Preparation method of cholesterol oxidase and cholesterol esterase modified by PEG

Abstract

The invention relates to a preparation method of cholesterol oxidase and cholesterol esterase modified by PEG, belonging to the technical field of clinical in-vitro detection. The preparation method comprises the following basic steps: first, preparing a buffer solution, then adding two substances including p-aminobenzene sulfonic acid and AEO-9 as activating agents, then adding the carrier PEG20000(sigma), adding a protective agent, an emulsifier and ionic substance, carrying out uniform stirring, sequentially adding cholesterol oxidase and cholesterol esterase, carrying out stirring for 10min at the low and medium speed, carrying out centrifugalizing by adopting a centrifugal machine with low rotation sped, and taking the supernate, thus obtaining the PEG-modified enzyme with the same effect. According to the formula, the ingredients are as follows: PIEPES, p-aminobenzene sulfonic acid, AEO-9, PEG20000, the protective agent, the emulsifier, the ionic substance, cholesterol oxidase, cholesterol esterase and MIT. The method is easy to operate and low in cost, and has good compatibility with detection for high density lipoprotein cholesterol adopting the PEG-modified enzyme method.

Description

technical field [0001] The invention relates to a preparation method of PEG-modified cholesterol oxidase and cholesterol lipase, belonging to the technical field of clinical in vitro detection. Background technique [0002] The PEG-modified enzyme method for detecting serum HDL cholesterol was first proposed by Sugiuchi in 1995. It is an important method for the homogeneous method to detect blood HDL cholesterol. The reaction principle of the method is the sulfuric acid α-ring in reagent 1. Dextrin and dextran sulfate form water-soluble complexes with LDL, VLDL and CM in the specimen. The PEG-modified enzyme system in Reagent 2 reacts to HDL to achieve the purpose of detection. The principle of this method is focused on: ① α-cyclodextrin sulfate has a shielding effect on the LDL component rich in apoB; ② PEG molecules in PEG-modified enzymes produce steric hindrance, which is not conducive to the contact between macromolecular substrates and enzymes. Without affecting the ...

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Problems solved by technology

[0003] However, due to the high technical difficulty, there is no product available in China. The technical problems mainly exist in the synthesis of modified enzymes and the screening of surfactants. The most difficult to control is the preparation of PEG-modified enzymes. The method established by Sugiuchi is to first use PEG6000 N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCCI) were activated, and cholesterol oxidase and cholesterol lipase were added to 0.1mol / L HEPES PH=8.5 buffer solution. After the dissolution was complete, the The activated PEG6000 is added to the enzyme solution, so that the PEG6000 and the enzyme are covalently bonded together, it takes at least 30 minutes, and then the PEG6000 is filtered out using a filter, and then the HPLC method is used to detect the binding of the enzyme. Activity, through a large number of experiments, the author found that this method has the following problems: ①In the process of combining PEG6000 with enzymes, even if PEG6000 is very excessive, there is still a small amount of enzymes that are not combined, which is the problem when using it. The specificity of the modifying enzyme increases the difficulty; ② If the two substances activated by PEG6000 are not cleaned properly, it will affect the subsequent detection of HDL-C; The detection of C is inaccurate. ④ The process of modifying enzymes is too complicated, requiring high technical and experimental levels, and the cost of obtaining modified enzymes is high.

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