A Rapid Method for Direct Quantification of Antibody Binding-Site Concentration in Serum

• Published in: ACS omega Vol. 7; no. 30; pp. 26812 - 26823
• Main Authors: Abucayon, Erwin G., Whalen, Connor, Torres, Oscar B., Duval, Alexander J., Sulima, Agnieszka, Antoline, Joshua F. G., Oertel, Therese, Barrientos, Rodell C., Jacobson, Arthur E., Rice, Kenner C., Matyas, Gary R.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.08.2022
• ISSN: 2470-1343; 2470-1343
• DOI: 10.1021/acsomega.2c03237

The quantitation of the available antibody binding-site concentration of polyclonal antibodies in serum is critical in defining the efficacy of vaccines against substances of abuse. We have conceptualized an equilibrium dialysis (ED)-based approach coupled with fluorimetry (ED-fluorimetry) to measure the antibody binding-site concentration to the ligand in an aqueous environment. The measured binding-site concentrations in monoclonal antibody (mAb) and sera samples from TT-6-AmHap-immunized rats by ED-fluorimetry are in agreement with those determined by a more established equilibrium dialysis coupled with ultraperformance liquid chromatography tandem mass spectrometry (ED-UPLC-MS/MS). Importantly, we have shown that the measured antibody binding-site concentrations to the ligand by ED-fluorimetry were not influenced by the sample serum matrix; thus, this method is valid for determining the binding-site concentration of polyclonal antibodies in sera samples. Further, we have demonstrated that under appropriate analytical conditions, this method resolved the total binding-site concentrations on a nanomolar scale with good accuracy and repeatability within the microliter sample volumes. This simple, rapid, and sample preparation-free approach has the potential to reliably perform quantitative antibody binding-site screening in serum and other more complex biological fluids.