Fluid Phase EIA

The fluid-phase EIA for HIT antibodies (Newman et al., 1998) is an adaptation of a staphylococcal protein A antibody-capture EIA method (Nagi et al., 1993). By permitting antibody-antigen interactions to occur in a fluid phase, problems of protein (antigen) denaturation inherent in solid-phase assays are avoided.

Add substrate Color

Add substrate Color

Antibody Antigen Interactions

with biotinylated all IgG with quantitates biotin-PF4-

PF4-heparin protein G agarose heparin complexes that coprecipitate with HIT-IgG

with biotinylated all IgG with quantitates biotin-PF4-

PF4-heparin protein G agarose heparin complexes that coprecipitate with HIT-IgG

FIGURE 3 Schematic figure of fluid-phase PF4-heparin-EIA. Abbreviations: EIA, enzyme immunoassay; HIT, heparin-induced thrombocytopenia; PF4, platelet factor 4.

FIGURE 3 Schematic figure of fluid-phase PF4-heparin-EIA. Abbreviations: EIA, enzyme immunoassay; HIT, heparin-induced thrombocytopenia; PF4, platelet factor 4.

PF4 (5% biotinylated) is mixed with an optimal concentration of heparin, and this antigen mixture is incubated with diluted patient serum or plasma (Fig. 3). Subsequently, the antigen-antibody mixture is incubated with protein G-Sepharose in a microcentrifuge tube. Biotinylated antigen-antibody complexes become bound to the protein G-Sepharose by antibody Fc, and the complexes are separated from unbound antigen by centrifugation and washing. The amount of biotin-PF4-H-antibody complexes immobilized to the beads is measured using peroxidase substrate after initial incubation with streptavidin-conjugated peroxidase.

The fluid-phase EIA appears to have a lower rate of false-positive reactions. This may be because in the solid-phase-EIA, nonspecific binding of IgG to the microtiter wells can occur. Furthermore, the cryptic antigen site of PF4 can be exposed when the molecule comes into close contact with the plastic surface, even in the absence of heparin (Newman and Chong, 1999). The fluid-phase assay avoids these problems by first precipitating all reactive IgG antibodies, then detecting the antigen specifically bound to the IgG. Thus, higher concentrations of patient serum or plasma can be tested without increasing nonspecific reactivity. The advantages of this assay in performing in vitro cross-reactivity are discussed later. Because antibody is bound using protein G-Sepharose, IgM and IgA anti-PF4-H antibodies are not detected in this assay.

Wang and coworkers (1999a,b) used protein A to capture IgG antibodies from HIT patient serum. The immobilized antibodies were then incubated with normal serum (presumed to contain PF4) and fluorescence-labeled heparin. The amount of fluorescence dye bound to the protein A sepharose was used to detect HIT antibodies. The major drawbacks of this approach include the initial capturing of IgG other than HIT-IgG, as well as the unpredictable PF4/heparin ratios.

Particle Gel Immunoassay (DiaMed ID-PaGIA Heparin/PF4 antibody test)

-test serum containing positive or negative HIT IgG

is mixed with red polystyrene beads coated with PF4-heparin O -the test mixture is then added to the particle gel immunoassay tube containing anti-human IgG

Negative result

Positive result

Heparin Pf4 Test

sephacryl beads and anti-human IgG

sephacryl beads and anti-human IgG

Positive result

Schematic Magnetic Particle Test Result

FIGURE 4 Schematic drawing showing the PaGIA using gel centrifugation technology. A secondary anti-antibody is used to facilitate particle gel agglutination. Unlike the EIA, which reaches an equilibrium state of antibody-antigen reaction, the PaGIA is a dynamic immunoassay. Abbreviations: HIT, heparin-induced thrombocytopenia; PaGIA, particle gel immunoassay; PF4, platelet factor 4. Source: From Warkentin and Sheppard, 2006a.

No aggregates (red color at bottom)

Bead aggregates (red color at top)

FIGURE 4 Schematic drawing showing the PaGIA using gel centrifugation technology. A secondary anti-antibody is used to facilitate particle gel agglutination. Unlike the EIA, which reaches an equilibrium state of antibody-antigen reaction, the PaGIA is a dynamic immunoassay. Abbreviations: HIT, heparin-induced thrombocytopenia; PaGIA, particle gel immunoassay; PF4, platelet factor 4. Source: From Warkentin and Sheppard, 2006a.

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