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Vitamin B12 immunization assay method and reagent thereof

A technology of reagents and preservatives, applied in the field of medical immunology in vitro diagnosis, to achieve the effect of improving practicability, improving specific recognition ability and high immunogenicity

Inactive Publication Date: 2018-09-04
BEIJING DIAGREAT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, there is a lack of highly sensitive and specific VB12 detection reagents on the market, especially reagents suitable for automated testing

Method used

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  • Vitamin B12 immunization assay method and reagent thereof
  • Vitamin B12 immunization assay method and reagent thereof
  • Vitamin B12 immunization assay method and reagent thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Synthesis and structure confirmation of VB12 derivatives

[0037] The biochemical structures of the VB12 derivatives used in the following examples are as follows: figure 1 Shown:

[0038] The specific synthetic steps of this VB12 derivative are as follows: figure 2 Shown:

[0039] 1) Weigh 23.7mg of VB12 and 22mg of succinic anhydride, add 3.0mL of anhydrous pyridine, reflux at 80°C for 2h, remove pyridine by rotary evaporation, add 4mL of ultrapure water, extract with 5mL×4 ethyl acetate, combine the organic phases, and subtract Press and spin dry to obtain a light red solid, namely the VB12 derivative (Formula 4).

[0040] Structural identification of the above light red solid purified product

[0041] 1) The obtained derivatives were analyzed and identified by chromatography / mass spectrometry (LCMS). Agilent's tandem quadrupole mass spectrometer LC / MSD1200 series was used, and the ion source was in positive or negative ionization mode. The chromatog...

Embodiment 2

[0043] Example 2: RSA-VB12 immunogen synthesis

[0044] The RSA-VB12 immunogen is composed of rabbit protein RSA and -CO-(CH 2 )n-COO-group connected, in the present embodiment, take n=2 as an example to describe the synthetic method of this immunogen in detail, and concrete steps are as follows:

[0045] 1) Dissolve 60mg of RSA in 6ml of 0.1M PBS with pH8.3, and place the above solution in beaker A;

[0046] 2) Mix 50mg of VB12 derivatives, 0.6ml of dimethylamide DMF, 0.5ml of ethanol, 1ml of 10mM MES buffer at pH5.0, 68mg of 1-ethyl-3-(-3-dimethylaminopropyl) The imine and 22 mg of N-hydroxysulfosuccinimide were dissolved in beaker B, and the reaction was stirred at room temperature for 1 hour.

[0047] 3) Slowly add the solution in beaker B to beaker A dropwise to obtain a mixed solution, and stir overnight at 2-8°C; dialyze and purify the above stirred mixed solution with neutral PBS buffer to obtain RSA-VB12 immune Original, stored at -70°C.

[0048] Similarly, when n...

Embodiment 3

[0050] Embodiment three: the preparation of anti-VB12 specific antibody

[0051] The RSA-VB12 immunogen that above-mentioned makes adopts conventional method to immunize New Zealand white rabbit, and after strengthening immunization, antiserum is taken, and concrete steps are as follows:

[0052] The RSA-VB12 immunogen synthesized above was diluted to 2.0 mg / ml with PBS to obtain an antigen solution, and then 1.0 ml of the antigen solution was mixed with Freund's complete adjuvant to subcutaneously immunize the back of New Zealand white rabbits at multiple points.

[0053] After 2 to 3 weeks, the New Zealand white rabbits were immunized once with 1.0 ml of the same antigen solution and Freund's incomplete adjuvant, and then immunized every three weeks for a total of 4 immunizations.

[0054] Blood was collected from the above New Zealand white rabbits, and the anti-VB12 specific antibody was obtained by separation and purification.

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Abstract

The invention discloses a method for immunological detection of a homogeneous enzyme assay technology and a reagent thereof. The method is used for assaying the content of a vitamin B12 (Vitamin B12,VB12) in a sample. The method is capable of adopting an anti-VB12 specific antibody and an enzyme-VB12 compound, and using an immunology reaction of the enzyme-VB12 compound and the anti-VB12 specificantibody, after forming an antigen-antibody compound, inhibiting the activity of the enzyme-VB12 compound, and through assaying the change of the enzymatic activity in a reaction system, acquiring the number of combined enzyme markers, thereby acquiring the content of the VB12 in the sample to be tested. The advantage of the method is that the immunology assay can be realized as a general biochemistry enzyme method, so that multiple examples are simultaneously assayed in a full-automatic biochemical analyzer.

Description

technical field [0001] The invention relates to the directional connection of small molecules and proteins, small molecule components and detection methods, and belongs to the field of medical immune in vitro diagnosis. It is a homogeneous immunological enzymatic assay. In particular, it relates to a detection method and the preparation of a kit, especially a VB12 detection reagent and its preparation and detection method. Background technique [0002] Vitamin B12, also known as cyanocobalamin, cobalamin, exogenous factor, and animal protein factor, is the only vitamin containing minerals. It is an essential nutrient obtained by the human body through diet, participates in various metabolic processes, and promotes red blood cells. maturation and maintenance of the normal function of the nervous system. Its deficiency can cause various clinical manifestations such as fatigue, dizziness and taste loss, but because the symptoms are scattered and easily overlooked, the detecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82
CPCG01N33/82
Inventor 许秀丽吴鸣月王艳新
Owner BEIJING DIAGREAT BIOTECH CO LTD
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