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


METHODS
HPA-1a glycoprotein (GP) IIb-IIIa was immobilized and mixed with recalcified anti-HPA-1a-positive plasma overnight at 4 degrees 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 microg 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 microg 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 microg per mL.


CONCLUSIONS
Using immobilized HPA-1a1a, antibodies against HPA-1a has been purified, and a quick and simple quantitative assay has been developed.
Bessos
Hagop
H
Immunihaematology Research Group, R & D Directorate, Scottish National Blood Transfusion Service, Edinburgh, Scotland. hagop.bessos@snbts.csa.scot.nhs.uk
Perez
Silvia
S
Armstrong-Fisher
Sylvia
S
Urbaniak
Stan
S
Turner
Marc
M
D000327:Adsorption; D002118:Calcium / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007518:Isoantibodies / Q000097:blood* / Q000302:isolation & purification; D008863:Microspheres; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007518:Isoantibodies; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D002118:Calcium