1. Measuring Antibody/Antigen Reactions Binding of antibody to antigen by itself is invisible: how can it be detected & quantitated ? One visible consequence of Ab/Ag binding may be precipitation, and one technique for detecting Ag/Ab reactions by precipitation in a semi-solid gel is the Ouchterlony assay.

2. Ouchterlony Analysis: 2-Dimensional Precipitation in Agarose Several distinct patterns of reactivity are shown above – the following animations illustrate how some of these patterns may be formed.

3. Ouchterlony: Double Diffusion in Agarose Ab = rabbit anti-HSA Remember that Ab/Ag binding is reversible; this line could move by dissolving along one edge and re-precipitating along the other, depending on the relative concentrations of Ab and Ag… Simple Precipitin Line Antigen (HSA) and antibody (Ab) each begin to diffuse radially away from their wells. Where they meet, a line of precipitation begins to develop.

4. Ouchterlony: Double Diffusion in Agarose  X,  Y YY Ab = rabbit anti-HSA Reaction of Partial Identity X X,Y The “αX+X” line (formed between “Ab” and “BSA”) cannot extend any further upward because the “αX” antibody cannot cross the other precipitin line (since that line also contains epitope “X”)