Chromatographic competitive binding immunoassays: a comparison of the sequential and simultaneous injection methods

Abstract

Two approaches for performing competitive binding immunoassays by HPLC and other flow-based systems are the simultaneous and sequential injection methods. Both these techniques make use of a column with a limited amount of antibody, onto which is injected a sample and a fixed amount of a labeled analyte analog. An indirect measure of the unlabeled analyte in the sample is then obtained by looking at the amount of analog in either the nonretained or retained peaks. In the simultaneous injection mode, the sample and labeled analog are applied at the same time to the column, while in the sequential mode the sample is injected first, followed by the analog. This results in a difference in the analytical characteristics of these two approaches. This study used chromatographic theory and previous data obtained for injections of human serum albumin (HSA) onto an anti-HSA antibody column to compare the response, detection limits, range, and sensitivity of these methods. Under equivalent conditions, it was found that the sequential method always provided the best lower limit of detection and sensitivity. However, the simultaneous mode had a broader dynamic range and higher upper limit of detection. From these observations, several guidelines were developed regarding the use and selection of such assays for new applications.

Analysis of Free Drug Fractions Using Near-Infrared Fluorescent Labels and an Ultrafast Immunoextraction/Displacement Assay

“…This value was calculated by using the relationship k = K A m L /V M , where K A is the association equilibrium constant for phenytoin with the antibodies, m L is the moles of active antibodies in the column, V M is the void volume of the immunoextraction layer, and (m L / V M ) is the effective concentration of active antibodies in this layer [48]. Further examination of the frontal results, as described in [28], gave a measured association rate constant (k a ) for phenytoin with the immobilized anti-phenytoin antibodies of 2.4 (± 0.4) × 10 6 M −1 s −1 at pH 7.4 and 37°C. From the relationship K A = k a /k d , the dissociation rate constant (k d ) for phenytoin from the immobilized antibodies was also determined, giving a value of 4.4 (± 0.8) × 10 −3 s −1 .…”

“…Other techniques (e.g., RAM) give only an incomplete separation of phenytoin's free and bound forms, making these methods difficult to use with real clinical samples [1,2,16,27]. This paper describes an alternative chromatographic-based approach for measuring free drug fractions based on an ultrafast immunoextraction/displacement assay (UFIDA), as illustrated in Figure 1 [28]. This approach uses an immunoextraction microcolumn that can bind a measurable amount of the free analyte (e.g., phenytoin) on a time scale that is sufficiently small to avoid dissociation of this analyte from its binding agents in the sample (e.g., HSA).…”

Chromatographic Immunoassays: Strategies and Recent Developments in The Analysis of Drugs and Biological Agents

“…The simultaneous injection method is a type of competitive binding chromatographic immunoassay in which a sample is mixed with a fixed amount of the label and both are applied together to a column containing a limited amount of an immobilized antibody or related binding agent for the target (see Figure 4) [7,8,62,63]. The target and label compete for this binding agent as the sample/label mixture is passed through the column.…”

“…These factors include the moles of label that are combined with the sample, the moles of the immobilized binding agent that are present, the injection flow rate and the adsorption kinetics of the column [8,62]. It is also important to use a label which can produce a signal that will not be significantly affected by the presence of the sample matrix, if the nonre-…”

“…The lowest detection limits in this method tend to occur at low-to-moderate flow rates and when using columns where the amount of active binding sites is in the same general range as the moles of target that are to be detected in the samples [8,62,63]. The fact that a labeled target analog is used in this method generally provides simultaneous injection methods with lower detection limits than can be obtained with chromatographic immunoassays that employ direct target detection [8].…”