CN109932516B - Analysis method for evaluating affinity of human follicle stimulating hormone receptor - Google Patents
Analysis method for evaluating affinity of human follicle stimulating hormone receptor Download PDFInfo
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Abstract
The invention discloses an analysis method for evaluating affinity of a human follicle stimulating hormone (hFSH) receptor by utilizing adherent cells-human ovarian granulosa cells (KGN cells). The method selects a specific anti-human follicle stimulating hormone receptor (hFSHR) antibody, and leads to the change of progesterone content by competitively binding hFSH and the anti-hFSHR antibody to hFSHR on the surface of KGN cells, thereby comparing the relative affinity of hFSH to hFSHR. The method avoids the radiation danger generated by using an isotope labeling method and the complicated operation process of flow cytometry, and can stably and effectively determine the relative affinity of hFSH for hFSHR.
Description
Technical Field
The invention belongs to the field of protein medicine biological engineering and technology, and particularly relates to a method for determining the relative affinity of hFSH to hFSHR by competitively binding hFSH and an anti-hFSHR antibody to hFSHR on the surface of human ovarian granulosa cells (KGN cells) to change the content of progesterone.
Background
Human follicle stimulating hormone (hFSH) is an important medicine for treating female infertility, and is mainly used for treating female anovulatory infertility, controlling sexual ovarian stimulation, assisting reproductive technology and the like. hFSH is a heterodimeric glycoprotein hormone produced by the anterior pituitary gland, which is linked by a non-covalent bond between an α subunit and a β subunit.
Human ovarian granulosa cells (KGN cells) are natural cells for expressing hFSHR, and the hFSHR expression condition on the surface of the KGN cells is determined by a flow cytometry method in a literature report, but the flow cytometry is complex in operation, high in analysis and detection cost, large in deviation and low in test repeatability. The use of isotopically labeled [ alpha ], [ beta ] -n-beta-cyclodextrin, or [ alpha ], [ beta ] -cyclodextrin, or [ beta ] -cyclodextrin125I]hFSH competition combination detection method to estimate hFSH and KGN cell specific combination ability and level; the isotope labeling method has radioactive hazards and higher requirements on operators, and the accuracy of the measurement result is greatly rechecked in an estimation mode. At present, there is a need to develop a simple, convenient, low-cost, safe and risk-free assay method for determining human fsh receptor affinity.
Disclosure of Invention
The inventor finds that after hFSH is combined with human follicle hormone receptor (hFSHR) on KGN cell membrane, a signal transduction pathway of cyclic adenosine monophosphate (cAMP) is stimulated, so that expression of a target gene is transcriptionally activated, aromatase is activated, synthesis and secretion of steroid steroids such as progesterone (progasterone), estradiol (estradiol) and the like are caused, and the receptor affinity of hFSH is evaluated by detecting progesterone secreted to the outside of KGN cells by hFSH stimulation. If the affinity is high, binding of hFSH to hffshr is strong, which further stimulates priming of downstream pathways, thereby enhancing the activity of hFSH.
To confirm the specificity of the response of KGN cells to hFSH and the affinity of the self-prepared and reference drugs for hffshr on the KGN cell surface, a specific anti-hffshr Antibody (Human FSHR Antibody) was selected, and incubated with KGN cells in advance to allow the anti-hffshr Antibody to bind specifically to the hffshr on the cell surface, and then hFSH was added to the cell culture medium to allow hFSH and the anti-hffshr Antibody to compete for binding to hffshr on KGN cells, thereby changing the progesterone content secreted into the culture supernatant, and the relative affinity of hFSH for hFSH was calculated.
The invention provides a method for determining the relative affinity of hFSH to hFSHR by determining the relative affinity of hFSH to hFSHR through the competitive binding of human follicle stimulating hormone (hFSH) and anti-human follicle stimulating hormone receptor (hFSHR) antibodies to hFSHR on the surface of human ovarian granulosa cells (KGN cells) to produce changes in progesterone content.
In order to realize the technical purpose of the invention, the invention adopts the following technical scheme:
an assay for evaluating the affinity of human follicle stimulating hormone (hFSH) receptor using KGN cells, the method comprising the steps of:
(1) cell recovery and passage;
(2) plating cells;
(3) incubating and combining the anti-hFSHR antibody with KGN cells;
(4) the test hFSH competes for binding to hffshr on KGN cells;
(5) the progesterone content was determined.
Further, the specific operation steps are as follows:
(1) cell recovery and passage
Taking out the cell strain from the liquid nitrogen tank, quickly putting the cell strain into a water bath at 37 ℃, and slightly shaking to quickly melt the cell strain; moving the suspension liquid into a centrifuge tube in a super clean bench, adding DMEM/F-12 (DF-12 for short) containing Fetal Bovine Serum (FBS) to culture the suspension cells; centrifuging at 800rpm for 5min, discarding supernatant, adding fresh cell culture medium, blowing, mixing, and transferring to cell culture bottle; the bottle wall is marked with the cell name, the operation date and the cell generation times;
(2) cell plating
Digesting human ovarian granulosa cell tumor cells (KGN) in logarithmic growth phase by using trypsin, discarding the trypsin, adding FBS + DF12 culture medium to terminate digestion, and re-suspending the cells; adjusting the cell concentration after counting by using a cell counter; adding 100 mul/well cells into a 96-well cell culture plate, and performing cell culture at 37 ℃ by 5% CO2Culturing overnight under the condition;
(3) incubation binding of anti-hFSHR antibody to KGN cells
Taking one anti-hFSHR Antibody (Human FSHR Antibody, 500 mu g/Antibody, sterile freeze-dried powder), carrying out resuspension by using sterile PBS (phosphate buffer solution) to fully dissolve the Antibody, then preparing anti-hFSHR antibodies with different concentrations by using FBS + DF12 culture medium, and incubating 100 mu l of the anti-hFSHR Antibody and KGN cells for 1-5 h;
(4) test hFSH (self-prepared drug and reference drug) competes for binding to hFSHR on KGN cells
Gradient dilution of hFSH test article using FBS + DF12 medium; adding the prepared test sample into the 96-well plate in the step (3), so that the test sample hFSH and the anti-hFSHR antibody compete to bind to hFSHR on KGN cells at the temperature of 37 ℃ and 5% CO2Culturing for more than 48 hours under the condition;
(5) the method for measuring the content of the progesterone comprises the following steps:
adding 25-50 mu l of culture supernatant obtained in the step (4) into a coated 96-well plate according to the operation steps of a progesterone ELISA kit (manufacturer: ALPHA DIAGNOSTIC, batch number: 1955), adding 100-200 mu l/well of HRP ligase, and incubating at room temperature for 60 min; after washing for 3 times by using a washing solution, adding a TMB substrate at 100-200 mul/hole, and after incubating for 20min at room temperature, adding 50-100 mul of stop solution into each hole; measuring absorbance at 450nm by using an enzyme-labeling instrument; using SoftMax software, EC of the self-prepared product and reference drug were calculated respectively50Values, compare the receptor affinity identity.
Wherein, the cells used in the step (1) are human ovarian granulosa cells (KGN cells) and are used for measuring the affinity of the hFSH receptor.
Wherein the culture medium in the step (1) is FBS + DF12 culture medium with the concentration of 8-12%.
When the cells are plated in the step (2), firstly, 8-12% of FBS + DF12 culture medium is adopted to stop digestion, and then the cells are cultured overnight at the concentration, so that the cells can grow adherent to the walls rapidly; and then replacing the medium with 1% -5% of FBS + DF12 medium.
Wherein the culture medium adopted in the step (3) is FBS + DF12 culture medium with the concentration of 1-5%.
Wherein, in the step (3), the anti-hFSHR antibody and the KGN cell are incubated for 1-5h, so that the anti-hFSHR antibody and the KGN cell can compete with hFSH to combine with FHSR on the KGN cell, and the concentration range is as follows: 0.1-20000 ng/ml.
Wherein, in the step (4), the hFSH test sample is diluted in a gradient manner by using FBS + DF12 culture medium with the concentration of 1% -5%.
Wherein, the concentration range of the hFSH test sample in the step (4) is as follows: 0.001 ng/ml-600ng/ml, and the dilution gradient is 2-5 times. The time for stimulating the cells to generate the progesterone by loading is 48-120 h.
Wherein, the progesterone content in the step (5) adopts a commercial ELISA kit.
In one embodiment, hFSH binds to hffshr on KGN cells, activating the downstream pathway, allowing the KGN cells to secrete progesterone. After the anti-hFSHR antibody with a series of concentrations is incubated with the KGN cells, the anti-hFHS antibody is combined with the hFHSR on the KGN cells; then adding hFSH with fixed concentration, and making it and anti-hFSHR antibody competitively bind to hFSHR on the KGN cell surface, so that the downstream pathway of KGN cell stimulated by hFSH is inhibited, the progesterone secretion amount is reduced, which shows that hFSH can specifically bind to hFSHR on KGN cell, therefore, the in vitro receptor affinity of hFSH can be effectively and specifically evaluated by KGN cell.
In another embodiment, the affinity of the self-preparation and the reference drug for the receptor (hFSHR) is examined by the analysis method of the present invention, and the affinity of the self-preparation and the reference drug for the FSHR on the surface of KGN cells is confirmed by examining the trend of the curves of action of the self-preparation and the reference drug in promoting progesterone production in the presence of a fixed concentration of anti-hFSHR antibody through comparison studies using multiple batches of self-preparation and multiple batches of reference drug.
The method has the advantages that based on the established method for determining the activity of hFSH by using KGN cells, the anti-hFSHR antibody is adopted, and the hFSH and the anti-hFSHR antibody are competitively combined with the hFSHR on the surface of the human ovarian granulosa cells (KGN cells), so that the progesterone content is changed, the relative affinity of the hFSH to the hFSHR can be compared, and the radiation risk caused by an isotope labeling method and the complicated operation process of flow cytometry are avoided.
Drawings
FIG. 1: competitive antagonism of hFSH binding ability of anti-FSHR antibodies with hFSH.
FIG. 2: effect of anti-hFSHR antibodies on the progesterone production-promoting effect of various batches of the self-prepared or reference drugs-curve 1.
FIG. 3: effect of anti-hFSHR antibodies on the progesterone production-promoting effect of different batches of the self-prepared or reference drug-curve 2.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in the form of examples. It should not be construed that the scope of the invention is limited to the following examples. All technical solutions realized based on the content of the present invention belong to the scope of the present invention. It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention, according to the common technical knowledge and common practice in the field.
Example 1 evaluation of the specificity of hFSH binding to the surface of KGN cells.
Example 1 was divided into three groups: group 1, non-neutralized control group: KGN cells + various concentrations of hFSH; group 2. test group: KGN cells + different concentrations of anti-hffshr antibody +10 ng/ml hFSH; group 3. irrelevant antibody groups: KGN cells + different concentrations of irrelevant antibody (anti-LMF antibody) +10 ng/ml hFSH.
(1) Cell recovery and passage
Taking out the KGN cell strain from the liquid nitrogen tank, quickly putting the KGN cell strain into a water bath at 37 ℃, and slightly shaking to quickly melt the KGN cell strain; transferring the suspension into a centrifuge tube in an ultra-clean bench, and adding 10% FBS + DF12 culture medium (hereinafter referred to as DF12-10% FBS) for resuspension of cells; centrifuging at 800-1000 rpm for 5min, removing the supernatant, adding a cell culture medium, blowing, uniformly mixing, and transferring to a cell culture bottle; the bottle wall is marked with the cell name, the operation date and the cell generation times.
(2) Cell plating
After digesting KGN cells in the logarithmic growth phase with trypsin, discarding the trypsin, adding DF12-10% FBS to terminate the digestion, and resuspending the cells; after counting with a cell counter, the cell concentration was adjusted to 2X 105Per ml; adding 100 mul/well of cells into a 96-well plate, and adding 5% CO at 37 DEG C2Culturing overnight under the condition; DF12 medium (hereinafter referred to as DF12-1% FBS) with the FBS concentration of 1% was replaced by 100 μ l/well.
(3) Incubation binding of anti-hFSHR antibody to KGN cells
Group 1: KGN cells were incubated in DF12-1% FBS for 2 h.
Group 2: one anti-hFSHR Antibody (Human FSHR Antibody, 500. mu.g/Antibody, sterile lyophilized powder) was resuspended in sterile PBS and dissolved thoroughly, and then serially diluted 5-fold with DF12-1% FBS to prepare anti-hFSHR antibodies (20000, 4000, 800, 160, 32, 6.4, 1.28, 0.256 ng/ml) at different concentrations, and 100. mu.l of the serially diluted anti-hFSHR antibodies was incubated with KGN cells for 2 h.
Group 3: irrelevant antibodies (anti-LMF antibodies) were diluted in a gradient with DF12-1% FBS to concentrations of 20000, 4000, 800, 160, 32, 6.4, 1.28, 0.256 ng/ml and 100. mu.l of serially diluted irrelevant antibodies were incubated with KGN cells for 2h in advance.
(4) Test hFSH competes for binding to hFSHR on KGN cells
Group 1: adding hFSH with different concentrations into the 96-well plate of the group 1 in the step (3). hFSH at various concentrations was obtained by gradient dilution of an hFSH solution with an initial concentration of 200ng/ml with DF12-1% FBS, 3-fold dilution gradient, and 8 concentrations were prepared by dilution altogether. Duplicate wells of 100. mu.l/well per sample were plated as an unneutralized control. 96 well plates at 37 ℃ 5% CO2Culturing for 72h in an incubator.
Group 2: subjecting group 2 of step (3)10ng/ml hFSH was added to a 96-well plate at 100. mu.l/well. 96 well plates at 37 ℃ 5% CO2Culturing for 72h in an incubator.
Group 3: 10ng/ml hFSH was added to the 96-well plate of group 3 in step (3) at 100. mu.l/well. 96 well plates at 37 ℃ 5% CO2Culturing for 72h in an incubator.
(5) Method for determining progesterone content
The plates were incubated at 37 ℃ with 5% CO2Culturing for 72h in an incubator. According to the operation steps of the progesterone ELISA kit, 25 mul of cell culture supernatant is taken and placed in a coated 96-well plate, then 100 mul/well HRP ligase is added, and incubation is carried out for 60min at room temperature; after washing with the washing solution for 3 times, adding a TMB substrate according to 150 mul/hole, and after incubating at room temperature for 20min, adding 50 mul of stop solution into each hole; absorbance at 450nm was measured with a microplate reader.
Absorbance at 450nm (OD) using SOFT MAX software with FSH/anti-hFSHR antibody/irrelevant antibody concentration as abscissa450) For the ordinate, the dose-response curve of the sample was plotted and fitted using a four parameter equation.
The results are shown in FIG. 1, where group 1 is the profile of normal hFSH action without the addition of anti-hFSHR antibody. Group 2 is the antagonism curve of the anti-hFSHR antibody against hFSH, and in the presence of 10ng/ml hFSH, the antagonism against hFSH is gradually shown with the increase of the concentration of the anti-hFSHR antibody, and it can be seen that the anti-hFSHR antibody can competitively bind to hFSHR on KGN cells with hFSH. Group 3 was an irrelevant antibody control, and in the presence of 10ng/ml of hFSH, irrelevant antibodies at various concentrations had no effect on binding of hFSH to hffsh on the surface of KGN cells, indicating that irrelevant antibodies had no effect on the effects of hFSH. The above results demonstrate that hFSH can be efficiently and specifically evaluated for its receptor affinity in vitro by KGN cells.
Example 2 evaluation of the affinity of the self-preparation for the receptor (hFSHR) with the reference drug
The test sample used in example 2 was homemade hFSH and reference drug, and the self-prepared product obtained according to the present invention was highly similar to the reference drug in terms of primary structure, higher structure, physicochemical properties, biological activity, etc., but was superior to the reference drug in terms of related impurities and purity.
(1) Cell recovery and passage
Taking out the KGN cell strain from the liquid nitrogen tank, quickly putting the KGN cell strain into a water bath at 37 ℃, and slightly shaking to quickly melt the KGN cell strain; transferring the suspension into a centrifuge tube in an ultra-clean bench, and adding 10% FBS + DF12 culture medium (hereinafter referred to as DF12-10% FBS) for resuspension of cells; centrifuging at 800-1000 rpm for 5min, removing the supernatant, adding a cell culture medium, blowing, uniformly mixing, and transferring to a cell culture bottle; the bottle wall is marked with the cell name, the operation date and the cell generation times.
(2) Cell plating
After digesting KGN cells in the logarithmic growth phase with trypsin, discarding the trypsin, adding DF12-10% FBS to terminate the digestion, and resuspending the cells; after counting with a cell counter, the cell concentration was adjusted to 2X 105Per ml; adding 100 mul/well of cells into a 96-well plate, and adding 5% CO at 37 DEG C2Culturing overnight under the condition; DF12 medium (hereinafter referred to as DF12-1% FBS) with the FBS concentration of 1% was replaced by 100 μ l/well.
(3) Incubation binding of anti-hFSHR antibody to KGN cells
Taking one anti-hFSHR Antibody (Human FSHR Antibody, 500. mu.g/branch, sterile lyophilized powder), resuspending with sterile PBS, dissolving completely, diluting with DF12-1% FBS to 10. mu.g/ml, adding 100. mu.l of anti-hFSHR Antibody per well, and adding 5% CO at 37 deg.C2Incubate for 2h under conditions.
(4) Test hFSH competes for binding to hFSHR on KGN cells
The 4 batches of the self-preparation and 4 batches of the reference drug were gradient diluted to 8 concentrations of 200, 66.7, 22.2, 7.4, 2.5, 0.8, 0.3, 0.1ng/ml using DF12-1% FBS, respectively. The gradiently diluted self-preparation and reference drug were added to the cell plates at 37 ℃ in 5% CO2Culturing for 72h in an incubator.
Further, the hFSH physicochemical control was diluted with DF12-1% FBS in a gradient of 200ng/ml for the initial concentration, and 3-fold serial dilutions were carried out for a total of 8 concentrations, and plating was carried out at 100. mu.l/well for 2 replicate wells per sample, thereby obtaining a control for non-neutralization.
(5) Method for determining progesterone content
The plates were incubated at 37 ℃ with 5% CO2Culturing for 72h in an incubator. According to the operation steps of the progesterone ELISA kit, 25 mul of cell culture supernatant is taken and placed in a coated 96-well plate, then 100 mul/well HRP ligase is added, and incubation is carried out for 60min at room temperature; after washing with the washing solution for 3 times, adding a TMB substrate according to 150 mul/hole, and after incubating at room temperature for 20min, adding 50 mul of stop solution into each hole; absorbance at 450nm was measured with a microplate reader.
Absorbance (OD) at 450nm, using the FSH concentration as the abscissa, using SOFT MAX software450) For the ordinate, the dose-response curve of the sample was plotted and fitted using a four parameter equation.
The results are shown in FIGS. 2 and 3. In the presence of the same concentration (10. mu.g/ml) of anti-hFSHR antibody, hFSH from different sources (self-preparation and reference) and from batch to batch were affected by the anti-FSHR antibody to the same extent, with the EC from reference and self-preparation from different batches50The average values are 15.878 ng/ml, 16.253 ng/ml and the EC of the two50There was no significant difference between the values, so the affinity of different batches of self-preparation and reference drug for hffshr was consistent.
Table 1: EC of 10. mu.g/ml anti-hFSHR antibody against different batches of self-prepared or reference drugs50Influence of (2)
(6) Detection of related substances
The relevant substances from the preparation and the reference drug were analyzed by HPLC detection, as shown in table 2:
Claims (6)
1. an assay for assessing the affinity of human fsh receptor, wherein a specific anti-human fsh receptor antibody is selected and the relative affinity of human fsh for human fsh receptor is determined by a change in the progesterone content resulting from competitive binding of human fsh to human fsh receptor antibody on the surface of cells of human ovarian granulosa, comprising the steps of:
(1) cell recovery and passage
Taking out the cell strain from the liquid nitrogen tank, quickly putting the cell strain into a water bath at 37 ℃, and slightly shaking to quickly melt the cell strain; moving the suspension liquid into a centrifuge tube in a super clean bench, and adding DMEM/F-12 culture medium containing fetal bovine serum to suspend cells; centrifuging at 800rpm for 5min, discarding supernatant, adding fresh cell culture medium, blowing, mixing, and transferring to cell culture bottle; the bottle wall is marked with the cell name, the operation date and the cell generation times;
(2) cell plating
Digesting the human ovarian granulosa cells in the logarithmic growth phase by using trypsin, discarding the trypsin, adding a DMEM/F-12 culture medium containing fetal bovine serum to terminate the digestion, and re-suspending the cells; adjusting the cell concentration after counting by using a cell counter; 100 μ l/well of cells were added to a 96-well cell culture plate at 37 ℃ in 5% CO2Culturing overnight under the condition;
(3) anti-human follicle-stimulating hormone receptor antibody and human ovarian granulosa cell incubation combination
Taking a piece of sterile lyophilized powder of the anti-human follicle-stimulating hormone receptor antibody, carrying out resuspension by using sterile PBS (phosphate buffer solution) to fully dissolve the sterile lyophilized powder, then preparing anti-human follicle-stimulating hormone receptor antibodies with different concentrations by using a DMEM/F-12 culture medium containing fetal calf serum, and incubating 100 mu l of the anti-human follicle-stimulating hormone receptor antibody and human ovarian granulosa cells for 1-5h to ensure that the anti-human follicle-stimulating hormone receptor antibody can compete with the human follicle-stimulating hormone to combine with the human follicle-stimulating hormone receptor on the human ovarian granulosa cells, wherein the concentration range is as follows: 0.1-20000 ng/ml;
(4) test human FSH competes for binding to human FSH receptor on human ovarian granulosa cells
Using DMEM/F-12 culture medium containing fetal calf serum to carry out gradient dilution on the human follicle-stimulating hormone test sample; adding the prepared test sample into the 96-well plate obtained in the step (3) to ensure that the test sample is human follicle-stimulating hormone and anti-human follicle-stimulating hormoneHormone receptor antibody competes for binding to human follicle stimulating hormone receptor on human ovarian granulosa cells at 100. mu.l/well, 37 ℃ 5% CO2Culturing for more than 48 hours under the condition;
(5) the method for measuring the content of the progesterone comprises the following steps:
adding 25-50 μ l of culture supernatant obtained in step (4) into coated 96-well plate, adding 100-; washing with a washing solution for 3 times, adding a TMB substrate in 200 mul/hole, incubating at room temperature for 20min, and adding 50-100 mul of stop solution into each hole; measuring absorbance at 450nm by using an enzyme-labeling instrument; using SoftMax software, EC of the self-prepared product and reference drug were calculated respectively50Values, compare the receptor affinity identity.
2. The assay for assessing the affinity of human fsh receptors according to claim 1, wherein the cells used in step (1) are human ovarian granulosa cells, and are used for the measurement of the affinity of human fsh receptors.
3. The assay for evaluating the affinity of human fsh receptors according to claim 1, wherein in the step (2), the cells are plated by first culturing overnight in 8% -12% DMEM/F-12 medium containing fetal bovine serum to allow the cells to grow adherent to the skin; then, the medium was changed to 1% -5% DMEM/F-12 medium containing fetal bovine serum.
4. The assay for evaluating the affinity of human FSH according to claim 1, wherein the culture medium used in step (3) is DMEM/F-12 medium containing fetal bovine serum at a concentration of 1% to 5%.
5. The assay for evaluating the affinity of human FSH according to claim 1, wherein the human FSH sample is diluted in step (4) in a gradient manner using 1% -5% DMEM/F-12 medium containing fetal bovine serum.
6. The assay for assessing the affinity of human fsh receptors according to claim 1, wherein the concentration of the human fsh sample in step (4) is in the range of: 0.001 ng/ml-600ng/ml, 2-5 times of dilution gradient, and 48-120h of progesterone production time by stimulating cells with sample.
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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 |
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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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