Instrumentation William R. Heineman H. Brian Halsall Department of Chemistry University of Cincinnati Cincinnati, Ohio 45221
StrategiesforElectrochemical Immunoassay Three -dimensional structure of an IgG molecule
A problem of paramount importance to analytical chemistry is selectivity, particularly at low detection limits where potential interferents might be present at substantially higher concentrations than the analyte(s) of interest. Remarkable progress in selectivity has been achieved over the past decades through the development of instrumental techniques. Striking examples of widely used techniques that provide excellent selectivity at the trace level are gas chromatography, mass spectrometry, and liquid chromatography for organic compounds and plasma emission spectroscopy for metals. Prior to the development of such powerful instrumental techniques, selectivity was generally achieved by means of selective chemical reactions. For example, a large body of literature is devoted to the study of metal complexation by ligands that can be used as selective titrants, chromophores, or masking agents for the determination of metal ions. However, the selectivity provided by chemical reactions of this type is generally limited. An extraordinarily selective and versatile reagent is provided by nature in the form of the antibody, Ab. As a part of the immune defense system in animals, antibodies with high specificity can be synthesized by an organism in reasonable quantity within weeks of injecting a foreign species called an antigen, Ag (1). The ability of organisms to recognize the presence of "nonself ' and to respond rapidly by 0003-2700/85/A357-1321 $01.50/0 © 1985 American Chemical Society
synthesizing antibodies that exhibit high binding constants for the different surface chemical features of the antigen is a remarkable feat of life that can be used to great advantage in analytical chemistry. Molecules generally designated as antibodies include a number of classes and subclasses of the immunoglobulins, whose physiological sites of action, specificities, and even molecular weights vary widely. The vast majority of immunoassays are performed with immunoglobulin G and the subclasses contained within it. For this article, "antibody" refers to this class of molecule, which is a glycoprotein (mol wt ~160,000) in which binding is accomplished at two identical sites. It is estimated that an animal can "synthesize" up to 10 7 -10 8 antibodies of different specificities (2). This variability is the source of the immense versatility in achieving high specificity of binding for a broad range of chemical features. Because antigens typically have a molecular weight greater than ~1500 to elicit efficient antibody formation, antibodies for smaller mole-
cules, such as the many therapeutic drugs, are obtained by chemically attaching the smaller molecule (hapten) to some carrier antigen, usually a large protein. The resulting conjugate will elicit the formation of antibodies, some of which will be directed toward the appended hapten and will therefore constitute a selective reagent for the hapten. Binding (formation) constants of up to 1011 are exhibited between haptens and antibodies. Immunoassay is based on the use of an antibody as a selective chemical reagent for an antigen or a hapten analyte. The development of immunoassay is one of the great success stories in bioanalytical chemistry. It is estimated that several hundred million immunoassays are performed in the world each year. The predominant immunoassay has been radioimmunoassay (RIA), which is based on the detection of a radioactive label. RIA has had a dramatic impact on the clinical laboratory as a result of the ease with which assays can be conducted by automated equipment at low concentration because of the very low detection limits of radioactivity. Minimal sample preparation is required because of antibody selectivity. A significant advantage of RIA is the absence of interfering radioactivity in clinical samples. However, the desire to avoid the inconveniences of handling radioactivity has stimulated the development of nonisotopic immunoassays that are based on other labeling strategies. Thus far, the most successful of
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985 · 1321 A
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