CN104034906A - Time-resolved immunofluorescence analysis method for beta-HCG (Human Chorionic Gonadotropin) detection and kit - Google Patents

Time-resolved immunofluorescence analysis method for beta-HCG (Human Chorionic Gonadotropin) detection and kit Download PDF

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CN104034906A
CN104034906A CN201410284459.9A CN201410284459A CN104034906A CN 104034906 A CN104034906 A CN 104034906A CN 201410284459 A CN201410284459 A CN 201410284459A CN 104034906 A CN104034906 A CN 104034906A
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hcg
antibody
beta
time
kit
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李永锋
陈国勇
傅汝毅
陶玲云
蓝文苑
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Guangxi Doctor Hai Yi Information Technology Co Ltd
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Guangxi Doctor Hai Yi Information Technology Co Ltd
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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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Abstract

The invention discloses a time-resolved immunofluorescence analysis method for beta-HCG (Human Chorionic Gonadotropin) detection and a kit. The method comprises the following steps: selecting an anti-beta-HCG monoclonal antibody as a coated antibody, and diluting the coated antibody with a sodium carbonate buffer solution to 1-10 ug/ml to obtain coating buffer; labeling anti-beta-HCG monoclonal antibody pairs by using the lanthanide ions as a labeled antibody; during experiment, diluting by a reaction buffer in a proportion of 1:20 for use; and on a reaction plate of the coated antibody, sequentially adding 25ul of beta-HCG standard substance or sample to be detected and the diluted labeled antibody in each hole, and carrying out fluorescence detection after incubation. The invention further provides the corresponding kit. The method has high sensitivity, specificity and stability, and an analysis system is highly automated. The speed of clinical test results can be increased, a personal error can be greatly reduced, and the reliability of detection results is improved.

Description

A kind of time-resolved fluoroimmunoassay and kit detecting for β-HCG
Technical field
The present invention relates to a kind of time-resolved fluoroimmunoassay and kit detecting for β-HCG, belong to time-resolved fluoroimmunoassay detection field.
Background technology
Human chorionic gonadotrophin (HCG) exists in a variety of forms in human body, the peptide junction, 44-45 position that wherein free β subunit is HCG β subunit in metabolic processes disconnects institute and forms, and is a kind of blood serum designated object that is applied to Prenatal Screening.In the gestational period female serum, β-HCG level is bimodal curve, and the 1st peak, before and after gestation 12 weeks, occurs physiological decline after 15 weeks, within 20～32 weeks, remains low and steady level reaches minimum in 26 weeks.The 2nd peak, in pregnant 37 weeks, is starkly lower than the 1st peak, during to full-term pregnancy, slightly declines.In pregnant 11 weeks, the content of serumβ-HCG and pregnant age are closely related, with gestation progress, linearly rise, and pregnant age, every increase was 1 day, and β-HCG on average increases 1.53IU/ml, and a minute puerperium sharply declines, and is down to 1IU/ml 4 days postpartum, basic disappearance in 9 days postpartum.
The level that Bargot in 1987 finds to nourish the hCG in the pregnancy serum of Down's syndrome (DS) fetus is 2 times of normal pregnancies.With pregnant woman age, detect in conjunction with indexs such as hCG in serum, the examination rate of DS can reach 63% again.1988, the Wald of London Bartholomews hospital proposed the screening method of AFP, β-HCG, uE3 tri-, is used widely, and uses so far always.When fetus suffers from Down's syndrome, placental weight is lower than normal level, histoorgan hypoplasia, the dysfunction of liver, placenta, therefore in pregnancy serum, hCG concentration declines than late about 3 weeks of the pregnant woman of bosom normal fetus, cause the hCG concentration in the female serum of second trimester (about approximately 20 weeks) to be high-level state, β-HCG concentration ratio normal pregnancies at least high 2 times (general > 2.5MOM) in the Down's syndrome youngster pregnancy serum of bosom.Therefore, in pregnancy serum β-HCG concentration can be used as screening pregnant woman pregnant youngster whether suffer from an auxiliary characteristics of Down's syndrome.
The β of serum-HCG detects euzymelinked immunosorbent assay (ELISA) (ELISA), the immune radiating method (IRMA) of mainly containing at present.Owing to being subject to the restriction of tracer agent proterties, the sensitivity of ELISA is not high, and sensing range is limited, and IRMA kit is subject to the impact of isotope half life period, and the term of validity is short, also has in addition certain radioactive contamination.Time resolution immunofluorescence analysis (time-resolved fluoroimmunoassay, TRFIA) utilize the lanthanide series with unique fluorescent characteristic, replace enzyme, isotope etc., as a kind of important method in luminescence immunoassay, have highly sensitive, tracer stable, be not subject to many advantages such as the interference of sample natural fluorescence, no radioactivity pollute.
Summary of the invention
The object of this invention is to provide a kind of time-resolved fluoroimmunoassay and kit detecting for β-HCG, mainly solve that the sensitivity that prior art exists is low, poor stability, operation is loaded down with trivial details and the technical matters such as contaminated environment.The present invention adopts europium label, and europium labelling kit is provided, to improve sensitivity and the stability of method.
Technical scheme of the present invention is: before carrying out time resolution immunofluorescence analysis detection method, needed β-HCG to become following preliminary work:
First preparation is coated with plate, and coated plate used is the coated plate of specific antibody, and the preparation of β-HCG coated slab comprises the following steps:
(1) Β-HCG monoclonal antibody of purifying is diluted with carbonate buffer solution, then add in each hole of coated slab, after adsorbing, wash, seal, being dried, obtain β-HCG monoclonal antibody coated slab;
(2) pack β-HCG monoclonal antibody coated slab into special-purpose aluminum foil sack, seal and refrigerate standby.
Next is the preparation of β-HCG europium mark, comprises the following steps:
(1) get antibody to be marked and add in bag filter, put into the carbonate buffer solution of the PH 9.5 preparing, room temperature dialysed overnight (repeatedly changing dislysate therebetween);
(2) next day, take out antibody, be diluted to 0.2-5 ug/mL, be in mass ratio 2: 1 ratio (DTTA-Eu: antibody) vibration limit in limit adds DTTA-Eu, under room temperature in shaken overnight, then in the dark standing 48 hours;
(3) antibody that mark is good is separated by Sephadex G50 chromatographic column (1 * 30cm) with free DTTA-Eu, nucleic acid-protein instrument monitoring for detachment process; Separated eluent used is pH7.8 50mmol/L Tris-HCl damping fluid.
(4) with small test tube successively segmentation, accept effluent, with spectrophotometer, at 280nm place, survey absorbance, survey fluorescence intensity simultaneously, calculate Eu labeling antibody concentration;
(5) with the filter of 0.22um, filter;
(6) add 0.5% BSA, 2-8 ℃ of preservation.
β-HCG time-resolved fluoroimmunoassay, comprises the following steps:
1. insolubilized antibody preparation: will resist β-HCG monoclonal antibody to be diluted to 1-10ug/mL as coating buffer with sodium carbonate buffer solution, coated reaction plate, and seal with confining liquid;
2. lanthanide ion labelled antibody preparation: select the anti-β-HCG monoclonal antibody lanthanide ion mark of pairing;
3. on the reaction plate with insolubilized antibody 1. making in step, every hole adds β-HCG standard items or testing sample, and hatches with the labelled antibody of reaction buffer dilution, finally carries out fluorometric assay.
Step 1. in coating buffer the concentration of monoclonal antibody be 1-10ug/mL, step 2. in labelled antibody with reaction buffer, take volume ratio and diluted as 1: 20.The lanthanide ion of step in is 2. preferably Eu 3+.The reaction buffer of step in is 3. 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%NaN3; And 3. this step automatically completes on time resolved fluoro-immunoassay instrument.Enhancing liquid used is fluorescence enhancement solution.
β-HCG immue quantitative detection reagent box, it is characterized in that kit comprises: the damping fluid of coated antibody, confining liquid, reaction buffer, washing lotion, enhancing liquid, and as anti-β-HCG monoclonal antibody of coated antibody, anti-β-HCG monoclonal antibody of lanthanide ion mark and β-HCG standard items.
The preparation method of described β-HCG standard items is: first respectively β-HCG freeze-dried antigen dissolved and is diluted to required maximum concentration, then adopting coubling dilution to be diluted to desired concn, obtaining β-HCG standard items.
Standard items contain 6%BSA for using, 4% sucrose, and 0.1%NaN3,50mmol/L Tris-HCl damping fluid is mixed with standard items by prolactin(PRL albumen.
The invention has the beneficial effects as follows: the present invention uses double antibody sandwich method, reactions steps comprises: reagent is prepared, application of sample reacts, washing pats dry, adds and strengthens liquid, survey fluorescent value, sensitivity for analysis high (0.3 IU/L), and detection time is short, specificity is good, little with the cross reaction of interfering material.
 
accompanying drawing explanation:
Fig. 1 is reaction principle figure of the present invention
The present invention adopts double-antibody sandwich single stage method.Anti-human β-HCG monoclonal antibody is coated in the glimmering plate of exempting from 96 holes, the β-HCG in calibration object or sample and coated monoclonal antibody and europium ion (Eu 3+) monoclonal antibody of mark is in micropore inside surface generation immune response, the sandwich immunoassay compound of micropore surface is separated by washing with free label monoclonal antibody.Eu in micropore surface immune complex 3+after being dissociated by fluorescence enhancement solution, form stable fluorescence complex, the proportional example of β-HCG concentration in fluorescence intensity and calibration object or sample, can draw β-HCG concentration in sample by dose-response curve.
 
embodiment:
Below with reference to Fig. 1, by embodiment, the invention will be further described.
 
Embodiment 1: carry out having needed β-HCG to become following preliminary work before time resolution immunofluorescence analysis detection method:
First preparation is coated with plate, and coated plate used is the coated plate of specific antibody, and the preparation of β-HCG coated slab comprises the following steps:
(1) β-HCG monoclonal antibody of purifying is diluted with carbonate buffer solution, then add in each hole of coated slab, after adsorbing, wash, seal, being dried, obtain β-HCG monoclonal antibody coated slab;
(2) pack β-HCG monoclonal antibody coated slab into special-purpose aluminum foil sack, seal and refrigerate standby.
Next is the preparation of β-HCG europium mark, comprises the following steps:
(1) get antibody to be marked and add in bag filter, put into the carbonate buffer solution of the pH 9.5 preparing, room temperature dialysed overnight (repeatedly changing dislysate therebetween);
(2) next day, take out antibody, be diluted to 0.2-5 ug/mL, be in mass ratio 2: 1 ratio (DTTA-Eu: antibody) vibration limit in limit adds DTTA-Eu, under room temperature in shaken overnight, then in the dark standing 48 hours;
(3) antibody that mark is good is separated by Sephadex G50 chromatographic column (1 * 30cm) with free DTTA-Eu, nucleic acid-protein instrument monitoring for detachment process; Separated eluent used is pH7.8 50mmol/L Tris-HCl damping fluid.
(4) with small test tube successively segmentation, accept effluent, with spectrophotometer, at 280nm place, survey absorbance, survey fluorescence intensity simultaneously, calculate Eu labeling antibody concentration;
(5) with the filter of 0.22um, filter;
(6) add 0.5% BSA, 2-8 ℃ of preservation.
 
Embodiment 2: β-HCG measures
1, standard items preparation
With containing 6%BSA, 4% sugar, 0.1%NaN 3, 50mmol/L Tris-HCl damping fluid is mixed with 0,2,10,50,250,500 IU/mL titers by β-HCG albumen, with national β-HCG standard items, proofreaies and correct.Packing, in+2～+ 8 ℃ of preservations.
2, operation steps
1) preparation of reagent
A) cleansing solution: the concentrated washing lotion of 40mL and 960mL deionized water are mixed in clean bottle for handling liquid toilet or cosmetic substance, standby as work cleansing solution.
B) europium mark: use in last hour, press 1: 20 dilution Europium label by analysis buffer.Reaction buffer 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%NaN3.
C) by kit analysis buffer, testing sample, calibration object and and the micropore reaction bar balance of requirement to room temperature (20～25 ℃).
2) draw calibration object or the sample of 25 μ l, be sequentially added into corresponding micropore bottom.
3) in each micropore, add the europium label solution of oneself dilution of 100 μ l, under room temperature, in slow shelves, vibrate 30 minutes.
4) wash plate 6 times, lath is patted dry on clean thieving paper.
5) in each micropore, add the enhancing liquid of 200 μ l, under room temperature, in slow shelves, vibrate 5 minutes.
6) with time resolution fluor tester, detect.
The sensitivity of calculating β-HCG kit by typical curve has reached 0.3 IU/L
3, specificity
β-HCG kit detects 500uU/ml hTSH, 250U/L hLH, and 250U/L hFSH, β-HCG apparent value is all lower than 2.0 U/L.

Claims (6)

1. the time-resolved fluoroimmunoassay and the kit that for β-HCG, detect, is characterized in that: described analytic approach comprises the following steps:
1. insolubilized antibody preparation: will resist β human chorionic gonadotrophin (β-HCG) monoclonal antibody to be diluted to 1-10ug/mL with sodium carbonate buffer solution
As coating buffer, coated reaction plate, and seal with confining liquid;
2. lanthanide ion labelled antibody preparation: select the anti-β-HCG monoclonal antibody lanthanide ion mark of pairing;
3. on the reaction plate with insolubilized antibody 1. making in step, every hole adds β-HCG standard items or testing sample, and hatches with the labelled antibody of reaction buffer dilution, finally carries out fluorometric assay.
2. β-HCG time-resolved fluoroimmunoassay according to claim 1, is characterized in that the lanthanide ion during step is 2. Eu 3+.
3. β-HCG time-resolved fluoroimmunoassay according to claim 1, is characterized in that the reaction buffer during step is 3. the 50mmol/L Tris-HCl of PH 7.8, includes 0.9%NaCl, 1%BSA, 0.05% N of IgG, 200uM DTPA, 0.05%Tween20 and 0.1%NaN3; And 3. this step automatically completes on time resolved fluoro-immunoassay instrument.
4. β-HCG time-resolved fluoroimmunoassay as claimed in claim 1, is characterized in that during step is 2. that the preparation of lanthanide ion labelled antibody is to adopt with the following method to carry out:
(1) get antibody to be marked and add in bag filter, put into the carbonate buffer solution of the PH 9.5 preparing, room temperature dialysed overnight, repeatedly changes dislysate therebetween;
(2) next day, take out antibody, be diluted to 0.2-5 ug/mL, DTTA-Eu: antibody is that the ratio arm vibration limit of 2: 1 adds DTTA-Eu in mass ratio, under room temperature in shaken overnight, then in the dark standing 48 hours;
(3) antibody that mark is good is separated by the Sephadex G50 chromatographic column of 1 * 30cm with free DTTA-Eu, nucleic acid-protein instrument monitoring for detachment process; Separated eluent used is pH7.8 50mmol/L Tris-HCl damping fluid;
(4) with small test tube successively segmentation, accept effluent, with spectrophotometer, at 280nm place, survey absorbance, survey fluorescence intensity simultaneously, calculate Eu++ labeling antibody concentration;
(5) with the filter of 0.22um, filter;
(6) add 0.5% BSA, 2-8 ℃ of preservation.
5. β-HCG immue quantitative detection reagent box, it is characterized in that kit comprises: the damping fluid of coated antibody, confining liquid, reaction buffer, washing lotion, enhancing liquid, and as anti-β-HCG monoclonal antibody of coated antibody, anti-β-HCG monoclonal antibody of lanthanide ion mark and β-HCG standard items.
6. a kind of β-HCG immue quantitative detection reagent box according to claim 5, is characterized in that standard items are with containing 6%BSA, 4% sucrose, and 0.1%NaN3,50mmol/L Tris-HCl damping fluid is mixed with standard items by β-HCG albumen.
CN201410284459.9A 2014-06-24 2014-06-24 Time-resolved immunofluorescence analysis method for beta-HCG (Human Chorionic Gonadotropin) detection and kit Withdrawn CN104034906A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105891463A (en) * 2014-12-31 2016-08-24 川北医学院 Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence
CN108896773A (en) * 2018-08-23 2018-11-27 潍坊市康华生物技术有限公司 A kind of gastrin 17 detection kit and its preparation and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105891463A (en) * 2014-12-31 2016-08-24 川北医学院 Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence
CN108896773A (en) * 2018-08-23 2018-11-27 潍坊市康华生物技术有限公司 A kind of gastrin 17 detection kit and its preparation and application

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Application publication date: 20140910