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A real-time assay method and kit for methyltransferase activity

A methyltransferase and real-time assay technology, which is applied to measuring devices, instruments, scientific instruments, etc., can solve problems such as product instability, detection value deviation, and many influencing factors, and achieve accurate MT activity determination and accurate and timely results. Effect

Active Publication Date: 2019-02-12
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

In fact, whether HPLC or LC-MS / MS is used to determine the content of SAH or methylated products, it is very inaccurate for evaluating MT activity.
HPLC and LC-MS / MS samples need special treatment. After a long time and many steps, SAH or methylated products will inevitably be lost. In addition, the products of the synthesis reaction may be unstable or even degrade in a short time. If it is not detected in time, it will cause the deviation of the detection value
All of these factors can make the measured values ​​very inaccurate, let alone trying to reflect the kinetic reaction of the enzyme
[0014] As for the multi-enzyme cycle method, two or more enzyme reactions are often involved, the process is complicated, and there are many influencing factors, and the detection method requires the use of large and expensive instruments such as biochemical analyzers

Method used

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  • A real-time assay method and kit for methyltransferase activity
  • A real-time assay method and kit for methyltransferase activity
  • A real-time assay method and kit for methyltransferase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Anti-SAH antibody cross-reacts with homocysteine ​​methyltransferase substrate SAM and Hcy

[0067] Experimental Materials

[0068] HRP enzyme-labeled anti-SAH antibody: Arthus Biosystems, MAH00301; bovine serum albumin BSA-SAH: Arthus Biosystems, ACT00301; S-adenosyl homocysteine ​​(SAH) standard: Arthus Biosystems, AST00301;

[0069] S-adenosylmethionine: Sigma, A2408,; Homocysteine ​​methyltransferase (HMT): Abt-P-005 of Beijing Aibison Biotechnology Co., Ltd.; BSA, Tris-Cl, NaCl, NaH 2 PO 4 , Na 2 HPO 4 , Na 2 HPO 4 .12H 2 O: Beijing Huamei Jiacheng Biotechnology Co., Ltd.; KCl: Tianjin Damao Chemical Reagent Factory; Homocysteine ​​(Hcy), ProClin 300: Sigma; TMB Chromogenic Solution: Huzhou Yingchuang Biotechnology Co., Ltd.; Sulfuric acid: Hunan Kangdu Pharmaceutical Co., Ltd.; 96-well enzyme-linked plate: American Corning high-adsorption enzyme-linked plate.

[0070] Reagent preparation:

[0071] According to the initial experiment, the BSA-SAH c...

Embodiment 2

[0080] Embodiment 2: In vitro determination of the most suitable buffer system for HMT activity

[0081] Take the ELISA plates coated with BSA-SAH antigen, and use 20mM Tris buffer of pH7.79, pH8.04, pH8.23, pH8.39 and pH7.0, pH7.4, pH7.8, pH8.0 respectively 100mM PB buffer was used to prepare the HMT system reaction solution containing 20uM SAM, 20uM Hcy and 1ug / ml HMT, the reaction solution was 50ul per well, and the HRP-labeled antibody was 50ul per well. React at 37°C for 20min, add 100ul TMB to each well after washing the plate, develop color at 37°C for 15min, add 50ul of stop solution and read. Among them, SAH standard curve uses 0, 0.3125, 0.625, 1.25, 2.5, 5, 10uM standard products, and is prepared with pH 7.8 PB buffer and pH 8.25 Tris buffer respectively. The detected curves were fitted, and the production of SAH in each experimental group was calculated respectively. The results are shown in the table below:

[0082] Table 3 Comparison of SAH formation catalyzed...

Embodiment 3

[0094] Embodiment 3: Enzyme concentration and substrate concentration of measuring HMT in vitro

[0095] Take the ELSIA plate coated with BSA-SAH antigen, prepare SAH standard 0, 0.3125, 0.625, 1.25, 2.5, 5, 10uM and 4uM quality control with enzyme reaction buffer pH7.8. And prepare different concentrations of SAM, Hcy and HMT enzyme formulations. Standards and enzyme reactions are 50ul per well, and HRP-labeled antibodies are 50ul per well. The reaction time at 37°C was 20min, 30min, and 40min. After washing the plate, 100ul TMB was allowed to develop color at 37°C for 15min, and read after 50ul of stop solution.

[0096] Table 6 Comparison of SAH formation catalyzed by HMT under different substrate concentrations and HMT concentrations

[0097]

[0098] According to the results in the table above, an increase in the concentration of SAM will cause an increase in the background value, which is caused by the cross-reaction between SAM and anti-SAH monoclonal antibody. At...

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Abstract

The invention discloses a method for measuring the activity of transmethylase in real time and a kit. The method comprises two reactions carried out at the same time in a reaction system; according to the first reaction, in a buffer system where it is guaranteed that S-ademetionine is adopted as a methyl donor and the transmethylase has the biological activity, a sample containing the transmethylase (MT), the methyl donor and a corresponding substrate are added, or the S-ademetionine methyl donor and the corresponding substrate are directly added into the liquid sample containing the transmethylase, and a reaction is carried out so that an S-adellosyl homocysteine (SAH) product can be generated, wherein the substrate is receptor matter of methyl; according to the second reaction, an immunological method is adopted for detecting the content of SAH to determine the activity of the transmethylase in the reaction system. The biochemical reaction of MT catalysis and the immunoreactions for measuring the product SAH are carried out at the same time, the two processes are organically combined, and great significance is especially achieved on accurate measurement of SAH with extremely unstable molecularity.

Description

technical field [0001] The invention relates to the technical field of methyltransferase detection, in particular to a method for real-time determination of the biological activity of methyltransferase using S-adenosylmethionine as a methyl donor and a detection kit. Background technique [0002] Methyltransferase (Methyltransferase, MT) is a class of enzymes that catalyze methylation reactions, and generally uses S-adenosyl methionine (S-Adenosyl Methionine, SAM) as a methyl donor. Methylation reactions widely exist in organisms, and their substrate range covers almost all biologically active molecules in cells, including nucleic acids, proteins, polysaccharides, lipids, and many small molecules. Therefore, many important physiological links such as gene expression Inhibition or shutdown, repair of DNA damage, and synthesis and degradation of intermediates in physiological processes in microorganisms, animals and plants are all involved in the regulation of MT. [0003] Ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/558G01N33/532
CPCG01N33/532G01N33/558
Inventor 郝秀娟周敏
Owner 泰州汇丰合泰生物技术有限公司
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