CN103575914B - The enzyme linked immunological kit of detection vitamin B12 and application thereof - Google Patents
The enzyme linked immunological kit of detection vitamin B12 and application thereof Download PDFInfo
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Abstract
The invention provides a kind of enzyme linked immunological kit detecting vitamin B12, it contains: be coated with the ELISA Plate of vitamin B12 coupled antigen, enzyme labelling anti antibody working solution, vitamin B12 specific antibody working solution, vitamin B12 standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, redissolution liquid.The invention also discloses a kind of method applying above-mentioned enzyme linked immunological kit detection vitamin B12, it includes: first carries out sample pre-treatments, then detects with test kit, ultimate analysis testing result.The enzyme linked immunological kit that the present invention provides can be used for detecting cereals (Semen Maydis flour, soy noodle, Semen setariae powder, rice flour), milk, the content of vitamin B12 in milk powder, it is easy and simple to handle, low cost, highly sensitive, can on-site supervision and the examination of applicable great amount 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 detecting vitamin B12, it is the mensuration of vitamin B12 be applicable to cereals (Semen Maydis flour, soy noodle, Semen setariae powder, rice flour), milk, milk powder.
Technical background
Vitamin is a class low-molecular-weight organic compound necessary to various biological maintenance normal physiological function, they great majority are the main coenzyme during biochemical metabolism, participate in vivo, regulate, control many synthesis and catabolism, when organism is deficient in vitamin, will releaser dysbolismus, cause multiple disease.Vitamin B12 is again cobalamine, and its main Physiological Function is to participate in manufacturing erythrocyte, prevents pernicious anemia, prevents cerebral nerve from being destroyed.Vitamin B12 is for promoting growth of animals or poultry particularly promoting that effect is notable in terms of young stock growth promoter, mainly play a role with participation one carbon unit and propanoic acid metabolism in poultry body, affect the synthesis of hereditary material, moreover it is possible to maintain the integrity of function of nervous system, be the indispensable nutrient raw material of poultry.During breeding production, the vitamin B12 in conventional feed is generally as safety using amount, and extra appropriate vitamin B12 of adding can improve livestock and poultry production performance, brings considerable economic benefit.
In recent years, remaining Lin Liang etc. reports and measures biotin with HPLC, and Deng Fuliang etc. measures biotin and folic acid in Vitamin H with HPLC simultaneously, and Ren Yiping, dragon Chaoyang etc. are with the folic acid in HPLC mensuration food, and Yang Yi etc. measures vitamin B12 with HPLC.The Ion-pair HPLC content of vitamin B12 and folic acid such as Sui Yurong, H.B.Li, Heudi different wave length determines biotin, folic acid and vitamin B12 simultaneously, Song Jinchun etc. are by optimizing chromatographic condition, and under the conditions of establishing Same Wavelength, HPLC measures biotin in Vitamin H, folic acid and the method for vitamin B12 content simultaneously.Additionally the assay method of vitamin B12 mainly has colorimetry, electrochemical methods, ion exchange, laser resonant Raman spectroscopy etc., and these methods are all first to measure Co content in sample, cobalt content be converted into the content of VB12, and complex operation, selectivity is poor.Liu Baosheng etc. establish the new method utilizing AO-R6G energy transfer fluorescent quencher method to measure vitamin B12.Immunological detection method is also applied in the detection of vitamin B12, uses the method that indirect competitive ELISA method measures vitamin B12 content, easy, quick, highly sensitive, and good stability can meet the rapid screening of the on-the-spot batch samples of vitamin B12.
Summary of the invention
Present invention aims to a kind of enzyme linked immunological kit for vitamin B12 assay of above-mentioned not enough offer, the screening of its applicable on-the-spot batch samples simple to operate.
Test kit of the present invention, it includes:
(1) ELISA Plate of vitamin B12 coupled antigen it is coated with；
(2) vitamin B12 specific antibody；
(3) enzyme labelling anti antibody；
(4) vitamin B12 standard solution；
(5) substrate nitrite ion；
(6) stop buffer；
(7) concentrated cleaning solution；
(8) redissolution liquid.
Described vitamin B12 coupled antigen is the conjugate of vitamin B12 hapten and carrier protein, and described carrier protein can be ovalbumin, bovine serum albumin, hemocyanin, thyroprotein, Mus serum albumin, human albumin.
The haptenic structure of described vitamin B12 is as follows:
。
Described vitamin B12 hapten preparation process: 0.50g vitamin B12,0.10g succinic anhydrides and 1ml pyridine mixed liquor in 20mlDMSO, stirring reaction 40 hours at 80 DEG C, solvent is evaporated off, phthalic acid list vitamin B12 ester is obtained, for vitamin B12 hapten after ethanol-water system repeatedly recrystallization.
The marker enzyme of described enzyme labelling anti antibody is horseradish peroxidase；Marker enzyme and anti antibody are carried out coupling and obtain by enzyme labelling anti antibody employing Over-voltage protection.Described anti antibody is sheep anti mouse anti antibody.
Described vitamin B12 specific antibody is vitamin B12 monoclonal antibody, is that immunogen immune animal obtains with vitamin B12 coupled antigen；Described vitamin B12 specific antibody can be Mus source, Ma Yuan, Yang Yuan or rabbit source antibody, and described vitamin B12 monoclonal antibody is preferably vitamin B12 mouse monoclonal antibody.
For more convenient on-site supervision and great amount of samples examination, described test kit also includes vitamin B12 standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, redissolution liquid.
Described stop buffer is sulphuric acid or the hydrochloric acid solution of 1 ~ 2mol/L, and described substrate nitrite ion is made up 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 0.5% ~ 1.0% tween 20 and the phosphate buffer of 0.1 ~ 0.2mol/L of the sodium azide preservatives of 0.01 ‰ ~ 0.05 ‰, described percentage ratio is weight percentage.
Described redissolution liquid be pH value be 7.0 ~ 7.2, containing 5% ~ 10% casein, the phosphate buffer of 0.02mol/L, described percentage ratio is weight percentage.
The buffer that is coated that wherein ELISA Plate is used in preparation process is pH9.0 ~ 9.6, and 0.1 ~ 0.2mol/L carbonate buffer solution, confining liquid used is pH7.2 ~ 7.6,0.1mol/L phosphate buffer, and described percentage ratio is weight percentage.
In the present invention, the preparation process of ELISA Plate is: with being coated buffer, envelope antigen is diluted to 0.1 ~ 0.2 μ g/ml, every hole adds 100 μ l, 37 DEG C of incubation 2h or 4 DEG C overnight, incline and are coated liquid, wash 2 times with cleaning mixture, 30s every time, pat dry, in every hole, then add 150 ~ 200 μ l confining liquids, 37 DEG C of incubation 1 ~ 2h, the liquid in hole that inclines pats dry, and dried sealing by aluminum film vacuum preserves.
The Cleaning Principle of the present invention is:
When pre-coated vitamin B12 coupled antigen on capillary strip, after adding sample solution or standard solution, add vitamin B12 specific antibody solution, vitamin B12 in sample and coated vitamin B12 coupled antigen competition vitamin B12 specific antibody in ELISA Plate, add enzyme labelling anti antibody and be amplified effect, developing the color with substrate nitrite ion, sample light absorption value is negative correlation with the content of vitamin B12, compares with standard curve and can draw the content of vitamin B12 in sample.Simultaneously according to the depth of color in ELISA Plate, the comparison with the vitamin B12 standard solution color of series concentration can the concentration range of vitamin B12 in judgement sample roughly.
Present invention also offers a kind of method applying above-mentioned enzyme linked immunological kit detection vitamin B12, it includes step:
(1) sample pre-treatments；
(2) detect with test kit；
(3) testing result is analyzed.
The present invention detects the enzyme linked immunological kit of vitamin B12 and mainly uses the content of vitamin B12 in indirect competitive ELISA method detection sample；Pre-treatment to sample requires low, and sample pretreatment process is simple, can quickly detect batch samples simultaneously；Using the vitamin B12 monoclonal antibody of high specific, main agents provides with the form of working solution, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.The enzyme linked immunological kit of the present invention, simple in construction, easy to use, carrying convenience, detection method efficiently, accurately, easy, be suitable to batch samples screening.
Accompanying drawing explanation
Fig. 1: vitamin B12 hapten synthesis figure
The canonical plotting of Fig. 2: test kit
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate the present invention, and be not limited to the scope of the present invention.
The preparation of embodiment 1 reagent constituents
1, prepared by vitamin B12 hapten
0.50g vitamin B12,0.10g succinic anhydrides and 1ml pyridine mixed liquor in 20mlDMSO, at 80 DEG C, stirring reaction 40 hours, are evaporated off solvent, obtain phthalic acid list vitamin B12 ester, for vitamin B12 hapten after ethanol-water system repeatedly recrystallization.
2, the preparation of antigen
Immunogen is prepared vitamin B12 hapten and is obtained immunogen with bovine serum albumin (BSA) coupling
1) take 20mg hapten, be dissolved in 1mlN, in dinethylformamide (DMF)；
2) take 30mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS) fully dissolve with 0.2ml water after add above-mentioned 1) in, stir 24h under room temperature, i.e. can get reactant liquor A；
3) weigh BSA50mg, be allowed to be substantially dissolved in 3.8mL0.01mol/LPBS(pH7.2) in, reactant liquor A is dropwise slowly dropped in protein solution, and stirs 24h at room temperature；
4) dialyse 3 days with 0.01mol/LPBS4 DEG C, change 3 dialysis solution every day, to remove unreacted small-molecule substance.Subpackage, saves backup in-20 DEG C.
Coating antigen is prepared vitamin B12 hapten and is obtained coating antigen with ovalbumin (OVA) coupling
BSA50mg ibid, is replaced with OVA50mg by preparation method.
3, the preparation of vitamin B12 monoclonal antibody
A. animal immune
Immunizing antigen above-mentioned steps obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/ so that it is produce antiserum.
B. cell merges and cloning
Take immunity Balb/c mouse boosting cell, in 9:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, screening obtains the vitamin B12 monoclonal hybridoma strain of stably excreting vitamin B12 monoclonal antibody.
C. cell cryopreservation and recovery
Hybridoma frozen stock solution is made 1 × 109The cell suspension of individual/ml, preserves in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
D. the preparation of monoclonal antibody and purification
Increment culture method: be placed in cell culture medium by hybridoma, cultivates under the conditions of 37 DEG C, is purified by the culture fluid obtained by octanoic acid-saturated ammonium sulfate method, obtains monoclonal antibody ,-20 DEG C of preservations.
4, the preparation of sheep anti mouse anti antibody
Using sheep as immune animal, for immunogen, pathogen-free domestic sheep is carried out immunity with Mus source antibody, obtain sheep anti mouse anti antibody.
5, the preparation of ELISA Plate
With being coated buffer, envelope antigen is diluted to 0.1 ~ 0.2 μ g/ml, every hole adds 100 μ l, 37 DEG C of incubation 2h or 4 DEG C overnight, incline and are coated liquid, wash 2 times with cleaning mixture, 30s every time, pat dry, in every hole, then add 150 ~ 200 μ l confining liquids, 37 DEG C of incubation 2h, the liquid in hole that inclines pats dry, and dried sealing by aluminum film vacuum preserves.
6, the preparation of enzyme labelling sheep anti mouse anti antibody
Over-voltage protection after anti antibody is used improvement with horseradish peroxidase (HRP) carries out coupling.Traditional Over-voltage protection requires that in reaction system, the molar concentration rate of enzyme and anti antibody is 4:1；Owing to horseradish peroxidase produces many sites being combined with anti antibody under the effect of Strong oxdiative, so horseradish peroxidase molecule of activation act as connecting the bridge of each molecule, reduce the enzymatic activity of enzyme marker, make the conjugate of preparation is mixed with many polymers, in order to solve this problem, traditional method is improved by we, it may be assumed that
1) closed process of amino is eliminated, because the amino that can produce the connection of self amino is actual seldom.
2) reducing horseradish peroxidase: the molar concentration ratio of anti antibody to 2:1, the method after improvement is easier than traditional method, and the loss to the activity of enzyme reduces.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of vitamin B12
Set up the enzyme linked immunological kit of detection vitamin B12 so that it is comprise following component:
(1) ELISA Plate of vitamin B12 coupled antigen it is coated；
(2) vitamin B12 monoclonal antibody working solution；
(3) with the sheep anti mouse anti antibody of horseradish peroxidase-labeled；
(4) vitamin B12 standard solution 5 bottles, 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 made up 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 sulphuric acid；
(7) concentrated cleaning solution is pH7.2 ~ 7.6, and containing 0.5% ~ 1.0% tween 20 and the phosphate buffer of 0.1 ~ 0.2mol/L of the sodium azide preservatives of 0.01 ‰ ~ 0.05 ‰, described percentage ratio is weight percentage.
(8) redissolve liquid be pH value be 7.0 ~ 7.2, containing 5% ~ 10% casein, the phosphate buffer of 0.02mol/L, described percentage ratio is weight percentage.
The detection of vitamin B12 in embodiment 3 sample
1. sample pre-treatments
The pre-treatment of sample is primarily to obtain vitamin B12 solution from sample, thus is used for follow-up detection.The pre-treating method of sample be presented herein below:
(1) corn (Semen Maydis flour, soy noodle, Semen setariae powder, rice flour)
Weigh the equal pledge of 1.0g ± 0.05g in 10ml polystyrene centrifuge tube, add 4ml0.8mol/L phosphate buffered solution, be centrifuged 5min by agitator 3min, more than 3000g room temperature of vibrating；Take 100 μ l supernatant to add 400 μ l and redissolve liquid, vibrate 3min with agitator, mixing；Take 50 μ l for analyzing.
(2) milk powder
Weigh the equal pledge of 1.0g ± 0.05g in 10ml polystyrene centrifuge tube, add 4ml10%NaCl solution, vibrate 3min with agitator immediately；Make it be completely dissolved；Take the 100 above-mentioned lysates of μ l to add 400 μ l and redissolve liquid, vibrate 3min with agitator, mixing；Take 50 μ l for analyzing.
(3) raw milk
Directly put plate analysis.
(4) finished milk
Take 50 μ l milk samples and add 450 μ l redissolution liquid mixings；Take 50 μ l for analyzing.
2. detect with test kit
nullVitamin B12 standard solution/sample 50 μ l is added in the ELISA Plate micropore be coated with vitamin B12 coupled antigen，Add vitamin B12 monoclonal antibody working solution 50 μ l，With cover plate molding plate，25 DEG C of lucifuge reaction 30min，Every hole adds 250 μ l cleaning mixture，Liquid in hole is poured out after 30s，Repeat operation and wash plate 4-5 time altogether，Pat dry with absorbent paper，Add horseradish peroxidase-labeled sheep anti mouse anti antibody working solution 100 μ l，Vibrate gently mixing，With cover plate molding plate，25 DEG C of calorstats react 30min，Pour out liquid in hole，Every hole adds 250 μ l cleaning mixture，Liquid in hole is poured out after 30s，Repeat operation and wash plate 4-5 time altogether，Pat dry with absorbent paper，Every hole adds substrate solution A liquid urea peroxide 50 μ l，Substrate solution B liquid tetramethyl benzidine (TMB) 50 μ l，Vibrate gently mixing，25 DEG C of calorstat lucifuge colour developing 15min，Every hole adds 2mol/L stop buffer sulphuric acid 50 μ l，Vibrate gently mixing，It is set at 450nm with microplate reader wavelength，Measure every hole absorbance (OD value).
3. Analysis of test results
With the absorbance values (B) of the standard solution of each concentration obtained divided by the absorbance (B of first standard solution (0 standard)0) it is multiplied by 100% again, obtain percentage absorbance.With the logarithm value of vitamin B12 standard concentration (μ g/L) as X-axis, percentage absorbance is Y-axis, draws canonical plotting.Calculate the percentage absorbance of sample solution, the concentration of each sample corresponding by same way, then can read the content of vitamin B12 from standard curve.
Embodiment 4 vitamin B12 enzyme linked immunological kit sensitivity, preci-sion and accuracy, storage life experiment
1. test kit sensitivity and detection limit
Conventionally measure test kit sensitivity test, test kit sensitivity is 0.2 μ g/L, respectively 20 parts of dummies (cereals, milk, milk powder) are detected, the concentration corresponding to each percentage absorptance is found from standard curve, detection limit is represented plus 3 times of standard deviations with the meansigma methods of 20 parts of sample vitamin B12 concentration, result obtains the method and cereals (Semen Maydis flour, soy noodle, Semen setariae powder, rice flour) detection is limited to 4 μ g/kg, milk powder detection is limited to 4 μ g/kg, detection to raw milk is limited to 1 μ g/L, and the detection to finished milk is limited to 2 μ g/L.
2. test kit accuracy and precision
Using the testing result coefficient of variation (CV%) of a certain concentration samples of replication as precision evaluation index.Using the response rate as accuracy estimating index.Coefficient of variation CV% computing formula is: CV(%)=SD/X × 100%；Wherein SD is standard deviation, and X is the meansigma methods of determination data.Response rate computing formula is: the response rate (%)=practical measurement value/theoretical value × 100%.The wherein interpolation concentration of theoretical value analog sample.
By 8 μ g/kg, two concentrations of vitamin B12 of 16 μ g/kg, Semen Maydis flour, soy noodle, Semen setariae powder, rice flour, powdered milk sample are added reclaiming and measure, it is added reclaiming mensuration to raw milk sample by 2 μ g/L, two concentrations of vitamin B12 of 4 μ g/L, it is added reclaiming mensuration to finished milk sample by 4 μ g/L, two concentrations of vitamin B12 of 8 μ g/L, each sample do 4 parallel, being measured with three batches of different test kits, the average recovery rate and the precision result that calculate sample see table.
Table 1 precision and accuracy test
Being added cereals (Semen Maydis flour, soy noodle, Semen setariae powder, rice flour) sample with the vitamin B12 of 8,16 two concentration of μ g/kg, average recovery rate is between 78.5% ~ 93.6%；Being added powdered milk sample with the vitamin B12 of 8,16 two concentration of μ g/kg, average recovery rate is between 76.5% ~ 85.3%；Being added raw milk sample with the vitamin B12 of 2,4 two concentration of μ g/L, average recovery rate is between 76.8% ~ 88.3%；Being added finished milk sample with the vitamin B12 of 4,8 two concentration of μ g/L, average recovery rate is between 86.6% ~ 90.8%；The coefficient of variation is respectively less than 20%.The preci-sion and accuracy of testing result all meets relevant criterion requirement.
3. test kit storage life test
Test kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of test kit, 50% inhibition concentration were all within normal range.Considering, during transport and using, to have improper preservation condition and occur, being placed 7 days under 37 DEG C of preservation conditions by test kit, be accelerated senile experiment, result shows that this test kit indices complies fully with requirement.Occurring in view of test kit freezing situation, test kit is put into-20 DEG C of refrigerator freezings 7 days, measurement result also indicates that test kit indices is the most normal.Can show that test kit can be able to preserve 12 months at 2 ~ 8 DEG C from result above.
Claims (7)
1. the enzyme-linked immunologic detecting kit detecting vitamin B12, it is characterised in that include:
(1) ELISA Plate of vitamin B12 coupled antigen it is coated with；
(2) vitamin B12 specific antibody；
(3) enzyme labelling anti antibody；
(4) vitamin B12 standard solution；
(5) substrate nitrite ion；
(6) stop buffer；
(7) concentrated cleaning solution；
(8) redissolution liquid；
Wherein, described vitamin B12 coupled antigen is to be obtained with carrier protein couplet by vitamin B12 hapten；The haptenic preparation method of described vitamin B12 mainly comprises the steps: 0.50g vitamin B12,0.10g phthalic anhydride and 1mL pyridine mixed liquor in 20mLDMSO, stirring reaction 40 hours at 80 DEG C, solvent is evaporated off, obtain phthalic acid list vitamin B12 ester after ethanol-water system repeatedly recrystallization, be vitamin B12 hapten；Described vitamin B12 hapten structure is as follows:
2. test kit as claimed in claim 1, it is characterised in that described vitamin B12 specific antibody is vitamin B12 monoclonal antibody.
3. test kit as claimed in claim 1, it is characterised in that the marker enzyme of described enzyme labelling anti antibody is horseradish peroxidase；Enzyme labelling anti antibody is to use Over-voltage protection to be obtained with anti antibody coupling by marker enzyme.
4. test kit as claimed in claim 1, it is characterized in that: described stop buffer is sulphuric acid or the hydrochloric acid solution of 1～2mol/L, described substrate nitrite ion is made up 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.
5. test kit as claimed in claim 1, it is characterized in that described concentrated cleaning solution is pH7.2～7.6, the phosphate buffer of 0.1～0.2mol/L containing the tween 20 that percentage by weight is 0.5%～1.0% and sodium azide preservatives that percentage by weight is 0.01 ‰～0.05 ‰, described redissolution liquid be pH value be 7.0～7.2, containing the casein that percentage by weight is 5%～10%, the phosphate buffer of 0.02mol/L.
6. test kit as claimed in claim 1, it is characterised in that described vitamin B12 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.
7. detecting a method for vitamin B12 content in sample, key step includes:
1) testing sample is carried out pre-treatment；
2) detect with the enzyme linked immunological kit described in any one of claim 1-6；
3) testing result is analyzed.
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| CN106841017B (en) * | 2017-01-22 | 2019-05-07 | 耐斯检测技术服务有限公司 | A method of evaluation irradiation is heat-treated on cobalamin-protein binding body influence |
| 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 |
| 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 |
| CN111665241B (en) * | 2020-06-12 | 2023-03-28 | 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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| 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 |
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