Postcolumn immunodetection

The technique of postcolumn immunodetection involves the use of an lAC column that is attached to the exit of an analytical HPLC system. The lAC column in this approach serves to collect and retain a specific analyte from the HPLC column eluent for later detection. Both the on-off mode and immunoassay formats of I AC have been used as strategies for postcolumn immunodetection. The most common of these approaches is the one-site immunometric assay. One reason for this is that the immobilized analog columns in one-site immunometric assays often have a much more flexible selection of elution conditions than immobilized antibody columns. Another reason is that one-site immunometric columns can usually be used for many sample injections before they are eluted and regenerated, which helps to decrease the overall analysis time associated with their use in postcolumn detection. 

One factor that must be considered in postcolumn immunodetection is the need to adjust the eluent of the... 

Shahdeo, K. March, C. Kames, H.T. Postcolumn immunodetection following conditioning of the HPLC mobile phase by on-Hne ion-exchange extraction. Anal. Chem. 1997,69,4278. 

Monitoring a liquid chromatographic effluent by means of an immunoassay provides sensitive and se-leetive deteetion in combination with the separation of eross-reaetive compounds [1,2]. When implementing the immunoassay as a postcolumn reaction detection system after liquid chromatography, it is frequently referred to as immunodetection [3,4]. Automation and assay speed are the main advantages of immunodetection over off-line eoupling of immunoassays to liquid ehromatography by means of fraction collection [5,6]. 

Requirements with respect to the label used to mark one of the immunoreagents are comparable to those in other postcolumn reaction detection systems [4]. The label should preferably allow sensitive and rapid detection and be nontoxic, stable, and commercially available. So far, mainly fluorescence labels have been employed (e.g., fluorescein), although, in principle, also liposomes, time-resolved fluorescence, and electrochemical or enzymatic labels are feasible. On the other hand, labels providing a slow response, including radioactive isotopes and glow-type chemiluminescence, are less suitable for immunodetection. 

The attractiveness of immunodetection consists in its on-line coupling to a separation step, such as hquid chromatography. Parameters to consider are band broadening caused by the postcolumn reaction and interference of the liquid chromatographic mobile phase with the immunoreaction [4,7]. 

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