使用抗破伤风类毒素重链片段C g(t1b)[72b9]抗体的使用方法

来自美国食品和药物管理局FDA实验室Marjorie Shapiro 博士的使用方法建议

请参考以下文献：

对于交叉阻断，纯化 5E4、35F7、72B9、81H10、87C10、18.1.7 和 18.2.12.6 mAb 并用生物素标记。 为每个生物素化 mAb 生成稀释曲线 (1:1000±1:512,000)，并将位于曲线线性段中间的稀释液用于后续 ELISA。 如下进行交叉阻断实验：ELISA如上所述进行，使用1mg/ml的rHC作为包被抗原。 将在 PBS-T 中从 10 mg mAb/ml 到 4.8 ng mAb/ml 的杂交瘤上清液的两倍系列稀释液添加到孔中（每个稀释液 100 ml），在室温下一式三份，持续 1 小时。 （杂交瘤 18.1.7 和 18.2.12.6 的融合配偶体 P3-X63Ag8 表达内源性抗体。因此，这些上清液中 HC 特异性 IgG 的确切浓度未知。）洗涤后，将 100 ml 适当的 在室温下将每种生物素标记抗体的稀释液添加到每个孔中 20 分钟，然后以 1:5000 (Amersham Life Sciences, Piscataway, NJ, USA) 在室温下添加 100 ml 链霉亲和素-碱性磷酸酶（Amersham Life Sciences, Piscataway, NJ, USA）20 分钟 . 如上所述清洗和显影孔。 含有和不含自身竞争剂的生物素化抗体用作对照。 还包括单克隆抗体 12C11 作为每个平板上的阴性对照。 该 mAb 与 HC 特异性 mAb 产自相同的 BALB/c 小鼠，并结合 TT 但不结合 HC。 还用来自用商业破伤风疫苗接种的豚鼠的多克隆抗破伤风血清进行ELISA。 该血清由 CBER 生成，用于中和试验，作为疫苗效力的衡量标准，是 Christine Anderson (CBER/OCBQ) 的礼物。 除了使用 1:4 至 1:1024 的豚鼠血清系列稀释液作为竞争剂外，该测定与上述相同。 结果表示为抑制百分比，使用每个生物素标记的 mAb 的 1:1024 稀释度值作为 0% 抑制（与不含血清的对照相比无抑制）。 每种稀释液一式三份加入孔中。

For cross blocking, 5E4, 35F7, 72B9, 81H10, 87C10, 18.1.7, and 18.2.12.6 mAbs were puri®ed and labeled with biotin. A dilution curve (1:1000±1:512,000) was generated for each biotinylated mAb and a dilution that resided in the middle of the linear segment of the curve was used for subsequent ELISAs. Cross blocking experiments were performed as follows: ELISAs were performed as described above with rHC at 1 mg/ml as coating antigen. Two-fold serial dilutions of hybridoma supernatants from 10 mg mAb/ ml to 4.8 ng mAb/ml in PBS-T were added to wells (100 ml of each dilution) in triplicate for 1 h at room temperature. (The fusion partner for hybridomas 18.1.7 and 18.2.12.6, P3-X63Ag8, expresses endogenous antibody. Therefore, the exact concentration of HC-speci®c IgG in these supernatants is not known.) After washing, 100 ml of the appropriate dilution of each biotin-labeled antibody were added to each well for 20 min at room temperature followed by the addition of 100 ml streptavidin-alkaline phosphatase at 1:5000 (Amersham Life Sciences, Piscataway, NJ, USA) for 20 min at room temperature. Wells were washed and developed as described above. Biotinylated antibodies with and without self competitor were used as controls. Monoclonal antibody 12C11 was also included as a negative control on each plate. This mAb was generated from the same BALB/c mouse as the HC-speci®c mAbs and binds TT but not HC. ELISAs were also performed with polyclonal antitetanus serum from guinea pigs which were vaccinated with a commercial tetanus vaccine. This serum was generated at CBER for use in neutralizing assays that serve as a measure of the potency of the vaccine and was a gift from Christine Anderson (CBER/OCBQ). The assay was the same as described above except that guinea pig serum serial dilutions from 1:4 to 1:1024 were used as a competitor. Results are presented as the percent inhibition using the value of the 1:1024 dilution for each biotin-labeled mAb as 0% inhibition (no inhibition compared to controls without serum). Each dilution was added to wells in triplicate.

这种小鼠 IgG2b 抗体针对破伤风类毒素产生并可识别破伤风类毒素。

强调：

• 与破伤风类毒素反应
• 与表位 VH 36-60 结合；VK4,5，识别线性表位
• 适用于蛋白质印迹和 ELISA 应用

破伤风也称为牙关紧闭症，是由破伤风梭菌感染引起的，这种细菌常见于土壤、唾液、灰尘和粪便中。细菌通常通过皮肤的裂口进入，例如被污染物体割伤或刺伤。它们会产生干扰正常肌肉收缩的毒素。破伤风的其他症状可能包括发烧、出汗、头痛、吞咽困难、高血压和心率加快。它可以通过免疫接种来预防。

克拉克 KC、菲茨西蒙斯 SP、夏皮罗·马乔里、威尔克森·拉沙德。通过识别不同表位的单克隆抗体抑制破伤风毒素片段 C 与神经节苷脂 GT1B 的结合。疫苗。2001；19：114-121。全文下载地址见下方。