Description
The LRG (leucine-rich alpha-2-glycoprotein) ELISA is a sandwich enzyme immunoassay for the quantitative determination of LRG in human serum and plasma samples. Samples must be pre-diluted 1:4000 prior to assaying, see chapter 5) Reagent and Sample Preparation. Standards and controls are used undiluted. In a first step, standards, controls and pre-diluted samples are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal sheep anti-human LRG antibody. LRG present in the standard/control/sample binds to the pre-coated antibody in the well. All non-specific unbound material is removed in a washing step and the detection antibody (CONJ, polyclonal sheep anti-LRG-HRPO) is pipetted into the wells. After another washing step, the substrate (TMB, tetramethylbenzidine) is added. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of LRG present in the sample. This color change is detectable with a standard microplate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) using the values obtained from the standards versus the standard concentration is generated. The concentration of LRG in the sample is determined from the dose response curve. This sample concentration must be multiplied by the dilution factor used for sample preparation to obtain final sample concentration (e.g. if dilution factor is 1:4000 the concentration obtained from the response curve must be multiplied by 4000)