CN102998467B - β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof - Google Patents
β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box, described kit comprises: β-hCG calibration object; Coupling has the magnetic particle suspending liquid of Streptavidin; Biotin labeled β-hCG antibody; β-hCG abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; β-hCG 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 of the present invention 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 β human chorionic gonadotrophin (β-hCG) magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof.
Background technology
Human chorionic gonadotrophin (Human Chorionic Gonadotropin, HCG) is the glycoprotein hormones that a kind of placenta becomes trophocyte to secrete, special in pregnancy period secretion, becomes the tumour of trophocyte's pathology and some incomplete differentiation also can secrete.Its physiological action is the degeneration delaying corpus luteum, thus maintains the physiological level of progesterone, plays an important role at Early placental phase.HCG also affects pregnancy period immune tolerance, ensures that fetus is not attacked by mother's immune system.In addition, HCG is also in the news relevant with embryo nidation and morning sickness.
HCG is the early pregnancy Testing index of current widespread use, is used to indicate the embryo that whether there is implantation.At first week of pregnancy, HCG level just started sharply to raise, and continued the time raising about 8 weeks, started subsequently to decline, reached maintenance level to about 20 weeks, was maintained until a point puerperium disappearance.
Human chorionic gonadotrophin is by α, the different subunit composition of β two, the FSH(follicular stimulating hormone of α subunit and pituitary), LH(lutropin) and TSH(thyrotropic hormone) etc. basic simlarity, cross reaction can be there is each other, and each dissmilarity of the structure of β subunit.β-HCG is close in β-LH structure, but last 24 amino acid extension do not exist in β-LH.So the stronger β subunit of specificity becomes the first-selected target spot in detection, total β-hCG can substitute the indicative function of total HCG to a great extent.
The method of detection β-hCG conventional at present has radiating immuning analysis technology (RIA) and enzyme-linked immunosorbent assay (ELISA), but RIA exist radioactive contamination, label half life period short, to operator, there is radioactive damage, and complex operation, the shortcomings such as the time is long; And ELISA sensitivity is low, sensing range is narrow; Along with developing rapidly of immuno-labelling technique, various new detection method emerges in an endless stream, wherein chemiluminescence and EIA enzyme immunoassay are combined in this and technically grow up by chemiluminescence immune assay (CLIA), current microimmuno-assays the most responsive, but detect sensitivity and accuracy still not high.
Magnetic particle has superparamagnetism, and tool magnetic field responsiveness under magnetic field, is applied to the solid phase of immune detection by magnetic particle, will greatly improve the surface area of reaction, more easily carries out solid phase, liquid phase separation, can improve the sensitivity of detection.Adopt small magnetic particle can increase bag by surface area as solid phase, thus increase the adsorbance of antigen or antibody, both accelerated reaction velocity, also make cleaning be separated easier.
Magneto separate-enzyme labeling-chemiluminescence is the new development of enzyme labeled immunoassay technology, this technology with micron order magnetic particle for carrier, the carboxyl-reactive group utilizing surface organic matter to provide and protein amino covalent bond, adopt antibody to carry out " bridging " and become immune magnetic particle, can antigen, antibody response be carried out.The novel part of this technology has: (1) utilizes paramagnetic iron particulate for solid phase carrier, expoeridium monoclonal antibody, the contact area of increase antigen, antibody and substrate light-emitting area, improve the sensitivity of reaction, and adopt rotating magnetic field to make magnetic particle play beating action and be separated conjugated antigen-antibody and free antibodies.(2) in liquid phase reactor, use luminescence enhancer, the luminescence sites of hydrone from luminous substrate is arranged, also can shorten luminous peak time simultaneously.(3) monoclonal antibody, improves the specificity of reaction.
Existing external chemical luminescence immune analysis reagent box is closed full automatic chemiluminescence measuring system, needs expensive Full-automatic chemiluminescence measuring instrument, thus limits and promote the use of, and cannot be widely used in clinical diagnosis and research work.Especially also medical field is not widely used in about β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box.
Summary of the invention
The problem to be solved in the present invention chemiluminescence immunoassay immue quantitative detection reagent box being to provide β-hCG 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, the defect that cost is high.
For solving the problems of the technologies described above, the technical solution used in the present invention is: β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box, comprising: β-hCG calibration object; Coupling has the magnetic particle suspending liquid of Streptavidin; Biotin labeled β-hCG antibody; β-hCG abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; β-hCG quality-control product; Chemical luminescence for liquid A liquid and B liquid; 20 times of concentrated washing lotions; Reaction tube.
Further, the principal ingredient of described luminescent solution A liquid is luminol, and the principal ingredient of B liquid is urea peroxide.A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L TrisHCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water.
Further, described magnetic particle is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 1-2um.
Further, described β-hCG quality-control product comprises low value quality-control product and high level quality-control product.
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 β-hCG calibration object:
The calibration object diluent preparing of β-hCG antigen containing 50% cow's serum is become the dense liquid storage of calibration object, calibrates with enterprise's calibration object, by the calibration object diluted containing 50% cow's serum to working concentration, be respectively 0,5,25,100,250,1000mIU/mL;
The compound method of above-mentioned β-hCG company standard product: the calibration object dilution of β-hCG antigen containing 50% cow's serum is made into the dense liquid storage of calibration object, be its definite value by national standard, according to national standard calibration value, the calibration object diluted of dense for calibration object liquid storage containing 50% cow's serum is become working concentration each point, then working concentration each point national standard is demarcated; Packing, labels, and is stored in-20 DEG C.β-hCG national standard lot number is: 150535, and specification is: 0.21IU/ props up.
(2) preparation of β-hCG quality-control product:
The calibration object diluted of β-hCG antigen containing 50% cow's serum is made into dense liquid storage, with enterprise's calibration object to after its definite value, is diluted to 8 ~ 12mIU/mL and 400 ~ 600mIU/mL respectively with calibration object dilution.
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
Get 100mL0.1mol/L MES solution (MES solution), add 10mg magnetic-particle and 3mg streptavidin (SA) successively, stir 30min, then 10mg/mL carbodiimide hydrochloride solution (1-ethyl-(3-dimethylaminopropyl is added, EDC) 3.5uL, after reaction 1h, uses magnetic frame absorption, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash 3 times altogether, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin labeled β-hCG antibody
Get 0.5mg β-hCG antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 25ug biotin, and add dimethyl sulfoxide (DMSO), ultimate density is 10% simultaneously, and lucifuge reaction 3h, slowly vibrates; 250uL1mol/L ammonium chloride solution is added, reacting at normal temperature without light 30 ~ 60min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01mol/L PBS solution, period changes liquid 3 ~ 5 times;
(5) preparation of β-hCG abzyme bond
After adopting sodium periodate oxidation that β-hCG antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:4000 with enzyme dilution, and added 5-20% enzyme stabilizers, be stored in 2 ~ 8 DEG C; Enzyme stabilizers is a kind of reagent that protein can be kept to keep natural folding to conceive under freeze-drying or solution, is conducive to the preservation of antigen or antibody, avoids extraneous factor as temperature, pH, salt, metallic ion and its stability of other pollutant effects.
The preparation of (6) 20 times of concentrated washing lotions
20 concentrated washing lotions comprise 58gL sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 1 ‰ 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 TrisHCl 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, this kit adopts the β-hCG in sandwich method principle mensuration serum or blood plasma, biotin-β-hCG antibody conjugates is added in Avidin-magnetic particle suspending liquid, by the compatible reaction of Avidin and biotin, form magnetic particle-Avidin-Biotin-β-hCG antibody complex, after adding sample and enzyme, antigen-antibody reaction can be passed through, define magnetic particle-Avidin-Biotin-β-hCG antibody-β-hCG-β-hCG 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 β-hCG concentration of sample are proportionate.β-hCG concentration in sample is carried out quantitatively according to the LogX-LogY mathematical model set up by calibration object β-hCG concentration and corresponding RLU, thus detects the β-hCG content in human serum, blood plasma.
β human chorionic gonadotrophin (β-hCG) the magnetic microparticle chemiluminescence immunological quantitative determining kit of invention, has the following advantages: (1) reaction fast, can judge testing result in 40 minutes; Easy and simple to handle, pollution-free.(2) highly sensitive, the sensitivity for analysis of this kit is not higher than 2mIU/mL.(3) high specificity, is less than 1% with the cross reaction coefficient of thyrotropic hormone (TSH), interstitialcellstimulating hormone (ICSH) (LH), follicle-stimulating hormone (FSH) (FSH).(4) precision is good, and precision 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 that Bo Aosaisi chemical luminescence reagent kit of the present invention mensuration β-hCG measures the measurement result comparison diagram of β-hCG with radioimmunological kit, wherein ordinate is the β-hCG value that Bo Aosaisi records, horizontal ordinate is that radioimmunological kit measures β-hCG value, two kinds of method related coefficient (r)=0.9891, straight-line equation y=0.9393x-24.714.
Embodiment
Embodiment 1: preparation β human chorionic gonadotrophin (β-hCG) magnetic microparticle chemiluminescence immunological quantitative determining kit I
(1) preparation of β-hCG calibration object:
The calibration object diluent preparing of β-hCG antigen containing 50% cow's serum is become the dense liquid storage of calibration object, calibrates with enterprise's calibration object, by calibration object diluted to working concentration, be respectively 0,5,25,100,250,1000mIU/mL;
The compound method of above-mentioned β-hCG company standard product: the β-hCG antigen calibration object dilution of 50% cow's serum is made into the dense liquid storage of calibration object, be its definite value by national standard, according to national standard calibration value, dense for calibration object liquid storage calibration object diluted is become working concentration each point, then working concentration each point national standard is demarcated; Packing, labels, and is stored in-20 DEG C.β-hCG national standard lot number is: 150535, and specification is: 0.21IU/ props up.
(2) preparation of β-hCG quality-control product:
The calibration object diluted of β-hCG antigen containing 50% cow's serum is made into dense liquid storage, with enterprise's calibration object to after its definite value, is diluted to the low value quality-control product of 8mIU/mL and the high level quality-control product of 600mIU/mL respectively with calibration object dilution.
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
Get 100mL0.1mol/L MES solution (MES solution), add 10mg magnetic-particle and 3mg streptavidin (SA) successively, stir 30min, then 10mg/mL carbodiimide hydrochloride solution (1-ethyl-(3-dimethylaminopropyl is added, EDC) 3.5uL, after reaction 1h, uses magnetic frame absorption, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash 3 times altogether, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin labeled β-hCG antibody
Get 0.5mg β-hCG antibody, to dialyse at 2 ~ 8 DEG C 1h with borate buffer solution; Antibody after dialysis is added 25ug biotin, and add dimethyl sulfoxide (DMSO), ultimate density is 10% simultaneously, and lucifuge reaction 3h, slowly vibrates; 250uL1mol/L ammonium chloride solution is added, reacting at normal temperature without light 60min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01mol/L PBS solution, period changes liquid 3 times;
(5) preparation of β-hCG abzyme bond
Adopt after β-hCG antibody and horseradish peroxidase carries out coupling by sodium periodate oxidation, be diluted to working concentration 1:4000 with enzyme dilution, and add 20% enzyme stabilizers and be stored in 2 ~ 8 DEG C; Enzyme stabilizers uses the protein stabiliser product of SurModics InVitro Technologies.
The preparation of (6) 20 times of concentrated washing lotions
20 concentrated washing lotions comprise 58gL sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 1 ‰ 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 TrisHCl 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.
Illustrate:
(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 β-hCG 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 0-1000mIU/mL concentration range is not less than 0.9900.
(4) sensitivity for analysis: kit assay sensitivity is not higher than 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 precision (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, and the allowed band of QcL and QcH is respectively 8 ~ 12mIU/mL and 400 ~ 600mIU/mL.
(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 2: preparation β human chorionic gonadotrophin (β-hCG) magnetic microparticle chemiluminescence immunological quantitative determining kit II
(1) preparation of β-hCG calibration object:
With the preparation of embodiment 1 β-hCG calibration object.
(2) preparation of β-hCG quality-control product:
The calibration object diluted of β-hCG antigen containing 50% cow's serum is made into dense liquid storage, with enterprise's calibration object to after its definite value, is diluted to the low value quality-control product of 10mIU/mL and the high level quality-control product of 500mIU/mL respectively with calibration object dilution.
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
Get 100mL0.1mol/L MES solution (MES solution), add 10mg magnetic-particle and 3mg streptavidin (SA) successively, stir 30min, then 10mg/mL carbodiimide hydrochloride solution (1-ethyl-(3-dimethylaminopropyl is added, EDC) 3.5uL, after reaction 1h, uses magnetic frame absorption, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash 3 times altogether, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin labeled β-hCG antibody
Get 0.5mg β-hCG antibody, to dialyse at 2 ~ 8 DEG C 2h with borate buffer solution; Antibody after dialysis is added 25ug biotin, and add dimethyl sulfoxide (DMSO), ultimate density is 10% simultaneously, and lucifuge reaction 3h, slowly vibrates; 250uL1mol/L 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 0.01mol/L PBS solution, period changes liquid 4 times;
(5) preparation of β-hCG abzyme bond
Adopt after β-hCG antibody and horseradish peroxidase carries out coupling by sodium periodate oxidation, be diluted to working concentration 1:4000 with enzyme dilution, and add 5% enzyme stabilizers and be stored in 2 ~ 8 DEG C; Enzyme stabilizers uses the protein stabiliser product of SurModics InVitro Technologies.
The preparation of (6) 20 times of concentrated washing lotions
20 concentrated washing lotions comprise 58gL sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 1 ‰ 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 TrisHCl 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 3: preparation β human chorionic gonadotrophin (β-hCG) magnetic microparticle chemiluminescence immunological quantitative determining kit III
(1) preparation of β-hCG calibration object:
With the preparation of embodiment 1 β-hCG calibration object
(2) preparation of β-hCG quality-control product:
The calibration object diluted of β-hCG antigen containing 50% cow's serum is made into dense liquid storage, with enterprise's calibration object to after its definite value, is diluted to the low value quality-control product of 12mIU/mL and the high level quality-control product of 400mIU/mL respectively with calibration object dilution.
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
Get 100mL0.1mol/L MES solution (MES solution), add 10mg magnetic-particle and 3mg streptavidin (SA) successively, stir 30min, then 10mg/mL carbodiimide hydrochloride solution (1-ethyl-(3-dimethylaminopropyl is added, EDC) 3.5uL, after reaction 1h, uses magnetic frame absorption, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash 3 times altogether, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin labeled β-hCG antibody
Get 0.5mg β-hCG antibody, to dialyse at 2 ~ 8 DEG C 3h with borate buffer solution; Antibody after dialysis is added 25ug biotin, and add dimethyl sulfoxide (DMSO), ultimate density is 10% simultaneously, and lucifuge reaction 3h, slowly vibrates; 250uL1mol/L ammonium chloride solution is added, reacting at normal temperature without light 30min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01mol/L PBS solution, period changes liquid 5 times;
(5) preparation of β-hCG abzyme bond
Adopt after β-hCG antibody and horseradish peroxidase carries out coupling by sodium periodate oxidation, be diluted to working concentration 1:4000 with enzyme dilution, and add 15% enzyme stabilizers and be stored in 2 ~ 8 DEG C; Enzyme stabilizers uses the protein stabiliser product of SurModics InVitro Technologies.
The preparation of (6) 20 times of concentrated washing lotions
20 concentrated washing lotions comprise 58gL sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 1 ‰ 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 TrisHCl 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 4: the using method of kit of the present invention
Kit to be checked is balanced 30 minutes by 1 under room temperature (18 ~ 25 DEG C).
2 preparation washing lotions: 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 preparation luminescent solutions: use first 5 minutes and get appropriate luminescent solution A and mix with luminescent solution B equal-volume.
Reaction tube is numbered by 4, 10-50uL calibration object or serum specimen is added successively in test tube, 100uL magnetic-particle-Streptavidin suspending liquid, 100uL biotin-β-hCG antibody conjugates, 100uL β-hCG enzyme conjugates, oscillating reactions 10-30min 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 200-400uL 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 β-hCG can be calculated according to the luminous value of serum specimen.
Embodiment 5: the evaluation of methodology result of this kit
Embodiment 6: the clinical comparison experiment of this kit
The kit of invention has carried out clinical examination, total sample number 120 example of this clinical testing, first with after the test of β-hCG radioimmunoassay kits, the kit of invention (chemiluminescence) is used to measure again, result shows, straight-line equation is y=0.9393x+-24.714, and related coefficient is R
2=0.9783.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 β-hCG value of two kinds of method mensuration is conspicuousnesses, and the β-hCG that visible two kinds of methods measure is worth closely related.Sensitivity (True Positive Rate) is 97.32%, specificity (true negative rate) is 96.52%, all higher; And false positive rate (misdiagnosis rate) be 3.20%, false negative rate (rate of missed diagnosis) is 2.69%, 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 98.12%, 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 475 portions of normal human serums, plasma sample, result shows that the reference value (term of reference) of this kit is 0 ~ 5.0mIU/mL.
Claims (2)
1. β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box, is characterized in that, described kit comprises:
1) β-hCG calibration object; The calibration object diluent preparing of β-hCG sterling containing 50% cow's serum is become the dense liquid storage of calibration object, calibrates with enterprise's calibration object, by calibration object diluted to working concentration, be respectively 0,5,25,100,250,1000mIU/mL;
2) coupling has the magnetic particle suspending liquid of Streptavidin; Described magnetic particle is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 1 ~ 2um; Preparation process is for getting 100mL0.1mol/L MES solution, add 10mg magnetic-particle and 3mg streptavidin successively, stir 30min, then add 10mg/mL carbodiimide hydrochloride solution 3.5 μ L, after reaction 1h, use magnetic frame adsorbs, and static 10min, removes liquid, add 10mL 0.01mol/L PBS, repeat said process, wash 3 times altogether, be finally settled to 1L with 0.01mol/L PBS;
3) biotin labeled β-hCG antibody; Preparation method gets 0.5mg β-hCG antibody, to dialyse 1 ~ 3h with borate buffer solution at 2 ~ 8 DEG C; Antibody after dialysis is added 25 μ g biotins, and add dimethyl sulfoxide (DMSO), ultimate density is 10% simultaneously, and lucifuge reaction 3h, slowly vibrates; 250 μ L1mol/L ammonium chloride solutions are added, reacting at normal temperature without light 30 ~ 60min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01mol/L PBS solution, period changes liquid 3 ~ 5 times;
4) β-hCG abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; Preparation method, after adopting sodium periodate oxidation that β-hCG antibody and horseradish peroxidase are carried out coupling, is diluted to working concentration 1:4000 with enzyme dilution, and is added 5-20% enzyme stabilizers, be stored in 2 ~ 8 DEG C;
5) β-hCG quality-control product, described β-hCG quality-control product comprises low value quality-control product and high level quality-control product; The concentration of low value quality-control product is the concentration of 8mIU/mL and high level quality-control product is 600mIU/mL; The calibration object diluted of β-hCG antigen containing 50% cow's serum is made into dense liquid storage, with enterprise's calibration object to after its definite value, is diluted to 8mIU/mL and 600mIU/mL respectively with calibration object dilution;
6) chemical luminescence for liquid A liquid and B liquid; Described A liquid is 0.7g/L luminol, 0.165g/L p-iodophenol, and damping fluid is the 5mmol/L TrisHCl 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;
7) 20 times of concentrated washing lotions; 20 concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 1 ‰ Proclin300;
8) reaction tube; The material of described reaction tube is transparent polystyrene, transparent polyethylene, transparent polypropylene or clear glass.
2. prepare a method for the kit of described claim 1, it is characterized in that comprising the following steps:
(1) preparation of β-hCG calibration object:
The calibration object diluent preparing of β-hCG sterling containing 50% cow's serum is become the dense liquid storage of calibration object, calibrates with enterprise's calibration object, by calibration object diluted to working concentration, be respectively 0,5,25,100,250,1000mIU/mL;
(2) preparation of β-hCG quality-control product:
The calibration object diluted of β-hCG antigen containing 50% cow's serum is made into dense liquid storage, with enterprise's calibration object to after its definite value, is diluted to 8mIU/mL and 600mIU/mL respectively with calibration object dilution;
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
Get 100mL0.1mol/L MES solution, add 10mg magnetic-particle and 3mg streptavidin successively, stir 30min, then add 10mg/mL carbodiimide hydrochloride solution 3.5 μ L, after reaction 1h, use magnetic frame adsorbs, and static 10min, removes liquid, add 10mL 0.01mol/L PBS, repeat said process, wash 3 times altogether, be finally settled to 1L with 0.01mol/LPBS;
(4) preparation of biotin labeled β-hCG antibody
Get 0.5mg β-hCG antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 25 μ g biotins, and add dimethyl sulfoxide (DMSO), ultimate density is 10% simultaneously, and lucifuge reaction 3h, slowly vibrates; 250 μ L1mol/L ammonium chloride solutions are added, reacting at normal temperature without light 30 ~ 60min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C by 0.01mol/L PBS solution, period changes liquid 3 ~ 5 times;
(5) preparation of β-hCG abzyme bond
After adopting sodium periodate oxidation that β-hCG antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:4000 with enzyme dilution, and added 5-20% enzyme stabilizers, be stored in 2 ~ 8 DEG C;
The preparation of (6) 20 times of concentrated washing lotions
20 concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 1 ‰ 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 TrisHCl 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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