title: Establishment of immunoassay for platelet-derived soluble glycoprotein VI, a novel platelet marker.
abstract: Soluble Glycoprotein VI (GPVI) is an attractive marker for disorders marked by platelet activation, such as thrombotic microangiopathy, myocardial infarction, and stroke. Several groups have already developed an immunoassay for soluble GPVI; however, there are several discrepancies between the groups' assays. In this study, we prepared the two types of recombinant soluble GPVI, the monomeric form GPVI (GPVI-His) and the dimeric form of GPVI (GPVI-Fc), moreover, we generated four anti-GPVI antibodies, F1232-7-1 (7S1), F1232-10-2 (10S2), F1232-19-1 (19D1), and F1232-21-1 (21D1). The former 2 antibodies (7S1 and 10S2) had a high affinity for both GPVI-His and GPVI-Fc, while the latter 2 antibodies (19D1 and 21D1) showed a high affinity for GPVI-Fc but low affinity for GPVI-His. All of the antibodies comparably recognized surface GPVI on resting platelets. Furthermore, we established two immunoassays for soluble GPVI, 7S1/10S2-HRP and 19D1/21D1-HRP (capture antibody/detection antibody). 7S1/10S2-HRP showed equivalent reactivity with GPVI-His and GPVI-Fc, whereas 19D1/21D1-HRP had high affinity for GPVI-Fc but low reactivity with GPVI-His. In terms of reactivity with platelet-derived soluble GPVI, 7S1/10S2-HRP demonstrated sensitive detection whereas 19D1/21D1-HRP was nonreactive. Taken together, 7S1/10S2-HRP is a suitable candidate for a reliable soluble GPVI immunoassay as it has a high affinity for monomeric GPVI.
authors: Biology Laboratory, Discovery Research, Mochida Pharmaceutical Co., LTD., 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan. Electronic address: katsuki.naitoh@mochida.co.jp.
authors: Katsuki
authors: K
authors: Naitoh
authors: Biology Laboratory, Discovery Research, Mochida Pharmaceutical Co., LTD., 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan.
authors: Yoshitaka
authors: Y
authors: Hosaka
authors: Biology Laboratory, Discovery Research, Mochida Pharmaceutical Co., LTD., 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan.
authors: Kamon
authors: K
authors: Shirakawa
authors: Biology Laboratory, Discovery Research, Mochida Pharmaceutical Co., LTD., 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan.
authors: Shoji
authors: S
authors: Furusako
mesh_terms: D000906:Antibodies / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D001792:Blood Platelets / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay; D010980:Platelet Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000906:Antibodies; D015415:Biomarkers; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C098341:platelet membrane glycoprotein VI
keywords: Anti-GPVI antibody; Glycoprotein VI (GPVI); Platelet; Soluble GPVI