Human Anti-Lysophosphatidic acid Recombinant Antibody (clone LT3015)

Overlay of four KinExA equilibrium affinity experiments with constant antibody (1 nM) and varying concentrations of FAF-BSA: blue 3 µM, green 30 µM, orange 150 µM, and pink 300 µM show the response curves shifting towards higher lipid concentrations in response to the BSA sequestering LPA(18:1). Naively fitting any of these curves would result in an incorrect estimation of the LT3015–LPA(18:1) K d value.

Fleming, J. K., Glass, T. R., Lackie, S. J., & Wojciak, J. M. (2016). A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay. Journal of lipid research, 57(9), 1737-1747.

Global curve fitting of the three affinity experiments used to determine the equilibrium dissociation constants for the individual LPA species (panels A-E) or S1P (panel F) binding the antibody or FAF-BSA in solution. The percentage of antigen-free binding sites on the antibody (in duplicate) is plotted as a function of the lysophospholipid concentration in each sample. The LT3015 and FAF-BSA concentrations used in each LPA experiment are as follows: blue curve, 0.5 nM LT3015 and 13 nM FAFBSA; green, 10 nM LT3015 and 13 nM FAF-BSA; orange, 10 nM LT3015 and 10 µM FAF-BSA. For the S1P, the LT1009 and FAF-BSA concentrations are as follows: blue, 1 nM LT1009 and 1 µM FAF-BSA; green 10 nM LT1009 and 1 µM FAF-BSA; orange, 10 nM LT1009 and 500 µM FAF-BSA.

Fleming, J. K., Glass, T. R., Lackie, S. J., & Wojciak, J. M. (2016). A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay. Journal of lipid research, 57(9), 1737-1747.

(b) Saturation binding assay of biotinylated-LPA (18:0) to surface-anchored LT3015 and LT1009 antibodies and Fab fragments. (c) Competition assays in which unlabeled LPA (14:0) or (18:2) were used to displace biotinylated-LPA (18:0) from LT3015 whole antibody (IgG) or Fab fragment (Fab) complexes.

Fleming, J. K., Wojciak, J. M., Campbell, M. A., & Huxford, T. (2011). Biochemical and structural characterization of lysophosphatidic Acid binding by a humanized monoclonal antibody. Journal of molecular biology, 408(3), 462-476.

(a) LPA binding affinity measured as in Figure 1B for native LT3015 (WT) and three LT3015 single point mutations. (b) In comparison to native LT3015 (WT), the introduction of mutations in the LT3015 heavy chain (AsnH52Tyr/SerH52Tyr) or light chain (AnsL30Arg) does not significantly increase LPA affinity. However, their combination (AsnH52Tyr/SerH54Tyr/AsnL30Arg) results in a modified version of the antibody with increased LPA binding affinity.

Fleming, J. K., Wojciak, J. M., Campbell, M. A., & Huxford, T. (2011). Biochemical and structural characterization of lysophosphatidic Acid binding by a humanized monoclonal antibody. Journal of molecular biology, 408(3), 462-476.

(a) Competition binding assays were carried out on surface-anchored biotin-LPA (18:0):LT3015 complexes with the lipids: sphingosine-1-phosphate (S1P), lysophosphatidylcholine (18:0 LPC), phosphatidic acid (PA), phosphatidiylcholine (PC), and platelet-activating factor (PAF) in comparison with LPA (18:1 LPA). (b) Competition binding assays were carried out on surface-anchored biotin-LPA (18:0):LT3015 complexes with the lipids: 1-alkyl-lysophosphatidic acid (C18:1 ether LPA), monoacylglyceral (18:1 sn-1 ester MG), and lysophosphatidic acid cyclic phosphodiester (18:1 ester cLPA) in comparison with LPA (18:1 ester LPA). Other than native LPA, only cLPA shows appreciable binding to LT3015.

Fleming, J. K., Wojciak, J. M., Campbell, M. A., & Huxford, T. (2011). Biochemical and structural characterization of lysophosphatidic Acid binding by a humanized monoclonal antibody. Journal of molecular biology, 408(3), 462-476.