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During methods evaluation, standard curves should be plotted in several ways, since different plots reveal different reaction characteristics. For example, the bound/free versus concentration plot is very steep at the low concentration end, and may be used to calculate the detection limit of the assay. The sigmoidal plots of bound/total against log concentration clearly show regions of insensitivity (low and high concentration ends) that might not be clearly apparent in the logit log curve. The low detection limits of immunoassays depend on the typically high af nities of antibodies for haptens and antigens, as well as the detection limits of the labels used. The detection limit is usually de ned as the concentration that yields a signal that is equal to the mean of the blank signal plus two or three standard deviations. This establishes the con dence range for the zero response. For this calculation, the bound/free versus concentration plot is used. While detection limits allow comparisons of different immunoassay methods at the lower concentration end, they say nothing about assay reliability; for this reason, both detection limits and precision pro les should be compared. Accuracy can be de ned as the fundamental ability any assay to measure the true concentration of an analyte. Pure standards in a valid matrix are often dif cult to obtain, and the biological reactivity of less pure standards may not parallel their immunoreactivity in the assay. On the other hand, standards may be pure, but the assay antibody may react with similar molecules or fragments; this cross-reactivity is discussed in detail below. Accuracy must be con rmed by comparison with an established method(s) using real samples. Cross-reactivity (an indicator of assay speci city) has critical importance for immunoassay methods in which a particular analyte is assayed in the presence of very similar species; for example, in the monitoring of a therapeutic drug in serum where various metabolites of the drug are also present. For the sake of uniformity, cross-reactivity is usually reported as the mass or concentration of interferent
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Figure 6.11. Cross-reactivity of an interferent in a competitive immunoassay.
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required to displace 50% of the label, as shown in Figure 6.11. % C 100 S 50 C 50
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Cross-reactivity is evaluated by comparing the ability of each potential cross-reacting material to displace label. The percent cross-reactivity is equal to 100 times the concentration of analyte at 50% response divided by the concentration of interferent at 50% response. Cross-reactivity depends on the selectivity of an antibody for a particular epitope, and can be controlled to some extent by the careful design of the immunogen used to produce antibodies. Both the site of attachment and the nature of the hapten linkage to the carrier protein are critical for the selectivity of the assay. Additionally, the hapten carrier conjugate should be prepared in the same way as the hapten label conjugate used in the assay, so that similar epitopes are available for antibody binding. Antibodies raised against a hapten conjugated to a carrier protein through a linking group sill often recognize the linking group; for example, the hapten 2,4-dinitrophenol can be linked to a carrier protein by the reaction of 2,4dinitro uorobenzene (Sanger s reagent) with the primary amine groups of lysine residues, yielding an antibody that preferentially binds 2,4-dinitrophenyllysine over 2,4-dinitrophenol.25 Heterology is the term used to describe the use of different hapten derivatives for the preparation of the immunogen and the preparation of the labeled hapten. There are three types of heterology that are recognized as being important to assay selectivity.26 Hapten heterology occurs when different haptens are used to produce the immunogen and the labeled species; for example, an estradiol assay that produces the immunogen by linking estradiol to the carrier protein through the 11-hydroxy group, while the enzyme-labeled species was produced by linking esterone to peroxidase through the 11-hydroxy group, using the same linking reagent. Bridge heterology occurs when, for example, a succinyl linking group is used in the immunogen, and a glutaryl group is used for the hapten-label conjugate. Finally, site heterology occurs when the same linking group is connected to different sites on the hapten, such as the 11-hydroxy and 17-hydroxy groups on estrogen. In the absence of nonspeci c interferences, any heterologous combination of immunogen and labeled hapten result in better detection limits, often as much as 100-fold lower than those achievable with no heterology. The reason is that heterology reduces the association constant for the labeled hapten with the antibody, and free hapten will displace the labeled hapten more readily. Lower concentrations of free hapten are thus more readily detected. While heterology improves the detection limits of an assay, it signi cantly increases the cross-reactions that occur with similar analytes. It has been found that antibodies with the best selectivity are produced when immunogens are prepared by coupling haptens at a site far from the regions where interfering species have slight structural differences. In an assay for morphine (Fig. 6.12), for example, the immunogen and the enzyme hapten conjugate were prepared in exactly the
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