During antibody development, two well-defined mechanisms, somatic recombination and hypermutation, serve to enhance antibody affinity to its antigen from a moderate to high binding. An alternative strategy, metal coordination, has not been widely considered. This is surprising considering the unique coordination properties that can be utilized by the incorporation of metals into the antigen-binding site. Recent characterization of an IgG antibody (LT1009) by X-Ray crystallography revealed the coordination of two interfacial Ca__ ions to specifically bind the phosphate group on sphingosine-1-phosphate (S1P). This brings forth interesting questions regarding processes of B-Cell development and the binding interactions that promote the downstream signals that eventually trigger B-cell affinity maturation. This study investigates this metal coordination property of the murine version of LT1009, called LT1002, in the absence of the S1P antigen. To do so we implement Flame Atomic Absorption, Induced-Coupled Spectrometry and Isothermal Titration Calorimetry to enable us to gain a fundamental understanding of the specificity of this metal coordination and the relative strength of the interaction. We incorporated several bioinformatic tools to iterate through thousands of immunoglobulin genes to see if the metal coordination motif is evolutionarily conserved and whether this antibody preserves a specific function. Lastly, we made initial advances on the expression of the genome encoded Fab region of LT1002 in BL21(DE3) E. coli cells in attempt to gather if this antibody retains its metal-binding capability.
