Q.31 The affinity of an antibody can be determined quantitatively by
(A) MALDI–TOF MS (B) isoelectric focusing
(C) SDS–PAGE (D) equilibrium dialysis
Answer: (D) equilibrium dialysis
Equilibrium dialysis quantitatively measures antibody affinity (Kd) by establishing a binding equilibrium across a semi-permeable membrane separating free ligand from antibody-antigen complexes. Free ligand concentration equals that inside the antibody compartment at equilibrium, enabling Scatchard plot construction: r/[L] vs r yields Kd = -1/slope and Bmax = x-intercept.
Option Analysis
(A) MALDI-TOF MS: Incorrect—matrix-assisted laser desorption measures mass-to-charge ratio for molecular weight determination, not binding affinity.
(B) Isoelectric focusing: Incorrect—separates proteins by pI (net charge zero point) via pH gradient; reveals charge heterogeneity, not Kd.
(C) SDS-PAGE: Incorrect—denaturing gel electrophoresis sizes polypeptide chains under reducing conditions; assesses purity/mass, not functional affinity.
(D) Equilibrium dialysis: Correct—gold standard solution-based method directly measures free vs bound ligand concentrations for thermodynamic affinity constants.
Introduction: Antibody Affinity Equilibrium Dialysis
Antibody affinity quantitative determination via equilibrium dialysis remains the reference method in immunochemistry. This technique directly measures dissociation constants (Kd) by partitioning free antigen from antibody-bound complexes, essential for therapeutic monoclonal antibody development and biotech exam preparation.
Technique Comparison Table
| Method | Principle | Measures Affinity? | Quantitative Kd? |
|---|---|---|---|
| Equilibrium Dialysis | Free/bound ligand partitioning | Yes | Direct |
| MALDI-TOF MS | Mass spectrometry | No | No |
| Isoelectric Focusing | pH gradient migration | No | No |
| SDS-PAGE | Size-based electrophoresis | No | No |
Equilibrium Dialysis Protocol
- Setup: Antibody in dialysis chamber vs serial antigen dilutions outside membrane.
- Equilibrium: 24-48h diffusion until [free Ag] equalizes across membrane.
- Analysis: Measure [Ag]free (outside) = [Ag]free (inside); calculate bound via mass balance.
- Scatchard: r/[L] vs r·Kd plot yields linear affinity quantification.
Remains critical for high-precision biophysical characterization in biochemical engineering and molecular biology research.
