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Affinity diffusion II. Comparison between thermodynamic data obtained by affinity diffusion and precipitation in

C J van Oss, D R Absolom, P M Bronson

    Immunological Communications
    |January 1, 1982
    PubMed
    Summary
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    This study compared two methods for measuring antigen-antibody interactions, finding affinity diffusion captures specific binding, while precipitation assays reflect total system energy. Both methods show positive entropy, but affinity diffusion is isothermic, suggesting van der Waals forces dominate.

    Area of Science:

    • Immunochemistry
    • Biophysical Chemistry

    Background:

    • Accurate quantification of antigen-antibody interactions is crucial in immunology and diagnostics.
    • Different experimental methods may yield varying results due to distinct measurement principles.

    Purpose of the Study:

    • To determine and compare association constants (Ka) and thermodynamic parameters of the bovine serum albumin (BSA)-anti-BSA system using two distinct methods: affinity diffusion and precipitation in tubes.
    • To elucidate the underlying interactions (e.g., van der Waals, electrostatic) driving these associations under different conditions.

    Main Methods:

    • Affinity diffusion was employed to measure Ka and thermodynamic parameters (ΔF, ΔH, ΔS) of BSA-anti-BSA interactions.
    • Precipitation in tubes at optimal ratios was used as a comparative method for determining the same parameters.

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  • Experiments were conducted at varying temperatures and pH to probe the nature of the interactions.

    • Main Results:

    • Affinity diffusion yielded Ka values of 0.6–1.1 x 10^5 L/M, while precipitation in tubes gave Ka values of 3.3–11.2 x 10^7 L/M.
    • Thermodynamic analysis revealed differences: affinity diffusion showed ΔF ≈ -6 to -7 kcal/M, ΔH ≈ 0, and ΔS ≈ +23 entropy units.
    • Precipitation in tubes indicated ΔF ≈ -10.2 to -10.7 kcal/M, ΔH ≈ -4.6 to -7.6 kcal/M, and ΔS ≈ +10 to +20 entropy units.
    • Affinity diffusion was observed even at pH 9.5 where liquid-phase precipitation is abolished, suggesting a dominant role for van der Waals interactions.

    Conclusions:

    • The significant differences in Ka and thermodynamic profiles between the two methods are attributed to affinity diffusion measuring interactions of precipitating components with the highest dissociation constants, whereas precipitation in tubes reflects the total system's association energy.
    • Both methods demonstrate a positive entropy contribution, but affinity diffusion represents an isothermic process, contrasting with the exothermic nature of the total system measured by precipitation.
    • Affinity diffusion appears to primarily involve van der Waals interactions, as it remains effective at high pH where electrostatic interactions are minimized.