Iodinated versus fluorescent labelling in the radioallergosorbent test (RAST) for the determination of serum IgE levels

Abstract

Experiments were performed to determine the feasibility of replacing the radioactive 125I tag in the radioallergosorbent test (RAST) with a new fluorescent compound, 2-methoxy-2, 4-diphenyl-3 (2H)-furanone (MDPF). It was possible to discriminate a grass-allergic from a grass-non-allergic patient using the traditional RAST procedure with 125I-ant-IgE. However, when a fluorescent label, MDPF was attached to IgE using the same RAST procedure, it was not possible to discriminate between allergic and non-allergic responses. Additional experiments, using BSA and anti-BSA as a model, showed that it was possible to detect 0.025 ppm antigen using iodinated antiserum while the fluorescent tag had a lower limit of antigen detection of 0.20 ppm. It was possible to distinguish between allergic and non-allergic responses using 125I-N-succinimidyl-3(4-hydroxyphenyl) propionate (SHPP) which binds to protein in a manner similar to MDPF. These results suggested that the unsatisfactory fluorescence results were not due to an alteration of protein structure as a result of MDPF labelling. Although the fluorescence work was performed using a fluorimeter with optics and electronics optimized for detection of MDPF, sensitivity of the method was limited by high fluorescent background.