Biotinylated FAB

Figure 7.3.2 Synkinetic membrane assembly on a water surface the biotin lipid forms a monolayer, which adsorbs streptavidin from the aqueous subphase. A biotinylated Fab fragment (see Sec. 9.6.9) then binds to the remaining binding sites of streptavidin. (From Herron et al., 1992.)...

As is briefly summarized in Fig. 13 this regeneration approach can be taken also for more complex architectures envisaged in biosensor configurations After binding of the streptavidin layer a layer of biotinylated Fab-fragments of Ad = 2.8nm is formed followed by a layer of HCG-antibodies of Ad = l.Snm. Again injection of free biotin removes the whole complexes and regenerates the free desthiobiotin surface (Fig. 13, left side). 

Fig. 1 Labeiing singie moiecuies on the ceii surface with QDs. Single membrane proteins are recognized by a primary antibody, which in turn is recognized by a biotinylated Fab fragment of a secondary antibody. A streptavidin-coated QD then binds to the biotin on the molecule/antibody complex...

Biotinylated Fab fragment of secondary antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA). ... 

Notes The label may also be biotin or any fluorophore. For antibody biotinylation, we use biotin-SP-conjugated AffiniPure Fab Fragment Goat Antimouse IgG (H+L) (Jackson ImmunoResearch Labs, Code Number 115-067-003). 

BPE-phenytoin donor or phenytoin in a sample is bound by biotinylated antiphenytoin antibody. The donor is quenched by a Texas Red-avidin acceptor. The assembly is small enough to fit into a 23-gauge needle. Cycle time is from 5 to 30 min, with excellent reproducibility. The sensor has been further optimized and the reagents have been characterized by the determination of association and dissociation constants/120, 121) Intact antibody as well as Fab fragments are used, resulting in a sensor which responds well over the clinically useful concentration range of phenytoin. 

Table n. Kinetics of Fab binding to biotinylated IL-1PK138C and mutant 1 ... 

Figure 6.5 Biotin head groups of a surface monolayer bind strongly to the protein streptavidin containing four biotin binding centres. A crystalline protein layer can now be observed by electron microscopy. The other binding centres can now be used to form a third monolayer with biotinylated proteins, e.g. the FAB fragment of F immunoglobulins. ...

There are, however, some specific characteristics and limitations for these variants. When anti-Ig antibodies are used, they should not be reactive with the immobilized antibody, i.e., for the more sensitive indirect methods (variants B, E and G) primary antisera from two different species should be available. This is not always practical or feasible and various ways have been developed to circumvent this problem (De Jong, 1983 Section 14.2). In variants C and D, the solid phase is coated with Fab or F(ab )2 and the complete antibody is applied in the third layer, together with enzyme-conjugated anti-lg antibodies (C) or enzyme-labeled SpA (which is Fc specific D) (Barbara and Clark, 1982 Koenig and Paul, 1982). Although this variant requires strictly Fc-specific antibodies, most anti-IgG antibodies, produced against complete IgG, are rather Fc-specific. If this is not the case, the anti-IgG serum could always be absorbed with an immunosorbent prepared with Fab or F(ab )2. In variants F and G biotinylated antibody and biotinylated enzyme are used... 

Similarly, Coutlee et al. (1989a) developed a sandwich assay in which (i) hybrids formed in solution between the biotinylated hapten probe and the target were collected by anti-biotin antibodies immobilized in wells of a microtiter plate and (ii) detected by /3-galactosi-dase-Fab fragment (of monoclonal antibody to RNA DNA) by conversion of a substrate to a fluorescent product. Optimum conditions included hybridization for 16 h at 75°C in 2 X SSC, 10 mM HEPES, using 0.1 xg/ml probe with 7% biotinylation. 

Reagents. Recombinant aequorin (Aq) with a free cys residue was from Chisso Corporation (Yokohama, Japan). Thermostable biotinylated firefly luciferase (b-Luc) was obtained from Kikkoman Corporation (Chiba, Japan). Aq labeled anti-Dig Fab fragment and b-Luc/streptavidin complex were produced by previously reported.1 HRP-labeled anti-DNP was purchased from LSL Co. (Tokyo). 

Ito K, Nishimura W, Maeda M, et al. Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase. Anal Chim Acta 2007 588 245-51. 

Eight-channel multiplexed Fab was site-specifically biotinylated from single engineered cysteine... 

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