CN103575914A - Enzyme linked immunosorbent assay kit for detecting vitamin B12 and application thereof - Google Patents
Enzyme linked immunosorbent assay kit for detecting vitamin B12 and application thereof Download PDFInfo
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- CN103575914A CN103575914A CN201210273343.6A CN201210273343A CN103575914A CN 103575914 A CN103575914 A CN 103575914A CN 201210273343 A CN201210273343 A CN 201210273343A CN 103575914 A CN103575914 A CN 103575914A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention provides an enzyme linked immunosorbent assay (ELISA) kit for detecting vitamin B12. The enzyme linked immunosorbent assay kit comprises an ELISA plate coated with a vitamin B12 coupling antigen, an enzyme labeling ant-antibody working solution, a vitamin B12 specific antibody working solution, a vitamin B12 standard substance solution, substrate developing liquid, a stop solution, a concentration scrubbing solution and redissolution liquid. The invention also discloses a method for detecting the vitamin B12 by using the enzyme linked immunosorbent assay kit. The method comprises the following steps: firstly carrying out sample pretreatment, then detecting by using the kit, and finally analyzing detection results. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of vitamin B12 in cereals (maize meal, soybean meal, millet meal and rice meal), milk and milk powder, is simple and convenient to operate, low in cost and high in sensitivity, can realize on-site monitoring and is suitable for screening lots of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detection of cobalamin, it is applicable to the mensuration of cobalamin in cereals (corn flour, soy noodle, little rice and flour, rice flour), milk, milk powder.
Technical background
Vitamin is that various biologies maintain the necessary class low-molecular-weight organic compound of normal physiological function, their great majority are the main coenzyme in biochemical metabolism process, participate in vivo, regulate, control much synthetic and kalabolism, when biosome is deficient in vitamin, will releaser dysbolism, cause various diseases.Cobalamin is again cobalamin, and its main Physiological Function is to participate in manufacturing erythrocyte, prevents pernicious anaemia, prevents that cerebral nerve is damaged.Cobalamin for promote growth of animals or poultry particularly promote cub grow aspect effect remarkable, in livestock and poultry body, mainly to participate in one carbon unit and propionic acid metabolism, play a role, affecting the synthetic of inhereditary material, can also maintain the integrality of nervous function, is the indispensable nutrient raw material of livestock and poultry.In breeding production process, the cobalamin in conventional feed, generally as safety using amount, additionally adds appropriate vitamin B12 and can improve livestock and poultry production performance, brings considerable economic benefit.
In recent years, Yu Linliang etc. have reported with HPLC and have measured biotin, Deng Fuliang etc. measure biotin and folic acid in Vitamin H with HPLC simultaneously, and the folic acid in food is measured with HPLC in arbitrary flat, imperial Chaoyang etc., and Yang Yi etc. measure cobalamin with HPLC.Sui Yurong etc. with Ion-pair HPLC the content of cobalamin and folic acid, H. B.Li, Heudi have measured biotin, folic acid and cobalamin with different wave length simultaneously, Song Jinchun etc. are by optimizing chromatographic condition, have set up the method that HPLC under Same Wavelength condition measures biotin in Vitamin H, folic acid and cobalamin content simultaneously.The assay method of cobalamin mainly contains colourimetry, electrochemical methods, ion exchange process, laser resonant Raman spectroscopy etc. in addition, these methods are all Co content in first working sample, by cobalt content, be converted into the content of VB12, complex operation, selectivity is poor.Liu Baosheng etc. have set up and have utilized AO-R6G energy to shift the new method that fluorescence quenching method is measured cobalamin.Immunological detection method is also applied in the detection of cobalamin, adopts indirect competitive ELISA method to measure the method for cobalamin content, easy, quick, highly sensitive, and good stability can meet the rapid screening of the on-the-spot batch samples of cobalamin.
Summary of the invention
The object of the invention is to provides a kind of enzyme linked immunological kit for cobalamin assay, the screening of its applicable on-the-spot batch samples simple to operate for above-mentioned deficiency.
Kit of the present invention, it comprises:
(1) be coated with the ELISA Plate of cobalamin coupled antigen;
(2) cobalamin specific antibody;
(3) enzyme labeling antiantibody;
(4) cobalamin standard solution;
(5) substrate nitrite ion;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) redissolution liquid.
Described cobalamin coupled antigen is the conjugate of cobalamin haptens and carrier protein, and described carrier protein can be ovalbumin, bovine serum albumin(BSA), hemocyanin, thyroprotein, mouse haemocyanin, human albumin.
The haptenic structure of described cobalamin is as follows:
Described cobalamin haptens preparation process: 0.50g cobalamin, 0.10g succinic anhydride and the mixed liquor of 1ml pyridine in 20ml DMSO, at 80 ℃, stirring reaction is 40 hours, steaming desolventizes, ethanol-water system repeatedly obtains phthalic acid list cobalamin ester after recrystallization, is cobalamin haptens.
The marker enzyme of described enzyme labeling antiantibody is horseradish peroxidase; Enzyme labeling antiantibody adopts sodium periodate method that marker enzyme and antiantibody are carried out to coupling and obtains.Described antiantibody is sheep anti mouse antiantibody.
Described cobalamin specific antibody is cobalamin monoclonal antibody, with cobalamin coupled antigen, is that immunogen immune animal obtains; Described cobalamin specific antibody can be mouse source, Ma Yuan, Yang Yuan or rabbit source antibody, and described cobalamin monoclonal antibody is preferably cobalamin mouse monoclonal antibody.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises cobalamin standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, redissolution liquid.
Described stop buffer is sulfuric acid or the hydrochloric acid solution of 1 ~ 2mol/L, and described substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is urea peroxide solution, and substrate solution B liquid is tetramethyl biphenyl amine aqueous solution.
Described concentrated cleaning solution is pH7.2 ~ 7.6, and containing the phosphate buffer of 0.1 ~ 0.2mol/L of 0.5% ~ 1.0% Tween-20 and 0.01 ‰ ~ 0.05 ‰ sodium azide antiseptic, described number percent is weight percentage.
Described redissolution liquid is that pH value is 7.0 ~ 7.2, the phosphate buffer that contains 5% ~ 10% casein, 0.02mol/L, and described number percent is weight percentage.
Wherein ELISA Plate coated damping fluid used in preparation process is pH9.0 ~ 9.6,0.1 ~ 0.2mol/L carbonate buffer solution, and confining liquid used is pH7.2 ~ 7.6,0.1mol/L phosphate buffer, described number percent is weight percentage.
In the present invention, the preparation process of ELISA Plate is: with coated damping fluid, envelope antigen is diluted to 0.1 ~ 0.2 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 150 ~ 200 μ l confining liquids, 37 ℃ of incubation 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
Detection principle of the present invention is:
When pre-coated cobalamin coupled antigen on capillary strip, add after sample solution or standard solution, add again cobalamin specific antibody solution, cobalamin coupled antigen coated in cobalamin in sample and ELISA Plate is competed cobalamin specific antibody, add enzyme labeling antiantibody to carry out amplification, with the colour developing of substrate nitrite ion, the content of sample light absorption value and cobalamin is negative correlation, relatively can draw the content of cobalamin in sample with typical curve.Simultaneously according to the depth of color in ELISA Plate, with the comparison of the cobalamin standard solution color of the series concentration concentration range of cobalamin in judgement sample roughly.
The present invention also provides a kind of method that above-mentioned enzyme linked immunological kit detects cobalamin of applying, and it comprises step:
(1) sample pre-treatments;
(2) with kit, detect;
(3) analyzing and testing result.
The enzyme linked immunological kit that the present invention detects cobalamin mainly adopts indirect competitive ELISA method to detect the content of cobalamin in sample; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously fast detecting batch samples; The cobalamin monoclonal antibody that adopts high specific, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, carrying convenience, detection method be efficient, accurate, easy, be suitable for batch samples screening.
Accompanying drawing explanation
Fig. 1: cobalamin haptens composite diagram
Fig. 2: the canonical plotting of kit
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only for the present invention is described, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 kit components
1, cobalamin haptens preparation
0.50g cobalamin, 0.10g succinic anhydride and the mixed liquor of 1ml pyridine in 20ml DMSO, at 80 ℃, stirring reaction is 40 hours, steaming desolventizes, and ethanol-water system repeatedly obtains phthalic acid list cobalamin ester after recrystallization, is cobalamin haptens.
2, the preparation of antigen
Immunogene preparation---cobalamin haptens and bovine serum albumin(BSA) (BSA) coupling obtains immunogene
1) get 20mg haptens, be dissolved in 1ml DMF (DMF);
2) get after 30mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS) fully dissolve with 0.2ml water and add above-mentioned 1) in, under room temperature, stir 24 h, can obtain reactant liquor A;
3) take BSA50mg, make it to be fully dissolved in 3.8mL 0.01mol/L PBS(pH 7.2) in, reactant liquor A is dropwise slowly added drop-wise in protein solution, and stirs 24 h under room temperature;
4) with 4 ℃ of dialysis of 0.01mol/L PBS 3 days, change dislysate every day 3 times, to remove unreacted small-molecule substance.Packing, saves backup in-20 ℃.
Coating antigen preparation---cobalamin haptens and ovalbumin (OVA) coupling obtains coating antigen
Preparation method is the same, and BSA 50mg is replaced with to OVA 50mg.
3, the preparation of cobalamin monoclonal antibody
A. animal immune
The immunizing antigen that above-mentioned steps is obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, in 9:1(quantitative proportion) ratio and SP2/0 myeloma cell's fusion, screening obtains the cobalamin monoclonal hybridoma strain of stably excreting cobalamin monoclonal antibody.
C. cell cryopreservation and recovery
Hybridoma is made to 1 * 10 with cryopreserving liquid
9the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.During recovery, take out cryopreservation tube, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
D. the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in to cell culture medium, cultivates under 37 ℃ of conditions, by sad-saturated ammonium sulfate method, the nutrient solution obtaining is carried out to purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
4, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, and the mouse source antibody of take carries out immunity as immunogene to pathogen-free domestic sheep, obtains sheep anti mouse antiantibody.
5, the preparation of ELISA Plate
With coated damping fluid, envelope antigen is diluted to 0.1 ~ 0.2 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 150 ~ 200 μ l confining liquids, 37 ℃ of incubation 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
6, the preparation of enzyme labeling sheep anti mouse antiantibody
Adopt the sodium periodate method after improvement to carry out coupling antiantibody and horseradish peroxidase (HRP).In traditional sodium periodate method requirement reaction system, the molar concentration rate of enzyme and antiantibody is 4:1; Because horseradish peroxidase produces many sites of being combined with antiantibody under the effect of strong oxidation, the horseradish peroxidase molecule of activation has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, in the conjugate that makes to prepare, be mixed with many condensates, in order to address this problem, we improve traditional method, that is:
1) saved amino closed process, because can produce self amino amino reality connecting seldom.
2) reduced horseradish peroxidase: the volumetric molar concentration ratio of antiantibody is to 2:1, and the method after improvement is easier than traditional method, the active loss of enzyme has been reduced.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of cobalamin
Set up the enzyme linked immunological kit that detects cobalamin, make it comprise following component:
(1) ELISA Plate of coated cobalamin coupled antigen;
(2) cobalamin monoclonal antibody working fluid;
(3) use the sheep anti mouse antiantibody of horseradish peroxidase-labeled;
(4) cobalamin standard solution is 5 bottles, and concentration is respectively 0 μ g/L, 0.2 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L;
(5) substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is urea peroxide, and substrate solution B liquid is tetramethyl benzidine;
(6) stop buffer is 2mol/L sulfuric acid;
(7) concentrated cleaning solution is pH7.2 ~ 7.6, and containing the phosphate buffer of 0.1 ~ 0.2mol/L of 0.5% ~ 1.0% Tween-20 and 0.01 ‰ ~ 0.05 ‰ sodium azide antiseptic, described number percent is weight percentage.
(8) redissolution liquid is that pH value is 7.0 ~ 7.2, the phosphate buffer that contains 5% ~ 10% casein, 0.02mol/L, and described number percent is weight percentage.
The detection of cobalamin in embodiment 3 samples
1. sample pre-treatments
The pre-treatment of sample is mainly in order to obtain cobalamin solution from sample, thereby for follow-up detection.The pre-treating method of sample below:
(1) cereal (corn flour, soy noodle, little rice and flour, rice flour)
Take the equal pledge of 1.0g ± 0.05g to 10ml polystyrene centrifuge tube, add 4ml 0.8mol/L phosphate buffered solution, with the oscillator 3min that vibrates, the centrifugal 5min of the above room temperature of 3000g; Get 100 μ l supernatants and add 400 μ l to redissolve liquid, with the oscillator 3min that vibrates, mix; Get 50 μ l for analyzing.
(2) milk powder
Take the equal pledge of 1.0g ± 0.05g to 10ml polystyrene centrifuge tube, add 4ml 10%NaCl solution, immediately with the oscillator 3min that vibrates; It is dissolved completely; Get the above-mentioned lysate of 100 μ l and add 400 μ l to redissolve liquid, with the oscillator 3min that vibrates, mix; Get 50 μ l for analyzing.
(3) former milk
Directly put plate analysis.
(4) finished milk
Getting 50 μ l milk samples adds 450 μ l and redissolves liquid and mix; Get 50 μ l for analyzing.
2. with kit, detect
To being coated with in the ELISA Plate micropore of cobalamin coupled antigen, add cobalamin standard solution/sample 50 μ l, add again cobalamin monoclonal antibody working fluid 50 μ l, with cover plate mould shrouding, 25 ℃ of lucifuge reaction 30min, every hole adds 250 μ l cleansing solutions, after 30s, pour out liquid in hole, repeat operation altogether and wash plate 4-5 time, with thieving paper, pat dry, add horseradish peroxidase-labeled sheep anti mouse antiantibody working fluid 100 μ l, vibration mixes gently, with cover plate mould shrouding, in 25 ℃ of constant temperature ovens, react 30min, pour out liquid in hole, every hole adds 250 μ l cleansing solutions, after 30s, pour out liquid in hole, repeat operation altogether and wash plate 4-5 time, with thieving paper, pat dry, every hole adds substrate solution A liquid urea peroxide 50 μ l, substrate solution B liquid tetramethyl benzidine (TMB) 50 μ l, vibration mixes gently, 25 ℃ of constant temperature oven lucifuge colour developing 15min, every hole adds 2mol/L stop buffer sulfuric acid 50 μ l, vibration mixes gently, by microplate reader wavelength set at 450nm place, measure every hole absorbance (OD value).
3. Analysis of test results
Absorbance (B with the absorbance mean value (B) of the standard solution of each obtained concentration divided by first standard solution (0 standard)
0) be multiplied by again 100%, obtain percentage absorbance.The logarithm value of cobalamin standard items concentration (μ g/L) of take is X-axis, and percentage absorbance is Y-axis, drawing standard curve map.By the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, can read from typical curve the content of cobalamin.
Embodiment 4 cobalamin enzyme linked immunological kit sensitivity, preci-sion and accuracy, storage life experiment
1. kit sensitivity and detectability
According to conventional method, measure kit sensitivity test, kit sensitivity is 0.2 μ g/L, respectively to 20 parts of dummy (cereals, milk, milk powder) detect, from typical curve, find the concentration corresponding to each percentage absorptance, mean value with 20 increment aetiocobalamin concentration adds that 3 times of standard deviations represent detectability, result obtains the method to cereals (corn flour, soy noodle, little rice and flour, rice flour) detect and be limited to 4 μ g/kg, milk powder is detected and is limited to 4 μ g/kg, the detection of former milk is limited to 1 μ g/L, the detection of finished milk is limited to 2 μ g/L.
2. kit accuracy and precision
The testing result coefficient of variation (CV%) of a certain concentration sample of the replication of usining is as precision evaluation index.Using the recovery as accuracy estimating index.Coefficient of variation CV% computing formula is: CV(%)=SD/ X * 100%; Wherein SD is standard deviation, the mean value that X is determination data.Recovery computing formula is: the recovery (%)=practical measurement value/theoretical value * 100%.The interpolation concentration that wherein theoretical value is analog sample.
By 8 μ g/kg, two concentration cobalamins of 16 μ g/kg, corn flour, soy noodle, little rice and flour, rice flour, powdered milk sample are added to reclaim and measure, by 2 μ g/L, two concentration cobalamins of 4 μ g/L, former milk sample is added to reclaim and measure, by 4 μ g/L, two concentration cobalamins of 8 μ g/L, finished milk sample is added to reclaim and measure, each sample do 4 parallel, with three batches of different kits, measure, average recovery rate and the precision of calculation sample the results are shown in following table.
Table 1 precision and accuracy test
Cobalamin with two concentration of 8,16 μ g/kg adds cereals (corn flour, soy noodle, little rice and flour, rice flour) sample, and average recovery rate is between 78.5% ~ 93.6%; Cobalamin with two concentration of 8,16 μ g/kg adds powdered milk sample, and average recovery rate is between 76.5% ~ 85.3%; Cobalamin with two concentration of 2,4 μ g/L adds former milk sample, and average recovery rate is between 76.8% ~ 88.3%; Cobalamin with two concentration of 4,8 μ g/L adds finished milk sample, and average recovery rate is between 86.6% ~ 90.8%; The coefficient of variation is all less than 20%.The preci-sion and accuracy of testing result all meets relevant criterion requirement.
3. kit storage life test
Kit preservation condition is 2 ~ 8 ℃, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration were all within normal range.Consideration, in transportation and use procedure, has improper preservation condition and occurs, kit is placed 7 days under 37 ℃ of preservation conditions, carries out accelerated deterioration experiment, and result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 ℃ of refrigerator freezings 7 days, measurement result also shows that kit indices is completely normal.From above result, can show that kit can preserve 12 months at 2 ~ 8 ℃.
Claims (10)
1. detect an enzyme-linked immunologic detecting kit for cobalamin, it is characterized in that including:
(1) be coated with the ELISA Plate of cobalamin coupled antigen;
(2) cobalamin specific antibody;
(3) enzyme labeling antiantibody;
(4) cobalamin standard solution;
(5) substrate nitrite ion;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) redissolution liquid.
2. kit as claimed in claim 1, is characterized in that described cobalamin coupled antigen is to be obtained by cobalamin haptens and carrier protein couplet, and described cobalamin haptens structure is as follows:
3. kit as claimed in claim 2, is characterized in that: described cobalamin is haptenic preparation method mainly comprise the steps:
0.50g cobalamin, 0.10g succinic anhydride and the mixed liquor of 1ml pyridine in 20ml DMSO, at 80 ℃, stirring reaction is 40 hours, and steaming desolventizes, and ethanol-water system repeatedly obtains phthalic acid list cobalamin ester after recrystallization.
4. kit as claimed in claim 1, is characterized in that described cobalamin specific antibody is cobalamin monoclonal antibody.
5. kit as claimed in claim 1, the marker enzyme that it is characterized in that described enzyme labeling antiantibody is horseradish peroxidase; Enzyme labeling antiantibody is to adopt sodium periodate method that marker enzyme and antiantibody coupling are obtained.
6. kit as claimed in claim 1, it is characterized in that: the sulfuric acid that described stop buffer is 1 ~ 2mol/L or hydrochloric acid solution, described substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, and described substrate solution A liquid is urea peroxide solution, and substrate solution B liquid is tetramethyl biphenyl amine aqueous solution.
7. kit as claimed in claim 1, is characterized in that coated damping fluid used is pH9.0 ~ 9.6,0.1 ~ 0.2mol/L carbonate buffer solution, and confining liquid used is pH7.2 ~ 7.6,0.1mol/L phosphate buffer, described number percent is weight percentage.
8. kit as claimed in claim 1, it is characterized in that described concentrated cleaning solution is pH7.2 ~ 7.6, phosphate buffer containing 0.1 ~ 0.2mol/L of 0.5% ~ 1.0% Tween-20 and 0.01 ‰ ~ 0.05 ‰ sodium azide antiseptic, described redissolution liquid is that pH value is 7.0 ~ 7.2, the phosphate buffer that contains 5% ~ 10% casein, 0.02mol/L, described number percent is weight percentage.
9. kit as claimed in claim 1, is characterized in that described cobalamin standard solution concentration is respectively 0 μ g/L, 0.2 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L.
10. a method that detects cobalamin content in sample, key step comprises:
1) testing sample is carried out to pre-treatment;
2) with the enzyme linked immunological kit described in claim 1-9 any one, detect;
3) analyzing and testing result.
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| CN106841017A (en) * | 2017-01-22 | 2017-06-13 | 耐斯检测技术服务有限公司 | It is a kind of to evaluate irradiation or be heat-treated the method influenceed on cobalamin protein binding body |
| CN110441536A (en) * | 2019-08-17 | 2019-11-12 | 宁波奥丞生物科技有限公司 | Detect the chemical luminescence reagent kit of vitamin B12 |
| CN110452968A (en) * | 2019-08-22 | 2019-11-15 | 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) | A method of food vitamins B12 is detected based on quantitative DNA technique |
| CN111665241A (en) * | 2020-06-12 | 2020-09-15 | 苏州良辰生物仪器试剂有限公司 | Tyrosine detection test strip and preparation method and application thereof |
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4082738A (en) * | 1975-02-25 | 1978-04-04 | Rohm And Haas Company | Cyanocobalamin derivatives |
| WO1993014405A1 (en) * | 1992-01-06 | 1993-07-22 | Baxter Diagnostics Inc. | Binding protein capture assay |
| US5332678A (en) * | 1989-01-11 | 1994-07-26 | Boehringer Mannheim Gmbh | Process for liberating an analyte from its binding protein |
| US5451508A (en) * | 1989-01-11 | 1995-09-19 | Boehringer Mannheim Gmbh | Method and monoclonal antibodies for vitamin B12 determination |
| US6942977B1 (en) * | 1991-04-09 | 2005-09-13 | Bio-Rad Laboratories, Inc. | Immunoassays for determining vitamin b12, and reagents and kits therefor |
| CN1766629A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting chloramphenicols in animal derived food |
| CN1766624A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting furazolidone metabolites and detection method thereof |
| CN1766618A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting penicillin G and detection method thereof |
| CN101021536A (en) * | 2007-03-12 | 2007-08-22 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunological kit for detecting neomycin drug and method |
-
2012
- 2012-08-02 CN CN201210273343.6A patent/CN103575914B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4082738A (en) * | 1975-02-25 | 1978-04-04 | Rohm And Haas Company | Cyanocobalamin derivatives |
| US5332678A (en) * | 1989-01-11 | 1994-07-26 | Boehringer Mannheim Gmbh | Process for liberating an analyte from its binding protein |
| US5451508A (en) * | 1989-01-11 | 1995-09-19 | Boehringer Mannheim Gmbh | Method and monoclonal antibodies for vitamin B12 determination |
| US5614394A (en) * | 1989-01-11 | 1997-03-25 | Boehringer Mannheim Gmbh | Method and monoclonal antibodies for vitamin B12 determination |
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