CN103267866B - A kind of FSH nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof - Google Patents
A kind of FSH nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof Download PDFInfo
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- 229940028334 follicle stimulating hormone Drugs 0.000 claims abstract description 140
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims abstract description 134
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims abstract description 134
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of follicle-stimulating hormone (FSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, described kit comprises: follicle-stimulating hormone (FSH) calibration object; Coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Biotin labeled follicle-stimulating hormone (FSH) antibody; Follicle-stimulating hormone (FSH) abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; Follicle-stimulating hormone (FSH) quality-control product; Chemical luminescence for liquid A liquid and B liquid; 20 times of concentrated washing lotions; Reaction tube.The invention also discloses the preparation method of kit in addition; Kit of the present invention is easy and simple to handle compared with available reagent box, safe non-environmental-pollution.In addition, the present invention also has the advantages such as the concentration range detecting sample is wide, cost is low, good stability.
Description
Technical field
The present invention relates to field of immunoassay medicine, concrete, the invention provides a kind of FSH (FSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof.
Background technology
Follicle-stimulating hormone (FSH) (follicle-stimulating hormone, FSH) is a kind of glycoprotein promoting sexual gland hormone being synthesized by anterior pituitary basophil cell and secrete, and enters blood and urine by blood circulation.As the same with HCG in LH, TSH as other glycoprotein, FSH forms with the subunit α of non-covalent bond combination and β subunit by two, molecular weight is 24000 ~ 35 000, α-subunit is similar with HCG structure to LH, TSH, for tethelin is common, beta subunit be FSH special, therefore, the β subunit of its uniqueness is depended in the biology of these hormones and the difference of immunological characteristic.For the male sex, its function promotes the maturation of convoluted tubule of testis and the generation of sperm.For women, then can promote Follicle development and maturation, promote granulosa cell proliferation, cause liquor folliculi secrete, and with LH synergic adjustment and impel fully-developed ovarian follicle to secrete estrogen and ovulation, the formation of participation menorrhea.Its generation is subject to ovary female hormone (E by the control of hypothalamus gonadotropin releasing hormone simultaneously
2) feedback regulation.FSH plays a decisive role to the property of men and women's both sexes, reproductive function.Gonadotropin-releasing hormone (GnRH) is produced by hypothalamus, controls the release of FSH in anterior pituitary.HFSH promotes that stratum granulosum of ovarian follicle hyperplasia is broken up, and promotes that whole ovary is grown up.Act on convoluted tubule of testis and can promote spermiogenesis tail.Injection FSH only increases Follicle number, there is no effect to follicle maturity.The follicle stimulating hormone releasing hormone of hypothalamus secretion controls the secretion of follicular stimulating hormone, and in the menstrual cycle, in blood, FSH concentration and every day are changed with mechanical periodicity by the amount of the FSH of homaluria.After menelipsis, blood and FSH discharge rate increase in urinating.
The method of detection interstitialcellstimulating hormone (ICSH) conventional at present has radiommunoassay (RIA) method, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immune detection (ECLIA) method, chemiluminescence immune assay (CLIA), but there is radioactive contamination in RIA, and complex operation, less application in clinical examination; And ELISA sensitivity is low, sensing range is narrow; Chemiluminescence and EIA enzyme immunoassay are combined in this and technically grow up by chemiluminescence immune assay (CLIA), many employings is reaction plate (luminous plaque at present, the patent No.: 200910200386.X), or fluorescein system, stabilization of kit is poor, the sensitivity detected is not high, measured value accuracy is not high, and automaticity is poor.
Summary of the invention
The problem to be solved in the present invention chemiluminescence immunoassay immue quantitative detection reagent box being to provide follicle-stimulating hormone (FSH) and preparation method thereof, the reagent term of validity avoiding radioimmunoassay is short, there is the shortcoming such as radioactive contamination, complex operation, and it is low to solve sensitivity, sensing range is narrow, measured value is accurate not, the defect that cost is high.
For solving the problems of the technologies described above, the technical solution used in the present invention is: follicle-stimulating hormone (FSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, comprising: follicle-stimulating hormone (FSH) calibration object; Coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Biotin labeled follicle-stimulating hormone (FSH) antibody; Follicle-stimulating hormone (FSH) abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; Follicle-stimulating hormone (FSH) quality-control product, quality-control product comprises the low value quality-control product of concentration 10mIU/mL and the high level quality-control product of 200mIU/mL; Chemical luminescence for liquid A liquid and B liquid, A liquid is the Tris-HCl damping fluid of 5mmol/L, pH8.6, and containing final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is 0.675g/L urea peroxide; 20 times of concentrated washing lotions; Reaction tube.A liquid keeps in Dark Place, and B liquid process water is prepared.
Further, described nano magnetic particulate is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 10-50nm.
Further, described follicle-stimulating hormone (FSH) quality-control product comprises low value quality-control product and high level quality-control product, and the concentration of low value quality-control product is 10mIU/mL, and the concentration of high level quality-control product is 200mIU/mL.
Further, the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.
The preparation method of kit, comprises the following steps:
(1) preparation of follicle-stimulating hormone (FSH) calibration object:
The buffer of follicle-stimulating hormone (FSH) antigen containing 40% cow's serum is become the dense liquid storage of calibration object, calibrates with national calibration object, by calibration object diluted to working concentration, be respectively 0,5,25,100,250,1000mIU/mL, be each calibration object point concentration.
(2) preparation of follicle-stimulating hormone (FSH) quality-control product:
With the damping fluid containing 40% cow's serum, the dense liquid storage of above-mentioned preparation is diluted to 10mIU/mL and 200mIU/mL, calibrate with national calibration object, using 10mIU/mL as low value quality-control product, 200mIU/mL is as high level quality-control product;
(3) preparation of nano magnetic particle-Streptavidin suspending liquid:
Prepared by A, ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1. by FeCl
36H
2o and FeCl
24H
2o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2. add 0.5M ammoniacal liquor under nitrogen atmosphere in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3., after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl groups
Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10% polyglycol PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, move into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, adds crosslinking chemical N, N '-methylene-bisacrylamide, consumption can not exceed 5% of magnetic fluid; Under the protection of nitrogen, be warming up to 60 ± 1 DEG C, constant temperature stirs 30min, add benzoyl peroxide, styrene, acrylic acid successively afterwards, benzoyl peroxide consumption is magnetic fluid consumption 3%, and volume of styrene is with magnetic fluid solution, acrylic acid volume is 1/4 of magnetic fluid solution, and stirring rate is about 500rpm and keeps stream of nitrogen gas, and all the other and above-mentioned condition remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses distilled water cyclic washing, then regulates pH=1 with hydrochloric acid, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe
3o
4magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that surface is associated with carboxyl;
The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES (MES) damping fluid, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mL carbodiimide (EDC) solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally dissolved to 1L with 0.01M PBS;
(4) preparation of biotin labeled follicle-stimulating hormone (FSH) antibody
Get 1mg follicle-stimulating hormone (FSH) antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 45ug biotin, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) final mass concentration be 5-10%, lucifuge reaction 3h, slowly vibrates; 250uL1M ammonium chloride solution is added, reacting at normal temperature without light 50min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01M PBS solution, period changes liquid 5 times;
(5) preparation of follicle-stimulating hormone (FSH) abzyme bond
After adopting improvement sodium periodate oxidation that follicle-stimulating hormone (FSH) antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:4500-6000 with enzyme dilution, and added 15% enzyme stabilizers, be stored in 2 ~ 8 DEG C;
Improvement sodium periodate oxidizing process step comprises:
A:HRP activates
1) 10mg/mL HRP solution is configured;
2) 12.8mg/mL sodium periodate NaIO is configured
4solution;
3) by above-mentioned 1) and) the 1:1 mixing by volume of 2 obtain solutions, 4 DEG C of lucifuge reaction 30min;
4) configuration concentration is the glycol water of 20uL/mL, mixes, reacting at normal temperature without light 20min with above-mentioned solution 3 with same volume, and namely activation completes, and puts-20 DEG C of preservations (holding time is no more than 3 months).
B, follicle-stimulating hormone (FSH) antibody labeling
1) raw material to be marked is loaded in bag filter, with the carbonate buffer solution of 0.05M pH9.6, dialysis 30min;
2) by the HRP of mark raw material and activation in mass ratio 1:2 mix, during 4 DEG C are dialysed 24h(, change liquid 2-3 time with 0.05M carbonate buffer solution afterwards);
3) configuration concentration is the NaBH of 2mg/mL
4aqueous solution, adds by 1mgHRP the NaBH that 80uL prepares
4the ratio of aqueous solution mixes, and in 4 DEG C of lucifuge reaction 2h;
4) by above-mentioned steps 3) the marking fluid 0.01M PBS that completes in 4 DEG C of dialysis 24h, add equal-volume glycerine ,-20 DEG C of preservations.
Enzyme dilution comprises 10mL/L2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L glucosan T-2000(Dextran T-2000), 1.05g/L Triton X-100 (Triton X-100), 2.5mL/L gentamicin sulphate, 1mL/L is carmine, 2g/L Tween-20(available from Sigma), 1mL/L ProClin300;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L Tris-HCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; The 5min mixing before use of A liquid and B liquid;
(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C;
(9) to adopt the party legal system kit carry out physical examination, measured value and the stability of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product measure.
Principle of the present invention is, adopt the follicle-stimulating hormone (FSH) in sandwich method for determining serum or blood plasma, biotin-follicle-stimulating hormone (FSH) antibody conjugates is added in Avidin-nano magnetic microparticle suspending liquid, by the compatible reaction of Avidin and biotin, form nano magnetic particulate-Avidin-Biotin-follicle-stimulating hormone (FSH) antibody complex, add sample and enzyme, pass through antigen-antibody reaction, form nano magnetic particulate-Avidin-Biotin-follicle-stimulating hormone (FSH) antibody-follicle-stimulating hormone (FSH)-follicle-stimulating hormone (FSH) antibody-HRP compound, with magnetic field, compound is adsorbed on bottom test tube, wash free composition, add substrate working fluid, under oxygenant effect, HRP catalysis luminol generates the aminophthalic acid ion being in excited state, when it returns to ground state, discharge the photon of 425nm, the luminous value RLU of each well is measured in the 5th minute.RLU and the sample follicle-stimulating hormone (FSH) concentration of sample are proportionate.Follicle-stimulating hormone (FSH) concentration in sample is according to the Log(X set up by calibration object follicle-stimulating hormone (FSH) concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus the follicle-stimulating hormone (FSH) content in detection human serum, blood plasma.
The follicle-stimulating hormone (FSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit of invention, has the following advantages: (1) reaction fast, can judge testing result in 30 minutes; Easy and simple to handle, pollution-free.(2) highly sensitive, the sensitivity for analysis of this kit is not higher than 0.2mIU/mL.(3) high specificity, is less than 1% with the cross reaction coefficient of thyrotropic hormone (TSH), luteinizing principle (LH), human chorionic gonadotrophin (HCG).(4) precision is good, and in batch, imprecision is not higher than 5%, and between batch, imprecision is not higher than 10%.(5) have good stability, this product can deposit more than 7 days at 37 DEG C, can deposit 1 year at 2 ~ 8 DEG C.(6) cost is low, and compare with like product on market, this kit is functional, and cost is low, has clinical value.
Accompanying drawing explanation
Fig. 1 is the measurement result comparison diagram of kit measurement follicle-stimulating hormone (FSH) of the present invention and Abbott Laboratories' kit measurement follicle-stimulating hormone (FSH), wherein ordinate is the follicle-stimulating hormone (FSH) value that kit of the present invention records, horizontal ordinate is Abbott Laboratories' kit measurement follicle-stimulating hormone (FSH) values, two kinds of method correlation coefficient r=0.9873, straight-line equation y=0.9732x+0.3553.
Embodiment
Embodiment 1: prepare follicle-stimulating hormone (FSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay quantitative determination reagent kit
(1) preparation of follicle-stimulating hormone (FSH) calibration object:
The buffer of follicle-stimulating hormone (FSH) antigen (production of Fitzgerald company) containing 40% cow's serum is become the dense liquid storage of calibration object, with national calibration object (lot number: 150533-0212, specification: 450mIU/ props up) calibrate, by calibration object diluted to working concentration, be respectively 0,5,25,100,250,1000mIU/mL, is each calibration object point concentration.
(2) preparation of follicle-stimulating hormone (FSH) quality-control product:
With the damping fluid containing 40% cow's serum, the dense liquid storage of above-mentioned preparation is diluted to 10mIU/mL and 200mIU/mL, calibrate with national calibration object, using 10mIU/mL as low value quality-control product, 200mIU/mL is as high level quality-control product;
(3) preparation of nano magnetic particle-Streptavidin suspending liquid:
Prepared by A, ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl
36H
2o and FeCl
24H
2o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor under nitrogen atmosphere in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl groups
Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, move into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, adds crosslinking chemical N, N '-methylene-bisacrylamide, consumption can not exceed 5% of magnetic fluid; Under the protection of nitrogen, be warming up to 60 ± 1 DEG C, constant temperature stirs 30min, add benzoyl peroxide, styrene, acrylic acid successively afterwards, benzoyl peroxide consumption is magnetic fluid consumption 3%, and volume of styrene is with magnetic fluid solution, acrylic acid volume is 1/4 of magnetic fluid solution, and stirring rate is about 500rpm and keeps stream of nitrogen gas, and all the other and above-mentioned condition remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses distilled water cyclic washing, then regulates pH=1 with hydrochloric acid, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe
3o
4magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that surface is associated with carboxyl;
The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally dissolved to 1L with 0.01M PBS;
(4) preparation of biotin labeled follicle-stimulating hormone (FSH) antibody
Get 1mg follicle-stimulating hormone (FSH) antibody (purchased from Fitzgerald company), to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 45ug biotin, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) final mass concentration be 5-10%, lucifuge reaction 3h, slowly vibrates; 250uL1M ammonium chloride solution is added, reacting at normal temperature without light 50min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01M PBS solution, period changes liquid 5 times;
(5) preparation of follicle-stimulating hormone (FSH) abzyme bond
After adopting improvement sodium periodate oxidation that follicle-stimulating hormone (FSH) antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:4500-6000 with enzyme dilution, and added 15% enzyme stabilizers, be stored in 2 ~ 8 DEG C;
Enzyme dilution comprises 10mL/L2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000(available from Sigma), 1.05g/LTriton X-100(available from Sigma), 2.5mL/L gentamicin sulphate, (famille rose is powder solid to 1mL/L famille rose, be mixed with concentration 40mg/mL to use later), 2g/L Tween-20(available from Sigma), 1mL/L ProClin300(available from Sigma);
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L Tris-HCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; The 5min mixing before use of A liquid and B liquid;
(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C;
(9) to adopt the party legal system kit carry out physical examination, measured value and the stability of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product measure.
Embodiment 2: the inspection of kit of the present invention
(1) physical examination: liquid component should be clarified, without precipitation or floccus; Other components should without packages in damaged condition.
(2) accuracy: kit calibration object and company standard product series are carried out analysis simultaneously and measured, and use double-log Model fitting, requires two not obvious parallel deviates of dose-response curve (t check, | t|<2.447); With follicle-stimulating hormone (FSH) company standard product for reference substance, use double-log Model fitting, the measured value of kit calibration object and the mean value of sign value ratio should in 0.90 ~ 1.10 scopes.
(3) dose-response curve is linear: with two reading Model fitting, dose-response curve correlation coefficient r absolute value in 1.2 ~ 200mIU/mL concentration range is not less than 0.9900.
(4) sensitivity for analysis: kit assay sensitivity is not higher than 0.2mIU/mL.
(5) precision: 10 hole replicate determination high level and low value quality-control product, calculates the mean concentration of measurement result
with standard deviation (SD), imprecision in batch
use 3 batches of products to carry out 3 tests, calculate the mean concentration of measurement result
with standard deviation (SD), imprecision between batch
result should meet batch interior imprecision (CV%) should not higher than 5%; Between batch, imprecision (CV%) should not higher than 10%.
(6) measured value of quality-control product: the quality-control product of replicate determination 10 hole high level and low value, with Log (X)-Log (Y) Model fitting, quality-control product measured value should in allowed band.
(7) specificity:
Cross reaction meets following table and requires:
(8) stability: place 7 days for 37 DEG C, measured value should meet above-mentioned requirements.
Embodiment 3: the using method of kit of the present invention
(1) kit to be checked is balanced 30 minutes under room temperature (18 ~ 25 DEG C).
(2) washing lotion is prepared: washing lotion will be concentrated by 1:20 dilution (1mL washing lotion adds 19mL distilled water) with distilled water.If concentrated washing lotion has crystallization, dilute again after concentrated washing lotion can being placed in room temperature or 37 DEG C of dissolvings to be crystallized.
(3) luminescent solution is prepared: use first 5 minutes and get appropriate luminescent solution A and mix with luminescent solution B equal-volume.
(4) reaction tube is numbered, 50uL calibration object or serum specimen is added successively in test tube, 50uL nano magnetic particulate-Streptavidin suspending liquid, 50uL biotin-follicle-stimulating hormone (FSH) antibody conjugates, 50uL follicle-stimulating hormone (FSH) abzyme bond, oscillating reactions 30min at 37 DEG C, test tube rack is placed on magnetic separator and is separated 5min, then supernatant is poured out, add 500uL washing lotion, after abundant mixing, be separated on magnetic separator, pour out washing lotion, repeat 3 times, Chemoluminescent substrate 100uL is added in each pipe, abundant mixing, secretly put 5min, tube-type chemical light-emitting appearance measures the luminous value (RLU) of each pipe, with the Log value of calibration object concentration for horizontal ordinate, with the Log of luminous value for ordinate, drawing standard curve, the concentration of follicle-stimulating hormone (FSH) can be calculated according to the luminous value of serum specimen.
Embodiment 4: the evaluation of methodology result of this kit
Sensing range: scope is 0.2 ~ 1000mIU/mL, measures after the sample being greater than 1000mIU/mL for concentration should first dilute again.
Sensitivity: 0.2mIU/mL.
Precision: be less than 5%.
Accuracy: the mean value of the recovery is in 0.90 ~ 1.10 scope.
Specificity: be less than 1% with the cross reaction coefficient of thyrotropic hormone (TSH), interstitialcellstimulating hormone (ICSH) (LH), human chorionic gonadotrophin (HCG).
Quality-control product measured value: the measured value of low value quality-control product (QcL) and high level quality-control product (QcH) is all in allowed band.
Stability: reagent component each in kit is placed 7d at 37 DEG C, has good stability.
Embodiment 5: the clinical comparison experiment of this kit
The kit of invention has carried out clinical examination, total sample number 118 example of this clinical testing, first with after the kit test of follicle-stimulating hormone (FSH) Abbott Laboratories, the kit of invention (chemiluminescence) is used to measure again, result shows, straight-line equation is y=0.9732x+0.3553, and related coefficient is R=0.9873.Kit prepared by visible this method and hospital's measured value have good consistance.With SPSS13.0 statistical analysis software, t inspection (inspection level α=0.05) is carried out to related coefficient, P<0.001, the related intimate degree of the follicle-stimulating hormone (FSH) value of two kinds of method mensuration is conspicuousnesses, and the follicle-stimulating hormone (FSH) value that visible two kinds of methods measure is closely related.Sensitivity (True Positive Rate) is 97.90%, specificity (true negative rate) is 97.89%, all higher; And false positive rate (misdiagnosis rate) be 2.10%, false negative rate (rate of missed diagnosis) is 2.12%, all lower, as seen the measured value of this kit and the matching degree of actual value (former measured value) good.The ability of crude agreement reflection kit diagnosis patient and non-patient, the crude agreement of this kit is 98.33%, close to 1, illustrates that the diagnosis capability of kit is stronger.
In order to determine the clinical reference value of this kit, adopt this kit to detect to 385 portions of normal human serums, plasma sample, result shows that the reference value (term of reference) of this kit is the male sex: 1 ~ 12mIU/mL; Women: follicular phase: 3.7 ~ 13.0mIU/mL, the onset of ovulation: 5.0 ~ 20.0mIU/mL, luteal phase: 1.6 ~ 13.0mIU/mL, climacteric: 20 ~ 138mIU/mL.
Claims (1)
1. a follicle-stimulating hormone (FSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, described kit comprises:
1) follicle-stimulating hormone (FSH) calibration object;
2) coupling has the nano magnetic microparticle suspending liquid of Streptavidin, and described magnetic particle is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 20-50nm;
3) biotin labeled follicle-stimulating hormone (FSH) antibody;
4) follicle-stimulating hormone (FSH) abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL;
5) follicle-stimulating hormone (FSH) quality-control product; Quality-control product comprises the low value quality-control product of concentration 10mIU/mL and the high level quality-control product of 200mIU/mL;
6) chemical luminescence for liquid A liquid and B liquid; A liquid is the Tris-HCl damping fluid of 5mmol/L, pH8.6, and containing final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is 0.675g/L urea peroxide;
7) 20 times of concentrated washing lotions;
8) reaction tube, the material of described reaction tube is transparent polystyrene, transparent polyethylene, transparent polypropylene or clear glass;
It is characterized in that, be made up of following steps:
(1) preparation of follicle-stimulating hormone (FSH) calibration object:
The buffer of follicle-stimulating hormone (FSH) antigen containing 40% cow's serum is become the dense liquid storage of calibration object, calibrates with national calibration object, by calibration object diluted to working concentration, be respectively 0,5,25,100,250,1000mIU/mL, be each calibration object point concentration;
(2) preparation of follicle-stimulating hormone (FSH) quality-control product:
With the damping fluid containing 40% cow's serum, the dense liquid storage of above-mentioned preparation is diluted to 10mIU/mL and 200mIU/mL, calibrate with national calibration object, using 10mIU/mL as low value quality-control product, 200mIU/mL is as high level quality-control product;
(3) preparation of nano magnetic particle-Streptavidin suspending liquid:
Prepared by A, ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) join in distilled water by FeCl36H2O and FeCl24H2O with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor under nitrogen atmosphere in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl groups
Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, move into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, adds crosslinking chemical N, N '-methylene-bisacrylamide, consumption can not exceed 5% of magnetic fluid; Under the protection of nitrogen, be warming up to 60 ± 1 DEG C, constant temperature stirs 30min, add benzoyl peroxide, styrene, acrylic acid successively afterwards, benzoyl peroxide consumption is magnetic fluid consumption 3%, and volume of styrene is with magnetic fluid solution, acrylic acid volume is 1/4 of magnetic fluid solution, and stirring rate is about 500rpm and keeps stream of nitrogen gas, and all the other and above-mentioned condition remain unchanged, reaction 8-10h, products therefrom leaves standstill, and uses distilled water cyclic washing, then regulates pH=1 with hydrochloric acid, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe
3o
4magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that surface is associated with carboxyl;
The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally settled to 1L with 0.01M PBS;
(4) preparation of biotin labeled follicle-stimulating hormone (FSH) antibody
Get 1mg follicle-stimulating hormone (FSH) antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 45 μ g biotins, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) final mass concentration be 5-10%, lucifuge reaction 3h, slowly vibrates; 250 μ L1M ammonium chloride solutions are added, reacting at normal temperature without light 50min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01M PBS solution, period changes liquid 5 times;
(5) preparation of follicle-stimulating hormone (FSH) abzyme bond
After adopting improvement sodium periodate oxidation that follicle-stimulating hormone (FSH) antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:4500-6000 with enzyme dilution, and added 15% enzyme stabilizers, be stored in 2 ~ 8 DEG C; Enzyme dilution comprises 10mL/L 2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L glucosan T-2000,1.05g/L Triton X-100,2.5mL/L gentamicin sulphate, and 1mL/L is carmine, 2g/L Tween-20,1mL/L ProClin300;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L Tris-HCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; The 5min mixing before use of A liquid and B liquid;
(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C;
(9) to adopting the obtained kit of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability are measured.
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