Radioimmunoassay

There is frequently a need to measure concentrations of neuropeptides in tissue perfusates, tissue extracts, chromatographic column fractions, and so on. Since the concentrations of neuropeptides encountered are often low (usually the low fmol/mL range in perfusates and pmol/g range in tissue extracts), it is necessary to employ sensitive methods for then measurement (see Note 1 ). Adequate sensitivity usually equates with the use of immunoassays, although sensitive bioassays are available for some neuropeptides, and these can be made specific by the judicious use of high-affinity receptor antagonists (1 ). Since the advent of the liquid-phase radioimmunoassay, several modifications to the general concept have been employed, including the use of radiolabeled antibodies instead of peptide ligand (immunoradiometric assay), nonradioactive markers (enzyme immunoassays), and “sandwich assays” in which the ligand is trapped between one antibody coupled to a solid phase such as a microplate and a second antibody attached to some detection molecule. Refinements such as signal enhancement and the sandwich technique can improve both the sensitivity and the useful range of an assay, but at the expense of considerable method development. As a general rule, such investment is worthwhile in the development of assays with a clinical or other commercial need, but not for research. Indeed, for assays employing a single antiserum, the sensitivity accomplished is dependent on the characteristics of that antiserum. In such a system, it is hard to improve on a monoiodinated ligand molecule for sensitivity and convenience. This chapter describes the radioimmunoassay technique as it could be applied for the measurement of a newly isolated neuropeptide.