Three novel beta3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency beta3-HPA antibodies.
Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging.



We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens.



The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens.



Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes.



These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.
Stafford
P
P
Department of Haematology, University of Cambridge and National Health Service Blood and Transplant, Cambridge, UK.
Garner
S F
SF
Huiskes
E
E
Kaplan
C
C
Kekomaki
R
R
Santoso
S
S
Tsuno
N H
NH
Watkins
N A
NA
Ouwehand
W H
WH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D016824:Antigens, Human Platelet / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001792:Blood Platelets / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D039661:Integrin beta3 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007518:Isoantibodies / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000276:immunology*; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017384:Sequence Deletion; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000175:diagnosis / Q000276:immunology*
C057260:4a alloantigen, human; D000911:Antibodies, Monoclonal; D016824:Antigens, Human Platelet; D000939:Epitopes; C528405:HPA-8bw antigen, human; D039661:Integrin beta3; D007518:Isoantibodies; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins