The effect of pH on the aggregation of biotinylated antibodies and on the signal-to-noise observed in immunoassays utilizing biotinylated antibodies

Technical Aspects of Immunohistochemistry

“…108 Another cause of increased hydrophobicity of Igs is biotinylation of Abs, which can modify their pI. 154 Several methods can reduce hydrophobic binding of Igs and tissue proteins, including diluent buffers with a pH different from the pI of the Ab (particularly for monoclonal Abs); diluents with low ionic strength (low salt concentration); addition of nonionic detergents (e.g., Tween 20, Triton X) or ethylene glycol to the diluent; or raising the pH of the diluent used for polyclonal Abs. 11,69,108 However, probably the most common method to reduce background from hydrophobic interactions is the use of blocking proteins prior to incubation of the primary Ab (Fig.…”

Digoxigenylated primary antibodies for sensitive dual-peroxidase labelling of neural markers

“…The modification of primary antibodies with a digoxigenin-bearing active ester is a mild and non-laborious procedure, and might be performed analogous to the recently reported preparation of biotinylated primary antibodies for immunocytochemistry (H~tig et al 1994a). No advantage was gained by modifying the primary antibodies with double the amount of dig-ONSu (200 gg/mg IgG); in fact, it is noteworthy that the tagging of antibodies with larger amounts of haptens might cause problems in bioanalytical procedures (Wadsley and Watt 1987). Moreover, unspecific immunofluorescence labelling is often ascribed to the creation of acidic immunoglobulins due to the blocking of primary amino groups (mainly lysyl residues) after using inappropriate fiuorochrome/protein ratios for haptenization procedures.…”

Monoclonal Antibodies

“…Non-specific binding of this material was recorded in coated ELISA wells (Figures 6.5 and 6.9) but not in uncoated wells ( Figure 6.9B), which showed that the binding was caused by protein-protein interactions and not by adsorption onto the polystyrene surface under the conditions of the assay. Non-specific binding of biotin-MAbs (Figures 6.8,6.10A and 6.13B) was reportedly a pH-related phenomenon since biotinylation shifted the isoelectric pH of MAbs towards more acidic values, causing decreased solubilities and therefore higher non-specific binding at pH values of 7.0 or less, so that the highest signal-to-noise ratios were obtained at pH 8.0-9.0 (Wadsley and Watt, 1987). All of the two-site AVP ELISAs were done at pH 7.5, and the effect of pH was not investigated, but non-specific binding might have been lower at pH 8.0.…”

“…However, no detectable two-site binding occurred between ESVP 1 and ESVP 3, irrespective of whether ESVP 3 was in the liquid phase ( Figure 6.8A) or coated onto the solid phase ( Figure 6.8B). This was probably due to the IgM isotype of ESVP 3, which would have caused much greater steric hindrance than an IgG antibody, although the high non-specific binding of the biotinylated MAbs possibly obscured a specific binding response that might have been revealed under different solution conditions (Wadsley and Watt, 1987).…”