The detection methods of homocysteine assay kit mainly include enzyme cycling method and enzyme-linked immunosorbent assay (ELISA), etc. The following is a specific introduction:
Enzyme cycling method: Principle: Based on small molecule capture technology (SMT), the cyclic enzyme method amplifies the detection signal through a series of enzymatic reactions to accurately determine the content of homocysteine (Hcy). Oxidative Hcy is converted to free Hcy under the action of triethoxycarboxyethylphosphine (TCEP), and 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 generated Hcy cycle participates in the reaction, amplifying the detection signal. Adenosine is hydrolyzed into hypoxanthine and ammonia, which converts NADH to NAD+under the action of glutamate dehydrogenase. The Hcy concentration in the sample is directly proportional to the change in NADH.
Operation steps: Collect fasting serum or plasma samples, store them at low temperature, and centrifuge them.
. Using a fully automatic biochemical analyzer, set the measurement temperature to 37 ℃, wavelength to 340nm, colorimetric cup diameter to 1.0cm, measurement mode to rate method, and measurement time to 120 seconds. Add reagent 1 and reagent 2 in proportion to the instrument, start the measurement after adding the sample, and the instrument will automatically complete the reaction and detection and output the results.Enzyme linked immunosorbent assay (ELISA): Principle: Using a double antibody one-step sandwich method, Hcy content is determined through antigen antibody reaction and enzymatic colorimetric reaction.
. Pre coated with human homocysteine (Hcy) capture antibody, the sample, standard, and HRP labeled detection antibody are sequentially added to the coated micropores to form an antibody antigen enzyme-linked antibody complex. After incubation and washing, substrate TMB was added for color development. TMB was converted to blue under peroxidase catalysis and yellow under acid action. The color depth is positively correlated with the Hcy content in the sample, and the concentration is calculated by measuring the absorbance (OD value) using an enzyme-linked immunosorbent assay (ELISA) reader.Operation steps: Set blank wells, standard wells, and test sample wells, add standard samples or test samples respectively, and then add biotin labeled antibody working solution and horseradish peroxidase labeled avidin working solution.
. Incubate the reaction plate in a 37 ℃ constant temperature incubator, wash the plate to remove unbound reagents, add color reagent and incubate in the dark, and finally add termination solution to terminate the reaction. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at 450nm wavelength and draw a standard curve.source:http://en.zjikon.com/news1075570.html