Constructs that react with all HNA-3a antibodies are needed to fully define the HNA-3a epitope. STUDY DESIGN AND METHODS HEK293 cells were transfected with cDNA encoding full-length CTL2 linked to green fluorescent protein (GFP). anti-HNA-3a. Antibodies specific for the leukocyte antigen HNA-3a are prone to cause severe, often fatal, transfusion-related acute lung injury (TRALI)1,2 when transfused with blood products.3C5 Unfortunately, it has not been practical to test blood donors routinely for antibodies realizing this antigen because it was thought to be specific for neutrophils, a cell difficult to use for donor screening, and the protein carrier for the antigen was unknown. Recently, two groups individually showed the HNA-3a/b antigens are carried on choline-transporterClike protein 2 (CTL2), are indicated on lymphocytes and platelets in addition to neutrophils, and appear to be determined by a single nucleotide substitution in the CTL2 gene expected to encode an arginine-glutamine polymorphism at CTL2 amino acid residue 154.6,7 Recognition of the carrier protein for HNA-3a/b suggests fresh approaches toward developing assays to display blood donors for the related antibodies Ginsenoside Rb2 on a large scale. However, the 66-kDa CTL2 protein is expected to span the cell membrane 10 instances and to possess five extracellular loops.8 Owing to this complex structure, intact CTL2 is unlikely to give itself to detergent solublization, purification, and immobilization to create a target suitable for antibody detection. As an alternative, we previously synthesized numerous CTL2 peptides comprising R154 or Q154 and SLI analyzed their reactions with anti-HNA-3a. The most adequate peptide proved to be a 36-mer (D131-K166) comprising R154, but it was identified in preference to the Q154 Ginsenoside Rb2 version by only 10 of 20 HNA-3a antibodies.9 Berthold and colleagues10 produced CTL2 fragments as GST fusion proteins in and analyzed their reactions with anti-HNA-3a in European blotting. None of the peptides possessing R154 was identified in preference to its Q154 counterpart by more than 9 of 21 examples of anti-HNA-3a.10 To analyze whether a larger recombinant version of CTL2 would mimic the native protein structure sufficiently well to react with all examples of anti-HNA-3a, we indicated the two alleles (R/Q154) of full-length CTL2 in HEK293 cells and analyzed their reactions having a panel of Ginsenoside Rb2 HNA-3aCspecific antibodies. MATERIALS AND METHODS Manifestation of CTL2-green fluorescent protein in HEK293 cells cDNA related to full-length CTL2 isoform p211 was a gift from Dr T. Carey (University or college of Michigan, Ann Arbor, MI). This cDNA, coding for CTL2 (R154), was subcloned into manifestation vector pcDNA3.1/CT-GFP-TOPO (Invitrogen, Carslbad, CA). cDNA encoding Q154 was produced having a site-directed mutagenesis kit (Quickchange II XL, Agilent Systems, Inc., Carlsbad, CA). cDNAs encoding the two versions of CTL2 were transfected into HEK293 cells using transfection reagent (Fugene HD, Roche, Atlanta, GA). Stable cell lines were selected using Dulbeccos revised Eagles medium supplemented with 10% fetal bovine serum, 2 mg/mL G418, and 25 g/mL gentamicin sulfate. Cells were selected for green fluorescent protein (GFP) expression using a cell sorter (BD FACS Aria IIu, BD Bioscience, San Jose, CA). Cells enriched for GFP were then cloned by limiting dilution to obtain monoclonal cell lines expressing high levels of HNA-3a or HNA-3b antigen. Confocal microscopy Cells were allowed to abide by cells tradition grade plastic over night. After being washed two times with phosphate-buffered saline, cells were examined on a multiphoton laser scanning microscope (Olympus Fluoview FV1000 MPE, Olympus America, Center Valley,.
