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LT1009 is a humanized version of murine LT1002 IgG1 that employs two bridging Ca²⁺ ions to bind its antigen, the biologically active lipid sphingosine-1-phosphate (S1P). We crystallized and determined the X-ray crystal structure of the LT1009 Fab fragment in 10 mM CaCl₂ and found that it binds two Ca²⁺ in a manner similar to its antigen-bound state. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds Ca²⁺ in solution and inductively-coupled plasma-mass spectrometry (ICP-MS) revealed that, although Ca²⁺ is preferred, LT1002 can bind Mg²⁺ and, to much lesser extent, Ba²⁺. Isothermal titration calorimetry (ITC) indicated that LT1002 binds two Ca²⁺ ions endothermically with a measured dissociation constant (<i>K</i>_D) of 171 μM. Protein and genome sequence analyses suggested that LT1002 is representative of a small class of confirmed and potential metalloantibodies and that Ca²⁺ binding is likely encoded for in germline variable chain genes. To test this hypothesis, we engineered, expressed, and purified a Fab fragment consisting of naïve murine germline-encoded light and heavy chain genes from which LT1002 is derived and observed that it binds Ca²⁺ in solution. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved across diverse mammalian genomes.