Conclusion

An excellent antiserum (as the one used here) is an absolute prerequisite for the specific and sensitive measurement of physiological concentrations of plasma MEL. However, the accuracy of direct measurements is strongly influenced by individual plasma factors, which act in a way distinct from common cross-reactions. In the present work the observed individual variations in interference by these plasma factors could be kept very low by the use of a polar 125I-tracer and the supplementation of the incubation mixture with PEG and additional plasma. The accuracy of this direct RIA is documented by the high correlation of assay results with those obtained by the C18-extraction RIA and by GCMS. Later studies have shown that under the RIA conditions described here human serum and porcine serum behaved very similarly with respect to non-specific matrix effects. Therefore, a more recent version of the direct

0 50 100 150 200 250 300 RIA

Melatonin (pg/ml)

Figure 3. Comparison of gas chromatographic-mass spectrometry (GCMS) and RIA. Melatonin concentrations of 93 plasma samples were measured. The correlation coefficient is based on 90 samples, i.e. excludes the 3 outliers. Data kindly provided by Dr. R. Sack, Oregon Health Science University, Portland, Oregon.

RIA is an assay for MEL in human serum, which uses C18-porcine serum as standard matrix and buffer supplement. Efforts are ongoing to make the ELISA suitable for direct measurement of MEL in serum, too.

For the direct measurement of melatonin in human serum we have developed highly specific antibodies in rabbits against a melatonin-BSA-conjugate, in which the melatonin was bridged to the protein at the C2-position via the Mannich reaction (1). This coupling strategy is advantageous over others, since it results in the production of antibodies which do not accept any changes in the sidechains of melatonin, but tolerate modifications at or near C2. Initially we used 2-Iodo-melatonin (2) as tracer, which was bound well by the antiserum and which produced sensitive standard curves. However, its use in the direct assay of serum samples was usually associated with complications, presumably due to its highly hydrophobic nature. Therefore we have constructed a polar tracer molecule, in which melatonin at the Nrposition was coupled to Gly-His over a short spacer, followed by iodination of the latter via the chloroamine T-procedure. The combination of this tracer with the above antiserum allowed the direct measurement of the very low day time serum levels (detection limit 1.5pg/ml). Variations in individual serum matrix effects could be abolished by the addition of C18-stripped porcine serum and polyethylene glycol to the incubation mixture. Measurements by RIA and GCMS showed a good correlation (r = 0.95).

By analogy to the RIA we have developed a competitive ELISA using the same antiserum and a biotinylated tracer molecule, in which the melatonin was linked to a biotin derivative at the Ni-position. Microtiter plates were coated with a second antibody (goat anti rabbit) and streptavidin-HRP and the chromogen TMB were used in the detection system. This assay is routinely used for the measurement of melatonin in culture media and extracted samples of other sources in a concentration range of 0.1 to 20 pg/well.

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