Simultaneous assay of pepsinogen I and pepsinogen II in serum by bioluminescent enzyme immunoassay using two kinds of Luciola lateralis luciferase Original Research Article

We developed a simultaneous bioluminescent enzyme immunoassay of serum pepsinogen I (PG I) and pepsinogen II (PG II) in which two kinds of biotinylated luciferase (Luciola lateralis) as labeled enzyme producing yellow-green light (λmax=559 nm) and red light (λmax=607 nm) were used. In the proposed method, PG I and PG II in serum were simultaneously captured in a sandwich type immunereaction between anti-PG I and anti-PG II monoclonal antibody-coated magnetic particles, and streptavidin biotinylated luciferase biotinylated anti-PG I and anti-PG II monoclonal antibodies triplex, respectively. After bound/free (B/F)separation by washing, bound enzyme activities of luciferase were measured twice as the bioluminescent intensity in the presence of luciferin, ATP, Mg2+ and molecular oxygen. The value of PG I was obtained by calculation based on the bioluminescent intensity at λmax=607 nm. The value of PG II (λmax=559 nm) was obtained by the method using a band pass filter. The calibration range of PG I was from 2 to 200 ng ml−1 and that of PG II was from 1 to 100 ng ml−1. The coefficients of variation for PG I and PG II determination were from 3.4% to 10.2% and from 4.0% to 8.6%, respectively. The correlation of PG I values in serum between the proposed method and a PG I specific bioluminescent immunoassay (single assay), and PG II values in serum between the proposed method and a PG II specific bioluminescent immunoassay were satisfactory. The regressions were y(proposed method)=0.9952x(single assay for PY I)+0.8411, r=0.9837 and y(proposed method)=1.0545x(single assay for PY II)+1.4693, r=0.9738, respectively. The proposed method is concise, accurate and rapid for the determination of PG I and PG II in serum, and is useful for clinical application.