期刊
ACS OMEGA
卷 7, 期 30, 页码 26812-26823出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsomega.2c03237
关键词
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资金
- National Institutes of Health (NIH) [UG3DA048351, 1DP1DA034787]
- Henry M. Jackson Foundation for the Advancement of Military Medicine
- U.S. Army Medical Research and Medical Command (MRMC)
- KCR in the Drug Design and Synthesis Section
- Intramural Research Programs of the National Institute on Drug Abuse
- National Institute of Alcohol Abuse and Alcoholism
We proposed 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 by ED-fluorimetry are in agreement with those determined by a more established method. Importantly, the measured concentrations were not affected by the sample matrix, indicating the validity of this method for determining the binding-site concentration of polyclonal antibodies in serum samples. This simple and rapid approach has the potential to reliably perform quantitative antibody binding-site screening in serum and other complex biological fluids.
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.
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