Time fluorometric immunoassay

Hemmila, I. (1988) Lanthanides as probes for time-resolved fluorometric immunoassays. Scand. J. Clin. Lab. Invest. 48, 389-400. 

E. Soini, Pulsed light, time-resolved fluorometric immunoassay, in Monocolonal Antibodies and New Trends in Immunoassays (Ch. A. Bixollon, ed.) pp. 197-208, Elsevier Science Publishers, Amsterdam (1984). 

Helmeste, D. M., Hammonds, R. G., Jr., and Li, C. H. (1986) Preparation of [I25I-Tyr27, Leu5]ph-endorphin and its use for cross-linking of opioid binding sites in human striatum and NG108-15 neuroblastoma-glioma cells. Proc. Natl. Acad. Sci. U.S.A. 83,4622—4625. Hemmila, I. (1988) Lanthanides as probes for time-resolved fluorometric immunoassays. Scand. J. Clin. Lab. Invest. 48,389—400. 

The dissociation-enhanced lanthanide fhtoroimmunoassay (DELFIA) technique is based on the principle of TRF. This theoretical concept was reduced to practice in the early 1970s [1 3] and was subsequently commercialized by the scientific equipment manufacturer, LKB/Wallac, as time-resolved fluorometric immunoassay methodology in the early 1980s [4 6]. DELFIA represents the first ultrasensitive nonisotopic immunoassay. This technology was reviewed in detail by Soini and Lovgren [7]. 

The enzyme attached to antibody 2 is critical for quantitative analysis. Figure 19-14 shows two ways in which the enzyme can be used. The enzyme can transform a colorless reactant into a colored product. Because one enzyme molecule catalyzes the same reaction many times, many molecules of colored product are created for each analyte molecule. The enzyme thereby amplifies the signal in the chemical analysis. The higher the concentration of analyte in the original unknown, the more enzyme is bound and the greater the extent of the enzyme-catalyzed reaction. Alternatively, the enzyme can convert a nonfluorescent reactant into a fluorescent product. Colorimetric and fluorometric enzyme-linked immunosorbent assays are sensitive to less than a nanogram of analyte. Pregnancy tests are based on the immunoassay of a placental protein in urine. 

The idea of the use of luminescent lanthanide chelates in bioanalysis can be traced back to the 1980s, when Hemmila (1985) reported a time-resolved fluorometric system using an Eu labeling reagent, which was commercially produced by Wallac Oy company. The system was intended for immunoassay and included the spectrometer for time-resolved measurements. The basic principle of the immunoassay system (DELFIA ) is shown in fig. 1. 

These three schemes of metabolism offer a unique perspective for discussing the analysis of metabolites in clinical chemistry labs. Traditionally, many laboratories have utilized the one analyte, one disease approach to testing. Hence, metabolite A in each of the scenarios would be a single measurement made in a laboratory, often by a relatively nonspecific test such as an immunoassay or fluorometric analysis. There are often numerous circumstances that can produce an elevation of a particular metabolite in addition to an enzyme deficiency produced by a metabolic disease. The result is a false positive. Measurement of more than one metabolite that is affected by a particular enzyme, Phe and Tyr as in the case of phenylketonuria (PKU), reduces the likelihood of a false positive as these amino acids are linked met-abolically. The case is similar in scenarios 2 and 3 where the pattern of metabolites may indicate one or more disorders that share common metabolic pathways. One final point regards scenario 2. Measurements of metabolites 2 or more steps away from the primary metabolic block, theoretically and in practice, are somewhat less reliable indicators of disease than the primary substrate. Often, however, no alternative is presented due to the primary substrate being chemically unstable or difficult to measure analytically. Generally, these metabolites are easier to detect in older infants as they accumulate over time. 

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