General, Label-Free Method for Determining K(d) and Ligand Concentration Simultaneously.
Some of the most commonly used affinity reagents (e.g., antibodies) are often developed and used in conditions where their input concentrations ([L]0) and affinities (K(d)) are not known. Here, we have developed a general approach to determine both [L]0 and K(d) values simultaneously for affinity reagents (small molecules, proteins, and antibodies). To do this, we perform quantitative equilibrium exclusion immunoassays with two different concentrations of target and fit the data simultaneously to determine K(d) and [L]0. The results give accurate and reproducible measures of both values compared to established methods. By performing detailed error analysis, we demonstrate that our fitting gives unique solutions and indicates where K(d) and [L]0 measures are reliable. Furthermore, we found that a divalent model of antibody binding gives accurate K(d) and [L]0 values in both the forward (antibody immobilized) and the reverse (target immobilized) assays-addressing the long-term problem of obtaining quantitative data from reverse assays.
Mork Family Department of Chemical Engineering and Materials Science, ‡Department of Chemistry, §Department of Molecular Computational Biology, and ∥USC Norris Comprehensive Cancer Center, University of Southern California , Los Angeles, California 90089-2905, United States.
Farzad
F
Jalali-Yazdi
Mork Family Department of Chemical Engineering and Materials Science, ‡Department of Chemistry, §Department of Molecular Computational Biology, and ∥USC Norris Comprehensive Cancer Center, University of Southern California , Los Angeles, California 90089-2905, United States.
Terry T
TT
Takahashi
Mork Family Department of Chemical Engineering and Materials Science, ‡Department of Chemistry, §Department of Molecular Computational Biology, and ∥USC Norris Comprehensive Cancer Center, University of Southern California , Los Angeles, California 90089-2905, United States.
Richard W
RW
Roberts
D000906:Antibodies / Q000032:analysis* / Q000737:chemistry; D001713:Biphenyl Compounds / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D008024:Ligands; D009596:Nitrophenols / Q000032:analysis* / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010879:Piperazines / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D013449:Sulfonamides / Q000032:analysis* / Q000737:chemistry; D013816:Thermodynamics
C501332:ABT-737; D000906:Antibodies; D001713:Biphenyl Compounds; D008024:Ligands; D009596:Nitrophenols; D010455:Peptides; D010879:Piperazines; D013449:Sulfonamides