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Preparation method for 25-hydroxyvitamin D3 antibody

A technology for the preparation of hydroxyvitamins and antibodies, which is applied in the field of cell engineering, can solve problems such as inability to obtain accurate tests, and achieve the effects of prevention and treatment of rickets, high potency, and strong binding force

Inactive Publication Date: 2019-01-04
NINGBO AUCHEER BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Now more and more companies are involved in the development of 25-hydroxyvitamin D3 detection kits. High-quality antibodies are the key technology and basis for the development of 25-hydroxyvitamin D3 detection kits, but the current 25-hydroxyvitamin D3 detection kits, no matter what Which technology, radioimmunoassay, colloidal gold, enzyme-linked immunoassay, chemiluminescent detection, magnetic particle immunoassay, cannot get the results comparable to the accurate test of HPLC, so it is necessary to further develop a 25-hydroxyvitamin D3 single Cloned antibody for use in immunoassay kits for rapid and accurate detection of 25-hydroxyvitamin D3

Method used

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Experimental program
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Effect test

Embodiment 1

[0017] A preparation method of 25-hydroxyvitamin D3 monoclonal antibody, comprising the steps of:

[0018] (1) Antigen preparation: Mix 100mg 25-hydroxyvitamin D3 protein with 1ml N, N dimethylamide, add 15ul triethylamine, mix well, react at 4°C for 2 hours, add 50ul isobutyl chloroformate, mix React at 4°C for 1 hour to obtain activated 25-hydroxyvitamin D3 protein; weigh 200mg OVA (ovalbumin) and dissolve it in 2ml ultrapure water, adjust the pH to 8.5, add 1ml N,N dimethylamide, mix well , react at 4°C for 2h to obtain activated ovalbumin; mix activated 25-hydroxyvitamin D3 protein with activated ovalbumin and react at 4°C for 4h, after the reaction is completed, dialyze with PBS buffer (50mM, pH=7.2) 6h, repeat twice, and then dialyze with ultrapure water for 10h, repeat twice to obtain 25-hydroxyvitamin D3 artificial antigen; because the molecular weight of 25-hydroxyvitamin D3 itself is too small, it is not immunogenic, and lacks T cell expression. 25-hydroxyvitamin D3...

Embodiment 2

[0030] Chromosomal analysis of hybridoma cells can be used to obtain one of the objective indicators of whether it is a real hybridoma cell. The chromosome number of hybridoma cells should be close to the sum of the chromosome numbers of the two parental cells. The specific method of hybridoma cell chromosome analysis is:

[0031] a. Passage the hybridoma cells 36-48 hours before adding colchicine;

[0032] b. Colchicine treatment: Add colchicine (100ug / ml, sterilized, stored at -20°C) to the culture flask to make the final concentration of colchicine 0.5ug / ml, continue to culture for 4 hours, and then pipette the cells , transferred to a centrifuge tube, centrifuged at 1000r / min for 10 minutes, discarded the supernatant;

[0033] c. Add 5ml of 0.075mol / L KCl solution preheated to 37°C, suspend and mix the precipitated cells, and bathe in 37°C water for 20 minutes;

[0034] d. Add 1ml of freshly prepared fixative to the cell suspension, mix well, then centrifuge at 1000r / min ...

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Abstract

The invention discloses a preparation method for a 25-hydroxyvitamin D3 antibody. The preparation method for the 25-hydroxyvitamin D3 antibody comprises the following steps: preparing artificial antigens, performing animal immunization and preparing spleen cells, myeloma cells and feeder cells, performing cell fusion, screening hybridoma cells and performing subcloning, purifying a 25-hydroxyvitamin D3 monoclonal antibody and the like, because the molecular weight of 25-hydroxyvitamin D3 is too small and the 25-hydroxyvitamin D3 has no immunogenicity, the 25-hydroxyvitamin D3 is coupled with ovalbumin to obtain the artificial antigens of the 25-hydroxyvitamin D3, the prepared 25-hydroxyvitamin D3 monoclonal antibody has the advantages of high titer, good affinity, strong binding force with25-hydroxyvitamin D3 antigens and the like, and can be used in immunofluorescence, enzyme-linked immunoassay or magnetic microparticle immunological techniques and the like to qualitatively or quantitatively detect the expression level of the 25-hydroxyvitamin D3 in human bodies, thereby being clinically assisted in prevention and treatment of rickets, osteomalacia and infantile tetany.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for preparing an antibody to 25-hydroxyvitamin D3. Background technique [0002] Serum 25-hydroxyvitamin D3 is the precursor for the synthesis of 25-OH-VD3 and the main circulating form of vitamin D. Its half-life is longer and its blood concentration is relatively stable, which can directly reflect the level of vitamin D in the body. Vitamin D participates in not only In recent years, vitamin D has been proved to have a wider range of biological and immunological effects, including the prevention and treatment of rickets, osteomalacia and infantile tetany, and the inhibition of the proliferation of various types of cells. , induce cell apoptosis and differentiation, regulate the function of the body's immune system, etc. Therefore, 25 hydroxyvitamin D3 in serum has become an important indicator of health checkup. [0003] Now more and more companies are invol...

Claims

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Application Information

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IPC IPC(8): C07K16/44C07K1/34C07K14/77C12N15/06
CPCC07K16/44C07K14/77C12N15/02
Inventor 唐静田青青陈星星周义正
Owner NINGBO AUCHEER BIOTECHNOLOGY CO LTD
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