Solid-Phase Synthesis of a Biotin Derivative and its Application to the Development of Anti-Biotin Antibodies

Abstract

A biotin derivative, namely biotin-aminocaproic acid-lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid-lysine "long-arm" was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its N(epsilon)-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic "lysine-dendrimer" was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten.

Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes

“…Anti‐biotin rabbit IgG was isolated from a rabbit antiserum raised against a synthetic biotin derivative conjugated to bovine thyroglobulin, as previously described (Papasarantos et al ., ). Pooled antiserum, obtained from four consecutive bleedings, with a high enzyme‐linked immunosorbent assay — titer value (1:50 000) was used as starting material.…”

Fast and Sensitive Determination of the Fungicide Carbendazim in Fruit Juices with an Immunosensor Based on White Light Reflectance Spectroscopy

“…The benzimidazole conjugate [ 17 ] used for chip coating is also schematically presented in Figure 1 b. Initially, a lysine-core peptidyl moiety was prepared using a fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis strategy previously described by us [ 18 ] with slight modifications. This moiety was then functionalized with 3-maleimidopropionic acid [ 19 ].…”

“…Finally, the potential of regeneration and re-use of the biofunctionalized sensor chips was investigated, as a means to reduce the total cost of analysis. The novelty of the present work is mainly based on the combined use of an in-house prepared antibody for carbendazim recognition [ 17 ] and a benzimidazole derivative as a coating conjugate [ 17 , 18 , 19 ] in the WLRS immunoassay for carbendazim in different fruit juices.…”

“…Finally, the potential of regeneration and re-use of the biofunctionalized sensor chips was investigated, as a means to reduce the total cost of analysis. The novelty of the present work is mainly based on the combined use of an in-house prepared antibody for carbendazim recognition [17] and a benzimidazole derivative as a coating conjugate [17][18][19] in the WLRS immunoassay for carbendazim in different fruit juices. For carbendazim determination, a competitive immunoassay involving the delivery of a mixture of standards or fruit juice samples with a carbendazim-specific antibody to a benzimidazole conjugate-modified chip was conducted, as it is schematically depicted in Figure 1a.…”