Coupling immunoassays with chromatographic separation techniques

Coupling immunoassays with HPLC separation techniques is becoming increasingly useful in the analysis of biological and nonbiological samples of both large and small molecules. This is because it provides both sensitivity and selectivity for molecular analysis at relatively low cost, low maintenance and with excellent potential for automation. This paper reviews application of this hyphenated approach both in the pre-column immunoextraction and post-column immunodetection modes. Systems in which immunoassays are interfaced to chromatographic separations in order to separate bound and free fractions of the immunoassay will not be included since these systems do not provide the enhanced selectivity common to hyphenated systems. Post-column immunodetection is based on various immunoassay formats such as direct detection, one-site, competitive and sandwich immunoassays. Homogeneous immunodetectors are more convenient than heterogeneous immunodectors since there are no separation and column regeneration steps involved in homogeneous immunoassays. On the other hand, heterogeneous immunoassays are generally more sensitive than homogeneous immunoassays since interfering substances are removed prior to immunodetection. Advantages and limitations for the various approachs will be discussed. Copyright © 2000 John Wiley & Sons, Ltd. Abbreviations used: Abs antibodies Ag antigen AChE acetylcholinesterases BODIPY 4,4-difluoro-5-(2-thienyl)-4-bora-3a, 4a-diaza-s-indacene-3-pro-pionic, acid, succinimidyl ester GCSF granulocyte colony stimulating factor GHRF growth hormone release factor LC Liquid chromatography LTC4, LTD4, LTE4 leukotriene C4, D4and E4 respectively PEG-GCSF GCSF modified with poly(ethylene glycol) RPLC reversed-phase liquid chromatography SPE solid-phase extraction uPA urokinase