Development of Standard and Competitive ELISA for Detection and Quantification of PRP-1 in Biological Fluids by Using anti-PRP-1 Polyclonal Antiserum

Abstract

This study aimed to establish a sensitive method for the detection of proline-rich polypeptide-1 (PRP-1) in biological fluids. PRP-1, also known as galarmin, is a fragment of neurophysin-vasopressin-associated glycoprotein synthesized by brain neurosecretory cells and consisting of 15 amino acid residues. An enzyme-linked immunosorbent assay (ELISA) was used for PRP-1 quantification. An ELISA system has been developed using polyclonal antibodies we raised against the synthetic PRP-1. According to the analysis, the concentration PRP-1 of 25 ng/mL was accepted as the main appropriate coating concentration for further experiments in 1:100 and 1:500 antibody dilutions. Then, a competitive ELISA was developed to quantify PRP-1 in the fluids. Based on the results, an appropriate condition was chosen to be the best condition for PRP-1 detection: the appropriate quantity of the immobilized PRP-1 (25 ng/mL); anti-rabbit primary antibodies against PRP-1 (1:500); anti-rabbit secondary antibodies conjugated to peroxidase (1:1000), and extravidin (1:1000); as a result, the minimum detectable amount of PRP-1 in the fluid was 1.5 ng/mL. Thus, this method provides a good detection limit and sensitivity and is easy to use. In addition, a large number of rats and human serum and plasma samples can be analyzed rapidly and simultaneously, which is what we intend to realize in the future.

Keywords

• prp-1
• galarmin
• elisa