Enzyme linked immunosorbent assay kit for detecting vitamin B12 and application thereof

An enzyme-linked immunoassay and vitamin technology, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of cumbersome operation and poor selectivity, and achieve the effects of high sensitivity, high accuracy and high precision

Active Publication Date: 2014-02-12
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the determination methods of vitamin B12 mainly include colorimetric method, electrochemical analysis method, ion exchange method, laser resonance Raman spectroscopy, etc. These methods are all to measure the Co content in the sample first, convert the cobalt content into the VB12 content, and operate cumbersome, less selective

Method used

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  • Enzyme linked immunosorbent assay kit for detecting vitamin B12 and application thereof
  • Enzyme linked immunosorbent assay kit for detecting vitamin B12 and application thereof
  • Enzyme linked immunosorbent assay kit for detecting vitamin B12 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Preparation of kit components

[0037] 1. Preparation of vitamin B12 hapten

[0038] A mixture of 0.50g vitamin B12, 0.10g succinic anhydride and 1ml pyridine in 20ml DMSO was stirred and reacted at 80°C for 40 hours, the solvent was evaporated, and phthalic acid monovitamin B12 was obtained after multiple recrystallizations in the ethanol-water system Ester, a vitamin B12 hapten.

[0039] 2. Antigen preparation

[0040] Immunogen preparation—vitamin B12 hapten coupled with bovine serum albumin (BSA) to obtain immunogen

[0041] 1) Take 20mg hapten and dissolve it in 1ml N,N-dimethylformamide (DMF);

[0042] 2) Take 30mg of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) fully dissolved in 0.2ml of water, add to the above 1), and stir at room temperature for 24 hours to obtain the reaction solution A;

[0043] 3) Weigh 50mg of BSA, fully dissolve it in 3.8mL 0.01mol / L PBS (pH 7.2), slowly add the reaction solution A to the protein solution drop by dro...

Embodiment 2

[0064] Example 2 Formation of ELISA Kit for Detecting Vitamin B12

[0065] Set up the ELISA kit for detecting vitamin B12 to include the following components:

[0066] (1) ELISA plate coated with vitamin B12-conjugated antigen;

[0067] (2) Vitamin B12 monoclonal antibody working solution;

[0068] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0069] (4) 5 bottles of vitamin B12 standard solution, the concentrations are 0μg / L, 0.2μg / L, 0.6μg / L, 1.8μg / L, 5.4μg / L;

[0070] (5) The substrate chromogenic solution is composed of substrate solution A and substrate solution B, the substrate solution A is carbamide peroxide, and the substrate solution B is tetramethylbenzidine;

[0071] (6) The stop solution is 2mol / L sulfuric acid;

[0072] (7) The concentrated washing solution is pH 7.2~7.6, 0.1~0.2mol / L phosphate buffer containing 0.5%~1.0% Tween-20 and 0.01‰~0.05‰ sodium azide preservative, so The stated percentages are percentages by weight.

[00...

Embodiment 3

[0074] Example 3 Detection of vitamin B12 in samples

[0075] 1. Sample pretreatment

[0076] The pretreatment of the sample is mainly to obtain vitamin B12 solution from the sample for subsequent detection. The following is the sample pretreatment method:

[0077] (1) Cereals (corn flour, soybean flour, millet flour, rice flour)

[0078] Weigh 1.0g±0.05g homogeneous substance into a 10ml polystyrene centrifuge tube, add 4ml 0.8mol / L phosphate buffer solution, shake with an oscillator for 3min, centrifuge at room temperature above 3000g for 5min; take 100μl supernatant and add 400μl complex Solution, oscillate with an oscillator for 3min, mix well; take 50μl for analysis.

[0079] (2) milk powder

[0080] Weigh 1.0g±0.05g of homogeneous material into a 10ml polystyrene centrifuge tube, add 4ml of 10% NaCl solution, and immediately shake it with an oscillator for 3 minutes; make it completely dissolved; take 100μl of the above solution and add 400μl of the complex solution,...

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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 [0001] The present invention relates to enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting vitamin B12, which is suitable for the detection of vitamin B12 in cereals (corn flour, soybean flour, millet flour, rice flour), milk and milk powder Determination. technical background [0002] Vitamins are a class of low-molecular organic compounds necessary for various organisms to maintain normal physiological functions. Most of them are the main coenzymes in the process of biochemical metabolism. They participate in, regulate, and control many synthesis and catabolism in the body. When organisms lack vitamins , It will cause substance metabolism disorders and cause various diseases. Vitamin B12 is also called cobalamin, and its main physiological function is to participate in the production of bone marrow red blood cells, prevent pernicious anemia, and prevent brain nerves from being damaged. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82G01N33/558
CPCG01N33/531G01N33/535G01N33/82
Inventor 万宇平吴鹏罗晓琴冯静杨秀贤韩雪倩顾蓉蓉蒲小容
Owner BEIJING KWINBON BIOTECH
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