Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations

被引:38
作者
Kawai, S
Minegishi, M
Ohashi, Y
Sasahara, Y
Kumaki, S
Konno, T
Miki, H
Derry, J
Nonoyama, S
Miyawaki, T
Horibe, K
Tachibana, N
Kudoh, E
Yoshimura, Y
Izumikawa, Y
Sako, M
Tsuchiya, S
机构
[1] Tohoku Univ, Inst Dev Aging & Canc, Dept Pediat Oncol, Aoba Ku, Sendai, Miyagi 9808575, Japan
[2] Tohoku Univ Hosp, Div Blood Transfus, Sendai, Miyagi, Japan
[3] Miyagi Prefectural Canc Ctr, Natori, Miyagi, Japan
[4] Univ Tokyo, Inst Med Sci, Dept Biochem, Tokyo 108, Japan
[5] Immunex Corp, Seattle, WA USA
[6] Tokyo Med & Dent Univ, Dept Pediat, Tokyo, Japan
[7] Toyama Med & Pharmaceut Univ, Fac Med, Dept Pediat, Toyama, Japan
[8] Nagoya Univ, Sch Med, Dept Pediat, Nagoya, Aichi 466, Japan
[9] Aomori Prefectural Cent Hosp, Dept Transfus Med & Pediat, Aomori, Japan
[10] Hirosaki Natl Hosp, Dept Pediat, Hirosaki, Aomori, Japan
[11] Shinjo Prefectural Hosp, Div Pediat, Shinjo, Japan
[12] Univ Ryukyus, Sch Med, Dept Pediat, Nishihara, Okinawa, Japan
[13] Osaka City Gen Hosp, Dept Pediat, Osaka, Japan
关键词
Wiskott-Aldrich syndromes; X-linked thrombocytopenia; WASP; monoclonal antibody; flow cytometry;
D O I
10.1016/S0022-1759(01)00549-X
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We have produced a novel monoclonal antibody (mAb) directed against Wiskott-Aldrich syndrome protein (WASP) by immunizing mice with the recombinant protein. The mAb designated 5A5 is highly specific to WASP and suitable for Western blot analysis and immunoprecipitation. A flow cytometric assay using the 5A5 mAb identifies expression of intracytoplasmic WASP in lymphocytes from normal individuals. Double staining analysis with cell surface CD3, CD19, and CD56, and intracytoplasmic molecules revealed WASP expression in each subpopulation. With regard to WASP expression in patients with Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), peripheral blood mononuclear cells (PBMCs) from nine patients and Epstein-Barr virus-transformed B-lymphoblastoid cell lines from seven patients examined did not show WASP expression by flow cytometric analysis. These results were confirmed by Western blot analysis. We conclude that WASP expression in lymphocyte subpopulations from patients with WAS and XLT can be more precisely evaluated by flow cytometry as compared with Western blot analysis. This flow cytometry method is important as a supplement to Western blots, but even more important as an alternative and powerful assay that can contribute to research on WASP as well as diagnosis in a clinical setting. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:195 / 205
页数:11
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