RADIOIMMUNOASSAY WITH HETEROLOGOUS ANTIBODY (HETERO-ANTIBODY RIA) - UTILIZATION OF HIGHLY CROSS-REACTIVE ANTIBODY PRESENT IN POLYCLONAL ANTISERUM

To develop a homologous radioimmunoassay (RIA) for a hormone of a smal l or rare animal often meets difficulty in collecting a large amount o f purified antigen required for antibody production. On the other hand , to employ a heterologous RIA to estimate the hormone often gives poo r sensitivity. To overcome this difficulty, a "hetero-antibody" RIA wa s studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a hetero logous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCG-beta or anti-ovine LH-beta was used as the het erologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine mo tilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fracti ons. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results s uggest that an antibody population having high affinity and cross-reac tivity is present in polyclonal antiserum and indicate that the popula tion can be used in hetero-antibody RIA at an appropriate concentratio n.