Radioimmunoassay of regulatory peptides in the presence of acetonitrile: marked improvement of cholecystokinin assays

Abstract

Radioimmunoassay has made it possible to measure the levels of many hormones. However, samples for some hormones, such as cholecystokinin (CCK), need to be purified by reverse phase chromatography before assay. Usually, samples are eluted from cartridges or HPLC columns in about 50% acetonitrile, dried on a vacuum centrifuge, and then reconstituted in buffer. Drying and reconstituting samples is time consuming and introduces additional sources of error and peptide loss. The present study investigated the effect of acetonitrile on radioimmunoassays for CCK to see if samples containing acetonitrile could be assayed directly. The non-specific binding of a radiolabeled peptide, the zero binding (B 0), and the fall in the presence of 2.5 fmol unlabeled CCK were determined in the presence of various proportions of acetonitrile with 0.1% TFA. Additionally, standard curves were compared in the presence and absence of 200μl of 50% acetonitrile, ( n=5). For assays using two separate CCK antisera, increasing amounts of acetonitrile gave progressively higher zero binding and fall, thereby increasing sensitivity and antibody titer. The use of 200μl 50% acetonitrile, chosen to represent typical sample conditions, increased antiserum titers by three to four-fold, as well as increasing sensitivity considerably. For one antiserum (CCK2), the IC 20 was 0.36±0.02 fmol CCK/tube in the presence of acetonitrile and 1.45±0.08 fmol/tube in its absence ( P<0.001). For the other antiserum (Dino 7), the IC 20 was 0.40±0.02 fmol CCK/tube in the presence of acetonitrile and 0.63±0.01 fmol/tube in its absence ( P<0.001). A similar increase in sensitivity was seen with a gastrin assay. However, no significant change in the gastrin antibody titer was evident. Assays for several other hormones were unaffected by 200 μl of 50% acetonitrile. At volumes encountered in samples following chromatography, acetonitrile did not adversely affect radioimmunoassays for a number of hormones, and the sensitivity and antibody titer of the CCK assays were improved. Measurement of CCK samples without drying and reconstitution increases assay efficiency and sensitivity.