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another approach that could be useful when only small amounts of sample are available and information on several clinical analytes is desired.Scheme for a theophylline flow-based simultaneous injection competitive binding immunoassay, using a label that consists of liposomes impregnated with carboxyfluorescein as a fluorescent marker.Other clinical analytes that have been measured by simultaneous injection competitive binding immunoassays include human chorionic gonadotropin (60), thyroid-stimulating hormone (60), HSA (61), IgG (62, 63), testosterone (64), and transferrin (61, 65); additional studies with theophylline have also been reported (66+-69).Other labels that have been reported include fluorescent tags such as fluorescein, Texas red, or lucifer yellow; chemiluminescent labels based on acridinium esters; and liposomes impregnated with fluorescent dye molecules (5).The easiest approach is to mix the sample with a labeled analyte analog (i.e., the label) and to inject the mixture simultaneously onto an immunoaffinity column that contains a relatively small amount of antibody.The detection of these labels generally is performed on line as they elute in the nonretained or retained peaks of the immunoaffinity column; however, fraction collection and off-line detection can also be used when required.6, in which theophylline was measured in serum by a flow-injection competitive binding immunoassay that used carboxyfluorescein-impregnated liposomes as the label.S-Octylglutathione has been reported as a ligand for the separation and analysis of glutathione S-transferase isoenzymes in human lung and liver samples (52, 53).Chromatographic Immunoassays One relatively new area of affinity chromatography that has received increasing attention in recent years has been the use of both low- and high-performance immunoaffinity columns to perform various types of immunoassays.6.

another approach that could be useful when only small amounts of sample are available and information on several clinical analytes is desired. columns based on miscellaneous ligands In addition to the ligands that have been mentioned already, several other types of ligands have also been used for the direct detection of clinical analytes by affinity chromatography. For example, an immobilized heparin column has been used for the determination of antithrombin III in human plasma (50, 51). S-Octylglutathione has been reported as a ligand for the separation and analysis of glutathione S-transferase isoenzymes in human lung and liver samples (52, 53). And finally, immobilized paminobenzamidine has been used for the separation of human plasminogen species, with the addition of an immobilized urokinase column for on-line detection (54). Chromatographic Immunoassays One relatively new area of affinity chromatography that has received increasing attention in recent years has been the use of both low- and high-performance immunoaffinity columns to perform various types of immunoassays. Such an approach is known as a ªchromatographic (or flow-injection) immunoassayº. This technique has been reviewed recently (5, 6, 55±58). This format is particularly valuable in the determination of trace analytes that by themselves may not produce a readily detectable signal when analyzed directly by affinity chromatography. This problem is overcome in chromatographic immunoassays by the use of a labeled antibody or analyte analog that can be used for indirect analyte detection. Many of the same labels that have been used in traditional immunoassays have also been used within chromatographic-based immunoassays. For example, enzyme labels such as horseradish peroxidase, alkaline phosphatase, and glucose oxidase have all been used in such methods. Other labels that have been reported include fluorescent tags such as fluorescein, Texas red, or lucifer yellow; chemiluminescent labels based on acridinium esters; and liposomes impregnated with fluorescent dye molecules (5). The detection of these labels generally is performed on line as they elute in the nonretained or retained peaks of the immunoaffinity column; however, fraction collection and off-line detection can also be used when required. competitive binding immunoassays There are several different methods for performing chromatographic immunoassays, but the most common method uses a competitive binding format. The easiest approach is to mix the sample with a labeled analyte analog (i.e., the label) and to inject the mixture simultaneously onto an immunoaffinity column that contains a relatively small amount of antibody. This format, known as a ªsimultaneous injection competitive binding immunoassayº, is the most common approach for chromatographic immunoassays. A specific example of this method is shown in Fig. 6, in which theophylline was measured in serum by a flow-injection competitive binding immunoassay that used carboxyfluorescein-impregnated liposomes as the label. The injected samples contained 50 L of serum diluted 1:100 and combined in a 1:2 ratio with a working solution of the label. The total cycle time between samples was 16 min, and good correlation was noted vs a fluorescence polarization immunoassay (59). Other clinical analytes that have been measured by simultaneous injection competitive binding immunoassays include human chorionic gonadotropin (60), thyroid-stimulating hormone (60), HSA (61), IgG (62, 63), testosterone (64), and transferrin (61, 65); additional studies with theophylline have also been reported (66±69). An alternative format that has been explored for clinical testing involves the application of only sample to the immunoaffinity column, followed later by a separate injection of the label. This method is known as a ªsequential injection competitive binding immunoassayº (61, 70). Fig. 6. Scheme for a theophylline flow-based simultaneous injection competitive binding immunoassay, using a label that consists of liposomes impregnated with carboxyfluorescein as a fluorescent marker. Reproduced with permission from Locascio-Brown et al. (59). Downloaded from https://academi