抗人中性粒细胞单克隆抗体的筛选、鉴定和抑制调理吞噬活性的检测

Screening and Identification of Monoclonal Antibodies against Human Neutrophil and Detection of Inhibiting Opsonic Phagocytic Activity

用淋巴细胞分离液梯度离心法,自健康人血液中分离出中性粒细胞,作为颗粒抗原,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞融合,获得分泌抗细胞膜单克隆抗体(单抗)杂交瘤细胞。用调理吞噬抑制试验,初步筛选出1株分泌抑制补体介导的吞噬功能的单抗杂交瘤细胞。单抗亚类检定结果为IgG_(2a)。

Using Ficoll-Hypaque gradi-ent centrifugation, we have successullyobtained the neutrophils from normalhuman donors, which can be used as par-ticle antigens for immunizing BALB/cmice. Hybridomas were produced thraough thefusion of SP2/0 myeloma cells and sple-nocytes from immunized BALB/c mice.The ratio of fusion was 1:5. The cellswere fused with 30% polyethylene glycol(MW 4000). The rate of fusion was 90%.The antibody producing colonies againstthe membrane of neutrophils in. HATmedium were selected by indirect immu-nofl uorescence assay. Antibody positiverate was 60%(102/170). Limiting dilution was used for coloniza-tion of the antibody-producing hybridomacells. After colonization for three times.one hybridoma cell line was obtained,which could inhibit complement-mediatedphagocytosis. Culture supernatant was used againstclass-and subclass- specific rabbit antisera(rabbit anti-mouse IgM, IgG, IgG_1, IgG_2?.IgG_(2b), IgG_3) in an agar immunodiffusionsystem;it was Shown to be IgG_(2?).