A simplified technique for the evaluation of antigen-binding cells

Abstract

A gamma counter technique was developed in an attempt to simplify the antigen-binding cell (ABC) assay for the detection of lymphocytes capable of binding 125I-human encephalitogenic basic protein ( 125I-HEProt), and for the detection of serum factors in the peripheral blood of multiple sclerosis (MS) patients capable of affecting the binding of 125I-HEProt to the ABC. Appropriate amounts of 125I-HEProt were added to sheep peripheral lymphocytes (SPL) and incubated at 0–4°C. The cells were counted for radioactivity and also smeared for autoradiography. Radioactivity (cpm) showed a positive linear relationship with increasing antigen dose from 2–80 ng of 125I-HEProt. Binding of sheep red blood cells (SRBC) was significantly lower (p < 0.01) than binding to SPL. Labelling of SPL was inhibited 80% by preincubation in excess unlabelled HEProt, but enhanced by preincubation in excess lysozyme, a basic protein of similar molecular weight and charge to HEProt. Thus, specificity of binding between SPL and 125I-HEProt was shown. Autoradiographic examination revealed that an increase in the dosage of labelled antigen did not result in an increase in the number of labelled cells but rather in an increase in the number or density of grains per cell. This increase bore a linear relationship to the increase in cpm noted by the gamma counter technique. There was no significant difference in cpm between cell preparations washed in 100% foetal calf serum (FCS), gradients of FCS and Eisen's balanced salt solution (EBSS) and those washed in EBSS alone. Further, five or more washes in either FCS, FCS gradients, or EBSS did not significantly reduce the cpm of the cell pellet below that obtained with three washes. In preliminary studies, serum from MS patients when preincubated with SPL was able to significantly (p < 0.01) inhibit the labelling of SPL by 125I-HEProt. Investigations are underway to determine whether the inhibitory factor is circulating HEProt or an antigen cross-reactive with HEProt, a blocking antibody, or an inhibitory protein in serum other than gamma globulin.