Micro ELISA for measuring E. Coli J-5 concentration using monoclonal antibody

Abstract

Spleen cells of mice immunized with E. Coli J-5 and myeloma cells (Ag8×63) were fused using 50% polyethyleneglycol. Consequently, three different monoclonal antibodies (MoAb) were obtained. These MoAb crossreacted with other gram negative rods such as pseudomonas and salmonella minnesota R595, and a positive reaction was found against lipid A in Dot blot analysis. 10ng/ml to 10μg/ml of E. Coli J-5 was found to be accurately measurable by the competition ELISA using these MoAb against lipid A. The %CV for each point on the standard curve using the competition ELISA ranged from 4.3 to 8.3% with an average of 6.2%. The recovery rate of E. Coli J-5 from urine samples was excellent. Using the competition ELISA, E. Coli J-5 could be measured without any interference from inhibitors and potentiators. We measured the E. Coli J-5 concentration of the plasma from a patient who died from sepsis, the urine sample of a patient with acute pyelonephritis and the dialysate at the inlet of the dialyzer during hemodialysis. The concentration was 540ng/ml, 40ng/ml and less than 10ng/ml, respectively.