Point: legitimate and illegitimate tests of free-analyte assay function.

In the history of clinical immunoassay, even up to the present day, no group of tests has been subjected to more scrutiny and controversy than analog-type freeanalyte assays (1– 4 ). Unfortunately, inappropriate design of many experiments has led users to an incorrect perspective of how analog assays work. An analysis of the important series of studies by Nelson and coworkers (5–10 ) shows that most experiments were conducted outside the valid working limits of analog-type or 2-step free-analyte assays. Here we explore the legitimacy of this viewpoint. In gold-standard methods of equilibrium dialysis and ultrafiltration, free analyte is first separated from that bound to serum proteins. Only then is the free analyte measured by what essentially is a high-sensitivity total analyte assay. For these methods, testing of assay parameters such as recovery of added analyte, linearity of response, and cross-reactivity are legitimate, applying equally to total analyte measurements. When the antibody probe is in direct contact with serum, however, the situation is radically different. For bound thyroxine (T4), serum contains a low-concentration, high-affinity binding protein (thyroxine-binding globulin) and high-concentration, low-affinity proteins (transthyretin and albumin). Free and total analyte are nonlinearly related (except with zero–thyroxine-binding globulin serum) (11 ). Recovery and linearity experiments in such conditions are thus not feasible, becoming increasingly less accurate at higher thyroxine-binding globulin concentrations (11 ). Analog-type and equilibrium dialysis/ultrafiltration methods must be calibrated differently. With gold-standard assays, direct calibration is done gravimetrically with known quantities of analyte, a method that is not possible with analog assays. Analog-type methods are calibrated by secondarily applying the values originally obtained from gold-standard methods to the same sera. The use of this calibration method is unavoidable, because much more analyte is bound to the antibody probe in the presence than the absence of the bound-analyte reservoirs of a given serum (12 ). The calibration of analog-type assays is analogous to water pressure measurement in a pipe from a reservoir, as illustrated in Fig. 1. The figure represents a reservoir that is connected to a thin pipe B of comparatively negligible volume ( 0.1% of the total). The reservoir size can vary greatly, with the flow through the pipe depending only on the pressure provided by height H of the water. The reservoir equates to analyte bound to variable amounts of serum proteins, and pipe B to free analyte entering the tissues. The higher the reservoir level, the greater is the flow and vice versa, equating to hyper or hypo analyte status in serum. If a thin manometer A is attached to the system, water will flow up the manometer to a height that is level with the reservoir. Owing to the negligible volume of both pipe B and manometer A, the reservoir’s water level scarcely changes (Fig. 1A). Thus, for various levels, calibrations can be marked on the manometer. This mirrors a free-analyte analog assay, the manometer being equivalent to added antibody probe and calibration marks equivalent to measured free analyte concentration. In an alternative situation, the pipe B is disconnected from the reservoir before attachment to manometer A (Fig. 1B). In this case, because there is no reservoir replenishing water in B as it enters the manometer, the final equilibrium position in the manometer rests much lower. This is equivalent to the performance of analog assays when only pure solutions of analyte are added (i.e., gross underestimation of the water pressure, and equivalently, the free analyte). The use of such pure solutions to analyze analog assays is invalid (5–10 ). If the reservoir level is unduly affected (lowered) by manometer sampling, invalidity also occurs, owing to overly large sequestration of fluid. Likewise, challenging overdiluted serum with a fixed amount of antibody results in undue disturbance of the natural equilibrium between free and bound analyte (12 ). The pipe B, disconnected from the reservoir before measurement of fluid by the manometer, is equivalent 1 North Lakes Clinical, West Yorkshire, United Kingdom, 2 Immunoassay consultant, Cardiff, Wales. * Address correspondence to this author at: North Lakes Clinical, Ben Rhydding, Ilkley, West Yorkshire LS29 8RX, Ilkley, UK. Fax 44 1943.602.052; e-mail [email protected]. Received December 12, 2008; accepted December 16, 2008. Previously published online at DOI: 10.1373/clinchem.2008.116525 Clinical Chemistry 55:3 439–441 (2009) Point/Counterpoint