Fig. 1.
Fig. 1. The DDAB ELISA detects pathophysiologically relevant antibodies. / (A) Detection of GP IIb/IIIa antagonist-dependent DDABs in chimpanzee. GP IIb/IIIa-coated wells were incubated with the indicated dilutions of chimpanzee A264 PPP in the presence or absence of L-739758 and analyzed using the initial ELISA version (see “Materials and methods”). The change of absorbance (mOD/min) of wells in the absence of drug (closed squares) and in the presence of GP IIb/IIIa antagonist (closed circles) is indicated. The change of absorbance in the presence of drug of a control chimpanzee PPP is indicated (open circles). (B) DDABs present in A264 PPP can be specifically depleted by platelets. A264 PPP was added to human DDAB− citrated whole blood in the absence (no drug) or presence (drug) of 50 nM L-739758 and incubated for 15 minutes at room temperature. EDTA (4.5 mM) was added to samples as indicated for an additional 15 minutes at room temperature. PPP was recovered and tested in the DDAB ELISA as in panel A (improved version). No drug wells were incubated with XP280 to prevent carry-over of L-739758. XP280 competes with L-739758 for GP IIb/IIIa binding but fails to elicit binding of chimpanzee A264 DDABs (Table 1). Black bars indicate mOD/min minus drug wells; open bars, mOD/min plus drug wells; gray bars, mOD/min L-739758 wells minus mOD/min XP280 wells. (C) Correlation between ELISA and FACS signal. (i) Wells were coated with GP IIb/IIIa in the absence (open circles) or presence (closed circles) of L-739758 and incubated with A264 PPP for 10 minutes, followed by PPP transfer to the next well. This depletion procedure was repeated 11 times. After 12 incubation steps, bound IgG on the depletion plate was detected (improved ELISA format). (ii) The ELISA-depleted PPPs were subsequently incubated with gel-filtered platelets in the presence of L-739758, followed by addition of FITC-labeled antihuman IgG and analyzed by FACS. The results are expressed as mean fluorescence of 10 000 events. Minus drug is A264 PPP incubated on ELISA plates in the absence of GP IIb/IIIa antagonist. Plus drug is A264 PPP incubated on ELISA plates in the presence of GP IIb/IIIa antagonist. Control PPP is ELISA DDAB− PPP from the platelet donor used for the FACS experiment.

The DDAB ELISA detects pathophysiologically relevant antibodies.

(A) Detection of GP IIb/IIIa antagonist-dependent DDABs in chimpanzee. GP IIb/IIIa-coated wells were incubated with the indicated dilutions of chimpanzee A264 PPP in the presence or absence of L-739758 and analyzed using the initial ELISA version (see “Materials and methods”). The change of absorbance (mOD/min) of wells in the absence of drug (closed squares) and in the presence of GP IIb/IIIa antagonist (closed circles) is indicated. The change of absorbance in the presence of drug of a control chimpanzee PPP is indicated (open circles). (B) DDABs present in A264 PPP can be specifically depleted by platelets. A264 PPP was added to human DDAB− citrated whole blood in the absence (no drug) or presence (drug) of 50 nM L-739758 and incubated for 15 minutes at room temperature. EDTA (4.5 mM) was added to samples as indicated for an additional 15 minutes at room temperature. PPP was recovered and tested in the DDAB ELISA as in panel A (improved version). No drug wells were incubated with XP280 to prevent carry-over of L-739758. XP280 competes with L-739758 for GP IIb/IIIa binding but fails to elicit binding of chimpanzee A264 DDABs (Table 1). Black bars indicate mOD/min minus drug wells; open bars, mOD/min plus drug wells; gray bars, mOD/min L-739758 wells minus mOD/min XP280 wells. (C) Correlation between ELISA and FACS signal. (i) Wells were coated with GP IIb/IIIa in the absence (open circles) or presence (closed circles) of L-739758 and incubated with A264 PPP for 10 minutes, followed by PPP transfer to the next well. This depletion procedure was repeated 11 times. After 12 incubation steps, bound IgG on the depletion plate was detected (improved ELISA format). (ii) The ELISA-depleted PPPs were subsequently incubated with gel-filtered platelets in the presence of L-739758, followed by addition of FITC-labeled antihuman IgG and analyzed by FACS. The results are expressed as mean fluorescence of 10 000 events. Minus drug is A264 PPP incubated on ELISA plates in the absence of GP IIb/IIIa antagonist. Plus drug is A264 PPP incubated on ELISA plates in the presence of GP IIb/IIIa antagonist. Control PPP is ELISA DDAB− PPP from the platelet donor used for the FACS experiment.

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