CN103018445B - CA50 magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof - Google Patents
CA50 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 CA50 magnetic microparticle chemiluminescence immune quantitative detection reagent box, described kit comprises: CA50 calibration object; Coupling has the magnetic particle suspending liquid of Streptavidin; Biotin labeled CA50 antibody; CA50 abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; CA50 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 CA50 (CA50) magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof.
Background technology
Cancer is also known as malignant tumour, it is a kind of disease of serious threat human health, according to statistics, annual new cancered patient about 1,600,000 people of China, every year because of number about 1,300,000 people of cancer mortality, by current medical level, early-stage cancer patient about has 80% ~ 90% can cure, greatly reduce the mortality ratio of cancer patient, the early detection of visible cancer patient, early diagnosis and early treatment are particularly important.
CA50 (CA50) is a kind of nonspecific broad-spectrum tumor mark, with a kind of gangliosides antigen that sialoprotein and sialic acid-carrying glycolipid are principal ingredient, it is that immunogene prepares CA50 monoclonal antibody first that nineteen eighty-three Lindholm etc. apply colon cancer cell.The form that CA50 combines with fat or lipoprotein is present in cell membrane, and generally do not exist in the normal tissue, when malignant change of cell, glycosylase is activated, and causes cell surface glycosyl structure change and become CA50 mark.CA50 has higher positive rate to multiple epithelium class malignant tumour, generally can observe CA50 content in serum to raise in the patient of tumor in digestive tract, with mark joint inspections such as CEA, CA125, CA199, can be the foundation that the examination in early stage of disease, auxiliary diagnosis, antidiastole, observation of curative effect, cancer metastasis and prognosis provide reference value.
Clinically, CA50 content < 20 μ g/L in the blood of normal person, all can raise in much malignant tumor patient blood, lung cancer as 66.6%, the liver cancer of 88.2%, the cancer of the stomach of 68.9%, the ovary of 88.5% or cervix cancer, 94.4% pancreas or cholangiocarcinoma, other as the carcinoma of the rectum, the dirty cancer of wing etc. all have more than 70% be raise, in addition, ulcerative colitis, cirrhosis, melanoma, lymthoma, autoimmune disease etc. also have CA50 to raise phenomenon.
The method of detection CA50 conventional at present has radiating immuning analysis technology (RIA) and enzyme-linked immunosorbent assay (ELISA), but there is many deficiencies in these two kinds of methods, such as 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), are current microimmuno-assays the most responsive.
Summary of the invention
The problem to be solved in the present invention chemiluminescence immunoassay immue quantitative detection reagent box being to provide CA50 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: CA50 magnetic microparticle chemiluminescence immune quantitative detection reagent box, comprise: CA50 calibration object, concentration be 0,5,10,25,50,100KU/mL, calibration object dilution is 50% cow's serum; Coupling has the magnetic particle suspending liquid of Streptavidin; Biotin labeled CA50 antibody; CA50 abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; CA50 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 CA50 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 CA50 calibration object:
CA50 sterling 50% cow's serum solution dilution is become graded series, concentration is respectively 0,5,10,25,50,100KU/mL.
(2) preparation of CA50 quality-control product:
By CA50 with preparing low value quality-control product high level quality-control product containing 50% cow's serum solution dilution, the allowed band of low value quality-control product (QcL) and high-quality quality-control product (QcH) is respectively 8 ~ 12KU/mL and 29.6 ~ 44.4KU/mL.
(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 CA50 antibody
Get 0.5mg CA50 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 CA50 abzyme bond
After adopting sodium periodate oxidation that CA50 antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:6000 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 58g/L 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, the present invention adopts the CA50 in sandwich method principle mensuration serum or blood plasma, biotin-CA50 antibody conjugates is added in Avidin-magnetic particle suspending liquid, by the compatible reaction of Avidin and biotin, form magnetic particle-Avidin-Biotin-CA50 antibody complex, after adding sample and enzyme, antigen-antibody reaction can be passed through, define magnetic particle-Avidin-Biotin-CA50 antibody-CA50-CA50 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.In the luminous value of sample and sample, CA50 concentration is proportionate.CA50 concentration in sample is according to the Log(X set up by calibration object CA50 concentration and corresponding RLU)-Log(Y) mathematical model is carried out quantitatively, thus the CA50 content in detection human serum, blood plasma.
The kit of invention preparation is by chemiluminescence immune assay and magnetic particle combine with technique, substantially increase sensitivity and the accuracy of detection, in addition, biotin-avidin system (the biotin-avidin system introduced in testing process, BAS) there is multistage signal amplification, and do not increase nonspecific interference, have highly sensitive, specificity is good, stability is high, the features such as the strong and experimental cost of applicability is low, this technology has the following advantages: (1) take magnetic particle as solid phase carrier, considerably increase effective package amount of antibody, save the consumption of antibody, (2) be solid phase carrier coated antibody with magnetic particle, add the contact area of Ag-Ab, and light-emitting area, improve the sensitivity of reaction, (3) reaction is carried out in the liquid phase, and utilizes rotating magnetic field to make its beating action of magnetic particle, substantially reduces the reaction time.
Accompanying drawing explanation
Fig. 1 is the measurement result comparison diagram that Bo Aosaisi chemical luminescence reagent kit of the present invention mensuration CA50 and radioimmunological kit measure CA50, wherein ordinate is the CA50 value that Bo Aosaisi records, horizontal ordinate is that radioimmunological kit measures CA50 value, two kinds of method related coefficient (r)=0.9928, straight-line equation y=0.9914x+0.1335.
Fig. 2 is CA50 typical curve.
Embodiment
Embodiment 1: prepare CA50 (CA50) magnetic microparticle chemiluminescence immunological quantitative determining kit I
(1) preparation of CA50 calibration object:
CA50 sterling 50% cow's serum solution dilution is become graded series, concentration is respectively 0,5,10,25,50,100KU/mL.
(2) preparation of CA50 quality-control product:
By CA50 with preparing low value quality-control product high level quality-control product containing 50% cow's serum solution dilution, low value quality-control product (QcL) concentration is 8KU/mL, and the concentration of high-quality quality-control product (QcH) is 38.2KU/mL.
(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 CA50 antibody
Get 0.5mg CA50 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 3 times;
(5) preparation of CA50 abzyme bond
After adopting sodium periodate oxidation that CA50 antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:6000 with enzyme dilution, and added 20% enzyme stabilizers, 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 58g/L 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 CA50 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 ~ 100KU/mL concentration range is not less than 0.9900.
4. sensitivity for analysis: kit assay sensitivity is not higher than 1.0KU/L.
5. precision: in batch and between criticizing, imprecision (CV%) should not higher than 10%.
6. the 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 ~ 12KU/mL and 29.6 ~ 44.4KU/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: prepare CA50 (CA50) magnetic microparticle chemiluminescence immunological quantitative determining kit II
(1) preparation of CA50 calibration object:
CA50 sterling 50% cow's serum solution dilution is become graded series, concentration is respectively 0,5,10,25,50,100KU/mL.
(2) preparation of CA50 quality-control product:
CA50 is prepared low value quality-control product, high level quality-control product with containing 50% cow's serum solution dilution, and low value quality-control product (QcL) concentration is 12KU/mL, and high-quality quality-control product (QcH) concentration is 29.6KU/mL.
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
With the preparation of embodiment 1 magnetic-particle-Streptavidin suspending liquid.
(4) preparation of biotin labeled CA50 antibody
Get 0.5mg CA50 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 360min 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 CA50 abzyme bond
After adopting sodium periodate oxidation that CA50 antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:6000 with enzyme dilution, and added 5% enzyme stabilizers, 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 58g/L 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: prepare CA50 (CA50) magnetic microparticle chemiluminescence immunological quantitative determining kit III
(1) preparation of CA50 calibration object:
CA50 sterling 50% cow's serum solution dilution is become graded series, concentration is respectively 0,5,10,25,50,100KU/mL.
(2) preparation of CA50 quality-control product:
By CA50 with preparing low value quality-control product high level quality-control product containing 50% cow's serum solution dilution, low value quality-control product (QcL) concentration is 10KU/mL, and high-quality quality-control product (QcH) concentration is 44.4KU/mL.
(3) preparation of magnetic-particle-Streptavidin suspending liquid:
With the preparation of embodiment 1 magnetic-particle-Streptavidin suspending liquid
(4) preparation of biotin labeled CA50 antibody
Get 0.5mg CA50 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 5 times;
(5) preparation of CA50 abzyme bond
After adopting sodium periodate oxidation that CA50 antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:6000 with enzyme dilution, and added 15% enzyme stabilizers, 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 58g/L 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-CA50 antibody conjugates, 100uLCA50 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 CA50 can be calculated according to the luminous value of serum specimen, must be Y=1.1667X+4.623 as the equation of linear regression of Fig. 2, relative coefficient is 0.9981.
Embodiment 5: the evaluation of methodology result of this kit
The clinical comparison experiment of embodiment 6 kits
The kit of invention has carried out clinical examination, total sample number 108 example of this clinical testing, first with after the test of CA50 radioimmunoassay kits, the kit of invention (chemiluminescence) is used to measure again, result shows, straight-line equation is y=0.9914x+0.1335, and related coefficient is R=0.9857.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 CA50 value of two kinds of method mensuration is conspicuousnesses, and the CA50 value that visible two kinds of methods measure is closely related.Sensitivity (True Positive Rate) is 96.12%, specificity (true negative rate) is 96.10%, all higher; And false positive rate (misdiagnosis rate) be 1.90%, false negative rate (rate of missed diagnosis) is 3.88%, 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 97.36%, 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 574 portions of normal human serums, plasma sample, result shows that the reference value (term of reference) of this kit is 0 ~ 30KU/L.
Claims (1)
1. CA50 magnetic microparticle chemiluminescence immune quantitative detection reagent box, is characterized in that, described kit comprises:
1) CA50 calibration object, concentration is 0,5,10,25,50,100KU/mL, calibration object dilution is 50% cow's serum;
2) coupling has the magnetic particle suspending liquid of Streptavidin;
3) biotin labeled CA50 antibody;
4) CA50 abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL;
5) CA50 quality-control product;
6) chemical luminescence for liquid A liquid and B liquid;
7) 20 times of concentrated washing lotions;
8) reaction tube;
Described kit preparation comprises the following steps:
(1) preparation of CA50 calibration object:
CA50 sterling 50% cow's serum solution dilution is become graded series, concentration is respectively 0,5,10,25,50,100KU/mL;
(2) preparation of CA50 quality-control product:
By CA50 with preparing low value quality-control product high level quality-control product containing 50% cow's serum solution dilution, low value quality-control product concentration is 10KU/mL, and high-quality quality-control product concentration is 44.4KU/mL;
(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/L PBS;
(4) preparation of biotin labeled CA50 antibody
Get 0.5mg CA50 antibody, to dialyse at 2 ~ 8 DEG C 2h 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 50min 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 CA50 abzyme bond
After adopting sodium periodate oxidation that CA50 antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:6000 with enzyme dilution, and added 15% 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
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 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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| CN103777019A (en) * | 2014-01-26 | 2014-05-07 | 东北林业大学 | Hybridoma for generating anti-CA50 monoclonal antibody and preparation of chemiluminescence immune assay kit |
| CN106124776A (en) * | 2016-06-30 | 2016-11-16 | 深圳市亚辉龙生物科技股份有限公司 | CA724 chemiluminescence immune detection reagent kit and preparation method thereof |
| CN107490695B (en) * | 2017-07-10 | 2019-10-25 | 江苏福隆生物技术有限公司 | The board-like chemoluminescence method detection kit and preparation method of Carbohydrate Antigen 50 |
| CN109324194B (en) * | 2018-12-19 | 2022-04-08 | 迈克生物股份有限公司 | Reagent combination for detecting carbohydrate antigen 72-4 |
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| CN112326971A (en) * | 2020-10-20 | 2021-02-05 | 成都海默云因医学检验实验室有限公司 | Novel method and kit for NMDAR antibody quantitative detection |
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