TY - JOUR
T1 - Analysis of antibody-antigen interactions using size-exclusion high- performance (pressure) liquid chromatography
AU - Sanny, Charles G.
AU - Price, Joseph A.
N1 - Funding Information:
The authors thank Oza M. McClain for her technical support in this study. Financial support was provided by the Of®ce of Research and the Department of Biochemistry/Microbiology of the Oklahoma State University College of Osteopathic Medicine and NIH Grant NIH R15 GM46117-01A1. We also thank C. Ownby for generously supplying the C. atrox venom.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 1997/3/1
Y1 - 1997/3/1
N2 - The development of methods to measure avidity of anti-sera reacting to antigen in solution using size-exclusion high-performance (pressure) liquid chromatography (SE-HPLC) was initiated using a model system composed of human serum albumin (HSA) and mouse anti-HSA IgG (mIgG). Mixtures containing varying concentrations of mIgG and HSA (mIgG, 0-2.4 μM, and HSA, 3.2 μM; mIgG, 0.80 μM, and HSA, 0-6.3 μM) were incubated for 1 h at 37°C. Mixture components were separated using a Bio-Sil TSK 250 size exclusion column (300 x 7.5 mm) equilibrated with 0.05 M sodium phosphate buffer (pH 6.8) containing 0.1 M sodium sulfate. Four separate peaks containing either HSA, mIgG, an intermediate molecular weight complex (C1) or a high-molecular- weight complex (C2) could be identified from the elution profiles at 280 nm. Plots of peak areas versus initial mIgG or HSA concentrations could be modeled mathematically using equations from the law of mass action. Apparent binding constants (1.1 x 107 to 5.2 x 107 liters/mol) and stoichiometry of binding are consistent with that reported in the literature.
AB - The development of methods to measure avidity of anti-sera reacting to antigen in solution using size-exclusion high-performance (pressure) liquid chromatography (SE-HPLC) was initiated using a model system composed of human serum albumin (HSA) and mouse anti-HSA IgG (mIgG). Mixtures containing varying concentrations of mIgG and HSA (mIgG, 0-2.4 μM, and HSA, 3.2 μM; mIgG, 0.80 μM, and HSA, 0-6.3 μM) were incubated for 1 h at 37°C. Mixture components were separated using a Bio-Sil TSK 250 size exclusion column (300 x 7.5 mm) equilibrated with 0.05 M sodium phosphate buffer (pH 6.8) containing 0.1 M sodium sulfate. Four separate peaks containing either HSA, mIgG, an intermediate molecular weight complex (C1) or a high-molecular- weight complex (C2) could be identified from the elution profiles at 280 nm. Plots of peak areas versus initial mIgG or HSA concentrations could be modeled mathematically using equations from the law of mass action. Apparent binding constants (1.1 x 107 to 5.2 x 107 liters/mol) and stoichiometry of binding are consistent with that reported in the literature.
UR - http://www.scopus.com/inward/record.url?scp=0031106709&partnerID=8YFLogxK
U2 - 10.1006/abio.1996.9995
DO - 10.1006/abio.1996.9995
M3 - Article
C2 - 9056176
AN - SCOPUS:0031106709
SN - 0003-2697
VL - 246
SP - 7
EP - 14
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -