A rapid one‐stage whole‐blood HPA‐1a phenotyping assay using a recombinant monoclonal IgG1 anti‐HPA‐1a

Abstract

Severe neonatal alloimmune thrombocytopenia and patients with HPA-1a-specific antibodies require transfusion of HPA-1a-negative platelets. Identifying HPA-1a-negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)-conjugated recombinant IgG1 anti-HPA-1a (CAMTRAN007) to develop a rapid and reliable enzyme-linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA-1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA-1a genotype demonstrated that HPA-1a-negative samples had OD values of < 0.266, whereas HPA-1a-positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut-off values and identified 45 HPA-1a-negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA-1a-negative sample had an OD value of 0.303. The HPA-1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence-specific primers (PCR- SSP).

Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia

“…Briefly, IgG2 residues from positions 233-236 (Δb) were substituted together with IgG4 residues 327, 330, and 331 (Δa) into IgG1 to generate G1Δab. The null allotype (Δn) mutations (Lys214 to Thr, Asp356 to Glu, and Leu358 to Met) were introduced in this template by sequential overlap extension PCR, using Pwo DNA polymerase (Boehringer Mannheim) to generate the IgG1Δnab constant region gene which was cloned as a BamHI-NotI fragment into pSVgptB2VHHuIgG2 (38) to yield the vector pSVgptB2VHHuIgG1Δnab. Thus the 2 full-length HPA-1a antibodies generated for this study had the same B2 variable region and were designated "B2G1" and "B2G1Δnab.…”

“…The generation of B2G1, a human IgG1λ version of an anti-HPA-1a single-chain Fv, has been previously described (38), as has the vector containing the modified IgG1 gene, pSVgptFog1VHHuIgG1Δab (39). Briefly, IgG2 residues from positions 233-236 (Δb) were substituted together with IgG4 residues 327, 330, and 331 (Δa) into IgG1 to generate G1Δab.…”

“…For each antibody, the heavy-chain vector was cotransfected with the B2 λ-chain expression vector (38) into a rat myeloma cell line, YB2/0, and stable transfectants secreting the highest levels of antibody were isolated as previously described (39). Cell culture supernatant containing monoclonal antibody IgG was passed through a 0.22-μm filter and IgG purified using a 5-ml Protein G Sepharose 4 Fast Flow column (Pharmacia).…”

“…The recombinant IgG1 antibody (B2G1) derived from this scFv was sufficiently specific for HPA-1a to permit its use as a routine phenotyping reagent (38).…”

Characterization of a Human Platelet Antigen-1a–Specific Monoclonal Antibody Derived from a B Cell from a Woman Alloimmunized in Pregnancy

“…2 individuals who are at risk for alloimmunization against HPA-1a (7,8). A prophylactic strategy to prevent FNAIT occurrence using anti-HPA-1a Abs derived from pooled donor plasma has been proposed (9).…”