Development of enzyme immunoassay for equine chromogranin A

Abstract

A region-specific enzyme immunoassay (EIA) system for equine chromogranin A (CgA) was developed using synthetic equine CgA (335-365), based on the amino acid sequence reported by Sato et al. (19). For developing the assay system, we used anti-equine CgA (335-365) serum raised in a rabbit, biotinyl-glycilglycil-equine CgA (335-365) as labeled antigen, and synthetic equine CgA (335-365) as standard. Standard displacement curve of this assay system was paralleled with the dilution curves of equine plasma and saliva. The minimum detection limit of the assay system was approximately 10 fmol/mL. The CgA-like immunoreactivity (IR-CgA) level of normal equine plasma and saliva were 0.38 ± 0.08 pmol/mL and 0.25 ± 0.15 pmol/mL, respectively. Gel filtration analysis of equine plasma and saliva on sephadex G75 column using PBS as eluent revealed the existence of major IR-CgA molecule having approximately 60 kDa. These results indicate that the present EIA system may be of great use for the measurement of IR-CgA in equine plasma and saliva.