Anti-beta 2 Microglobulin Anticorps [B2M-01] (Biotin) (A85547)

The application of optical biosensors in the study of macromolecular interactions requires immobilization of one binding partner to the surface. It is often highly desirable that the immobilization is uniform and does not affect the thermodynamic and kinetic binding parameters to soluble ligands. To achieve this goal, a variety of sensor surfaces, coupling strategies and surface chemistries are available. Previously, we have introduced a technique for determining the distribution of affinities and kinetic rate constants from families of binding and dissociation traces acquired at different concentrations of soluble ligand. In the present work, we explore how this affinity distribution analysis can be useful in the assessment and optimization of surface immobilization. With this goal, using an antibody-antigen interaction as a model system, we study the activity, thermodynamic and kinetic binding parameters, and heterogeneity of surface sites produced with different commonly used sensor surfaces, at different total surface densities and with direct immobilization or affinity capture.

Background: : The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and albumin from degradation and thus contributes to maintain high serum levels of these proteins. Analbuminemia is a rare autosomal recessive disorder characterized by clinically observed allelic albumin variants that are absent or found in very low concentrations in the blood circulation. Such variants may have altered FcRn binding properties that affect their half-life, biodistribution and thereby transport ability.

Methods: : We established an easy cloning, expression and purification strategy to obtain recombinant GST-tagged human serum albumin (HSA) variants for evaluation of pH dependent FcRn binding properties using an enzyme-linked immunosorbent assay (ELISA) and a real time surface plasmon resonance (SPR) biosensor system.

Results: : The strategy yielded purified GST-tagged albumin variants. A recombinant truncated HSA variant similar to a clinically observed splice mutant denoted Bartin, here abrogated HSA(Bartin), showed no detectable pH dependent FcRn binding compared to a fully functional albumin wild type variant, HSA(Wt), and a truncated HSA variant consisting of only the carboxy terminal domain III (HSA(DIII)).

Conclusions: : The approach described can be used to rapidly screen clinically observed truncated or otherwise mutant or modified HSA variants regarding their pH dependent FcRn binding properties. Here, we demonstrate that a recombinant truncated HSA variant, HSA(Bartin), does not interact with FcRn, which gives a molecular explanation for the low serum levels. In addition, DIII of HSA alone was shown to retain its FcRn binding property.