Particle counting immunoassay (PACIA). I. A general method for the determination of antibodies, antigens, and haptens

Abstract

By using a device designed for counting blood cells, it is possible to measure the agglutination of polystyrene beads (0.8 mu) with accuracy and great sensitivity, the agglutination resulting in a reduction in the number of particles. The latter coated with antigen can be used for determining IgM or IgG antibodies e.g. human rheumatoid factor or rabbit anti-bovine serum albumin antibodies. Macromolecules with multiple antigenic determinants agglutinate particles carrying specific antibodies. This system has been applied for determining HPL and alpha1-fetoprotein with a threshold of sensitivity of about 10 microgram/1. However the agglutination was decreased by serum factors which led to a 10-fold loss of sensitivity. The interference of rheumatoid factor which agglutinated the particles coated with rabbit or goat immunoglobulins could be avoided by reduction of the serum to be analyzed with 5mM dithiothreitol for 5 min. Haptens, i.e. DNP-lysine and T4, were determined by their inhibitory activities toward their specific antibodies, the agglutinator being a hapten-macromolecule conjugate or the antibodies themselves.