CN103267866A - Chemiluminescence immune quantitative detection kit for follicle-stimulating hormone nanometre magnetic particles, and preparation method thereof - Google Patents
Chemiluminescence immune quantitative detection kit for follicle-stimulating hormone nanometre magnetic particles, and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chemiluminescence immune quantitative detection kit for follicle-stimulating hormone nanometre magnetic particles. The kit comprises a follicle-stimulating hormone calibration product, a nanometre magnetic particle suspension coupled with streptavidin, a biotin-marked follicle-stimulating hormone antibody, a follicle-stimulating hormone abzyme conjugate, a follicle-stimulating hormone quality control product, chemiluminescence solution A and chemiluminescence solution B, 20-times concentrated washing solution, and a reaction tube, wherein the used enzyme is horse radish peroxidase having a purity RZ being not less than 3.0 and an activity being not less than 250 U/mL. Additionally, the invention further discloses a preparation method of the kit. Compared with the existing kit, the kit disclosed by the invention is simple and convenient to operate, safe, and free from environmental pollution; additionally, the kit disclosed by the invention further has the advantages of wide concentration range of the detected samples, low cost, good stability, and the like.
Description
Technical field
The present invention relates to the immunoassay medical domain, concrete, the invention provides a kind of short ovarian follicle and generate plain (FSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof.
Background technology
(follicle-stimulating hormone is a kind of glycoprotein promoting sexual gland hormone of and secretion synthetic by the anterior pituitary basicyte FSH) to follicle-stimulating hormone (FSH), can enter blood and urine by blood circulation.As other glycoprotein such as LH, TSH and HCG, FSH is made up of two α of subunit and β subunits with the non-covalent bond combination, molecular weight is 24 000～35 000, α-subunit and LH, TSH and HCG structural similarity, for tethelin common, beta subunit is that FSH is special, and therefore, the β subunit that it is unique is depended in the biology of these hormones and the difference of immunological characteristic.For the male sex, its function is to promote the maturation of convoluted tubule of testis and the generation of sperm.For the women, then can promote follicular development and maturation, promote granular cell propagation, cause the liquor folliculi secretion, and work in coordination with adjusting and impel fully-developed ovarian follicle secretion estrogen and ovulation with LH, participate in the formation of menorrhea.Its generation is subjected to the control of hypothalamus gonadotropin releasing hormone, is subjected to ovary female hormone (E simultaneously
2) feedback regulation.Property, the reproductive function of the men and women's both sexes of FSH play a decisive role.Gonadotropin-releasing hormone (GnRH) is produced by hypothalamus, is controlling the release of FSH in the anterior pituitary.The HFSH promotes stratum granulosum of ovarian follicle hyperplasia differentiation, promotes whole ovary to grow up.Acting on convoluted tubule of testis can promote sperm to form.Injection FSH only increases the ovarian follicle number, and follicle maturity be there is no effect.The secretion of the follicle stimulating hormone releasing hormone control follicular stimulating hormone of hypothalamus secretion, in the menstrual cycle, FSH concentration and every day are changed with the cycle by the amount of the FSH of homaluria in the blood.After menelipsis, the FSH discharge rate increases in blood and the urine.
The method of detection interstitialcellstimulating hormone (ICSH) commonly used has radiommunoassay (RIA) method, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immune detection (ECLIA) method, chemiluminescence immune assay (CLIA) at present, but there is radioactive contamination in RIA, and complex operation, less application in the clinical examination; And ELISA sensitivity is low, and sensing range is narrow; Chemiluminescence immune assay (CLIA) is combined in this technical growing up with chemiluminescence and EIA enzyme immunoassay, many employings is reaction plate (luminous plaque at present, the patent No.: 200910200386.X), or fluorescein system, the kit poor stability, the sensitivity that detects is not high, the measured value accuracy is not high, and automaticity is relatively poor.
Summary of the invention
The problem to be solved in the present invention provides quantitative detection kit of chemiluminescence immunoassay of follicle-stimulating hormone (FSH) and preparation method thereof, avoid the reagent term of validity of radioimmunoassay short, had shortcomings such as radioactive contamination, complex operation, and it is low to have solved sensitivity, sensing range is narrow, measured value is accurate inadequately, the defective 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) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box comprises: the follicle-stimulating hormone (FSH) calibration object; Coupling has the nanometer magnetic particle suspending liquid of Streptavidin; Biotin labeled follicle-stimulating hormone (FSH) antibody; Follicle-stimulating hormone (FSH) abzyme bond, used enzyme are horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL; Follicle-stimulating hormone (FSH) quality-control product, quality-control product comprise the low value quality-control product of concentration 10mIU/mL and the high value quality-control product of 200mIU/mL; Chemical luminescence for liquid A liquid and B liquid, A liquid are 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide; 20 times of concentrated washing lotions; Reaction tube.A liquid keeps in Dark Place, and B liquid is prepared with process water.
Further, described nanometer magnetic particle is the tri-iron tetroxide that the surface parcel has 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 value quality-control product, and the concentration of low value quality-control product is 10mIU/mL, and the concentration of high value 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 may further comprise the steps:
(1) preparation of follicle-stimulating hormone (FSH) calibration object:
Follicle-stimulating hormone (FSH) antigen is mixed with the dense liquid storage of calibration object with the damping fluid that contains 40% cow's serum, calibrates with national calibration object, to working concentration, be respectively 0,5,25,100,250,1000mIU/mL with the calibration object diluted, be each calibration object point concentration.
(2) preparation of follicle-stimulating hormone (FSH) quality-control product:
With the damping fluid that contains 40% cow's serum the dense liquid storage of above-mentioned preparation is diluted to 10mIU/mL and 200mIU/mL, with national calibration object calibration, as the low value quality-control product, 200mIU/mL is as high value quality-control product with 10mIU/mL;
(3) preparation of nano magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1. with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2. under blanket of nitrogen, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3. after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: ultrasonic being dispersed in the 10% polyglycol PEG8000 solution of nanometer magnetic particle of getting above-mentioned preparation, magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, immigration has stirrer, condenser pipe in the three-necked bottle of nitrogen inlet, adds crosslinking chemical N, N '-methylene-bisacrylamide, consumption can not surpass 5% of magnetic fluid; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide, styrene, acrylic acid afterwards successively, the benzoyl peroxide consumption is magnetic fluid consumption 3%, and volume of styrene is with magnetic fluid solution, the 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 the distilled water cyclic washing, regulates pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M 2-morpholino b acid (MES) damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mL carbodiimide (EDC) solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled follicle-stimulating hormone (FSH) antibody
Get 1mg follicle-stimulating hormone (FSH) antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 45ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, lucifuge reaction 3h, slowly vibration; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 50min; With 0.01M PBS solution 2～8 ℃ of down dialysis 2 days, during change liquid 5 times;
(5) preparation of follicle-stimulating hormone (FSH) abzyme bond
After adopting the improvement sodium periodate oxidation that follicle-stimulating hormone (FSH) antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4500-6000, and adds 15% enzyme stabilizers, be stored in 2～8 ℃;
Improvement sodium periodate oxidizing process step comprises:
The A:HRP activation
1) configuration 10mg/mL HRP solution;
2) configuration 12.8mg/mL sodium periodate NaIO
4Solution;
3) with above-mentioned 1) and) 2 obtain solutions 1:1 mixing by volume, 4 ℃ of lucifuges reaction 30min;
4) configuration concentration is the glycol water of 20uL/mL, mix with equal volume with above-mentioned solution 3, and reacting at normal temperature without light 20min, activation is namely finished, and puts-20 ℃ and preserves (holding time is no more than 3 months).
B, follicle-stimulating hormone (FSH) antibody labeling
1) raw material to be marked is packed in the bag filter, with the carbonate buffer solution of 0.05M pH9.6, dialysis 30min;
2) the mark raw material is mixed by mass ratio 1:2 with the HRP of activation, during 4 ℃ of dialysis 24h(, change liquid 2-3 time with the 0.05M carbonate buffer solution afterwards);
3) configuration concentration is the NaBH of 2mg/mL
4Aqueous solution adds the NaBH that 80uL prepares by 1mgHRP
4The ratio of aqueous solution is mixed, and in 4 ℃ of lucifuge reaction 2h;
4) with above-mentioned steps 3) marking fluid finished in 4 ℃ of dialysis 24h, adds equal-volume glycerine ,-20 ℃ of preservations with 0.01M PBS.
Comprise 10mL/L2M NaOH in the enzyme dilution, 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 famille rose, 2g/L Tween-20(is available from Sigma company), 1mL/L ProClin300;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) to adopt this method system kit carry out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Principle of the present invention is, adopt the follicle-stimulating hormone (FSH) in sandwich method for determining serum or the blood plasma, in Avidin-nanometer magnetic particle suspending liquid, add biotin-follicle-stimulating hormone (FSH) antibody conjugates, compatible reaction by Avidin and biotin, form nanometer magnetic particle-Avidin-biotin-follicle-stimulating hormone (FSH) antibody complex, add sample and enzyme, by antigen-antibody reaction, form nanometer magnetic particle-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 the test tube bottom, wash free composition, add the substrate working fluid, under the oxygenant effect, HRP catalysis luminol generates the aminophthalic acid ion that is in excited state, when it returns to ground state, discharge the photon of 425nm, measured the luminous value RLU of each well in the 5th minute.The RLU of sample and sample follicle-stimulating hormone (FSH) concentration are proportionate.Follicle-stimulating hormone (FSH) concentration in the sample is according to the Log(X that is set up by calibration object follicle-stimulating hormone (FSH) concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus detect the follicle-stimulating hormone (FSH) content in human serum, the blood plasma.
The follicle-stimulating hormone (FSH) nanometer magnetic microparticle chemiluminescence immunological quantitative determining kit of this patent invention has the following advantages: (1) reaction can be judged testing result fast 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, with the cross reaction coefficient of thyrotropic hormone (TSH), luteinizing principle (LH), human chorionic gonadotrophin (HCG) less than 1%.(4) precision is good, and imprecision is not higher than 5% in batch, and imprecision is not higher than 10% between batch.(5) have good stability, this product can be deposited more than 7 days at 37 ℃, can deposit 1 year at 2～8 ℃.(6) cost is low, compares with like product on the market, and this kit is functional, and cost is low, has clinical value.
Description of drawings
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), the follicle-stimulating hormone (FSH) value that records for kit of the present invention of ordinate wherein, 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: preparation follicle-stimulating hormone (FSH) nanometer magnetic microparticle chemiluminescence immunological quantitative determining kit
(1) preparation of follicle-stimulating hormone (FSH) calibration object:
Follicle-stimulating hormone (FSH) antigen (production of Fitzgerald company) is mixed with the dense liquid storage of calibration object with the damping fluid that contains 40% cow's serum, with national calibration object (lot number: 150533-0212, specification: 450mIU/ props up) calibrate, to working concentration, be respectively 0 with the calibration object diluted, 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 that contains 40% cow's serum the dense liquid storage of above-mentioned preparation is diluted to 10mIU/mL and 200mIU/mL, with national calibration object calibration, as the low value quality-control product, 200mIU/mL is as high value quality-control product with 10mIU/mL;
(3) preparation of nano magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under blanket of nitrogen, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nanometer magnetic particle of above-mentioned preparation, magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, immigration has stirrer, condenser pipe in the three-necked bottle of nitrogen inlet, adds crosslinking chemical N, N '-methylene-bisacrylamide, consumption can not surpass 5% of magnetic fluid; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide, styrene, acrylic acid afterwards successively, the benzoyl peroxide consumption is magnetic fluid consumption 3%, and volume of styrene is with magnetic fluid solution, the 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 the distilled water cyclic washing, regulates pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mLEDC solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled follicle-stimulating hormone (FSH) antibody
Get 1mg follicle-stimulating hormone (FSH) antibody (available from Fitzgerald company), with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 45ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, lucifuge reaction 3h, slowly vibration; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 50min; With 0.01M PBS solution 2～8 ℃ of down dialysis 2 days, during change liquid 5 times;
(5) preparation of follicle-stimulating hormone (FSH) abzyme bond
After adopting the improvement sodium periodate oxidation that follicle-stimulating hormone (FSH) antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4500-6000, and adds 15% enzyme stabilizers, be stored in 2～8 ℃;
Comprise 10mL/L2M NaOH in the enzyme dilution, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000(is available from Sigma company), 1.05g/LTriton X-100(is available from Sigma company), the 2.5mL/L gentamicin sulphate, (famille rose is powder solid to the 1mL/L famille rose, being mixed with concentration 40mg/mL uses later on), 2g/L Tween-20(is available from Sigma company), 1mL/L ProClin300(is available from Sigma company);
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) to adopt this method system kit carry out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Embodiment 2: the inspection of kit of the present invention
(1) physical examination: liquid component should be clarified, and does not have precipitation or floccus; Other components should not have packages in damaged condition.
(2) accuracy: kit calibration object and company standard product series are carried out assay determination simultaneously, with the match of double-log mathematical model, require two not obvious parallel deviates of dose-response curve (t check, | t|＜2.447); Be reference substance with follicle-stimulating hormone (FSH) company standard product, with the match of double-log mathematical model, the mean value of the measured value of kit calibration object and sign value ratio should be in 0.90～1.10 scope.
(3) linearity of dose-response curve: with two reading mathematical model matches, 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: the kit sensitivity for analysis is not higher than 0.2mIU/mL.
(5) precision: the high value of 10 hole replicate determinations and low value quality-control product, the mean concentration of calculating 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
The result should meet batch interior imprecision (CV%) should not be higher than 5%; Imprecision (CV%) should not be higher than 10% between batch.
(6) measured value of quality-control product: the quality-control product of the high value in replicate determination 10 holes and low value, with Log (X)-Log (Y) mathematical model match, the quality-control product measured value should be in allowed band.
(7) specificity:
Cross reaction meets following table and requires:
(8) stability: placed 7 days for 37 ℃, measured value should meet above-mentioned every requirement.
Embodiment 3: the using method of kit of the present invention
(1) kit to be checked was descended balance 30 minutes in room temperature (18～25 ℃).
(2) preparation washing lotion: will concentrate washing lotion by 1:20 dilution (the 1mL washing lotion adds 19mL distilled water) with distilled water.If concentrate washing lotion crystallization is arranged, can dilute again after washing lotion places room temperature or 37 ℃ of dissolvings to be crystallized concentrating.
(3) preparation luminescent solution: use and got an amount of luminescent solution A in preceding 5 minutes and mix with luminescent solution B equal-volume.
(4) reaction tube is numbered, in test tube, add 50uL calibration object or serum specimen successively, 50uL nanometer magnetic particle-Streptavidin suspending liquid, 50uL biotin-follicle-stimulating hormone (FSH) antibody conjugates, 50uL follicle-stimulating hormone (FSH) abzyme bond, 37 ℃ of following oscillating reactions 30min, test tube rack placed separate 5min on the magnetic separator, pour out supernatant then, add the 500uL washing lotion, fully behind the mixing, on magnetic separator, separate, pour out washing lotion, repeat 3 times, in each pipe, add chemical luminous substrate liquid 100uL, fully mixing is secretly put 5min, measures the luminous value (RLU) of each pipe at the tubular type Chemiluminescence Apparatus, Log value with calibration object concentration is horizontal ordinate, Log with luminous value is ordinate, and the drawing standard curve can calculate the concentration of follicle-stimulating hormone (FSH) 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 should diluting earlier greater than the sample of 1000mIU/mL for concentration again.
Sensitivity: 0.2mIU/mL.
Precision: less than 5%.
Accuracy: the mean value of the recovery is in 0.90～1.10 scope.
Specificity: with the cross reaction coefficient of thyrotropic hormone (TSH), interstitialcellstimulating hormone (ICSH) (LH), human chorionic gonadotrophin (HCG) less than 1%.
The quality-control product measured value: the measured value of low value quality-control product (QcL) and high value quality-control product (QcH) is all in allowed band.
Stability: each reagent component in the kit is placed 7d down in 37 ℃, have good stability.
Embodiment 5: the clinical comparison experiment of this kit
The kit of this patent invention has carried out clinical examination, total sample number 118 examples of this clinical testing, earlier with after the kit test of follicle-stimulating hormone (FSH) Abbott Laboratories, measure with the kit (chemiluminescence) of this patent invention again, the result shows, straight-line equation is y=0.9732x+0.3553, and related coefficient is R=0.9873.As seen kit and hospital's measured value of this method preparation have consistance preferably.With the SPSS13.0 statistical analysis software related coefficient is carried out t check (inspection level α=0.05), 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 are measured is closely related.Sensitivity (True Positive Rate) is 97.90%, specificity (true negative rate) is 97.89%, and is all higher; And false positive rate (misdiagnosis rate) is 2.10%, false negative rate (rate of missed diagnosis) is 2.12%, and all lower, as seen the matching degree of the measured value of this kit and actual value (former measured value) is good.Crude agreement reflection kit diagnosis patient and non-patient's ability, 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 parts of normal human serums, plasma samples, the 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 (4)
1. follicle-stimulating hormone (FSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box is characterized in that described kit comprises:
1) follicle-stimulating hormone (FSH) calibration object;
2) coupling has the nanometer magnetic particle suspending liquid of Streptavidin;
3) biotin labeled interstitialcellstimulating hormone (ICSH) antibody;
4) follicle-stimulating hormone (FSH) abzyme bond, used enzyme are horseradish peroxidase, horseradish peroxidase purity RZ 〉=3.0, activity 〉=250U/mL;
5) interstitialcellstimulating hormone (ICSH) quality-control product; Quality-control product comprises the low value quality-control product of concentration 10mIU/mL and the high value quality-control product of 200mIU/mL;
6) chemical luminescence for liquid A liquid and B liquid; A liquid is 5mmol/L, the Tris-HCl damping fluid of pH8.6, and contain final concentration 0.7g/L luminol and final concentration 0.165g/L p-iodophenol in this damping fluid; B liquid is the 0.675g/L urea peroxide;
7) 20 times of concentrated washing lotions;
8) reaction tube.
2. follicle-stimulating hormone (FSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box according to claim 1 is characterized in that, described magnetic particle is the tri-iron tetroxide that the surface parcel has amino or carboxyl reactive group, particle diameter 20-50nm.
3. interstitialcellstimulating hormone (ICSH) nanometer magnetic microparticle chemiluminescence immune quantitative detection reagent box according to claim 1 is characterized in that the material of described reaction tube is transparent polystyrene, tygon, polypropylene or glass.
4. a method for preparing the described kit of the arbitrary claim of claim 1-3 is characterized in that, may further comprise the steps:
(1) preparation of follicle-stimulating hormone (FSH) calibration object:
Follicle-stimulating hormone (FSH) antigen is mixed with the dense liquid storage of calibration object with the damping fluid that contains 40% cow's serum, calibrates with national calibration object, to working concentration, be respectively 0,5,25,100,250,1000mIU/mL with the calibration object diluted, be each calibration object point concentration.
(2) preparation of follicle-stimulating hormone (FSH) quality-control product:
With the damping fluid that contains 40% cow's serum the dense liquid storage of above-mentioned preparation is diluted to 10mIU/mL and 200mIU/mL, with national calibration object calibration, as the low value quality-control product, 200mIU/mL is as high value quality-control product with 10mIU/mL;
(3) preparation of nano magnetic particle-Streptavidin suspending liquid:
A, the preparation of ferriferrous oxide nano magnetic particle
Adopt the precipitation method to prepare the ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) with FeCl
36H
2O and FeCl
24H
2O joins in the distilled water with mol ratio 2:1, the vigorous stirring dissolving; 2) under blanket of nitrogen, add 0.5M ammoniacal liquor in above-mentioned iron salt solutions, transfer pH9-10,65 ℃ of temperature of reaction, reaction time 45min; 3) after reaction finishes, be washed with distilled water to neutrality, abandon supernatant, in 60 ℃ of oven dry, namely get the ferriferrous oxide nano magnetic particle of 10-50nm;
The coupling of B, nanometer magnetic bead surface carboxyl
Adopt dispersion copolymerization method to carry out coupling, concrete preparation method is as follows: get in the ultrasonic 10%PEG8000 of the being dispersed in solution of nanometer magnetic particle of above-mentioned preparation, magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume in the magnetic fluid solution, stir 30min after, immigration has stirrer, condenser pipe in the three-necked bottle of nitrogen inlet, adds crosslinking chemical N, N '-methylene-bisacrylamide, consumption can not surpass 5% of magnetic fluid; Under protection of nitrogen gas, be warming up to 60 ± 1 ℃, constant temperature stirs 30min, add benzoyl peroxide, styrene, acrylic acid afterwards successively, the benzoyl peroxide consumption is magnetic fluid consumption 3%, and volume of styrene is with magnetic fluid solution, the 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 the distilled water cyclic washing, regulates pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use the distilled water cyclic washing again, remove the Fe that does not coat
3O
4Magnetic is put into 50 ℃ of following dry 24h of vacuum drying chamber to the product that precipitates, and obtains the nanometer magnetic particle that the surface is associated with carboxyl;
The preparation of C, nanometer magnetic particle-Streptavidin suspending liquid, preparation 1L, method is as follows:
Get 100mL0.1M MES damping fluid, adding 10mg surface is associated with the nanometer magnetic particle of carboxyl, stirring at room 40min, add the 3.5mg Streptavidin afterwards, add 8mg/mLEDC solution then, behind the 2-8 ℃ of reaction 1h, with 0.01M PBS damping fluid washing 3 times, get final product to 1L with 0.01M PBS is fixed molten at last;
(4) preparation of biotin labeled follicle-stimulating hormone (FSH) antibody
Get 1mg follicle-stimulating hormone (FSH) antibody, with borate buffer solution dialysis 1～3h under 2～8 ℃; Antibody after the dialysis is added the 45ug biotin, add dimethyl sulfoxide (DMSO) simultaneously, making dimethyl sulfoxide (DMSO) final mass concentration is 5-10%, lucifuge reaction 3h, slowly vibration; In above-mentioned solution, add 250uL1M ammonium chloride solution, reacting at normal temperature without light 50min; With 0.01M PBS solution 2～8 ℃ of down dialysis 2 days, during change liquid 5 times;
(5) preparation of follicle-stimulating hormone (FSH) abzyme bond
After adopting the improvement sodium periodate oxidation that follicle-stimulating hormone (FSH) antibody and horseradish peroxidase are carried out coupling, with the enzyme dilution it is diluted to working concentration 1:4500-6000, and adds 15% enzyme stabilizers, be stored in 2～8 ℃;
The preparation of (6) 20 times of concentrated washing lotions
20 times of concentrated washing lotions comprise the 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;
(7) preparation of chemical luminescence for liquid A liquid and B liquid
A liquid is the 0.7g/L luminol, and 0.165g/L p-iodophenol, damping fluid are the 5mmol/L Tris-HCl of pH8.6, keep in Dark Place; B liquid is the 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid 5min before use mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 ℃;
(9) to adopt this method system kit carry out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
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| CN105092862A (en) * | 2014-05-05 | 2015-11-25 | 江苏泽成生物技术有限公司 | Follicle stimulating hormone (FSH) quantitative detection kit, preparation method and detection method thereof |
| CN110577243A (en) * | 2019-10-22 | 2019-12-17 | 江苏省农业科学院 | Modified nano iron oxide particles and application thereof in anaerobic fermentation |
| CN110628829A (en) * | 2019-10-22 | 2019-12-31 | 江苏省农业科学院 | Method for producing methane by anaerobic fermentation |
| CN113189217A (en) * | 2021-04-12 | 2021-07-30 | 西安交通大学 | Magnetic nano material with cell membrane reversely coated and preparation method and application thereof |
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| CN110628829A (en) * | 2019-10-22 | 2019-12-31 | 江苏省农业科学院 | Method for producing methane by anaerobic fermentation |
| CN110628829B (en) * | 2019-10-22 | 2023-03-21 | 江苏省农业科学院 | Method for producing methane by anaerobic fermentation |
| CN113189217A (en) * | 2021-04-12 | 2021-07-30 | 西安交通大学 | Magnetic nano material with cell membrane reversely coated and preparation method and application thereof |
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