Metalloimmunoassay Using Atomic Absorption Spectroscopy

who coined the term metalloimmunoassay, prepared a series of organometallic markers (metallohaptens) with the potential for use in immunoassay. These are derivatives of steroids (estradiol, estriol), mood-altering drugs (amphetamine and cocaine) or medications (morphine and barbiturates), labeled with ferrocene, cobaltocenium and cymantrene [57-59]. He then assessed the use of a Sepharose 4B column to separate the free and bound fractions of the tracer and quantification of the tracer by atomic absorption spectrometry [58]. 

The first ferrocene-based labels were quickly abandoned, as trace quantities of iron are found in the buffer solutions, and thus perturb the results of quantification by AAS. Cymantrene-based markers were next favored, since manganese is much less likely to occur naturally. Insert Fig. 8.4 shows the MIA of nortriptyline [62]. Antiserum dilution curves were obtained in the presence of 12.5-50.0 ng tracer 12 per tube (Fig. 8.4a). 

The feasibility of the assay was demonstrated by the calibration curve shown in Fig. 8.4b. The range of sensitivity for the assay is between 10 and 240 ng mL nortriptyline. This is entirely compatible with concentrations of medications in the blood, which are in the microgram mL range (cf Table 8.2). To increase the sensitivity of the assay, signal amplification was obtained using the biotin/ streptavidin system. The principle of this 4-step assay is shown in Fig. 8.5. This is 

MAC Maximum admissible concentration set by the European Community for drinking water. 

BSA Anti-BSA antibodies Biotinylated antibodies streptavidin Cy-BtoBn Fig. 8.5 Principle of a solid-phase streptavidin-cymantrenyl-biotin competitive immunoassay [65.  

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