Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.
Abstract
For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (exp...Continue Reading
References
Antibodies for phosphotyrosine: analytical and preparative tool for tyrosyl-phosphorylated proteins.
Immunogenicity of polylysine and polyornithine when complexed to phosphorylated bovine serum albumin
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Antibodies produced by B cells are highly specific for antigen as a result of random gene recombination and somatic hypermutation and affinity maturation. As the main effector of the humoral immune system, antibodies can neutralize foreign cells. Find the latest research on antibody specificity here.