ELISA Project—Detection, Optimizing, and Validation of Von Willebrand Factor

The enzyme-linked immunosorbent assay (ELISA) is standardized and validated in order to identify and quantify levels of plasma von Willebrand factor (vWF) for a routine check-up and diagnostic method for immune-mediated TTP patients. The kind of ELISA method in this project is a sandwich ELISA in plasma vWF that binds to a primary antibody (a polyclonal rabbit anti-human vWF antibody). It is coated with primary antibody in the plate and sandwiched in between with secondary antibody bond to fluorescent marker, horseradish peroxidase (HRP). In this project, the optimal absorption is recorded by a microplate ELISA reader at 450nm. The optimal concentration of primary antibody, plasma, and secondary antibody is 1:800, 1:40, and 1:8000 respectively. The linearity of the standard curve is established with an average R2 value of 0.99. The lower limit in the experiment is 3.13%/1:1280. The quality controls, QC1 and QC2, are within the reference range. Finally, it is noted that plasma samples are measured by ELISA. Due to time limitations, intra, inter-assay and the upper limits of the range are not analyzed thoroughly.