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Figure 6.2. Antigen calibration curves for (a) labeled antibody methods, and (b) labeled ligand methods, where the dashed lines show the analytically useful region of the curve.
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For example, a separation-based radioimmunoassay (RIA) has been developed for human placental lactogen (HPL),11 which is a protein hormone secreted by the placenta. The HPL levels have been used as indicators of normal placental function during pregnancy. It is antigenic, and antibodies may be produced by immunizing an animal with the human protein. The HPL is labeled by iodination with 125 I at its tyrosine residues, to give a labeled antigen, HPL*. This labeled species is a low-energy g-ray emitter. Figure 6.3 shows an outline of the assay procedure. A xed amount of HPL* and the unknown HPL are incubated with a xed, insuf cient quantity of anti-HPL antibodies. Competition for the Ab-binding sites ensues, and after a xed incubation period, the bound fraction is precipitated by the addition of ethanol. After centrifugation, the radioactivity in either the precipitate or the supernatant is measured.
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Figure 6.3. Outline of RIA for HPL.
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Standard curves are constructed by plotting radioactivity against log[HPL], as shown in Figure 6.4. If precipitate radioactivity is measured, then the measured signal decreases as HPL concentration increases, since fewer HPL* molecules will have access to the binding sites on the antibody. If supernatant radioactivity is quantitated, then the inverse will be true. Both curves exhibit a sigmoid shape, and the position of the midpoint of the curve on the log[HPL] axis depends on antibody af nity K0. For quantitative purposes, these sigmoid curves can be linearized using the logit transformation. First, the measured signals are normalized to values that lie between zero and one. The logit transformation is then performed, using logit y lnfy= 1 y g. If y is the fraction of the label in the supernatant, then a plot of logit(y) against log[HPL] will be linear with a negative slope, as detailed in 16. If the radioactivity of the precipitate is measured, then the logit transformation will yield a straight line with a positive slope.
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6.3.3. Radioisotopes Because radioisotopes were the rst labels used in immunoassay methods, there exists a vast array of radioimmunoassays. Radioisotopes are easily detected at low levels, and labeling procedures are simple. The label has virtually no effect on antibody antigen binding. However, radioisotopes are costly, hazardous, and require inconvenient monitoring and disposal procedures. In addition, isotopic decay necessitates the regular replacement of the labeled component. In general, 125 I is used as a label for large-protein antigen labeling.12 It has a half-life of 60 days, is a low-energy g-emitter, and thus requires only inexpensive instrumentation for detection. The isotope 3 H is commonly used for hapten labeling. It has a 12-year half-life and is a b-emitter, thus requiring a scintillation counter for detection. A further disadvantage of 3 H is that it has a low speci c activity, and yields poor detection limits relative to 125 I. Some specialized assays employ isotopes of Co, Fe, and Se. The RIA detection limits for antigens are $ 10 12 M, and are equalled only by the enzymatic labels, since they are capable of catalytic signal ampli cation.
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Figure 6.4. Calibration curves for HPL.
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6.3.4. Fluorophores Commonly used uorescent labels13 include uorescein, rhodamine, and umbelliferone derivatives (Fig. 6.5). Recently, a group of proteins isolated from algae have been used as immunoassay labels due to their high molar absorptivity (at least 10 times that of uorescein) and their quantum yields > 0.8; these are the phycobiliproteins, called phycoerythrin, phycocyanin, and allophycocyanin. Fluorescent labels are safe, and require no licensing for their use. Antigen detection limits of $ 10 10 M are normal with uorescent labels, two orders of magnitude higher than those of radioactive labels, as a result of scattering, quenching, and background uorescence from biological samples, especially those containing signi cant quantities of protein. Fluorescent labels offer many options for signal
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