Comparative assessment of immunization procedures for development of anti proinsulin antisera for radioimmunoassay

Two schedules of immunization were employed for developing anti proinsulin antisera for radioimmunoassay. Biosynthetic human proinsulin-h P I (Elli Lilly. US), was injected subcutaneously in guinea pigs in multiple sites. In the first schedule were used 50 u g of h P I and the booster injections were administered 4 weeks after the primary immunization and then at 3-week intervals. In the second schedule was used 250 u g of h P I and boosters were done 7, 9 and 18 weeks later. As the antisera were not sufficiently specific for h P I they were purified and assessed for kinetic of precipitation and avidity. Both immunization schedules gave comparable responses. The antisera generated by the use of 50 u g of h P I presented higher cross-reactivity with insulin while the reactivity with cpeptide was of the same order in both antiserum groups. The avidity was very variable in the two groups and the three most sensitive antisera required 24 h at 4o C for achieving maximum binding with the 125 I-h P I. However, only one antiserum (from the first group) was suitable for the radioimmunoassay. This study emphasizes the difficulties of making valid comparisons between different immunization procedures, especially in the cases when highest avidity is required. (author)