CN101614748A - Follicle-stimulating hormone time-resolved fluoroimmunoassay method and kit - Google Patents
Follicle-stimulating hormone time-resolved fluoroimmunoassay method and kit Download PDFInfo
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- CN101614748A CN101614748A CN200810043548A CN200810043548A CN101614748A CN 101614748 A CN101614748 A CN 101614748A CN 200810043548 A CN200810043548 A CN 200810043548A CN 200810043548 A CN200810043548 A CN 200810043548A CN 101614748 A CN101614748 A CN 101614748A
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Abstract
The invention discloses the time-resolved fluoroimmunoassay and the kit of follicle-stimulating hormone (FSH) (FSH), it selects for use anti-FSH monoclonal antibody as coated antibody, is diluted to 1-10ug/ml as coating buffer with sodium carbonate buffer solution; The anti-FSH monoclonal anti body and function lanthanide ion mark that the matches antibody that serves as a mark uses by dilution in 1: 20 with reaction buffer during experiment; On the reaction plate of coated antibody, every hole adds the FSH standard items of 25ul or the labelled antibody of testing sample and dilution successively, carries out fluoroscopic examination after hatching.The present invention also provides the reagent corresponding box.The present invention has higher sensitivity, specificity and stability, and analytic system is increasingly automated, can improve clinical examination result's speed, reduces personal error significantly and increases the reliability that detects the result.
Description
Technical field:
The present invention relates to follicle-stimulating hormone (FSH) (FSH) detection by quantitative kit, belong to the time-resolved fluoroimmunoassay detection range.
Background technology:
(Follicle-stimulating-hormone is a kind of glycoprotein by the anterior pituitary secretion FSH) to follicle-stimulating hormone (FSH), and by the non-covalent bond be combined into, molecular weight is 37000 by two polypeptied chains.FSH can strengthen the synthetic of Cytochrome P450, makes androgen be converted into estrogen, and FSH and LH regulating synthesizing of steroid hormone in the ovary jointly, brings into play crucial effects in the generation of the maturation of ovarian follicle and corpus luteum.Can promote follicle maturity at women FSH, ovulation induction, and in the menstrual cycle, change synchronously with LH, mainly promote the generation and the maturation of sperm at male sex FSH.The FSH level raises in climacteric, the depletion of the ripe early gamogenetic egg nest of oophorectomize postoperative, gives estrogen this moment and can make the FSH level recover normal, proves that it is the negative feedback regulation mechanism.
The detection of serum FSH at present mainly contains euzymelinked immunosorbent assay (ELISA) (ELISA), immune radiating method (IRMA).Because be subjected to the restriction of tracer agent proterties, the sensitivity of ELISA is not high, sensing range is limited, and the IRMA kit is subjected to the influence of isotope half life period, and the term of validity is short, also has certain radioactive contamination in addition.
Time resolution immunofluorescence analysis (time-resolved fluoroimmunoassay, TRFIA) utilize lanthanide series with unique fluorescent characteristic, replace enzyme, isotope etc., as a kind of important method in the luminescence immunoassay, have highly sensitive, tracer stable, be not subjected to many advantages such as the interference of sample natural fluorescence, "dead" pollution.
Summary of the invention:
The purpose of this invention is to provide time-resolved fluoroimmunoassay and kit that a kind of FSH of being used for detects, solve mainly that the sensitivity that prior art exists is low, poor stability, operation is loaded down with trivial details and technical matters such as contaminated environment.The present invention adopts europium label, and the europium labelling kit is provided, with the sensitivity and the stability of raising method.
Technical scheme of the present invention is: need finish following preliminary work before carrying out time resolution immunofluorescence analysis detection method:
At first the preparation bag is by plate, and used bag is the plate of specific antibody bag quilt by plate, and the preparation of FSH coated slab may further comprise the steps:
(1) with the FSH monoclonal anti body and function carbonate buffer solution dilution of purifying, adds then in each hole of coated slab, after absorption, washing, sealing, drying, obtain FSH monoclonal antibody coated slab;
The Aluminium Foil Package pack of (2) FSH monoclonal antibody coated slab being packed into special-purpose seals and refrigerates standby.Next is the preparation of FSH europium mark, may further comprise the steps:
(1) gets antibody to be marked and add in the bag filter, put into the carbonate buffer solution of the PH 9.5 for preparing, room temperature dialysed overnight (repeatedly changing dislysate therebetween);
(2) next day, take out antibody, be diluted to 0.2-5/mL, in mass ratio 2: 1 ratio (DTTA-Eu: antibody) add DTTA-Eu while vibrating, on the vibration plate machine, vibrated at a slow speed 1 hour under the room temperature, left standstill in the dark then 48 hours;
(3) (1.5 * 50cm) separate the antibody that mark is good, and detachment process is monitored with the nucleic acid-protein instrument by Sephadex G50 chromatographic column with free DTTA-Eu;
(4) accept effluent with small test tube segmentation successively, use spectrophotometer, survey absorbance at the 280nm place, survey fluorescence intensity simultaneously, calculate Eu labeling antibody concentration;
(5) add 0.3%BSA, 2-8 ℃ of preservation.
Follicle-stimulating hormone time-resolved fluoroimmunoassay method comprises the following steps:
1. insolubilized antibody preparation: monoclonal antibodies is diluted to 1-10ug/mL as coating buffer with sodium carbonate buffer solution, wraps, and seal with confining liquid by reaction plate;
2. lanthanide ion labelled antibody preparation: select for use the pairing monoclonal antibodies to carry out the lanthanide ion mark with conventional method;
3. on the reaction plate with insolubilized antibody that 1. step makes, every hole adds follicle-stimulating hormone (FSH) standard items or testing sample, and hatches with the labelled antibody of reaction buffer dilution, carries out fluorometric assay at last.
The concentration of monoclonal antibody is 1-10ug/mL in the 1. middle coating buffer of step, and the 2. middle labelled antibody of step is dilution in 1: 20 with reaction buffer with volume ratio.The lanthanide ion of step in 2. is preferably Eu
3+The reaction buffer of step in 3. is the 50mmol/L Tris-HCl of PH 7.8, includes 0.9%NaCl, 1%BSA, 0.05% N of IgG, 20uM DTPA, 0.08%Tween20 and 0.1%NaN
3And 3. this step is finished on the time resolved fluoro-immunoassay instrument automatically.Used enhancing liquid is fluorescence enhancement solution.
Follicle-stimulating hormone (FSH) detection by quantitative kit, it is characterized in that kit comprises: the damping fluid of coated antibody, confining liquid, reaction buffer, washing lotion, enhancing liquid, and as monoclonal antibodies, the monoclonal antibodies of lanthanide ion mark and the follicle-stimulating hormone (FSH) standard items of coated antibody.
The preparation method of described FSH standard items is: elder generation dissolves the FSH freeze-dried antigen respectively and is diluted to required maximum concentration, adopts coubling dilution to be diluted to desired concn again, obtains the FSH standard items.With containing 6%BSA, 4% sugar, 0.1%NaN
3, 50mmol/L Tris-HCl damping fluid is mixed with standard items with follicle-stimulating hormone (FSH) albumen.
The present invention uses double antibody sandwich method, and reactions steps comprises: reagent is prepared, application of sample reacts, washing pats dry, adds enhancing liquid, survey fluorescent value.
The invention has the beneficial effects as follows: sensitivity for analysis height (0.2mIU/mL), detection time short (65min), specificity is good, and is little with the cross reaction of interfering material.
Description of drawings:
Fig. 1 is reaction principle figure of the present invention
The present invention adopts the double-antibody sandwich single stage method.Anti-people FSH monoclonal antibody bag is by the glimmering plate of exempting from 96 holes, and the anti-monoclonal antibody that reaches europium ion (Eu3+) mark of FSH in calibration object or the sample and Sheet is in micropore inside surface generation immune response, and the sandwich immunoassay compound of micropore surface separates by washing with the free label monoclonal antibody.Eu3+ in the micropore surface immune complex forms the stable fluorescence complex after being dissociated by fluorescence enhancement solution, and the proportional example of FSH concentration in fluorescence intensity and calibration object or the sample can draw FSH concentration in the sample by dose-response curve.
Fig. 2 is an operational flowchart of the present invention
The first step: dilution europium label, second step: add calibration object and testing sample, the 3rd step: the europium mark after the adding dilution, the 4th step: hatched for the 5th step: wash plate, the 6th step: adding strengthens liquid, the 7th step: detect.
Embodiment:
The invention will be further described by embodiment below with reference to Fig. 1,2.
Embodiment:
Follicle-stimulating hormone (FSH) (FSH) is measured
1, standard items preparation
With containing 6%BSA, 4% sugar, 0.1%NaN3,50mmol/L Tris-HCl damping fluid with FSH albumen be mixed with 0,3,10,30,100, the 300mIU/mL titer, proofread and correct with national FSH standard items.Packing is in+2～+ 8 ℃ of preservations.
2, operation steps
1) preparation of reagent
A) cleansing solution: concentrated washing lotion of 40mL and 960mL deionized water are mixed in clean bottle for handling liquid toilet or cosmetic substance, standby as the work cleansing solution.
B) europium mark: use in last hour, press 1: 20 dilution europium label with analysis buffer.
C) with kit analysis buffer, testing sample, calibration object and and the micropore reaction bar balance of requirement to room temperature (20～25 ℃).
2) calibration object or the sample of absorption 25 μ l are sequentially added into corresponding micropore bottom.
3) in each micropore, add the own europium label solution of diluting of 100 μ l, vibrate 60 minutes in slow shelves under the room temperature.
4) wash plate 6 times, lath is patted dry on the thieving paper of cleaning.
5) the enhancing liquid of adding 200 μ l in each micropore vibrates 5 minutes in slow shelves under the room temperature.
6) differentiating fluor tester with the time detects.
Follicle-stimulating hormone (FSH) (FSH) is measured flow process and is seen Table 1
Table 1 follicle-stimulating hormone (FSH) (FSH) is measured flow process
3, specificity sees Table 2
The cross reaction of table 2FSH TRIFMA and LH, TSH, HCG
#
| Cross reacting material | Concentration | Apparent FSH concentration |
| Thyrotropic hormone (TSH, Biodesign) | ??130mIU/L | ??0.2mIU/mL |
| Interstitialcellstimulating hormone (ICSH) (LH, country) | ??2000mIU/mL | ??2.6mIU/mL |
| Human chorionic gonadotrophin (HCG, country) | ??2000mIU/mL | ??0.3mIU/mL |
The cross reacting material that apparent FSH concentration is concentration shown in the last table detects resulting FSH concentration with this reagent.
Claims (6)
1, follicle-stimulating hormone time-resolved fluoroimmunoassay method is characterized in that comprising the following steps:
1. insolubilized antibody preparation: monoclonal antibodies is diluted to 1-10ug/mL as coating buffer with sodium carbonate buffer solution, wraps, and seal with confining liquid by reaction plate;
2. lanthanide ion labelled antibody preparation: select for use the pairing monoclonal antibodies to carry out the lanthanide ion mark with conventional method;
3. on the reaction plate with insolubilized antibody that 1. step makes, every hole adds follicle-stimulating hormone (FSH) standard items or testing sample, and hatches with the labelled antibody of reaction buffer dilution, carries out fluorometric assay at last.
2,, it is characterized in that the 2. middle labelled antibody of step is dilution in 1: 20 with reaction buffer with volume ratio according to the described follicle-stimulating hormone time-resolved fluoroimmunoassay method of claim 1.
3,, it is characterized in that the lanthanide ion during step 2. is Eu according to the described follicle-stimulating hormone time-resolved fluoroimmunoassay method of claim 1
3+
4, according to the described follicle-stimulating hormone time-resolved fluoroimmunoassay method of claim 1, it is characterized in that the reaction buffer during step is 3. is the 50mmol/L Tris-HCl of PH 7.8, include 0.9%NaCl, 1%BSA, 0.05% N of IgG, 200uM DTPA, 0.08%Tween20 and 0.1%NaN
3And 3. this step is finished on the time resolved fluoro-immunoassay instrument automatically.
5, follicle-stimulating hormone (FSH) detection by quantitative kit, it is characterized in that kit comprises: the damping fluid of coated antibody, confining liquid, reaction buffer, washing lotion, enhancing liquid, and as monoclonal antibodies, the monoclonal antibodies of lanthanide ion mark and the follicle-stimulating hormone (FSH) standard items of coated antibody.
6, follicle-stimulating hormone (FSH) detection by quantitative kit according to claim 5 is characterized in that standard items are with containing 6%BSA, 4% sugar, 0.1%NaN
3, 50mmol/L Tris-HCl damping fluid is mixed with standard items with follicle-stimulating hormone (FSH) albumen.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102236020A (en) * | 2010-04-20 | 2011-11-09 | 上海新波生物技术有限公司 | Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit |
| CN102236021A (en) * | 2010-04-20 | 2011-11-09 | 上海新波生物技术有限公司 | Human immunodeficiency virus (HIV) antibody time resolved fluoroimmunoassay method and kit |
| CN103293313A (en) * | 2012-02-24 | 2013-09-11 | 上海新波生物技术有限公司 | Carbohydrate antigen 15-3 time-resolved immunofluorescence assay and kit |
| CN103308683A (en) * | 2012-03-14 | 2013-09-18 | 上海新波生物技术有限公司 | Time resolution immunofluorescence analyzing method and kit for saccharides antigen 19-9 |
| CN105106939A (en) * | 2010-07-30 | 2015-12-02 | 辉凌公司 | Stabilization of FSH |
| CN110132929A (en) * | 2019-06-12 | 2019-08-16 | 上海雄图生物科技有限公司 | A kind of food safety time-resolved fluorescence quick and quantitative determination system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7410768B2 (en) * | 2000-04-03 | 2008-08-12 | Inverness Medical Switzerland Gmbh | Test methods and devices |
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2008
- 2008-06-25 CN CN200810043548A patent/CN101614748A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7410768B2 (en) * | 2000-04-03 | 2008-08-12 | Inverness Medical Switzerland Gmbh | Test methods and devices |
Non-Patent Citations (5)
| Title |
|---|
| J.I.J. VAN CASTEREN ET AL.: "Development of time-resolved immunofluorometric assays for rat follicle-stimulating hormone and luteinizing hormone and application on sera of cycling rats.", 《BIOLOGY OF REPRODUCTION》 * |
| M.JIMENEZ ET AL.: "Validation of an ultrasensitive and specific immunofluorometric assay for mouse follicle-stimulating hormone.", 《BIOLOGY OF REPRODUCTION》 * |
| 史庭燕等: "β-hCG时间分辨荧光免疫分析法的建立", 《生殖与避孕》 * |
| 吴英松等: "人促卵泡激素时间分辨荧光免疫分析检测试剂的研制", 《现代检验医学杂志》 * |
| 吴英松等: "人促卵泡激素时间分辨荧光分析检测试剂的研制", 《现代检验医学杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102236020A (en) * | 2010-04-20 | 2011-11-09 | 上海新波生物技术有限公司 | Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit |
| CN102236021A (en) * | 2010-04-20 | 2011-11-09 | 上海新波生物技术有限公司 | Human immunodeficiency virus (HIV) antibody time resolved fluoroimmunoassay method and kit |
| CN105106939A (en) * | 2010-07-30 | 2015-12-02 | 辉凌公司 | Stabilization of FSH |
| CN103293313A (en) * | 2012-02-24 | 2013-09-11 | 上海新波生物技术有限公司 | Carbohydrate antigen 15-3 time-resolved immunofluorescence assay and kit |
| CN103308683A (en) * | 2012-03-14 | 2013-09-18 | 上海新波生物技术有限公司 | Time resolution immunofluorescence analyzing method and kit for saccharides antigen 19-9 |
| CN103308683B (en) * | 2012-03-14 | 2015-08-26 | 珀金埃尔默医学诊断产品（上海）有限公司 | CA19-9 time-resolved fluoroimmunoassay and kit |
| CN110132929A (en) * | 2019-06-12 | 2019-08-16 | 上海雄图生物科技有限公司 | A kind of food safety time-resolved fluorescence quick and quantitative determination system |
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