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Expression and purification of functional recombinant epitopes for the
platelet antigens, PlA1 and PlA2
EA Barron-Casella, TS Kickler, OC Rogers and JF Casella
Department of Pediatrics, Johns Hopkins University School of Medicine,
Baltimore, MD 21205.
The platelet antigens, PlA1 and PlA2, are responsible for most cases of
posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia
(NAIT) in the caucasian population and are determined by two allelic forms
of the platelet glycoprotein GPIIIa gene. To study the interaction between
these antigens and their respective antibodies, we inserted the sequence
that encodes the signal peptide and the N- terminal 66 amino acids of the
PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2
antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2
allelic form. When transformed and induced in Escherichia coli, the two
constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion
proteins, one containing leucine at position 33 (PlA1), the other proline
(PlA2). These proteins are easily purified in milligram quantities using
glutathione-Sepharose and react specifically with their respective
antibodies by immunoblot and enzyme-linked immunosorbent assay.
Antigenicity of the PlA1 fusion protein in reduced glutathione increases
with time; moreover, the addition of oxidized glutathione accelerates this
process, presumably because of formation of the native disulfide bonds.
Neutralization assays indicate that the PlA1 fusion protein competes for
all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT
that is capable of interacting with the surface of intact platelets. This
study shows that the GST/N-terminal GPIIIa fusion proteins contain
conformational epitopes that mimic those involved in alloimmunization, and
that regions other than the amino terminal 66 amino acids of GPIIIa are not
likely to contain or be required for the development of functional PlA1
epitopes. Furthermore, these recombinant proteins can be used for the
affinity-purification of clinical anti-PlA1 antibodies and specific
antibody identification by western blotting, making them useful in the
diagnosis of patients alloimmunized to PlA1 alloantigens.
Volume 84,
Issue 4,
pp. 1157-1163,
08/15/1994
Copyright © 1994 by The American Society of Hematology
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