biotinyl-n-hydroxysuccinimide-ester and biotin-hydrazide

The biotinylation of surface proteins and the detection of immunoprecipitated protein(s) after transfer to nitrocellulose using chemiluminescence methods is a highly sensitive alternative to hazardous radioactive labelling procedures. Ligation of proteins using the common biotin-NHS-ester (N-hydroxysuccinimido-biotin) is often associated with a decrease in immunoreactivity. Here a new non-radioactive method of leukocyte surface glycoprotein labelling using biotin-LC-hydrazide is described. This technique is based on the labelling of glycoproteins after mild oxidation of carbohydrate hydroxyl groups to reactive aldehydes. Flow cytometric and immunoprecipitation analyses of selected leukocyte markers such as CD3, CD26 and CD65 indicated that the alteration in immunoreactivity achieved by NHS-mediated biotin ligation was different from that obtained with hydrazide-mediated biotin ligation. CD3 and CD26 immunoreactivity was diminished using NHS biotinylation but preserved by biotin-LC-hydrazide, whereas CD65 binding to monoclonal antibodies was completely abolished after treatment with biotin-hydrazide. However, the immunoreactivity of CD13 was found to be totally unaffected by both NHS and hydrazide biotinylation. The combination of different biotinylation methods for surface protein labelling offers a viable alternative to radioiodination in the biochemical analysis of membrane proteins.

Topics: Antibodies, Monoclonal; Antigens, CD; Biotin; Cell Line; Cells, Cultured; Flow Cytometry; Humans; Leukocytes, Mononuclear; Membrane Proteins; Precipitin Tests; Succinimides; T-Lymphocytes; Tumor Cells, Cultured

We studied the effects of biotinylation on three monoclonal antibodies (Mabs) that were raised against carrier protein conjugates of human defensin HNP-1, and of rabbit defensins NP-2 and NP-5 respectively. Before biotinylation, each Mab specifically bound to its peptide hapten. Biotinylation of these Mabs by the N-hydroxysuccinimide-biotin (NHS-biotin) resulted in crossreactivity of each Mab with the two irrelevant defensin peptides. In contrast, Mab specificity was preserved after biotinylation with biotin hydrazide, which links biotin to the glycan moiety of antibodies. The effects of NHS-biotinylation were in part mimicked by 2,4-dinitrofluorobenzene, another agent that modified primary amine groups of proteins, suggesting that this modification contributed to the change in antibody specificity. When a high degree of antigenic specificity against peptide immunogens is required, biotinylation on the glycan moiety of Mabs may be preferable.

Topics: alpha-Defensins; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Antifungal Agents; Antigen-Antibody Reactions; Biotin; Blood Proteins; Cross Reactions; Defensins; Dinitrofluorobenzene; Enzyme-Linked Immunosorbent Assay; Epitopes; Humans; Hybridomas; Molecular Sequence Data; Rabbits; Sequence Homology, Amino Acid; Succinimides

We describe a non-radioactive method for the labeling of platelet surface proteins, consisting of platelet protein biotinylation by means of N-hydroxysuccinimido-biotin (NHS-B) and biotin-hydrazide (H-B); NHS-B labels proteins amino residues while H-B binds to periodate-modified sialoglycoproteins. Washed platelets were biotinylated and protein bands were detected after SDS-electrophoresis and western-blot using avidin-peroxidase and luminol as substrate to enhance the signal which was then detected by X-ray film. Biotin-labeled platelet proteins were also immunoprecipitated with monoclonal antibodies against glycoproteins Ib and the IIb-IIIa complex. The use of periodate induced biotinylation is the method of choice for labeling platelet surface glycoproteins while NHS-B also labels internal proteins. The sensitivity of this new procedure is similar to that obtained with radiolabeling techniques; biotinylation does not interfere with the antigenic properties of Ib and IIb-IIIa glycoproteins.

Topics: Antibodies, Monoclonal; Antigens, Human Platelet; Biotin; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Periodic Acid; Platelet Membrane Glycoproteins; Precipitin Tests; Succinimides

Topics: Biotin; Chemical Phenomena; Chemistry; Indicators and Reagents; Molecular Structure; Structure-Activity Relationship; Succinimides