Homocysteine (HCY) detection reagent with strong stability

Abstract

The invention relates to a highly stable dual-reagent liquid homocysteine ​​(HCY) detection reagent. The reagent R1 mainly contains buffer, NADH, LDH, serine, preservatives, and TCEP (trichloroethyl phosphate) , protective agent, surfactant, edetate disodium, substrate stabilizer; Reagent 2 mainly contains buffer, preservative, protective agent, surfactant, CBS (cystathionine-β-synthase) , CBL (cystathionine‑β‑lyase). The present invention preferably selects the buffer solution with good effect, uses high-efficiency preservatives, and uses three different protective agents in R2 to synergistically protect the activity of enzymes in the reagents, thereby better ensuring the stability of the reagents, especially Thermal stability, convenient for clinical promotion and use.

Description

technical field [0001] The invention relates to a highly stable homocysteine ​​(HCY) detection reagent, which belongs to the technical field of clinical in vitro detection. Background technique [0002] Homocysteine ​​(HCY), also known as homocysteine, is a sulfur-containing amino acid formed after demethylation of methionine, which is an intermediate product of the methionine cycle; it is the transmethylation of methionine in cells An amino acid containing a sulfhydryl group produced during the process, Hcy is transported into the serum through circulation, and most of the Hcy in the serum combines with the albumin in the serum in the form of disulfide bonds in an oxidized form to form a Hcy-protein complex, and only a small amount It exists in the form of free form and disulfide bond-linked Hcy-SS-Hcy; homocysteine ​​​​forms cysteine ​​or methionine during metabolism, and Hcy is irreversible in the transsulfurization pathway of vitamin B6 Hcy is decomposed into cysteine, ...

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Problems solved by technology

[0003] At present, there are many methods for detecting homocysteine ​​in blood. The main one is the isotope detection method established by Refsum et al. in 1985. This method has high sensitivity and strong specificity. Due to the cumbersome operation and radioactive pollution, it has not been widely used even though it has been improved. ; In 1987, Stabler first reported the gas chromatography-mass spectrometry tHCY chromatographic detection method, which can be used for a large number of samples in terms of measurement accuracy and analysis speed, but the cost is high; more immunological methods and enzymatic methods are used at present. This method has the advantages of simple method, high accuracy and precision. Compared with the immunological method, the enzymatic method has lower cost and is more convenient to use. It can be combined with a fully automatic biochemical analyzer. With the current enzyme purification level in enzymatic reagents The cost of the detection reagents of this method is further reduced, and the enzymatic detection of serum homocysteine ​​is divided into two methods, one is to use S-adenosyl homocysteine ​​(SAH) hydrolase (SAHase) , so it is called the hydrolase method, and the other is to use cystathionine-β-synthetase (CBS), also called the cystathionine method. The cost of the former is higher, while the cost of the latter enzyme is lower, which is more conducive to popularization. The principle is that homocysteine ​​(oxidized form) is reduced to free homocysteine, which reacts with serine under the catalysis of cystathionine-β-synthase (CBS) to generate L-cystionine, L- Cystathionine is decomposed into homocysteine ​​and pyruvate by cystathionine-β-lyase, and pyruvate participates in the color reaction of NADH, and the generated homocysteine ​​participates in the first step reaction again, and so on. However, since the cystathionine cycle enzyme method needs to add NADH, cystathionine-β-synthase, cystathionine-β-lyase and other enzymes or coenzymes to detect serum homocysteine, these enzymes are easy to Attenuation, especially in a high temperature environment, is very easy to attenuate, so most of the reagents on the market are freeze-dried powder, which brings great inconvenience to clinical use, so it can solve the stability of the enzyme and improve the reagents. Stability, which has great significance for clinical promotion

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