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Coupling immunoassays with chromatographic separation techniques
Author(s) -
Tang Zhe,
Karnes H. Thomas
Publication year - 2000
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/1099-0801(200010)14:6<442::aid-bmc26>3.0.co;2-9
Subject(s) - chromatography , chemistry , immunoassay , homogeneous , ethylene glycol , high performance liquid chromatography , selectivity , chromatographic separation , antibody , biochemistry , organic chemistry , physics , biology , immunology , thermodynamics , catalysis
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 antibodiesAg antigenAChE acetylcholinesterasesBODIPY 4,4‐difluoro‐5‐(2‐thienyl)‐4‐bora‐3a, 4a‐diaza‐ s ‐indacene‐3‐pro‐pionic, acid, succinimidyl esterGCSF granulocyte colony stimulating factorGHRF growth hormone release factorLC Liquid chromatographyLTC 4 , LTD 4 , LTE 4 leukotriene C 4 , D 4 and E 4 respectivelyPEG‐GCSF GCSF modified with poly(ethylene glycol)RPLC reversed‐phase liquid chromatographySPE solid‐phase extractionuPA urokinase