CN102998462B - Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for procollagen III (PC III), and preparation method of kit - Google Patents
Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for procollagen III (PC III), and preparation method of kit Download PDFInfo
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Abstract
The invention discloses a quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for procollagen III (PC III). The kit comprises PC III calibrators, magnetic particle suspension coupled with streptavidin, a PC III antibody labeled with biotin, a PC III abzyme combination, a PC III quality controller, chemiluminescence liquor A, chemiluminescence liquor B, 20 times concentrated washing liquor, and a reaction tube, wherein the concentrations of the PC III calibrators include 0, 20ng/ml, 50ng/ml, 100ng/ml, 200ng/ml and 500ng/ml, and diluent of the calibrators is a physiological saline solution containing 5% of bovine serum; and enzyme adopted by the PC III abzyme combination is horse radish peroxidase with the purity RZ being more than or equal to 3.0 and the activity being more than or equal to 250U/ml. The invention also discloses a preparation method of the kit. Compared with the conventional kit, the quantitative detection kit is simple and convenient to operate, is safe, does not cause environment pollution, and also has the advantages of wide concentration range, low cost, good stability and the like of detection samples.
Description
Technical field
The present invention relates to immunoassay medical domain, concrete, the invention provides a kind of III procollagen type (PC III) magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof.
Background technology
In tissue, there are five kinds of collagens (I, II, III, IV, V type).Main containing I, III, IV, V procollagen type in normal liver, be the outer desmoplastic main collagen of interstitial of liver cell.PC III is secreted in blood after synthetic in cell, and P III P is the amino terminal peptide fragment after the degraded of PC III, only has 1/10 of PC III, molecular weight 45000, and when liver fibrosis, the synthetic PC III of fibroblast increases, and degraded reduces.When chronic active hepatitis and cirrhosis, liver fiber anabolism is vigorous, and blood-serum P C III raises, and while therefore having activity fiberization, blood-serum P C III raises obviously, but not necessarily raises when old liver fibrosis or end-age cirrhosis, hepatatrophy.Liver is in the time only having inflammation, necrosis, and PIIIP also can correspondingly raise.
(1) in PC III blood content with chronic persistent hepatitis, chronic active hepatitis, raise successively to cirrhosis (for the phase), no matter P III P or PC III serum-concentration and the liver biopsy fiberization order of severity are high-positive correlation, are the sensitive marks of diagnosing liver fibrosis and early diagnosis liver fibrosis.
(2) as the index of liver fibrosis classification.(cirrhosis that comprise after hepatitis, Alcoholic and other reasons causes);
(3) as the reliability index of cirrhosis (compensatory phase) early treatment and observation of curative effect;
(4) cirrhosis late period (losing the compensatory phase), blood PC III content can have decline.
The method of at present conventional detection III procollagen type (PC III) has radiating immuning analysis technology (RIA) and enzyme-linked immunosorbent assay (ELISA), but there is many deficiencies in these two kinds of methods, for example RIA exist radioactive contamination, label half life period short, operator is had to radioactive damage, and complex operation, the shortcomings such as time length; And ELISA sensitivity is low, sensing range is narrow; Along with developing rapidly of immuno-labelling technique, various new detection methods emerge in an endless stream, and wherein magnetic microparticle chemiluminescence immunoassay technology is current the most responsive skeptophylaxis determination method.
But magnetic microparticle chemiluminescence analytical approach is not also widely used in the detection of PC III, is particularly widely used in clinical.
Summary of the invention
The problem to be solved in the present invention is to provide magnetic microparticle chemiluminescence immune quantitative detection reagent box of PC III and preparation method thereof; avoid the reagent term of validity of radioimmunoassay short, had the shortcoming such as radioactive contamination, complex operation; and it is low to have solved 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: PC III procollagen type magnetic microparticle chemiluminescence immune quantitative detection reagent box, comprise: PC III calibration object, PC III calibration object concentration is 0,20,50,100,200,500ng/mL gradient concentration, calibration object dilution is the normal saline solution that contains 5% cow's serum; Coupling has the magnetic particle suspending liquid of Streptavidin; Biotin labeled PC III antibody; PC III abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; PC III quality-control product, described quality-control product comprises low value quality-control product and high value quality-control product, concentration is respectively 42.5 ~ 57.5ng/mL and 255 ~ 345ng/mL; 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 the 5mmol/L TrisHCl that damping fluid is 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 of surface parcel with amino or carboxyl reactive group, particle diameter 1-2um.
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 PC III calibration object: by the normal saline solution that contains 5% cow's serum for PC III sterling, concentration is respectively 0,20,50,100,200,500ng/mL.
(2) preparation of PC III quality-control product:
The allowed band that PC III antigen is diluted to respectively to low value quality-control product (QcL) and high value quality-control product (QcH) with the normal saline solution that contains 5% cow's serum, is respectively 42.5 ~ 57.5ng/mL and 255 ~ 345ng/mL.
(3) preparation of magnetic-particle-Streptavidin suspending liquid
Get 100mL0.1mol/L MES solution (MES solution), add successively 10mg magnetic-particle and 3mg streptavidin (SA), stir 30min, then add EDC solution (1-ethyl-(3-dimethylaminopropyl of 10mg/mL, have another name called: carbodiimide hydrochloride solution) 3.5uL, after reaction 1h, the absorption of use magnetic frame, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash altogether 3 times, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin-PC III antibody conjugates
1) get 0.5mgPC III antibody, 1～3h dialyses at 2～8 DEG C with borate buffer solution.
2) antibody after dialysis is added to 25ug biotin, add dimethyl sulfoxide (DMSO) (DMSO) simultaneously, its final concentration is 10%, lucifuge reaction 3h, slowly vibration.
3) in above-mentioned solution, add 250uL1mol/L ammonium chloride solution, reacting at normal temperature without light 30～60min.
4) with the 0.01M PBS solution 2d that dialyses, change during this time liquid 3～5 times at 2～8 DEG C.
(5) preparation of PC III abzyme bond
Adopt sodium periodate oxidation that PC III antibody and horseradish peroxidase are carried out after coupling, be diluted to 1:8000 with enzyme dilution, and add 5-20% enzyme stabilizers, be stored in 2 ~ 8 DEG C.Enzyme stabilizers is a kind of reagent that can keep protein to keep natural folding conception 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
According to the concentrated washing lotion of following formulated, 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20,1 ‰ Proclin300.
(7) preparation of chemical luminescence for liquid A liquid and B liquid
0.7g/L luminol, 0.165g/L p-iodophenol, damping fluid is 5mmol/L TrisHCl(pH8.6); B liquid is 0.675g/L urea peroxide, prepares with process water.
(8) assembling: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C.
(9) kit that adopts the party's legal system is carried out to evaluation of methodology.
Principle of the present invention is, this kit adopts sandwich method principle to measure the PC III in serum or blood plasma, in Avidin-magnetic particle suspending liquid, add biotin-PC III antibody conjugates, by the compatible reaction of Avidin and biotin, form magnetic particle-Avidin-Biotin-PC III antibody complex, add after sample and enzyme, can be by antigen-antibody reaction, form magnetic particle-Avidin-Biotin-PC III antibody-PC III-PC III antibody-HRP compound, compound is adsorbed on to test tube bottom with magnetic field, wash free composition, add substrate working fluid, under oxygenant effect, HRP catalysis luminol generates the aminophthalic acid ion 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.In the luminous value of sample and sample, PC III concentration is proportionate.PC III concentration in sample is according to the Log(X being set up by calibration object PC III concentration and corresponding RLU)-Log(Y) mathematical model carries out quantitatively, thus the PC III content in detection human serum, blood plasma.
This kit is by chemiluminescence immune assay and the combination of magnetic particle technology, compared with III procollagen type (PC III) chemical luminescence reagent kit taking microwell plate as solid phase carrier, there is following advantage: (1) is taking magnetic particle as solid phase carrier, greatly increase effective package amount of antibody, save the consumption of antibody, thereby saved cost; (2) taking magnetic particle as solid phase carrier coated antibody, increase the contact area of Ag-Ab, and light-emitting area, improve the sensitivity of reacting; (3) reaction is carried out in liquid phase, and utilizes rotating magnetic field to make its beating action of magnetic particle, has greatly shortened the reaction time; (4) in course of reaction, introduced biotin-avidin system (biotin-avidin system, BAS), it is a kind of novel reaction amplification system growing up the end of the seventies, owing to having and the strong bonded of labelled reagent high-affinity, multistage enlarge-effect, make it have feature highly sensitive, that specificity good, stability is high, become the new technology that is widely used in trace antigen, antibody qualitative and quantitative analysis and position observation research at present; (5) this kit supports the use with tube-type chemical light-emitting appearance, and in sample mensuration process, dirigibility is better.
Auxin (PC III) the magnetic microparticle chemiluminescence immunological quantitative determining kit of this patent 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 10ng/mL.(3) high specificity, the cross reaction coefficient of hyaluronic acid (HA), laminin (LN) is less than 1%.(4) precision is good, and precision is not higher than 10%.(5) have good stability, this product can be deposited more than 7 days at 37 DEG C, can deposit 1 year at 2～8 DEG C.(6) cost is low, and with like product comparison on market, this kit is functional, and cost is low, has clinical value.
Brief description of the drawings
Fig. 1 is the measurement result comparison diagram that Bo Aosaisi chemical luminescence reagent kit of the present invention is measured PC III and radioimmunological kit mensuration PC III, wherein ordinate is the PC III value that Bo Aosaisi records, horizontal ordinate is that radioimmunological kit is measured PC III value, two kinds of method related coefficients (r)=0.9945, straight-line equation y=0.9646x+3.1299
Embodiment
Embodiment 1: preparation III procollagen type (PC III) magnetic microparticle chemiluminescence immunological quantitative determining kit I
(1) preparation of PC III calibration object: by the normal saline solution that contains 5% cow's serum for PC III sterling, concentration is respectively 0,20,50,100,200,500ng/mL.
(2) preparation of PC III quality-control product:
By the normal saline solution preparation low value quality-control product and the high-quality quality-control product that contain 5% cow's serum for PC III antigen, QcL compound concentration 42.5ng/mL, QcH compound concentration 255ng/mL.
(3) preparation of magnetic-particle-Streptavidin suspending liquid
Get 100mL0.1mol/L MES solution (MES solution), add successively 10mg magnetic-particle and 3mg streptavidin (SA), stir 30min, then add EDC solution (1-ethyl-(3-dimethylaminopropyl of 10mg/mL, have another name called: carbodiimide hydrochloride solution) 3.5uL, after reaction 1h, the absorption of use magnetic frame, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash altogether 3 times, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin-PC III antibody conjugates
1) get 0.5mgPC III antibody, 3h dialyses at 2～8 DEG C with borate buffer solution.
2) antibody after dialysis is added to 25ug biotin, add dimethyl sulfoxide (DMSO) (DMSO) simultaneously, its final concentration is 10%, lucifuge reaction 3h, slowly vibration.
3) in above-mentioned solution, add 250uL1mol/L ammonium chloride solution, reacting at normal temperature without light 30min.
4) with the 0.01M PBS solution 2d that dialyses, change during this time liquid 3 times at 2～8 DEG C.
(5) preparation of PC III abzyme bond
Adopt sodium periodate oxidation that PC III antibody and horseradish peroxidase are carried out after coupling, be diluted to 1:8000 with enzyme dilution, and add 5% enzyme stabilizers, and being stored in 2 ~ 8 DEG C, enzyme stabilizers uses the protein stabiliser product of SurModics In Vitro Technologies.
The preparation of (6) 20 times of concentrated washing lotions
According to the concentrated washing lotion of following formulated, 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20,1 ‰ Proclin300.
(7) preparation of chemical luminescence for liquid A liquid and B liquid
0.7g/L luminol, 0.165g/L p-iodophenol, damping fluid is 5mmol/L TrisHCl(pH8.6); B liquid is 0.675g/L urea peroxide, prepares with process water.
(8) assembling: 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 the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Illustrate:
1) physical examination: liquid component should be clarified, without precipitation or floccus; Other components should be without packages in damaged condition.
2) accuracy: kit calibration object and company standard product series are analyzed mensuration simultaneously, use double-log Model fitting, requires two not obvious parallel deviates of dose-response curve (t inspection, | t|<2.447); Taking PC III company standard product as reference substance, use double-log Model fitting, 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 Model fittings, dose-response curve correlation coefficient r absolute value in 0-500ng/mL concentration range is not less than 0.9900.
4) sensitivity for analysis: kit sensitivity for analysis is not higher than 10ng/mL.
5) precision: precision (CV%) should be higher than 10%.
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) Model fitting, quality-control product measured value should be in allowed band, and the allowed band of QcL and QcH is respectively 42.5 ~ 57.5ng/mL and 255 ~ 345ng/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 III procollagen type (PC III) magnetic microparticle chemiluminescence immunological quantitative determining kit II
(1) preparation of PC III calibration object: by the normal saline solution that contains 5% cow's serum for PC III sterling, concentration is respectively 0,20,50,100,200,500ng/mL.
(2) preparation of PC III quality-control product:
By the normal saline solution preparation low value quality-control product and the high-quality quality-control product that contain 5% cow's serum for PC III antigen, QcL compound concentration is 57.5ng/mL, and QcH compound concentration is 345ng/mL.
(3) preparation of magnetic-particle-Streptavidin suspending liquid
Get 100mL0.1mol/L MES solution (MES solution), add successively 10mg magnetic-particle and 3mg streptavidin (SA), stir 30min, then add EDC solution (1-ethyl-(3-dimethylaminopropyl of 10mg/mL, have another name called: carbodiimide hydrochloride solution) 3.5uL, after reaction 1h, the absorption of use magnetic frame, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash altogether 3 times, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin-PC III antibody conjugates
1) get 0.5mgPC III antibody, 1h dialyses at 2～8 DEG C with borate buffer solution.
2) antibody after dialysis is added to 25ug biotin, add dimethyl sulfoxide (DMSO) (DMSO) simultaneously, its final concentration is 10%, lucifuge reaction 3h, slowly vibration.
3) in above-mentioned solution, add 250uL1mol/L ammonium chloride solution, reacting at normal temperature without light 60min.
4) with the 0.01M PBS solution 2d that dialyses, change during this time liquid 5 times at 2～8 DEG C.
(5) preparation of PC III abzyme bond
Adopt sodium periodate oxidation that PC III antibody and horseradish peroxidase are carried out after coupling, be diluted to 1:8000 with enzyme dilution, and add 5% enzyme stabilizers, and being stored in 2 ~ 8 DEG C, enzyme stabilizers uses the protein stabiliser product of SurModics In Vitro Technologies.
The preparation of (6) 20 times of concentrated washing lotions
According to the concentrated washing lotion of following formulated, 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20,1 ‰ Proclin300.
(7) preparation of chemical luminescence for liquid A liquid and B liquid
0.7g/L luminol, 0.165g/L p-iodophenol, damping fluid is 5mmol/L TrisHCl(pH8.6); B liquid is 0.675g/L urea peroxide, prepares with process water.
(8) assembling: 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 the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Embodiment 3: preparation III procollagen type (PC III) magnetic microparticle chemiluminescence immunological quantitative determining kit III
(1) preparation of PC III calibration object: by the normal saline solution that contains 5% cow's serum for PC III sterling, concentration is respectively 0,20,50,100,200,500ng/mL.
(2) preparation of PC III quality-control product:
By the normal saline solution preparation low value quality-control product and the high-quality quality-control product that contain 5% cow's serum for PC III antigen, QcL compound concentration is between 50.2ng/mL, and QcH compound concentration is 305ng/mL.
(3) preparation of magnetic-particle-Streptavidin suspending liquid
Get 100mL0.1mol/L MES solution (MES solution), add successively 10mg magnetic-particle and 3mg streptavidin (SA), stir 30min, then add EDC solution (1-ethyl-(3-dimethylaminopropyl of 10mg/mL, have another name called: carbodiimide hydrochloride solution) 3.5uL, after reaction 1h, the absorption of use magnetic frame, static 10min, remove liquid, add 10mL 0.01mol/L PBS, repeat said process, wash altogether 3 times, be finally dissolved to 1L with 0.01mol/L PBS.
(4) preparation of biotin-PC III antibody conjugates
1) get 0.5mgPC III antibody, 2h dialyses at 2～8 DEG C with borate buffer solution.
2) antibody after dialysis is added to 25ug biotin, add dimethyl sulfoxide (DMSO) (DMSO) simultaneously, its final concentration is 10%, lucifuge reaction 3h, slowly vibration.
3) in above-mentioned solution, add 250uL1mol/L ammonium chloride solution, reacting at normal temperature without light 50min.
4) with the 0.01M PBS solution 2d that dialyses, change during this time liquid 4 times at 2～8 DEG C.
(5) preparation of PC III abzyme bond
Adopt sodium periodate oxidation that PC III antibody and horseradish peroxidase are carried out after coupling, be diluted to 1:8000 with enzyme dilution, and added 20% enzyme stabilizers, be stored in 2 ~ 8 DEG C, enzyme stabilizers uses the protein stabiliser product of SurModics In Vitro Technologies.
The preparation of (6) 20 times of concentrated washing lotions
According to the concentrated washing lotion of following formulated, 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20,1 ‰ Proclin300.
(7) preparation of chemical luminescence for liquid A liquid and B liquid
0.7g/L luminol, 0.165g/L p-iodophenol, damping fluid is 5mmol/L TrisHCl(pH8.6); B liquid is 0.675g/L urea peroxide, prepares with process water.
(8) assembling: 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 the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
The using method of embodiment 4 kits of the present invention
1) kit to be checked is descended to balance 30 minutes in room temperature (18 ~ 25 DEG C).
2) preparation washing lotion: will concentrate washing lotion with distilled water and dilute (1mL washing lotion adds 19mL distilled water) by 1:20.If concentrated washing lotion has crystallization, can dilute again concentrating after washing lotion is placed in room temperature or 37 DEG C of dissolvings to be crystallized.
3) preparation luminescent solution: use first 5 minutes and get appropriate luminescent solution A and mix with luminescent solution B equal-volume.
4) reaction tube is numbered, in test tube, add successively 10-50uL calibration object or serum specimen, 100uL magnetic-particle-Streptavidin suspending liquid, 100uL biotin-PC III antibody conjugates, 100uL PC III enzyme conjugates, oscillating reactions 10-30min at 37 DEG C, test tube rack is placed in and on magnetic separator, separates 5min, then pour out supernatant, add 500uL washing lotion, after fully mixing, on magnetic separator, separate, pour out washing lotion, repeat 3 times, in each pipe, add Chemoluminescent substrate 200-400uL, fully mix, secretly put 5min, on tube-type chemical light-emitting appearance, measure the luminous value (RLU) of each pipe, taking the Log value of calibration object concentration as horizontal ordinate, taking the Log of luminous value as ordinate, drawing standard curve, can calculate the concentration of PC III according to the luminous value of serum specimen.
The evaluation of methodology result of 5 kits of embodiment
Embodiment 6: the clinical comparison experiment of this kit
The kit of this patent invention has carried out clinical examination, total sample number 108 examples of this clinical testing, first with after the test of PC III radioimmunoassay kit, measure with the kit (chemiluminescence) of this patent invention again, result shows, straight-line equation is y=0.9646x+3.1299, and related coefficient is R=0.9695.Kit prepared by visible this method and hospital's measured value have good consistance.With SPSS13.0 statistical analysis software, the PC III measured value of two kinds of methods is carried out to t inspection (inspection level α=0.05), P=0.839 > 0.05, two kinds of PC III value no difference of science of statistics that method is measured, the PC III value that visible two kinds of methods are measured is closely related.Sensitivity (True Positive Rate) is 95.06%, specificity (true negative rate) is 98.68%, all higher; And false positive rate (misdiagnosis rate) is 1.32%, false negative rate (rate of missed diagnosis) is 4.94%, all lower, the matching degree of the measured value of this kit and actual value (former measured value) is good as seen.Crude agreement reflection kit diagnosis patient and non-patient's ability, the crude agreement of this kit is 97.41%, 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 467 parts of normal human serums, plasma samples, result shows that the reference value (term of reference) of this kit is: 0 ~ 120ng/mL
Claims (2)
1.PC III procollagen type magnetic microparticle chemiluminescence immune quantitative detection reagent box, is characterized in that, described kit comprises:
1) PC III calibration object;
PC III calibration object concentration is 0,20,50,100,200,500ng/mL gradient concentration, and calibration object dilution is the normal saline solution that contains 5% cow's serum;
2) coupling has the magnetic particle suspending liquid of Streptavidin, and described magnetic particle is the tri-iron tetroxide of surface parcel with amino or carboxyl reactive group, particle diameter 1～2 μ m;
3) biotin labeled PC III antibody;
4) PC III abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL;
5) PC III quality-control product, described quality-control product comprises low value quality-control product and high value quality-control product, concentration is respectively 42.5ng/mL and 255ng/mL;
6) chemical luminescence for liquid A liquid and B liquid, luminescent solution A liquid principal ingredient is 0.7g/L luminol, B liquid principal ingredient 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, tygon, polypropylene or glass.
2. prepare a method for the kit of described claim 1, it is characterized in that comprising the following steps:
(1) preparation of PC III calibration object:
PC III sterling is diluted to serial gradient with the normal saline solution that contains 5% cow's serum, and concentration is respectively 0,20,50,100,200,500ng/mL;
(2) preparation of PC III quality-control product:
PC III sterling is diluted to respectively to low value quality-control product 42.5ng/mL with the normal saline solution that contains 50% cow's serum, high value quality-control product 255ng/mL;
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
Get 100mL0.1mol/L MES solution, add successively 10mg magnetic-particle and 3mg streptavidin, stir 30min, then add 10mg/mL carbodiimide hydrochloride solution 3.5 μ L, after reaction 1h, the absorption of use magnetic frame, static 10min, removes liquid, add 10mL 0.01mol/L PBS, repeat said process, wash altogether 3 times, be finally dissolved to 1L with 0.01mol/L PBS;
(4) preparation of biotin labeled PC III antibody
Get 0.5mg PC III antibody, 1～3h dialyses at 2～8 DEG C with borate buffer solution; Antibody after dialysis is added to 25 μ g biotins, add dimethyl sulfoxide (DMSO) simultaneously, ultimate density is 10%, lucifuge reaction 3h, slowly vibration; In above-mentioned solution, add 250 μ L1mol/L ammonium chloride solutions, reacting at normal temperature without light 30～60min; At 2～8 DEG C, dialyse 2 days with 0.01mol/L PBS solution, change during this time liquid 3～5 times;
(5) preparation of PC III abzyme bond
Adopt sodium periodate oxidation that PC III antibody and horseradish peroxidase are carried out after coupling, be diluted to 1:8000 with enzyme dilution, and add 5-20% enzyme stabilizers, be stored in 2～8 DEG C;
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/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 the 5mmol/L TrisHCl that damping fluid is pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; A liquid and B liquid before use 5min mix;
(8) assembling: mentioned reagent is assembled into box, is stored in 2～8 DEG C;
(9), to adopting the kit that makes of the method to carry out physical examination, measured value and the stability of linearity to accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
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| CN105277689A (en) * | 2015-11-17 | 2016-01-27 | 苏州浩欧博生物医药有限公司 | Reagent kit and method for detecting total serum IgE through nano magnetic particle chemiluminescence method |
| CN106855574A (en) * | 2016-06-30 | 2017-06-16 | 深圳市亚辉龙生物科技股份有限公司 | A kind of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent and preparation method thereof |
| CN109444430A (en) * | 2018-12-13 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of kit detecting III procollagen type N-terminal peptide |
| CN111679086B (en) * | 2020-08-11 | 2020-11-13 | 苏州康和顺医疗技术有限公司 | HBP magnetic particle chemiluminescence detection kit and preparation method thereof |
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| CN101533028A (en) * | 2008-03-14 | 2009-09-16 | 北京科美东雅生物技术有限公司 | Chemoluminescent immunoassay kit of hyaluronic acid and preparation method thereof |
| CN101598730B (en) * | 2009-05-11 | 2013-05-08 | 北京博生福生物技术有限责任公司 | In vitro diagnosis kit of hepatitis B virus E antibody through dual-antigen sandwich method and preparation method thereof |
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