Antigen Assays For Hit Antibodies A Solid Phase PF4H Enzyme Immunoassay

The solid-phase enzyme immunoassay (EIA) has been described in detail (Amiral et al., 1992; Visentin et al., 1994; Greinacher et al., 1994a; Amiral et al., 1995; Horsewood et al., 1996; Juhl et al., 2006). Methods differ in the way that PF4-H complexes are coated on the microtiter wells. A general scheme is shown in Figure 2. In this assay, stoichiometric concentrations of PF4 and heparin (e.g., 50 mL each of 20 mg/mL PF4 and 1 U/mL UFH) dissolved in phosphate buffer are added

Add serum or plasma

Add substrate

Color

Wash

Add substrate

Add alkaline phosphatase-conjugated goat antihuman lgG (Fc-specific)

Microtiter plates coated with PF4 and heparin in stoichiometric concentrations

Add alkaline phosphatase-conjugated goat antihuman lgG (Fc-specific)

FIGURE 2 Schematic figure of solid-phase PF4-heparin-EIA. Abbreviations: EIA, enzyme immunoassay; HIT, heparin-induced thrombocytopenia; PF4, platelet factor 4.

FIGURE 2 Schematic figure of solid-phase PF4-heparin-EIA. Abbreviations: EIA, enzyme immunoassay; HIT, heparin-induced thrombocytopenia; PF4, platelet factor 4.

together to the wells of a microtiter plate and incubated at 4°C overnight. After washing with phosphate buffer saline Tween 20 (PBS-Tw), the wells are "blocked" with a protein-containing solution such as PBS-Tw containing either 10% normal goat serum (NGS) or 20% fetal calf serum, followed by washing with PBS-Tw. To perform the assay, 50-100 mL of test or control plasma or serum diluted 1: 50 in PBS containing 2% NGS is added to duplicate wells for 1 h at room temperature. After thorough washing with PBS-Tw, bound immu-noglobulin is detected by adding alkaline phosphatase-conjugated goat antihuman immunoglobulin (e.g., affinity-purified goat antihuman IgG Fc diluted 1:1000 in PBS-Tw-2% NGS) followed by incubation for 1 h at room temperature. After thorough washing, p-nitrophenyl phosphate in 1 M diethanolamine buffer is added. After incubation in the dark, the reaction is stopped with 1 N NaOH, and absorbance is read at 405 nm using an automated microplate reader. The upper limit of the normal range is usually set at the mean +3 SD obtained using normal sera. Some laboratories set an indeterminate range for samples that are only minimally above the upper normal range (e.g., up to 1.0 units).

0 0

Post a comment