The development of a quantitative ELISA for antibodies against human platelet antigen type 1a

被引:7
作者
Bessos, H [1 ]
Perez, S
Armstrong-Fisher, S
Urbaniak, S
Turner, M
机构
[1] SNBTS, NSL, Immunohaematol Res Grp, R&D Directorate, Edinburgh EH17 7QT, Midlothian, Scotland
[2] Napier Univ, Edinburgh EH14 1DJ, Midlothian, Scotland
[3] Univ Aberdeen, Aberdeen, Scotland
[4] Univ Edinburgh, Edinburgh, Midlothian, Scotland
关键词
D O I
10.1046/j.1537-2995.2003.00322.x
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
BACKGROUND: Severe neonatal alloimmune thrombocytopenia is often due to antibodies against human platelet antigen type la (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 4degreesC. 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 mug 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 pg 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 mug per mL. CONCLUSION: Using immobilized HPA-1a1a, antibodies against HPA-1a has been purified, and a quick and simple quantitative assay has been developed.
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页码:350 / 356
页数:7
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