Drug-dependent
antibodies (DDAbs) that cause acute
thrombocytopenia upon
drug exposure are nonreactive in the absence of the
drug but bind tightly to a
platelet membrane glycoprotein, usually α(IIb)/β3
integrin (GPIIb/IIIa) when the
drug is present. How a
drug promotes binding of antibody to its target is unknown and is difficult to study with human DDAbs, which are poly-specific and in limited supply. We addressed this question using
quinine-dependent murine
monoclonal antibodies (mAbs), which, in vitro and in vivo, closely mimic
antibodies that cause
thrombocytopenia in patients sensitive to
quinine. Using surface plasmon resonance (SPR) analysis, we found that
quinine binds with very high affinity (K(D) ≈ 10⁻⁹ mol/L) to these mAbs at a molar ratio of ≈ 2:1 but does not bind detectably to an irrelevant mAb. Also using SPR analysis, GPIIb/IIIa was found to bind monovalently to immobilized mAb with low affinity in the absence of
quinine and with fivefold greater affinity (K(D) ≈ 2.2 × 10⁻⁶) when
quinine was present. Measurements of
quinine-dependent binding of intact mAb and fragment
antigen-binding (
Fab) fragments to platelets showed that affinity is increased 10 000- to 100 000-fold by bivalent interaction between antibody and its target. Together, the findings indicate that the first step in
drug-dependent binding of a
DDAb is the interaction of the
drug with antibody, rather than with
antigen, as has been widely thought, where it induces structural changes that enhance the affinity/specificity of antibody for its target
epitope. Bivalent binding may be essential for a
DDAb to cause
thrombocytopenia.