title: Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin.
abstract: Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems.
authors: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
authors: Younis
authors: Y
authors: Skaik
authors: Anja
authors: A
authors: Battermann
authors: Oliver
authors: O
authors: Hiller
authors: Oliver
authors: O
authors: Meyer
authors: Constanca
authors: C
authors: Figueiredo
authors: Abdulgabar
authors: A
authors: Salama
authors: Rainer
authors: R
authors: Blasczyk
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology; D016824:Antigens, Human Platelet / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D057809:HEK293 Cells; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D039661:Integrin beta3 / Q000096:biosynthesis / Q000235:genetics; D007518:Isoantibodies / Q000097:blood*; D011237:Predictive Value of Tests; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D014162:Transfection
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D016824:Antigens, Human Platelet; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007518:Isoantibodies; D011994:Recombinant Proteins; D006639:Histidine