CN102798726B - Cobalamin chemiluminescence immunoassay immue quantitative detection reagent box and preparation method thereof - Google Patents
Cobalamin chemiluminescence immunoassay immue quantitative detection reagent box and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of VB12 chemiluminescence immunoassay immue quantitative detection reagent box, described kit comprises VB12 antibody bag by plate, VB12 enzyme conjugates, VB12 calibration object, VB12 quality-control product, 20 times of concentrated washing lotion, luminescent solution A liquid and B liquid, sample treatment solution A liquid and B liquid.The invention also discloses a kind of preparation method of VB12 chemiluminescence immunoassay immue quantitative detection reagent box 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 and detects that the concentration range of sample is wide, the reagent term of validity long, good stability, low cost and other advantages.
Description
Technical field
The present invention relates to field of immunoassay medicine, be specifically related to cobalamin (VB12) chemiluminescence immunoassay immue quantitative detection reagent box and preparation method thereof.
Background technology
Cobalamin (vitamin B12, VB12) cobalamin is again, a kind of vitamin containing cobalt, all general names with the bioactive corrinoid of cyanocobalamin, the VB12 of occurring in nature is Microbe synthesis, high animals and plants can not manufacture VB12, and VB12 needs a kind of intestinal secretion thing (castle's intrinsic factor) to help the absorbed a kind of vitamin uniquely of ability.In addition, cobalamin is also unique containing must the vitamin of mineral matter, because taking on a red color containing cobalt, also known as red vitamin, is the coloured vitamin of minority.VB12 belongs to B group's vitamin, and storage capacity is little, and about 2 ~ 3mg is at liver.Body vitamin B12 requirement is few, as long as diet is normal, would not lack.Mainly discharge from urine, part is discharged from bile.
Its main Physiological Function comprises: 1. improve folic acid utilization factor, and when lacking VB12, the activity shifting methyl group from methyl tetrahydrofolate reduces, and makes folic acid become unavailable form, causes folic acid deficiency.2. metabolism and the function of neural myelin is safeguarded.When lacking VB12, neurological disorder, spinal cord degeneration can be caused, and serious mental symptom can be caused.VB12 lacks can cause peripheral neuritis.The Early manifestation that child lacks VB12 is abnormal feeling, have a dull expression on one's face, slow in reacting, finally cause anaemia.3. erythrocytic growth and maturation is promoted.Methylmalonyl CoA is changed into succinyl-coenzyme A, and participate in tricarboxylic acid cycle, wherein succinyl-coenzyme A is relevant with the synthesis of protoheme.4. participate in the synthesis of picodna (DNA), the metabolism of fat, carbohydrates and protein, increase the synthesis of nucleic acid and protein.
Cobalamin is required by body maintains eubolism, DNA synthesis and regeneration of erythrocytes.Vitamin B12 deficiency can not get correcting in time causing megaloblastic anemia and nonreversibility central lesion.Cobalamin or folic acid to finding out cobalamin, folic acid deficiency has diagnostic value, especially to the meaningful diagnostic method of the antidiastole of megaloblastic anemia
Current cobalamin clinically quantitatively detects common method to be had:
(1) radiating immuning analysis technology (RIA): clinical quantitative detecting method comparatively early, highly sensitive, specificity is high, but because there is radioactive contamination, and complex operation, market is atrophy gradually.
(2) enzyme-linked immunosorbent assay (ELISA): the analytical approach occurred after RIA, because of simple to operate, no radioactivity pollute, price is lower, has now certain market share, but ELISA sensitivity is not as RIA.
The advantages such as chemiluminescence immune assay (CLIA) is current microimmuno-assays the most responsive, has highly sensitive, good stability, pollution-free, have been widely applied to preclinical medicine and clinical medical every field at present, have become the one preferred technique replacing RIA.Chemiluminescence immune assay is started in the beginning of the eighties, fast development is applied to the nineties, principal feature is hypersensitivity, can measure that concentration range is wide, sample can detect without the need to dilution, the reagent term of validity is long, simple to operate, degree of changing into is high automatically, data automatic generating process ability is strong in detection, compatible good, the non-environmental-pollution of determining instrument etc., thus obtains and develops rapidly and application.But use the kit of chemiluminescence method detection VB 12 also less at present, clinical practice is less.
Summary of the invention
The problem to be solved in the present invention is to provide chemiluminescence immunoassay immue quantitative detection reagent box of cobalamin (VB12) and preparation method thereof, and solve sensitivity low, 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: VB12 chemiluminescence immunoassay immue quantitative detection reagent box, it is characterized in that, described kit comprises: VB12 antibody bag is by plate, VB12 antigen enzyme conjugates, VB12 calibration object, VB12 quality-control product, 20 times of concentrated washing lotion, luminescent solution A liquid and B liquid, sample treatment solution A liquid and B liquid.
Described VB12 antibody bag is 96 holes or the 48 holes white microwell plate that are coated with VB12 antibody by plate, and between hole, average variation (CV%) is not higher than 10%.
Described antigen enzyme conjugates enzyme used is horseradish peroxidase, purity requirement RZ >=3.0, activity >=250U/mL, available from Sigma.
VB12 antibody sources, in Fitzgerald company, answers clear in appearance, and without precipitation, purity answers>=90%, adopts doubling dilution to measure and tires and should be not less than 10
5doubly dilution, should guarantee sensitivity that VB12 measures, specificity and stability.
Described VB12 quality-control product comprises low value quality-control product (QcL) and high level quality-control product (QcH), and wherein the concentration range of low value quality-control product is 158.68 ~ 238.02pg/mL, and the concentration range of high level quality-control product is 1141.37 ~ 1712.05pg/mL.
Described VB12 chemiluminescence immunoassay immue quantitative detection reagent box, described luminescent solution A liquid comprises 0.7g/L luminol, 0.165g/L p-iodophenol; The urea peroxide aqueous solution of B liquid to be concentration be 0.675g/L.Sample treatment solution A liquid comprises 36g/L NaOH; Sample treatment solution B liquid comprises 10.66g/L MES(2-(N-morpholino) ethyl sulfonic acid), 1.03g/L DTT(dithiothreitol (DTT)) and, 1g/L Proclin300, pH5.5.
20 times of described concentrated washing lotions comprise 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/L Proclin300.
The preparation method of mentioned reagent box, comprises the following steps:
1) VB12 antibody bag is by the preparation of plate
VB12 antibody 0.05mol/L pH9.6 carbonate buffer solution is diluted to 1 ~ 10ug/mL, joins in white microwell plate, 37 DEG C of bags were by 2 hours; Discard liquid in hole, wash plate with pH7.4PBS damping fluid, then add the phosphate buffer closed porosity plate containing 0.5%BSA, close 2 hours for 37 DEG C; Discard liquid in hole, dry latter 37 DEG C and dry 4 hours; Load aluminium foil bag, add drying agent, sealing, labels, is stored in 2 ~ 8 DEG C;
2) adopt sodium periodate oxidation to prepare VB12 antigen-HRP enzyme conjugates, and in enzyme conjugates, add HRP stabilizing agent, label, be stored in 2 ~ 8 DEG C;
3) VB12 calibration object
VB12 1%BSA calibration object diluent preparing become concentration to be the calibration object of 0,100,200,500,1000,2000pg/mL, label, be stored in 2 ~ 8 DEG C;
4) preparation of VB12 quality-control product: normal human serum detects be negative (using the experimental technique through SFDA approval) through 56 DEG C of water-bath 30min deactivation and HBsAg, anti-HCV and anti-HIV after, and add Proclin300 as antiseptic.Low value quality-control product (QcL) and high level quality-control product (QcH), the mean value of its concentration is respectively 198.35pg/mL and 1426.71pg/mL;
5) luminescent solution A liquid and B liquid is prepared
A liquid is the 5mmol/LTrisHCl damping fluid of the pH8.6 containing 0.7g/L luminol and 0.165g/L p-iodophenol; The urea peroxide aqueous solution of B liquid to be concentration be 0.675g/L, prepares with process water;
6) 20 times of concentrated washing lotions are prepared
The formula of 20 times of concentrated washing lotions is as follows: 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300;
7) preparation of sample treatment solution
Sample treatment solution A comprises 36g/L NaOH; Sample treatment solution B comprises 10.66g/L MES(2-(N-morpholino) ethyl sulfonic acid), 1.03g/L DTT(dithiothreitol (DTT)) and, 1g/L Proclin300, pH5.5;
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.
ProClin300 is with its broad spectrum of activity, superior compatibility and stability and the hypotoxicity under working concentration thereof, and ProClin300 becomes the desirable efficient antiseptic for diagnostic reagent.Proclin300 antiseptic can within the longer time eradicate bacteria, fungi and yeast, thus extend storage time of product.It is water-soluble guarantees that it can dissolve in required reagent easily.Particularly, the anticorrosion function on most enzyme or antibody linked reaction of ProClin300 without impact, so can not interference test indicator.
Cobalamin (VB12) immue quantitative detection reagent box (chemoluminescence method) adopts competition law principle to measure VB12, sample is added in the batten micropore of the White-opalescent being coated with pig intrinsic factor, add humanized murine antibodies-horseradish peroxidase (HRP) bond again, if containing VB12 in sample, then form solid phase VB12 antibody-VB12-VB12 antibody-HRP compound, wash free composition off, 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 respectively adding sample aperture is measured in 5th ~ 15 minutes.RLU and the sample VB12 concentration of sample are negative correlation.VB12 concentration in sample is carried out quantitatively according to the mathematical model set up by calibration object VB12 concentration and corresponding RLU, thus detects the VB12 content in human serum, blood plasma.
Cobalamin (VB12) the chemiluminescent fluorescence immunoassay quantified detection kit of invention, adopts current the sensitiveest detection method---chemiluminescence immune assay, has advantage: (1) reaction fast, can judge testing result in 65 minutes; Easy and simple to handle, pollution-free.(2) highly sensitive, the sensitivity for analysis of this kit is not higher than 50pg/mL.(3) high specificity, is all less than 1% with the cross specificity of cobinamide.(4) precision is good, and withinrun precision is not higher than 15%, and betweenrun precision is not higher than 20%.(5) have good stability, this product can deposit more than 7 days at 37 DEG C, can deposit 1 year at 2 ~ 8 DEG C.(6) cost is low, and compare with like product on market, this kit is functional, and cost is low, has clinical value.
Accompanying drawing explanation
Fig. 1 is the measurement result comparison diagram that Bo Aosaisi chemical luminescence reagent kit of the present invention mensuration VB12 and radioimmunological kit measure VB12, wherein ordinate is the VB12 value that Bo Aosaisi records, horizontal ordinate is that radioimmunological kit measures VB12 value, two kinds of method related coefficient (r)=0.9571, straight-line equation y=1.049x-22.175.
Embodiment
Embodiment 1: preparation VB12 chemiluminescence immunoassay immue quantitative detection reagent box
VB12 chemiluminescence immunoassay immue quantitative detection reagent box, it is characterized in that, described kit comprises: VB12 antibody bag is by plate, VB12 antigen enzyme conjugates, VB12 calibration object, VB12 quality-control product, 20 times of concentrated washing lotion, luminescent solution A liquid and B liquid, sample treatment solution A liquid and B liquid.
VB12 chemiluminescence immunoassay immue quantitative detection reagent box is prepared by following method:
1) VB12 antibody bag is by the preparation of plate
VB12 antibody 0.05mol/L pH9.6 carbonate buffer solution is diluted to 1 ~ 10ug/mL, joins in white microwell plate, 37 DEG C of bags were by 2 hours; Discard liquid in hole, wash plate with pH7.4PBS damping fluid, then add the phosphate buffer closed porosity plate containing 0.5%BSA, close 2 hours for 37 DEG C; Discard liquid in hole, dry latter 37 DEG C and dry 4 hours; Load aluminium foil bag, add drying agent, sealing, labels, is stored in 2 ~ 8 DEG C;
2) adopt sodium periodate oxidation to prepare VB12 antigen-HRP enzyme conjugates, and in enzyme conjugates, add HRP stabilizing agent, label, be stored in 2 ~ 8 DEG C;
3) VB12 calibration object
VB12 1%BSA calibration object diluent preparing become concentration to be the calibration object of 0,100,200,500,1000,2000pg/mL, label, be stored in 2 ~ 8 DEG C;
4) preparation of VB12 quality-control product: normal human serum detects be negative (using the experimental technique through SFDA approval) through 56 DEG C of water-bath 30min deactivation and HBsAg, anti-HCV and anti-HIV after, and add Proclin300 as antiseptic.Low value quality-control product (QcL) and high level quality-control product (QcH), the mean value of its concentration is respectively 198.35pg/mL and 1426.71pg/mL;
5) luminescent solution A liquid and B liquid is prepared
A liquid is the 5mmol/LTrisHCl damping fluid of the pH8.6 having 0.7g/L luminol and 0.165g/L p-iodophenol; The urea peroxide aqueous solution of B liquid to be concentration be 0.675g/L, prepares with process water;
6) 20 times of concentrated washing lotions are prepared
The formula of 20 times of concentrated washing lotions is as follows: 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300;
7) preparation of sample treatment solution
Sample treatment solution A comprises 36g/L NaOH; Sample treatment solution B comprises 10.66g/L MES(2-(N-morpholino) ethyl sulfonic acid), 1.03g/L DTT(dithiothreitol (DTT)) and, 1g/L Proclin300, pH5.5;
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, linear, precision, the specificity of accuracy, dose-response curve, sensitivity and stability measure.
Illustrate: testing index
Physical examination: liquid component should be clarified, without precipitation or floccus; Other components should without packages in damaged condition.
Accuracy: kit calibration object and company standard product series are carried out analysis simultaneously and measured, with Log (X)-Logit (Y) Model fitting, require two dose-response curves remarkable parallel deviate (t checks, | t|<2.447); With company standard product for reference substance, with Log (X)-Logit (Y) Model fitting, the measured value of kit calibration object and the mean value of sign value ratio should in 0.90 ~ 1.10 scopes.
Enterprise's calibration object is that VB12 sterling calibration object dilution is made into series concentration, and concentration is respectively 0,100,200,500,1000,2000pg/mL, is its definite value, is stored in-20 DEG C according to VB12 Pharmacopoeial Reference Standards (Lot.O0H288).
Dose-response curve linear: with Log (X)-Logit (Y) Model fitting, dose-response curve correlation coefficient r in 0 ~ 2000pg/mL concentration range is not less than 0.9900.
Sensitivity for analysis: kit assay sensitivity is not higher than 50pg/mL.
Precision: in batch, imprecision (CV%) should not higher than 15%; Between batch, imprecision (CV%) should not higher than 20%.
Quality-control product measured value: the quality-control product of replicate determination 10 hole high level and low value, with Log (X)-Logit (Y) Model fitting, quality-control product measured value should in allowed band, and the allowed band of QcL and QcH is respectively 158.68 ~ 238.02pg/mL and 1141.37 ~ 1712.05pg/mL.
Specificity: cross reaction meets following table requirement;
Stability: place 7 days for 37 DEG C, measured value should meet above-mentioned requirements.
Embodiment 2: preparation VB12 chemiluminescence immunoassay immue quantitative detection reagent box
Except the white microwell plate that solid phase carrier is 48 holes, prepare kit of the present invention with method in the same manner as in Example 1.
Embodiment 3: the using method of kit of the present invention
1) kit to be checked is balanced 30 minutes under room temperature (18 ~ 25 DEG C).
2) washing lotion is prepared: washing lotion will be concentrated by 1:20 dilution (1mL washing lotion adds 19mL distilled water) with distilled water.If concentrated washing lotion has crystallization, dilute again after concentrated washing lotion can being placed in room temperature or 37 DEG C of dissolvings to be crystallized.
3) luminescent solution is prepared: use first 5 minutes and get appropriate luminescent solution A liquid and mix with B liquid equal-volume.
4) sample treatment solution is prepared: according to test hole count preparation sample treatment solution, every hole needs sample treatment solution A liquid 500 μ L, sample treatment solution B liquid 10 μ L.Can increase preparation in right amount, general polygamy system 10% is advisable.Get appropriate containers sample treatment solution A, B are mixed.The sample treatment solution prepared can deposit 24 hours at 2 ~ 8 DEG C.
5) sample process
A gets the centrifuge tube of suitable size, and often pipe adds calibration object, quality-control product or sample to be tested 50 μ L.
B often pipe adds sample treatment solution 500 μ L, vibration mixing.
C puts into boiling water bath (100 DEG C), reacts 15 ~ 20 minutes.
D takes out centrifuge tube, puts into normal temperature water-bath and cools 5 minutes, namely can be used for inspection.
6) experimentally need to take out appropriate coated slab.Blank 1 hole is set, each 2 holes of calibration object.Every hole adds VB12 calibration object, quality-control product or sample after process 100 μ L, and blank does not add calibration object, sample.
7) with cover plate film, plate hole is built, react 30 minutes at 37 DEG C.
8) throw off cover plate film, sucking-off or after pouring out reactant liquor, add washing lotion and wash five times, each every hole of washing lotion amount is no less than 300 μ L, soak time 10 seconds, sucking-off or pat dry after pouring out washing lotion.Also can with washing trigger washing.
9) every hole adds VB12 enzyme conjugates 100 μ L, except blank control wells.
10) with cover plate film, plate hole is built, react 30 minutes at 37 DEG C.
11) throw off cover plate film, sucking-off or after pouring out reactant liquor, add washing lotion and wash five times, each every hole of washing lotion amount is no less than 300 μ L, soak time 10 seconds, sucking-off or pat dry after pouring out washing lotion.Also can with washing trigger washing.
12) every hole adds luminescent solution 100 μ L, comprises blank control wells.
13) every hole adds luminescent solution 100 μ L, comprises blank control wells.
14) secretly 5min is put under room temperature (18 ~ 25 DEG C), chemical illumination immunity analysis instrument measures luminous value, then Log (X)-Logit (Y) Model fitting is adopted, obtain calibration object dose-response curve, VB12 concentration in sample and the relation between RLU are inversely proportional to, can from above-mentioned dose-response curve the anti-concentration value extrapolating VB12 in sample.
The clinical testing of embodiment 4 kits
The kit of invention has carried out clinical examination, total sample number 110 example of this clinical testing, first with after the test of VB12 radioimmunoassay kits, use the kit of invention (chemiluminescence) to measure again, and paired t-test, linear dependence analysis and Chi-square Test have been carried out to measurement result.Result shows, straight-line equation is y=1.049x-22.175, and linear regression coeffficient is 1.049, and related coefficient is R=0.9571.Linear regression coeffficient is 1.049 close to 1, illustrates that our kit and hospital's measured value have good consistance.Carry out t inspection (inspection level α=0.05) with SPSS13.0 statistical analysis software to related coefficient, P<0.001, the VB12 value of two kinds of method mensuration is closely related.Sensitivity (True Positive Rate) is 95.12%, specificity (true negative rate) is 97.10%, all higher; And false positive rate (misdiagnosis rate) be 2.90%, false negative rate (rate of missed diagnosis) is 4.88%, 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 96.36%, 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 522 portions of normal human serums, plasma sample, result shows that reference value (term of reference) (2.5% ~ 97.5%) of this kit is 175 ~ 880pg/mL.
Above-mentioned reference value is only for reference, advises that the clinical normal and pathology value scope of oneself should be set up in each laboratory.Sample near critical value should carry out second time and test confirmation, and carries out diplopore mensuration; Exceed the sample of this kit range of linearity, be the result of calculation drawn by calibration curve extension, result accurately be obtained, need measure after Sample Dilution.
Claims (2)
1.VB12 chemiluminescence immunoassay immue quantitative detection reagent box, described kit comprises: VB12 antibody bag is by plate, VB12 enzyme conjugates, VB12 calibration object, VB12 quality-control product, 20 times of concentrated washing lotions, luminescent solution A liquid and B liquid, sample treatment solution A liquid and B liquid, it is characterized in that: described VB12 antibody bag is 96 holes or the 48 holes white microwell plate that are coated with VB12 antibody by plate, between the hole of white microwell plate, average variation is not higher than 10%, bag is that VB12 antibody 0.05mol/L pH9.6 carbonate buffer solution is diluted to 1 ~ 10 μ g/mL by plate, join in white microwell plate, 37 DEG C of bags were by 2 hours, discard liquid in hole, wash plate with pH7.4PBS damping fluid, then add the phosphate buffer closed porosity plate containing 0.5%BSA, close 2 hours for 37 DEG C, discard liquid in hole, dry rear 37 DEG C dry 4 hours obtained, described antigen enzyme conjugates enzyme used is horseradish peroxidase HRP, purity requirement RZ>=3.0, activity>=250U/mL, described VB12 antibody purity>=90%, tires and is not less than 10
5doubly dilution, antigen enzyme conjugates adopts sodium periodate oxidation to prepare VB12 antigen-HRP enzyme conjugates, and in enzyme conjugates, add HRP stabilizing agent, described VB12 quality-control product comprises low value quality-control product and high level quality-control product, and wherein the concentration of low value quality-control product is 198.35pg/mL, the concentration 1426.71pg/mL of high level quality-control product, normal human serum after 56 DEG C of water-bath 30min deactivation and HBsAg, anti-HCV and anti-HIV detect and be negative, and is added Proclin 300 as antiseptic by the preparation of VB12 quality-control product, described luminescent solution A liquid is the 5mmol/LTrisH Cl damping fluid of the pH8.6 containing 0.7g/L luminol and 0.165g/L p-iodophenol, the urea peroxide aqueous solution of B liquid to be concentration be 0.675g/L, prepares with process water, sample treatment solution A liquid comprises 36g/L NaOH, sample treatment solution B liquid comprises 10.66g/L MES, 1.03g/L DTT, 1g/L Proclin300, pH5.5, 20 times of described concentrated washing lotions comprise 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300, VB12 calibration object becomes concentration to be respectively the calibration object of 0,100,200,500,1000,2000pg/mL VB12 1%BSA calibration object diluent preparing, labels, be stored in 2 ~ 8 DEG C.
2. prepare a method for the kit of described claim 1, it is characterized in that comprising the following steps:
1) VB12 antibody bag is by the preparation of plate
VB12 antibody 0.05mol/L pH9.6 carbonate buffer solution is diluted to 1 ~ 10 μ g/mL, joins in white microwell plate, 37 DEG C of bags were by 2 hours; Discard liquid in hole, wash plate with pH7.4PBS damping fluid, then add the phosphate buffer closed porosity plate containing 0.5%BSA, close 2 hours for 37 DEG C; Discard liquid in hole, dry latter 37 DEG C and dry 4 hours; Load aluminium foil bag, add drying agent, sealing, labels, is stored in 2 ~ 8 DEG C;
2) adopt sodium periodate oxidation to prepare VB12 antigen-HRP enzyme conjugates, and in enzyme conjugates, add HRP stabilizing agent, label, be stored in 2 ~ 8 DEG C;
3) VB12 calibration object
Become concentration to be respectively the calibration object of 0,100,200,500,1000,2000pg/mL VB12 1%BSA calibration object diluent preparing, label, be stored in 2 ~ 8 DEG C;
4) preparation of VB12 quality-control product: normal human serum after 56 DEG C of water-bath 30min deactivation and HBsAg, anti-HCV and anti-HIV detection are negative, and adds Proclin 300 as antiseptic; Low value quality-control product and high level quality-control product, the mean value of its concentration is respectively 198.35pg/mL and 1426.71pg/mL;
5) luminescent solution A liquid and B liquid is prepared
A liquid is the 5mmol/LTrisHCl damping fluid of the pH8.6 containing 0.7g/L luminol and 0.165g/L p-iodophenol; The urea peroxide aqueous solution of B liquid to be concentration be 0.675g/L, prepares with process water;
6) 20 times of concentrated washing lotions are prepared
The formula of 20 times of concentrated washing lotions is as follows: 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300;
7) preparation of sample treatment solution
Sample treatment solution A comprises 36g/L NaOH; Sample treatment solution B comprises 10.66g/L MES, 1.03g/L DTT, 1g/L Proclin300, pH5.5;
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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| CN108490195A (en) * | 2018-03-30 | 2018-09-04 | 北京丹大生物技术有限公司 | A kind of vitamin B12 immunization assay method and its reagent |
| CN110967493B (en) * | 2018-09-29 | 2022-08-02 | 深圳天辰医疗科技有限公司 | Vitamin B12 determination kit and preparation method thereof |
| CN110455785A (en) * | 2019-08-08 | 2019-11-15 | 郑州安图生物工程股份有限公司 | A kind of chemiluminescent substrate method of inspection |
| CN110452968B (en) * | 2019-08-22 | 2023-07-21 | 甘肃省食品检验研究院(甘肃省动物源性食品基因检测中心) | Method for detecting vitamin B12 in food based on quantitative DNA technology |
| CN113495158A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Sample pretreatment agent, vitamin B12 quantitative detection kit and use method |
| CN116718710A (en) * | 2023-08-09 | 2023-09-08 | 江苏硕世生物科技股份有限公司 | Kit and method for detecting folic acid and metabolite thereof |
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| Comparison of a Microbiological Assay and a fully automated chemiluminescent system for the determination of vitamin B12 in food;Fumino Watanabe,et al;《J.Agric.Food.Chem.》;19981231;第46卷;1433-1436 * |
| Enhancement of chemiluminescence for vitamin B12 analysis;Sagaya Selva Kumar,et al;《Analytical Biochemistry》;20090515;第388卷;312-316 * |
| 化学发光免疫分析法检测黄体生成素、维生素B12和癌胚抗原的性能评价;罗福东等;《江西医学检验》;20061031;第24卷(第05期);431-432 * |
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