To begin, centrifuge the coagulated blood-containing tubes at 626 to 1, 409 G for 20 minutes at two to eight degrees Celsius. Then carefully collect the supernatant in a new tube. Store the supernatant at minus 80 degrees Celsius.
Next, set up blank standard and sample wells. Pipette 50 microliters of the diluted standards into the ELISA plate. Then transfer 40 microliters of the diluted samples into the sample wells.
Add 10 microliters of serum into the sample wells. Now, seal the plate with a sealing film. Place the sealed plate in an incubator at 37 degrees Celsius for 30 minutes.
Dilute the 30 times concentrated wash solution with distilled water to obtain a single strength wash solution. Next, remove the sealing film from the plate and discard the liquid. Now, fill each well with 200 microliters of washing solution five times.
After discarding the supernatant for the last time, tap the plate dry. Pipette 50 microliters of the enzyme reagent into all wells other than the blank ones. Then seal the plate with a sealing film before placing it in an incubator at 37 degrees Celsius for 30 minutes.
After washing the plate five more times, add 50 microliters of each of the color developers, A and B.Shake the plate gently to mix well. Then incubate the plate at 37 degrees Celsius at low light for 10 minutes. Now, pipette 50 microliters of the stop solution into each well to terminate the reaction.
Measure the absorbance of each well sequentially at 450 nanometers. The plasma homocysteine concentration in the methionine group was twofold greater than in the control group. The plasma homocysteine concentrations were relatively lower in the treatment groups.
