Kim K J, Burnette W N, Sublett R D, Manclark C R, Kenimer J G
Division of Bacterial Products, Food and Drug Administration, Bethesda, Maryland 20892.
Infect Immun. 1989 Mar;57(3):944-50. doi: 10.1128/iai.57.3.944-950.1989.
To identify the neutralizing epitopes on the S1 subunit (A promoter) of pertussis toxin, we characterized anti-S1 monoclonal antibodies (MAbs) X2X5, 3CX4, and 6FX1. We confirmed by immunoblot analysis that these MAbs bind to the S1 subunit and not to the B oligomer of pertussis toxin and that they recognize different epitopes by a competitive binding enzyme-linked immunosorbent assay. These MAbs had differential abilities to neutralize the lymphocytosis-promoting factor activity of pertussis toxin in mice: 3CX4 and 6FX1 had partial neutralizing abilities, while MAb X2X5 had none. With these MAbs, the epitopes on the S1 subunit were examined by using trypsinized S1 peptides, recombinant truncated S1 molecules, and synthetic peptides. The non-neutralizing MAb X2X5 bound in immunoblots to tryptic peptides of various sizes as small as 1.5 kilodaltons; the neutralizing MAbs 3CX4 and 6FX1 bound only to a 24-kilodalton tryptic peptide band. Immunoblot studies with recombinant truncated S1 molecules demonstrated that amino acid residues 7 to 14 and 15 to 26 play an important role in the binding of neutralizing MAbs and the non-neutralizing MAb, respectively. The binding of these MAbs was not dependent upon the presence of C-terminal amino acid residues 188 to 234. To further define B-cell epitopes, the binding of the MAbs we tested to synthetic peptides representing the entire S1 subunit were examined. Neutralizing MAbs 3CX4 and 6FX1 bound to none of these peptides, further suggesting that these MAbs recognize conformational epitopes. The non-neutralizing MAb X2X5 bound to peptides 11 to 26 and 16 to 30, demonstrating that the major antigenic determinant recognized by this MAb is a linear epitope located within residues 16 to 26.
为了鉴定百日咳毒素S1亚基（A启动子）上的中和表位，我们对抗S1单克隆抗体（MAb）X2X5、3CX4和6FX1进行了特性分析。我们通过免疫印迹分析证实，这些单克隆抗体与S1亚基结合，而不与百日咳毒素的B寡聚体结合，并且通过竞争性结合酶联免疫吸附测定法表明它们识别不同的表位。这些单克隆抗体在中和小鼠体内百日咳毒素的淋巴细胞增多促进因子活性方面具有不同的能力：3CX4和6FX1具有部分中和能力，而单克隆抗体X2X5则没有。利用这些单克隆抗体，通过使用胰蛋白酶消化的S1肽、重组截短的S1分子和合成肽来检测S1亚基上的表位。非中和性单克隆抗体X2X5在免疫印迹中与小至1.5千道尔顿的各种大小的胰蛋白酶肽结合；中和性单克隆抗体3CX4和6FX1仅与一条24千道尔顿的胰蛋白酶肽带结合。用重组截短的S1分子进行的免疫印迹研究表明，氨基酸残基7至14和15至26分别在中和性单克隆抗体和非中和性单克隆抗体的结合中起重要作用。这些单克隆抗体的结合不依赖于C末端氨基酸残基188至234的存在。为了进一步确定B细胞表位，检测了我们测试的单克隆抗体与代表整个S1亚基的合成肽的结合情况。中和性单克隆抗体3CX4和6FX1与这些肽均不结合，进一步表明这些单克隆抗体识别构象表位。非中和性单克隆抗体X2X5与肽11至26和16至30结合，表明该单克隆抗体识别的主要抗原决定簇是位于残基16至26内的线性表位。
