In a recent radioimmunoassay for human β-melanocyte-stimulating hormone (βh-MSH) (Thody & Plummer, 1973) the dextran-coated charcoal technique (Herbert, Lau, Gottlieb & Bleicher, 1965) was used for the separation of antibody-bound and free hormone. We have now examined several other methods of separation.
Details of the antiserum and iodination of synthetic βh-MSH have been described (Thody & Plummer, 1973). Tubes were set up containing 25 pg 125I-labelled βh-MSH and diluted antiserum in 210 μl diluent buffer (0·05 m-phosphate buffer, pH 7·4 containing 0·5% human serum albumin and 0·02% thiomersal (Merthiolate)). The final dilution of antiserum was 1:8250, sufficient to bind approximately 50–70% of 125I-labelled βh-MSH (Thody & Plummer, 1973). Blanks containing no antiserum were set up in parallel. After incubation for 24 h at 4 °C antibody-bound and free 125I-labelled βh-MSH were separated. For separation by the second
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