the relative affinity of hFSHR.
Description
Technical field
The invention belongs to protein medical bioengineering and technical fields, and in particular to one kind passes through hFSH and anti-hFSHR
Thus the hFSHR on antibody competition combination Human ovarian granulosa cell (KGN cell) surface may be used so that progesterone content generates variation
HFSH is measured to the relative affinity of hFSHR.
Background technique
Human Fallicle-Stimulating Hormone (hFSH) is a kind of important drugs for treating female acyesis, is mainly used for treating women No-clay weak interbed
Property infertility, Controlled ovarian stimulation and assisted reproductive technology etc..HFSH is the glycoprotein hormones of a heterodimer, by hanging down
Body frontal lobe generates, and is connected by a α subunit and a β subunit through non-covalent bond.
Human ovarian granulosa cell (KGN cell) is the n cell for expressing hFSHR, has document report to pass through flow cytometer
Method determine the hFSHR expression of KGN cell surface, but flow cytometry operations are cumbersome, analysis detection it is at high cost,
Deviation is larger, and test repeatability is low.Also have document report using isotope labelling [125I] hFSH competitive binding detection method,
To estimate hFSH and KGN cell-specific binding ability and level；Isotope-labelling method has radiological hazard, wants to operator
It asks higher, makes the accuracy of measurement result there are larger check by way of estimation,.It being capable of letter currently, needing to develop one kind
The analysis method of single convenient, inexpensive, safe devoid of risk measures Human Fallicle-Stimulating Hormone's receptor affinity.
Summary of the invention
The inventors discovered that exciting cycli phosphate after the human follicle hormone receptor (hFSHR) on hFSH and KGN cell membrane combines
The signal transduction pathway of adenosine (cAMP), and then transcriptional activation destination gene expression activate aromatizing enzyme, and then cause steroids
The synthesis and secretion of steroidal such as progesterone (progesterone), estradiol (estradiol) etc. stimulate KGN by detection hFSH
Cell is secreted into extracellular progesterone to evaluate the receptor affinity of hFSH.Affinity is high, then the combination of hFSH and hFSHR is strong, into
One step can stimulate the excitation of downstream passages, to enhance the activity of hFSH.
For confirmation KGN cell to the specificity and own product of hFSH response and referring to medicine to KGN cell surface
The affinity size of hFSHR has selected the anti-hFSHR antibody of a species specificity (Human FSHR Antibody), by itself and KGN
Cell is incubated in advance, specifically binds anti-hFSHR antibody and cell surface hFSHR, backward cell culture fluid in add
HFSH, so that hFSHR of the hFSH in conjunction with anti-hFSHR antibody competition on KGN cell, so that being secreted into culture supernatant
Progesterone content changes, and thus can calculate hFSH to the relative affinity of hFSHR.
Summary of the invention
The present invention provides one kind to pass through Human Fallicle-Stimulating Hormone (hFSH) and anti-human Follicle Stimulating Hormone Receptors (hFSHR) antibody
Thus the hFSHR on competitive binding Human ovarian granulosa cell (KGN cell) surface can be measured so that progesterone content generates variation
Relative affinity of the hFSH to hFSHR out.
Technical purpose to realize the present invention, the invention adopts the following technical scheme:
A kind of analysis method using KGN cell evaluation Human Fallicle-Stimulating Hormone (hFSH) receptor affinity, this method contains as follows
Step:
(1) cell recovery and passage；
(2) plating cells；
(3) anti-hFSHR antibody is in conjunction with KGN cell incubation；
(4) hFSHR on test sample hFSH competitive binding KGN cell；
(5) progesterone content is measured.
Further, concrete operation step is as follows:
(1) cell recovery and passage
Cell strain is taken out from liquid nitrogen container, is put into rapidly in 37 DEG C of water-baths, gently shaking makes its fast melt；In super-clean bench
Move it into centrifuge tube, the DMEM/F-12(abbreviation DF-12 for containing fetal calf serum (FBS) be added) culture medium resuspension cell；
It is discarded supernatant after 800rpm centrifugation 5min, fresh cell culture medium is added, piping and druming is mixed, moved in Tissue Culture Flask；Bottle wall
Subscript clear-cells title, date of operation, cell generation；
(2) plating cells
By the Human ovarian granulosa cell tumour cell (KGN) in logarithmic growth phase using after trypsin digestion, pancreas egg is discarded
White enzyme is added FBS+DF12 culture medium and terminates digestion, and cell is resuspended；Cell concentration is adjusted after being counted with cell counter；
It is added in 96 porocyte culture plates in 100 μ l/ hole cells, 37 DEG C, 5%CO2Under the conditions of overnight incubation；
(3) anti-hFSHR antibody is in conjunction with KGN cell incubation
An anchorage hFSHR antibody (Human FSHR Antibody, 500 μ g/ branch, sterile lyophilized powder) are taken, with sterile PBS weight
It is outstanding, it dissolves it sufficiently, the anti-hFSHR antibody of various concentration is then prepared with FBS+DF12 culture medium, above-mentioned anti-hFSHR is resisted
100 μ l of body and 1 ~ 5h of KGN cell incubation；
(4) test sample hFSH(own product and referring to medicine) hFSHR on competitive binding KGN cell
Using FBS+DF12 culture medium by hFSH test sample gradient dilution；Prepared test sample is added to the 96 of step (3)
In orifice plate, so that hFSHR of the test sample hFSH in conjunction with anti-hFSHR antibody competition on KGN cell, 100 holes μ l/, 37 DEG C, 5%
CO2Under the conditions of cultivate 48 hours or more；
(5) progesterone content method is measured:
According to progesterone ELISA kit (producer: ALPHA DIAGNOSTIC, lot number: 1955) operating procedure, what is be coated with
25 ~ 50 μ l of culture supernatant in step (4) is added in 96 orifice plates, 100 ~ 200 hole μ l/ HRP ligases, incubation at room temperature is added
60min；After washing lotion is washed 3 times, TMB substrate is added, 100 ~ 200 holes μ l/, after being incubated at room temperature 20min, 50 ~ 100 μ are added in every hole
The terminate liquid of l；With absorbance at microplate reader measurement 450nm；Using SoftMax software, own product is calculated separately and referring to medicine
EC50Value, compares the consistency of its receptor affinity.
Wherein, cell used in step (1) is Human ovarian granulosa cell (KGN cell), affine for hFSH receptor
The measurement of power.
Wherein, the culture medium in step (1) is the FBS+DF12 culture medium that concentration is 8% ~ 12%.
Wherein, in step (2) when plating cells, digestion is terminated using 8% ~ 12% FBS+DF12 culture medium first, later
Overnight incubation at this concentration makes the rapid adherent growth of cell；It is changed to 1% ~ 5% FBS+DF12 culture medium later.
Wherein, use in step (3) culture medium be concentration for 1% ~ 5% FBS+DF12 culture medium.
Wherein, anti-hFSHR antibody and 1 ~ 5h of KGN cell incubation are used in step (3), can be competed and be tied with hFSH
Close the FHSR on KGN cell, concentration range are as follows: 0.1 ~ 20000ng/ml.
Wherein, the FBS+DF12 culture medium for the use of concentration being 1% ~ 5% in step (4) is by hFSH test sample gradient dilution.
Wherein, in step (4) hFSH test sample concentration range: 0.001ng/ml~600ng/ml, dilution gradient be 2 ~ 5
Times.The time that sample-adding stimulation cell generates progesterone is 48 ~ 120h.
Wherein, progesterone content uses the ELISA kit being commercialized in step (5).
In a specific embodiment, hFSH activates downstream passages, so that KGN is thin in conjunction with the hFSHR on KGN cell
Intracrine progesterone.And after a series of anti-hFSHR antibody of concentration and KGN cell incubation, so that anti-hFHS antibody and KGN cell
On hFHSR combine；The hFSH for adding fixed concentration later makes its KGN cell surface in conjunction with anti-hFSHR antibody competition
HFSHR so that the downstream passages of hFSH stimulation KGN cell are suppressed, the decline of progesterone secretion amount illustrates that hFSH can be special
HFSHR on anisotropic combination KGN cell, therefore the extracorporeal receptor of hFSH can effectively, be specifically evaluated by KGN cell
Affinity.
In another embodiment, through the invention the analysis method investigated own product with referring to medicine to receptor
(hFSHR) affinity, using multiple batches of own product and it is multiple batches of compare research referring to medicine, by investigate in fixed concentration
Anti- hFSHR antibody in the presence of own product and referring to medicine promote progesterone generate effect curves variation tendency, self-control is confirmed with this
Product and referring to medicine to the affinity of KGN cell surface FSHR, can efficiently, accurately be surveyed by using method of the present invention
HFSH is determined to the relative affinity of hFSHR.
The beneficial effects of the present invention are the active method of KGN raji cell assay Raji hFSH is used based on what is established before, adopt
With anti-hFSHR antibody, by hFSH in conjunction with anti-hFSHR antibody competition Human ovarian granulosa cell (KGN cell) surface
Thus hFSHR may compare hFSH to the relative affinity of hFSHR, avoid and use same position so that progesterone content generates variation
Radiological hazard caused by plain marked price method and the cumbersome operating process of flow cytometry.
Detailed description of the invention
The competitive antagonism of Fig. 1: anti-FSHR antibody and hFSH to hFSHR binding ability.
Fig. 2: anti-hFSHR antibody promotes the influence curve -1 of progesterone nucleus formation to different batches own product or referring to medicine.
Fig. 3: anti-hFSHR antibody promotes the influence curve -2 of progesterone nucleus formation to different batches own product or referring to medicine.
Specific embodiment
The specific embodiment of form is described in further detail the contents of the present invention by the following examples.But no
It is to be understood as the scope of the present invention is only limitted to following embodiment.All technical solutions realized based on the contents of the present invention are equal
Belong to the scope of the present invention.Obviously, content according to the present invention, according to the ordinary technical knowledge and customary means of this field,
Under the premise of not departing from basic fundamental thought of the invention, the modification, substitution and alteration of other diversified forms can also be made.
Embodiment 1, hFSH are to the Evaluation on specificity of the hFSHR combination of KGN cell surface.
Embodiment 1 is divided for three groups: 1. unneutralized effect control groups of group: KGN cell+various concentration hFSH；2. test of group
Group: anti-+ 10 ng/ml hFSH of hFSHR antibody of KGN cell+various concentration；3. irrelevant antibody groups of group: KGN cell+various concentration
Irrelevant antibody (anti-LMF antibody)+10 ng/ml hFSH.
(1) cell recovery and passage
KGN cell strain is taken out from liquid nitrogen container, is put into rapidly in 37 DEG C of water-baths, gently shaking makes its fast melt；Ultra-clean
It is moved it into centrifuge tube in platform, the FBS+DF12 culture medium (hereinafter referred to as DF12-10%FBS) that concentration is 10% is added and is resuspended carefully
Born of the same parents；It is discarded supernatant after 800 ~ 1000rpm centrifugation 5min, cell culture medium is added, piping and druming is mixed, moved in Tissue Culture Flask；Bottle
Wall subscript clear-cells title, date of operation, cell generation.
(2) plating cells
By the KGN cell in logarithmic growth phase using after trypsin digestion, trypsase is discarded, DF12-10%FBS is added
Digestion is terminated, and cell is resuspended；Cell concentration is adjusted after being counted with cell counter to 2 × 105A/ml；100 μ l/ of cell
Hole is added in 96 orifice plates, and 37 DEG C, 5%CO2Under the conditions of overnight incubation；Replace the DF12 culture medium of FBS concentration 1% (hereinafter referred to as
DF12-1%FBS), 100 μ l/hole.
(3) anti-hFSHR antibody is in conjunction with KGN cell incubation
Group 1: KGN cell is incubated for 2h in DF12-1%FBS.
Group 2: an anchorage hFSHR antibody (Human FSHR Antibody, 500 μ g/ branch, sterile lyophilized powder) are taken, with nothing
Bacterium PBS is resuspended, and dissolves it sufficiently, is then serially diluted the anti-hFSHR antibody for preparing various concentration by 5 times with DF12-1%FBS
(20000,4000,800,160,32,6.4,1.28,0.256 ng/ml), by the above-mentioned 100 μ l of anti-hFSHR antibody being serially diluted
With KGN cell incubation 2h.
Group 3: use DF12-1%FBS by irrelevant antibody (anti-LMF antibody) gradient dilution to concentration for 20000,4000,
800, the 100 μ l of irrelevant antibody being serially diluted and KGN cell are incubated for 2h by 160,32,6.4,1.28,0.256 ng/ml in advance.
(4) hFSHR on test sample hFSH competitive binding KGN cell
Group 1: the hFSH of various concentration will be added in step (3) in 96 orifice plates of group 1.The hFSH of various concentration passes through will be initial dense
The hFSH solution that degree is 200ng/ml carries out gradient dilution acquisition, 3 times of dilution gradients, altogether dilution preparation 8 with DF12-1%FBS
Concentration.According to 2 multiple holes of each sample, 100 holes μ l/ carry out bed board, compare as unneutralized effect.96 orifice plates are in 37 DEG C, 5%
CO272h is cultivated in incubator.
Group 2: 10 ng/ml hFSH will be added according to 100 holes μ l/ in 96 orifice plates of group 2 in step (3).96 orifice plates are in 37
DEG C, 5%CO272h is cultivated in incubator.
Group 3: 10 ng/ml hFSH will be added according to 100 holes μ l/ in 96 orifice plates of group 3 in step (3).96 orifice plates are in 37
DEG C, 5%CO272h is cultivated in incubator.
(5) progesterone content method is measured
By above-mentioned culture plate in 37 DEG C, 5%CO272h is cultivated in incubator.According to the operating procedure of progesterone ELISA kit, take
25 μ l cell culture supernatants add 100 hole μ l/ HRP ligases in 96 orifice plates being coated with, and are incubated at room temperature 60min；It washes
After liquid washs 3 times, TMB substrate is added according to 150 holes μ l/, after being incubated at room temperature 20min, the terminate liquid of 50 μ l is added in every hole；With
Microplate reader measures absorbance at 450nm.
Utilize SOFT MAX software, using the concentration of the anti-hFSHR antibody/irrelevant antibody of FSH/ as abscissa, corresponding 450nm
Under absorbance (OD450) it is ordinate, the dose-response curve of sample is drawn, is fitted using quadruplex parameters.
As a result as shown in Figure 1, group 1 is the effect curves for being not added with the normal hFSH of anti-hFSHR antibody.Group 2 is anti-hFSHR
Antibody to the antagonism curve of hFSH, in the case that 10ng/ml hFSH there are, with the increase of anti-hFSHR antibody concentration,
Gradually show its antagonism to hFSH, it is seen that anti-hFSHR antibody can on hFSH competitive binding KGN cell
hFSHR.Group 3 compares for irrelevant antibody, when 10ng/ml hFSH exists, irrelevant antibody under various concentration to hFSH and
KGN cell surface hFSHR is combined and is not had an impact, illustrates irrelevant antibody not to the effect of hFSH.Result above is said
The bright extracorporeal receptor affinity that hFSH can effectively, be specifically evaluated by KGN cell.
Embodiment 2, own product with referring to medicine to receptor (hFSHR) affinity Conformance Assessment
Test sample employed in embodiment 2 is for homemade hFSH and referring to medicine, and own product obtained is in level-one according to the present invention
Structure, higher structure, physicochemical properties, biological activity etc. and similar referring to medicine height, but in related impurities and pure
Degree aspect is better than referring to medicine.
(1) cell recovery and passage
KGN cell strain is taken out from liquid nitrogen container, is put into rapidly in 37 DEG C of water-baths, gently shaking makes its fast melt；Ultra-clean
It is moved it into centrifuge tube in platform, the FBS+DF12 culture medium (hereinafter referred to as DF12-10%FBS) that concentration is 10% is added and is resuspended carefully
Born of the same parents；It is discarded supernatant after 800 ~ 1000rpm centrifugation 5min, cell culture medium is added, piping and druming is mixed, moved in Tissue Culture Flask；Bottle
Wall subscript clear-cells title, date of operation, cell generation.
(2) plating cells
By the KGN cell in logarithmic growth phase using after trypsin digestion, trypsase is discarded, DF12-10%FBS is added
Digestion is terminated, and cell is resuspended；Cell concentration is adjusted after being counted with cell counter to 2 × 105A/ml；100 μ l/ of cell
Hole is added in 96 orifice plates, and 37 DEG C, 5%CO2Under the conditions of overnight incubation；Replace the DF12 culture medium of FBS concentration 1% (hereinafter referred to as
DF12-1%FBS), 100 μ l/hole.
(3) anti-hFSHR antibody is in conjunction with KGN cell incubation
An anchorage hFSHR antibody (Human FSHR Antibody, 500 μ g/ branch, sterile lyophilized powder) are taken, with sterile PBS weight
It is outstanding, it dissolves it sufficiently, is then diluted to 10 μ g/ml with DF12-1%FBS, the anti-hFSHR antibody of 100 μ l is added in every hole, in
37 DEG C, 5%CO2Under the conditions of be incubated for 2h.
(4) hFSHR on test sample hFSH competitive binding KGN cell
Using DF12-1%FBS by 4 batch own products and 4 batches referring to medicine distinguish gradient dilution to 200,66.7,22.2,7.4,
2.5,0.8,8 0.3,0.1ng/ml concentration.It the own product of gradient dilution and is added in cell plates referring to medicine, in 37 DEG C, 5%
CO272h is cultivated in incubator.
In addition hFSH physics and chemistry reference substance is subjected to gradient dilution with DF12-1%FBS, initial concentration 200ng/ml, 3 times
It is serially diluted, totally 8 concentration, according to 2 multiple holes of each sample, 100 holes μ l/ carry out bed board, in this, as unneutralized effect pair
According to.
(5) progesterone content method is measured
By above-mentioned culture plate in 37 DEG C, 5%CO272h is cultivated in incubator.According to the operating procedure of progesterone ELISA kit, take
25 μ l cell culture supernatants add 100 hole μ l/ HRP ligases in 96 orifice plates being coated with, and are incubated at room temperature 60min；It washes
After liquid washs 3 times, TMB substrate is added according to 150 holes μ l/, after being incubated at room temperature 20min, the terminate liquid of 50 μ l is added in every hole；With
Microplate reader measures absorbance at 450nm.
Absorbance (OD using SOFT MAX software, using the concentration of FSH as abscissa, under corresponding 450nm450) it is vertical
Coordinate is drawn the dose-response curve of sample, is fitted using quadruplex parameters.
As a result as shown in Figures 2 and 3.In the case that same concentrations (10 μ g/ml) anti-hFSHR antibody there are, separate sources
(own product with referring to medicine) is consistent by anti-FSHR antibody effect with the hFSH between batch, and different batches are referring to medicine, self-control
The EC of product50Average value is 15.878 ng/ml, 16.253 ng/ml, and the EC of the two50There was no significant difference between value, therefore not
It is consistent with referring to affinity of the medicine to hFSHR with the own product of batch.
The anti-hFSHR antibody of table 1:10 μ g/ml is to different batches own product or referring to the EC of medicine50Influence
(6) in relation to the detection of substance
By HPLC detection and analysis own product and referring to the related substance of medicine, as shown in table 2:
Claims (10)
1. a kind of analysis method for evaluating Human Fallicle-Stimulating Hormone's receptor body affinity, it is characterised in that this method has selected a kind of spy
Anisotropic anti-human Follicle Stimulating Hormone Receptors (hFSHR) antibody, by hFSH in conjunction with anti-hFSHR antibody competition KGN cell surface
HFSHR thus measure hFSH to the relative affinity of hFSHR so that progesterone content generates variation.
2. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 1, it is characterised in that
This method, which contains, to have the following steps:
(1) cell recovery and passage；
(2) plating cells；
(3) anti-hFSHR antibody is in conjunction with KGN cell incubation；
(4) hFSHR on test sample hFSH competitive binding KGN cell；
(5) progesterone content is measured.
3. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 2, it is characterised in that
This method, which contains, to have the following steps:
(1) cell recovery and passage
Cell strain is taken out from liquid nitrogen container, is put into rapidly in 37 DEG C of water-baths, gently shaking makes its fast melt；In super-clean bench
Move it into centrifuge tube, the DMEM/F-12(abbreviation DF-12 for containing fetal calf serum (FBS) be added) culture medium resuspension cell；
It is discarded supernatant after 800rpm centrifugation 5min, fresh cell culture medium is added, piping and druming is mixed, moved in Tissue Culture Flask；Bottle wall
Subscript clear-cells title, date of operation, cell generation；
(2) plating cells
By the Human ovarian granulosa cell tumour cell (KGN) in logarithmic growth phase using after trypsin digestion, pancreas egg is discarded
White enzyme is added FBS+DF12 culture medium and terminates digestion, and cell is resuspended；Cell concentration is adjusted after being counted with cell counter；
It is added in 96 porocyte culture plates in 100 μ l/ hole cells, 37 DEG C, 5%CO2Under the conditions of overnight incubation；
(3) anti-hFSHR antibody is in conjunction with KGN cell incubation
An anchorage hFSHR antibody (Human FSHR Antibody, 500 μ g/ branch, sterile lyophilized powder) are taken, with sterile PBS weight
It is outstanding, it dissolves it sufficiently, the anti-hFSHR antibody of various concentration is then prepared with FBS+DF12 culture medium, above-mentioned anti-hFSHR is resisted
100 μ l of body and 1 ~ 5h of KGN cell incubation；
(4) test sample hFSH(own product and referring to medicine) hFSHR on competitive binding KGN cell
Using FBS+DF12 culture medium by hFSH test sample gradient dilution；Prepared test sample is added to the 96 of step (3)
In orifice plate, so that hFSHR of the test sample hFSH in conjunction with anti-hFSHR antibody competition on KGN cell, 100 holes μ l/, 37 DEG C, 5%
CO2Under the conditions of cultivate 48 hours or more；
(5) progesterone content method is measured:
According to progesterone ELISA kit (producer: ALPHA DIAGNOSTIC, lot number: 1955) operating procedure, what is be coated with
25 ~ 50 μ l of culture supernatant in step (4) is added in 96 orifice plates, 100 ~ 200 hole μ l/ HRP ligases, incubation at room temperature is added
60min；After washing lotion is washed 3 times, TMB substrate is added, 100 ~ 200 holes μ l/, after being incubated at room temperature 20min, 50 ~ 100 μ are added in every hole
The terminate liquid of l；With absorbance at microplate reader measurement 450nm；Using SoftMax software, own product is calculated separately and referring to medicine
EC50Value, compares the consistency of its receptor affinity.
4. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 3, which is characterized in that
Cell used in step (1) is Human ovarian granulosa cell (KGN cell), the measurement for hFSH receptor affinity.
5. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 3, which is characterized in that
In step (2) when plating cells, 8% ~ 12% FBS+DF12 culture medium overnight incubation is used first, makes cell adherent life rapidly
It is long；It is changed to 1% ~ 5% FBS+DF12 culture medium later.
6. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 3, it is characterised in that
Use in step (3) culture medium be concentration for 1% ~ 5% FBS+DF12 culture medium.
7. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 3, which is characterized in that
Anti- hFSHR antibody and 1 ~ 5h of KGN cell incubation are used in step (3), can on hFSH competitive binding KGN cell
FHSR, concentration range are as follows: 0.1 ~ 20000ng/ml.
8. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 3, which is characterized in that
The FBS+DF12 culture medium for the use of concentration being 1% ~ 5% in step (4) is by hFSH test sample gradient dilution.
9. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 3, which is characterized in that
The concentration range of hFSH test sample in step (4): 0.001ng/ml~600ng/ml, dilution gradient are 2 ~ 5 times, and sample-adding stimulation is thin
The time that born of the same parents generate progesterone is 48 ~ 120h.
10. a kind of analysis method of appraiser's Follicle Stimulating Hormone Receptors affinity according to claim 3, feature exist
In progesterone content is using the ELISA kit being commercialized in step (5).
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4383034A (en) * | 1980-08-27 | 1983-05-10 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Process for the production of human follicle-stimulating hormone |
| CN1803838A (en) * | 2006-01-23 | 2006-07-19 | 南京医科大学 | Follicle stimulating hormone receptor(FSHR) specific binding region polypeptide, its expression vector, preparation method and application in drug preparation |
| CN104569440A (en) * | 2014-12-26 | 2015-04-29 | 北京泰德制药股份有限公司 | Analytical method for evaluating in-vitro biological activity of human follicle stimulating hormone |
| CN107255725A (en) * | 2017-07-07 | 2017-10-17 | 江西科技师范大学 | A kind of homologous competitive enzyme-linked immune analytic approach detected with quantifying Human Fallicle-Stimulating Hormone's In vitro biological activity |
-
2019
- 2019-03-19 CN CN201910208247.5A patent/CN109932516B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4383034A (en) * | 1980-08-27 | 1983-05-10 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Process for the production of human follicle-stimulating hormone |
| CN1803838A (en) * | 2006-01-23 | 2006-07-19 | 南京医科大学 | Follicle stimulating hormone receptor(FSHR) specific binding region polypeptide, its expression vector, preparation method and application in drug preparation |
| CN104569440A (en) * | 2014-12-26 | 2015-04-29 | 北京泰德制药股份有限公司 | Analytical method for evaluating in-vitro biological activity of human follicle stimulating hormone |
| CN107255725A (en) * | 2017-07-07 | 2017-10-17 | 江西科技师范大学 | A kind of homologous competitive enzyme-linked immune analytic approach detected with quantifying Human Fallicle-Stimulating Hormone's In vitro biological activity |
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