A study of protein A-gold resolution for immunoelectron microscopy

For the purpose of investigating a topographical correlation between antigen molecules and protein A-gold(PAG) particles which localized as an immunocytochemical probe, the simplest model on a localization pattern of antigen molecules, which were arranged two-dimensionally on a plane surface of the resin, was used. Ultrathin sections of a G-actin layer, which was adsorbed on epoxy resin and was re-embedded subsequently in JB-4 resin, was stained indirectly with rabbit anti-actin antibody and subsequently by PAG. From this immunoelectron microscopy, a histogram (relative frequency, denoted by y vs. relative length, denoted by x) was obtained using a computer-assisted method. For this histogram, a fitting curve was calculated by a least squares optimization and three parameters (H, U, and W) of the curve which could be useful for a study on the topographical organization of antigen molecules were estimated. Parameter H (maximum y of the curve) would reflect the maximum amount of epitopes at x = U. Half width W, which is the width of the curve at y = H/2, would reflect a breath of epitope masses. This fitting curve was separated into two overlapping curves whose Ws were different from each other. The one constituent curve of which value W was smaller than the other was regarded as a unit curve and the other constituent curve could be resolved into many unit curves whose W values are the same. From these unit curves, the resolution power of the immunoelectron microscopy, using a post-embedding procedure of ultrathin sections, was estimated as 58-66 A degrees.