Three novel β3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency β3-HPA antibodies

Abstract

Immunologic and structural analysis of eight novel domain-deletion b 3 integrin peptides designed for detection of HPA-1 antibodies. This issue, pp 366-75.Summary. Background: 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 b3 subunit of the aIIbb3 integrin. Antibody detection is generally based on platelet-derived aIIbb3 from HPA-genotyped donors. Recombinant allelic b3 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. Objectives: We aimed to generate three recombinant b3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency b3 alloantigens. Methods: The removal of the specificitydetermining loop from the bA domain and fusion of truncated b3 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. Results: Reactivity of the three peptides with b3-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. Conclusions: These data demonstrate that b3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.

Low‐avidity HPA‐1a alloantibodies in severe neonatal alloimmune thrombocytopenia are detectable with surface plasmon resonance technology

“…23,30 In this respect, the use of recombinant PLT alloantigens may help in the further development of the SPR technique for the detection of PLT antibodies. 11,31 In conclusion, our results showed that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies. Further studies are required to establish the impact of this new method in determining the binding characteristics of maternal antibodies as predictive parameter for the clinical severity of NAIT.…”

“…The question as to whether this technique is also applicable to other human PLT antigens, especially for “labile” HPAs (HPA‐2, ‐3, and ‐15), remains 23,30 . In this respect, the use of recombinant PLT alloantigens may help in the further development of the SPR technique for the detection of PLT antibodies 11,31 …”

Neonatal alloimmune thrombocytopenia: pathogenesis, diagnosis and management

“…Given that panel cells carrying low frequency HPA antigens are not readily available, sequencing of relevant exons encoding known low frequency HPA antigens in paternal DNA may be required to identify the probable sensitizing antigen. Transfected cell lines expressing low frequency antigens (Kroll et al , ) or recombinant glycoprotein fragments engineered to carry relevant epitopes (Stafford et al , ,b) that can be used to identify such antibodies may become available in the future. Although many low frequency antigens have been described (Table ), sensitization to these markers probably accounts for no more than a few percent of all cases of NAIT (Ghevaert et al , ).…”

A multicenter validation of recombinant β3 integrin–coupled beads to detect human platelet antigen‐1 alloantibodies in 498 cases of fetomaternal alloimmune thrombocytopenia

“…In addition, we have demonstrated in other proof-ofprinciple studies that recombinant proteins can be used for the detection of alloantibodies against HPA-2, 30 HPA-4, 31 HPA-5, and HPA-15 32 and several of the extremely low-frequency HPAs on the b3 integrin. 31,33 With the addition of other recombinant HPAs, the Luminex multiplex technique would be extremely useful in enabling the prompt diagnosis and management of alloimmune PLT disorders. Furthermore, large-scale screening for HPA antibodies becomes feasible with an affordable and simple-touse bead assay as described here.…”