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Therefore, it can be extrapolated that a minor variation of the biotinylation process could cause a detectable ECL change in the competitive assays

Therefore, it can be extrapolated that a minor variation of the biotinylation process could cause a detectable ECL change in the competitive assays. Although DLA-IgG was used as a reference in this work, it must be understood that it was not solely or specifically chosen for BA-PCT. It can serve the same purpose for other biotinylated species. receptors, and polypeptides. Introduction The strong noncovalent conversation of biotin and avidin or streptavidin has long been employed in many protein and nucleic acid assays and purification methodologies.1 The rapid formation and high stability in various solvents of a biotinC(strept)avidin complex and the small molecular weight of biotin have established biotinC(strept)avidin chemistry as a platform, specifically, for clinical immunoassays so far developed by many researchers and product manufacturers. 2 In immunoassays involving biotinC(strept)avidin chemistry, an analyte-specific antibody is usually labeled with biotin at its amino sites through amide bond formation. Since both the -amino groups of em N /em -terminals and a large number of -amino groups of lysine residues are readily available for biotinylation, the labeled amino sites are randomly distributed in the Fab and Fc fragments of an antibody. A question is usually then raised as to how the batch-to-batch variation can be evaluated for better control of the biotinylation process, especially when the biotinylated species are crucial ingredients in clinical reagents. This ITI214 question has long been resolved by analytical approaches focusing on quantitation of biotin or determination of an average molar conjugation ratio of biotin to protein. No matter what the reporting signal modalities were, these methods were based on replacement3,4 of 2-(4-hydroxyazobenzene)benzoic acid (HABA) by biotin or biotinylated species, competition5,6 of (strept)avidin binding sites between biotin and biotinylated species, liquid chromatography,7,8 and electrospray ionization mass spectrometry (ESI-MS).9 ITI214 In terms of quantifying the bound biotin, these methods give either an average conjugation ratio or a distribution profile of the conjugation ratios.9 While widely used, the HABA assay does not have sufficient sensitivity and reproducibility. On the other side, although HBEGF a new commercial product QuantTag claimed a better assay performance in quantitation of bound biotin10 and ESI-MS provided precise distribution profiles of conjugation ratios, the variation of reaction conditions or manufacturing parameters may change the distribution of the amino sites to which the biotin moieties are attached. The inconsistency in the distribution of the occupied amino sites can certainly cause inconsistent ITI214 performance of the biotinylated species (e.g., antibodies in immunoassays).9 A more reasonable characterization of a biotinylated antibody (BA) is its binding capability after biotinylation of the antibody. In clinical immunoassays, the biotinylated antibodies (BAs) are, in most cases, used for immobilization of immunologically active species (analyte and analytes specific antibodies) on a streptavidin-coated solid state surface. Therefore, the term binding is usually threefoldfirst, binding to the streptavidin-coated solid-state surface through biotinCstreptavidin chemistry; second, binding specifically to the analyte (antigen); and third, binding nonspecifically to other species in a sample. In this work, the authors report a method of characterizing the binding capability or activity of a BA toward streptavidin-coated magnetic beads. This method is based on a competitive biotinCstreptavidin reaction and electrochemiluminescence (ECL) detection. A reference antibody (or protein) dually labeled with biotin moieties and ECL luminophores is used in this method. When mixing the dually labeled antibody (DLA) and the BA of interest with streptavidin-coated magnetic beads (MBs), the conjugated biotin moieties on DLA and BA will compete for the binding sites of streptavidin on MBs. At the end of the competitive biotinCstreptavidin reaction, both the DLA and the BA of interest are immobilized on the surface of MBs (see Scheme 1). Open in a separate window Scheme 1 Dually Labeled Antibody (DLA) and a Biotinylated Antibody (BA) Competing for the Binding Sites of Streptavidin on the Surface of a Magnetic Bead (MB)The MBs attached with both antibodies were brought into a flow cell for magnetic separation and ECL measurement. When brought into an ECL measurement cell built in an automated ECL immunoassay analyzer, the ECL moieties, which are attached to the DLA immobilized on the surface of MBs, will generate an ECL signal under conditions described generally in a number of review papers11? 15 and specifically in a recent book chapter.16 In a competitive immunoassay, a stronger ECL signal implies a higher.