Detection hydrazide-avidin

These techniques have been used to target, detect, or assay glycoproteins in solution or on cell surfaces by using hydrazide-activated enzymes, avidin, or streptavidin (Chapter 23, Section 5) (Bayer and Wilchek, 1990 Bayer et al., 1987a, b, 1990) and to form conjugates with glycoproteins. 

Other molecules can be used in this type of assay approach. Hydrazide-modified (strept)avidin, lectins, biocytin, fluorescent probes and other detectable molecules can be used to detect specifically glycoconjugates in biological samples (Wilchek and Bayer, 1987). 

First, examples of fluorescence [235] and chemiluminescence-based biosensors [236], derived fix)m PTs, were reported by Tripathy and coworkers. Later, the synthesis of 119 containing pendant biotin units was described. A water-soluble copolymer 120 with sulfonate and biotin in the side chains [237] exhibits a deep violet color (Amax = 550 run) which turns yellow (A ax = 400 nm) on binding with avidin. An extension of this work based on the homopolymer 121 involved the preparation of monolayers of a biotinylated PT on an aminosilane-treated ITO surface by successive deposition of 121 and biocytin hydrazide in electrostatic interactions [238,239]. This ultrathin film modified electrode was shown to detect femtomoles of avidin in aqueous solution. Electrochemical and optical evidences for avidin binding were reported for a copolymer based on poly(terthiophene) 122 [240] and for the homopolymer 123 [198]. 

Several established protocols have been adapted for 96-well plate readers including catalase, hyaluronidase, acetylcholinesterase, protein phosphatases and membrane-bound ATPases (22-26). In several instances these have involved novel protocols that are well suited to the ELISA format. For example, a sensitive, rapid microtitre-based assay for hyaluronidase activity was described by Frost and Stem (23). The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtitre plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtitre plate, artefacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples, with an interassay variation of less than 5%. 

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