A sensitive radioimmunoassay method for quantitative determination of sea-squirt antigens in vitro was developed. A purified sea-squirt antigen, Ei-2, was labeled with ^<125>I and the labeled Ei-2 was further refined by means of affinity chromatography using an immobilized rabbit anti-Ei-2 antibody. The refined labeled Ei-2 had a high affinity to the antibody and the radioactivity was high enough to use as tracer. A mixture of 100 μl of appropriately diluted refined labeled Ei-2 as a tracer (about 10000 cpm) and 50 μl of a sample solution (0.5-20 μg antigen/ml) were reacted with 50 μl of diluted (1:10) rabbit anti-Ei-2 serum for 16 hr at room temperature. At the end of the reaction, 400 μl of 20% Na_2SO_4 was added and the mixture was allowed to stand for 1 hr at room temperature, then centrifuged at 3500 rpm for 5 min. After aspirating off the supernatant, the precipitate was counted for radioactivity. From the ratio of antibody-bound radioactivity (B) in the presence of unlabeled antigen to that obtained with the tracer alone (B_o), the amount of antigen in the sample was determined by the use of a standard curve (B/B_o-standard Ei-2 concentration). It was also found that the other antigen, Gi-2, cross-reacted to the anti-Ei-2 antibody. Therefor, this assay method was considered to be applicable not only to Ei-2 but also in general to sea-squirt antigens.
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