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An antibody is a protein that recognizes particular molecular targets through binding. Antibodies participate in immunity; laboratory assays such as Western blots exploit their selective binding to detect chosen proteins.

An IgG gives a concrete architecture: two immunoglobulin heavy chains and two immunoglobulin light chains, joined by disulfide bonds. Each arm ends in a paratope assembled from an antibody variable region of each kind of chain. The two paratopes recognize the same epitope in this conventional IgG; the flexible hinge allows different arm separations. The antibody constant regions form the remaining portions. This is an illustrative IgG, rather than the architecture of every antibody format. ^[ref]

Three questions must be kept separate. Antibody specificity asks which antigens are recognized. Antibody affinity describes one paratope–epitope interaction. Avidity describes the combined attachment when several contacts can cooperate. A selective antibody can have modest antibody affinity, and a monoclonal antibody can still show cross-reactivity. For an assay, the practical question is whether the observed readout identifies the intended antigen under the tested conditions; Western blot antibody specificity controls make that question concrete.

Antibody equilibrium occupancy counts occupied antibody binding sites, rather than retained assay label. For an original 1:1 model, let $A$ denote an unoccupied site, $E$ free antigen bearing one accessible epitope, and $AE$ their complex. At chemical equilibrium, writeSquare brackets mean concentrations. The dissociation constant $K_d$ here has concentration units. This is the same independent-site calculation as receptor binding occupancy, now applied to an antibody binding site. The last equality follows by substituting $[AE]=[A]c/K_d$ into the occupied fraction and cancelling $[A]$.

Assume identical independent sites, reversible 1:1 binding, dilute chemical solution behavior, fixed temperature and buffer solution, and enough time for chemical equilibrium. Exclude two-arm bridging, cross-reactivity and inaccessible sites from this model. Crucially, $c$ is free antigen concentration; substituting the amount supplied per volume requires negligible depletion by binding. Independent IgG arms could obey this model site by site, but it does not describe avidity from bridging a surface.

Stipulate $K_d=20\mathrm{nM}$, with $1\mathrm{nM}=10^{-9}\mathrm{mol}{\mathrm{L}}^{-1}$. Free antigen concentrations $2$, $20$ and $200\mathrm{nM}$ give $\theta =1/11$, $1/2$ and $10/11$. A second stipulated dissociation constant of $200\mathrm{nM}$ instead gives $\theta =1/11$ at $c=20\mathrm{nM}$. Both curves approach one at large $c$: a nearly saturated readout can conceal an antibody affinity difference. These are invented values, with no universal threshold for a useful antibody.

A Western blot readout additionally depends on Western blot transfer, accessible epitopes, loss during washing and Western blot antibody detection. Western blot response saturation can flatten the final signal even when binding itself remains responsive. An ELISA also reports retained label through an enzyme readout, rather than automatically reporting $\theta$. Thus half of an assay's maximum signal need not occur at $c=K_d$. Varying free antigen through concentrations near the candidate dissociation constant, while checking depletion and the readout's response, is more informative than comparing a single saturated point.

Cross-reactivity occurs when an antibody also recognizes another antigen beyond the one against which it was raised. This may involve related features, but obvious overall similarity is not required. ^[ref]

For an imagined example, a paratope contacts a small surface patch shared by two otherwise different proteins. Both can bind even though their remaining surfaces differ. Increasing antibody concentration can reveal weak binding that was previously undetectable. Conversely, an extra Western blot band need not prove cross-reactivity: the intended protein may occur in multiple forms, or detection reagents may contribute background. The target-deleted and no-primary antibody comparisons under Western blot antibody specificity controls test different explanations.

A monoclonal antibody preparation derives from one selected antibody-producing biological cell and its descendants, or reproduces their specified antibody sequences. Monoclonal describes the origin and composition of the preparation; it does not guarantee absence of cross-reactivity or establish antibody affinity. ^[ref]