Quantitative assay for immunoglobulin class-specific anti-RNP antibodies

Abstract

Serum anti-ribonucleoprotein (RNP) antibody levels were measured using an enzyme linked immunosorbent assay (ELISA) technique. Paper discs, activated by CNBr and coupled covalently with rabbit thymus extract (RTE), were used for the solid phase of ELISA.
Twenty serum samples containing antibodies specific only to the RNP antigen, determined by double immunodiffusion, were examined by ELISA. The sera were obtained from patients with MCTD, SLE and PSS. The samples were measured for anti-RNP antibody levels in each immunoglobulin class. It was found that most of the anti-RNP antibodies were IgG class and the rest were IgA class. No IgM or IgE anti-RNP antibodies were detected in this study.
To determine the absolute concentration of anti-RNP antibodies in sera, the IgG fraction of a sample from a patient with MCTD was labelled with N-succinimidyl [2, 3-³H] propionate ([³H] NSP). Then, Sepharose 4B coupled with RTE was reacted with the [³H] NSP-labelled IgG, and non-binding [³H] NSP-labelled IgG was removed by washing. The radioactivity of the Sepharose 4B obtained was measured and the absolute concentration of anti-RNP antibodies in serum was calculated from the binding ratio of the radioactivities of the entire [³H] NSP-labelled IgG to that bound with Sepharose 4B. Thus, the IgG anti-RNP anibody concentration of the IgG fraction was determined. The IgG fraction was sequentially diluted, and the anti-RNP antibody levels of the fractions were measured by ELISA. A dose response cure was obtaned from these values. The concentration of anti-RNP antibodies of twenty serum samples was determined from each antibody level by ELISA, using the dose response curve. The concentration of anti-RNP antibodies in sara tested ranged from 0.16 to 3.98mg/ml.