Detection principle of homocysteine assay kit

The detection principle of homocysteine (HCY) assay kit is mainly based on chemical reactions to quantitatively determine the content of homocysteine in serum. Here are several common detection principles:

1. Enzymatic method

Principle overview: Enzymatic method converts homocysteine (Hcy) into a detectable substance through a series of enzymatic reactions, and then determines its content.

Specific steps:

Oxidative Hcy is first converted into free Hcy.

Free Hcy reacts with serine under the catalysis of CBS (cystathionine beta synthase) to produce L-cysteine.

L-cystathionine is catalyzed by CBL (cystathionine lyase) to generate Hcy, pyruvic acid, and NH3.

The pyruvate generated by this cyclic reaction can be detected by lactate dehydrogenase (LDH) and NADH (reduced nicotinamide adenine dinucleotide).

The rate of NADH conversion to NAD+is directly proportional to the Hcy content in the sample, and the Hcy content can be calculated by detecting the absorbance change rate of NADH.

2. Circulating enzyme method

Principle overview: The cyclic enzyme method based on small molecule capture technology (SMT) amplifies the detection signal through a series of enzymatic reactions to accurately determine Hcy content.

Specific steps:

Oxidized homocysteine is converted to free Hcy under the action of TCEP (triethoxyethylphosphine).

Free Hcy reacts with covalent substrate S-adenosylmethionine (SAM) to form methionine and S-adenosyl-homocysteine (SAH).

SAH is hydrolyzed by SAH hydrolase into adenosine and Hcy, and the formed Hcy can be cyclically added to the reaction, thereby amplifying the detection signal.

The generated adenosine is immediately hydrolyzed into hypoxanthine and ammonia, which converts NADH to NAD+under the action of glutamate dehydrogenase. The Hcy concentration in the sample is proportional to the change in NADH.

3. ELISA method (enzyme-linked immunosorbent assay)

Principle overview: The ELISA method uses a double antibody one-step sandwich method to determine Hcy content through antigen antibody reaction and enzymatic colorimetric reaction.

Specific steps:

Add the sample, standard, and HRP (horseradish peroxidase) labeled detection antibody in sequence to the pre coated micropores containing human homocysteine (Hcy) capture antibodies.

After incubation and thorough washing, the substrate TMB (tetramethylbenzidine) was used for color development.

TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid.

The depth of color is positively correlated with Hcy in the sample. Measure the absorbance (OD value) at a specific wavelength using an enzyme-linked immunosorbent assay (ELISA) reader to calculate the sample concentration.

matters needing attention

The detection principles of different brands and models of reagent kits may vary slightly, and specific operations should refer to the reagent kit instructions.

Sample collection and processing should be strictly carried out in accordance with the requirements to ensure the accuracy of the test results.

The reagent kit should be stored under suitable temperature and humidity conditions to avoid expiration or deterioration that may affect the test results.

In summary, there are various detection principles for homocysteine assay kits, but the core is to convert Hcy into a detectable substance through chemical reactions and quantitatively determine its content through specific detection methods such as absorbance measurement.