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The development of a quantitative ELISA for antibodies against human platelet antigen type 1a
Author(s) -
Bessos Hagop,
Perez Silvia,
ArmstrongFisher Sylvia,
Urbaniak Stan,
Turner Marc
Publication year - 2003
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1046/j.1537-2995.2003.00322.x
Subject(s) - serial dilution , antibody , antigen , chemistry , platelet , chromatography , neonatal alloimmune thrombocytopenia , immunology , microbiology and biotechnology , medicine , biology , pregnancy , fetus , alternative medicine , pathology , genetics
BACKGROUND : Severe neonatal alloimmune thrombocytopenia is often due to antibodies against human platelet antigen type 1a (HPA‐1a). The aim of this study was to develop a quantitative ELISA for the measurement of antibodies against HPA‐1a. STUDY DESIGN AND METHODS : HPA‐1a glycoprotein (GP) IIb‐IIIa was immobilized and mixed with recalcified anti‐HPA‐1a‐positive plasma overnight at 4°C. The beads were washed, the antibodies against HPA‐1a were eluted, and the eluate pH level was promptly adjusted. The purified antibodies were dialyzed and used for the development of an ELISA incorporating HPA‐1a‐coated plates. RESULTS: Serial doubling dilutions of the purified antibodies resulted in consistent sigmoid standard curves with a sensitivity of 0.5 μg per mL. To determine the reproducibility of the ELISA, antibodies against HPA‐1a in five plasma samples (Samples A‐E) were measured at serial doubling dilutions in four separate assays. Three of the samples (Samples A‐C) contained antibodies against HPA‐1a. The mean amounts in μg per mL (± SD, percentage of CV) obtained in the four assays were as follows: Sample A, 133 (9.4, 7.1%); Sample B, 16.5 (1.7, 10%); and Sample C, 8 (0.8, 10%). The amounts in the two antibody‐negative controls (Samples D and E) were consistently less than 0.2 μg per mL. CONCLUSION : Using immobilized HPA‐1a1a, antibodies against HPA‐1a has been purified, and a quick and simple quantitative assay has been developed.