An enzyme-linked immunofiltration assay (ELIFA) was developed for detecting anti-human platelet factor 4 (hPF4)–heparin antibody in sera of patients with heparin-induced thrombocytopenia (HIT). The immunofiltration assay was developed to capture HIT antibody by hPF4–heparin complex adsorbed onto a positively charged nylon membrane, as an alternative to plastic bound hPF4. Of 75 sera with a positive serotonin-release assay (SRA), anti-PF4–heparin of the immunoglobulin (Ig)G class was detected in 72 (96%) sera. With SRA-negative sera from thrombocytopenic patients treated with heparin, anti-hPF4–heparin IgG and IgA were detected in 16% (n = 126) and 14% (n = 74) of sera respectively; 6% (n = 71) of SRA-negative sera contained both IgG and IgA anti-hPF4–heparin antibodies. The detection of anti-hPF4–heparin IgG in all HIT sera supports the assay of anti-PF4–heparin IgG as being a sensitive screening test for HIT. Alternatively, the absence of anti-hPF4–heparin IgA cannot be used as a test for excluding HIT, as it was detected in only 48% of SRA-positive HIT sera. However, it may be used to support the diagnosis of HIT, when HIT IgG is weak. This study emphasized the need to use different immobilizing media for the capture of anti-PF4–heparin antibody.