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Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses

A monoclonal antibody and adenosine isotype technology, applied in the field of immunology, can solve the problems of inapplicability, unsuitability for measuring methylation index, and inability to use antibodies to measure plasma samples, etc.

Active Publication Date: 2015-04-01
泰州汇丰合泰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lower limit of detection of the rabbit anti-SAH monoclonal antibody is higher than 260nM, which is relatively high, which limits the need for highly sensitive detection
This antibody cannot be used for the determination of plasma samples. In the competitive ELISA, we observed non-specific inhibitory reactions, making this antibody unusable for EDTA anticoagulated plasma samples
Therefore, this commercially available monoclonal antibody is not suitable for measuring methylation index due to the limitation of lower sensitivity and its inapplicability to plasma samples

Method used

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  • Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses
  • Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses
  • Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1: the preparation of antigen SAH-BSA

[0084] The chemical reagents used in this embodiment and the material purchase route are as follows:

[0085]SAH was purchased from Chengdu Novi Biotechnology Co., Ltd.; BSA, bovine serum albumin, was purchased from SIGMA; EDC.HCl was purchased from SIGMA; N-hydroxysuccinimide was purchased from SIGMA; 12000, purchased from Beijing Boao Tuoda Technology Co., Ltd.; and 10mMPBS phosphate buffer (pH 7.4) was self-prepared.

[0086] 5mgSAH, 28.8mgBSA, 14.96mgEDC.HCl, 5.3mgN-hydroxysuccinimide and 5mL (10mM, pH7.4)PBS were sequentially added into the reaction flask, and reacted for 6 hours under magnetic stirring. The reactant was put into a dialysis bag and dialyzed against pH 7.4, 10 mM PBS for 24 hours, during which the solution was changed 3 times. After the dialysis, the volume and concentration are determined, subpackaged, and frozen for future use, so as to prepare the BSA-SAH aqueous solution.

Embodiment 2

[0087] Example 2: Preparation of hybridomas and monoclonal antibodies

[0088] The 8-week-old female BALB / C mice used were purchased from the Institute of Zoology, Chinese Academy of Sciences.

[0089] The test reagents used: Freund's complete adjuvant, purchased from SIGMA Company; BSA, bovine serum albumin, purchased from SIGMA Company; BSA-SAH aqueous solution, prepared according to Example 1; RPMI1640 solution, purchased from GIBCO Company; PEG4000, Purchased from Beijing Baierdi Biological Company; SP2 / 0 myeloma cells, purchased from Shanghai Yanyu Biotechnology Co., Ltd.; polylysine-SAH conjugated antigen, purchased from Sigma; enzyme-linked plate, purchased from Beijing Bay Erdi Company; goat anti-mouse-HRP secondary antibody, which was purchased from Nanjing Shengxing Biotechnology; HAT selection medium, which was purchased from SIGMA Company; mineral oil, which was purchased from SIGMA Company; bovine serum albumin-conjugated SAH, which was purchased from Sigma Cor...

Embodiment 3

[0117] Embodiment 3: the cross reaction test of monoclonal antibody 301

[0118] The source of the chemical reagent used in the present embodiment is as follows:

[0119]S-adenosylhomocysteine ​​standard (SAHNa): purchased from Sigma-Aldrich; S-adenosylmethionine (SAM): purchased from Sigma-Aldrich; adenosine (Ade): purchased from Sigma-Aldrich company; Homocysteine ​​(H-Cys): purchased from Sigma-Aldrich company; L-cysteine ​​(L-Cys): glutathione (GST): purchased from Sigma- Aldrich Company; cystathionine (L-CTT): purchased from Sigma-Aldrich Company; BSA-SAH antigen: prepared according to the method of Example 1; HRP-coupled goat anti-mouse IgG antibody: 1:2000 diluted anti- SAH monoclonal antibody: obtained by diluting the anti-SAH monoclonal antibody prepared according to the method in Example 2.

[0120] The 96-well plate was coated with 0.5 μg / ml BSA-SAH antigen (BSA-SAH was dissolved in 50 mM carbonate buffer solution, pH 9.6, and 100 ul was added to the 96-well pla...

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Abstract

The invention provides a monoclonal antibody 301 specifically binding S-adenosyl-L-homocysteine or an antigen-binding site of the monoclonal antibody, a hybridomas generating the monoclonal antibody, a composition or a kit comprising the monoclonal antibody 301 or the antigen-binding site, and test paper comprising the monoclonal antibody 301 or the antigen-binding site of the monoclonal antibody. The invention also provides a method of achieving non-treatment or diagnosis objectives of detecting existence of the S-adenosyl-L-homocysteine or the level of the S-adenosyl-L-homocysteine in a sample by using the monoclonal antibody 301 or the antigen-binding site, the composition or the kit, and provides uses of the method. A cell strain and a product thereof allow an immune method to be capable of detecting the methylation index (namely a ratio of S-ademetionine to the S-adenosyl-L-homocysteine) in a metabolic activity process of life for the first time. The obtained anti-S-adenosyl-L-homocysteine monoclonal antibody cell strain will have high practicability and a good application prospect.

Description

technical field [0001] The present invention relates to the field of immunology. Specifically, the present invention mainly relates to monoclonal antibodies or functional fragments thereof, hybridomas, compositions, kits and uses thereof that can specifically recognize and bind to S-adenosylhomocysteine. Background technique [0002] 1. S-adenosylhomocysteine [0003] The methionine molecule contains a sulfur (S) methyl group. After reacting with ATP to generate -adenosyl-L-methionine (S-Adenosyl-L-methionine, SAM), it can be converted into about 50 kinds of known substances in the body through various transmethylation effects. Important physiologically active substances (DNA, RNA, proteins, phospholipids, hormones, neurotransmitters, etc.) provide methyl groups. SAM is the only methyl provider in the regulation of methylation of important physiologically active substances in these life activities. After S-adenosylmethionine transfers the methyl group to another substance...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 郝秀娟任会明肖平李惠军甘鸿杰周雅娴夏嘉志郝广钧
Owner 泰州汇丰合泰生物技术有限公司
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