measuring antibody/antigen reactions binding of antibody to antigen by itself is invisible: how can...
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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.
HTf
HTf HSA
BSAAb
completeidentity
non-identity
partialidentity
X
X
Y HSA
BSA
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.
Ab
HSA
Ab
HSA
Ab
HSA
Ab
HSA
Ab
HSA
Ab
HSA
Ouchterlony: Double Diffusion in Agarose
Ab = rabbit anti-HSA
Remember that Ab/Agbinding is reversible; this linecould move by dissolving alongone edge and re-precipitatingalong the other, dependingon the relative concentrationsof Ab and Ag…
Simple Precipitin Line
Antigen (HSA) and antibody(Ab) each begin to diffuse radially away from their wells.
Where they meet, aline of precipitationbegins to develop.
Ouchterlony: Double Diffusion in Agarose
HSA
Ab
BSA
HSA
Ab
BSA
Ab
HSA
BSA
Ab
HSA
BSA
Ab
HSA
BSA
Ab
HSA
BSA
aX,aY aY X
X
Y HSA
BSA
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”)