Principle of the ELISA test
This assay is based on the method of competitive enzyme linked immunoassays. The sample preparation includes the addition of an derivatization-reagent for analyte coupling. Afterwards, the treated samples and the polyclonal analyte-antiserum are incubated in wells of microplate coated with analyte-derivative (tracer). During the incubation period, the target analyte in the sample competes with the tracer immobilized on the wall of the microtiter wells for the binding of the polyclonal antibodies. The analyt in the sample displaces the antibodies out of the binding to the tracer. Therefore the concentration of the tracer-bound antibody is inverse proportional to the analyte concentration in the sample. During the second incubation step, a peroxidase-conjugated antibody is added to each microtiter well to detect the anti-analyte antibodies. After washing away the unbound components, tetramethylbenzidine (TMB) is added as a substrate for peroxidase. Finally, the enzymatic reaction is terminated by an acidic stop solution. The color changes from blue to yellow and the absorbance is measured in the photometer at 450 nm. The intensity of the yellow color is inverse proportional to the analyte concentration in the sample; this means high analyte concentration in the sample reduces the concentration of tracer-bound antibodies and lowers the photometric signal.
A dose response curve of absorbance unit (optical density, OD at 450 nm) vs. concentration is generated using the values obtained from the standard.
Analyte present in the patient samples is determined directly from this curve.
This assay is a photometric test intended for the determination of leucine, isoleucine and valine by enzymatic dehydration, in which NAD+ is transformed to NADH.
In this reaction the amino acids L‑leucine, L‑isoleucine, L‑valine are oxidized to the respective α-ketone bodies by reducing NAD+ to NADH. This reaction can be measured at 340 nm and it is proportional to the amount of oxidized BCAA.