Rodríguez Barbosa J I, Gutiérrez Martín C B, Tascón R I, Suárez J, Rodríguez Ferri E F
Department of Animal Pathology, Faculty of Veterinary Medicine, University of León, Spain.
Clin Diagn Lab Immunol. 1995 Sep;2(5):563-8. doi: 10.1128/cdli.2.5.563-568.1995.
Monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae serotype 4 (reference strain M62 and field isolate F6) were produced and characterized. Three hybridoma clones were raised against strain M62, and 13 were raised against strain F6. The predominant antibody class was immunoglobulin M (IgM), although IgG2a and IgG2b were also obtained. Three of the MAbs produced to field isolate F6 (5C5, 1E10, and 5H7) did not recognize the reference strain of serotype 4, another (6F7) was reactive with both reference strains of serotypes 4 and 7, and the remaining 12 MAbs reacted only with the reference strain of the homologous serotype. All epitopes recognized by MAbs, except for one (6F7), were sensitive to periodic acid oxidation, and all of them were resistant to boiling in the presence of sodium dodecyl sulfate and reducing conditions, as evidenced by immunodot. Enhanced chemioluminiscence-immunoblot assays revealed that 10 MAbs (3E12, 5B8, 7C3, 6F7, 7F5, 7E6, 5G4, 4F1, 7E10, and 4B8) recognized a ladder-like banding pattern, which is in accordance with the O side chain antigen of lipopolysaccharide (LPS), while the remaining 6 MAbs (5C5, 5H7, 1E10, 6D11, 6B4, and 5E4) blotted with high-molecular-weight regions composed of a single banding pattern. The suitability of MAbs for serotyping of 78 field isolates was also examined. A high correlation was found between the results previously established by indirect hemagglutination with polyclonal sera and those obtained by enzyme-linked immunosorbent assay with MAbs. According to the different immunoreactivity of MAbs, three groups were established: group I (MAbs 3E12, 5B8, 7C3, 6F7, and 7F5), group II (MAbs 7E6, 5G4, 4F1, 7E10, and 4B8), and group III (MAbs 5C5, 5H7, and 1E10). MAbs 6D11, 6B4, and 5E4 could not be included in any of the described above. At least six different immunodominant epitopes on the O antigen of the A. pleuropneumoniae serotype 4 LPS were identified. Finally, the implications of the effect of the O antigen of LPS in cross-reactions between serotypes 4 and 7 are clearly evidenced.
制备并鉴定了抗胸膜肺炎放线杆菌（嗜血杆菌）4型（参考菌株M62和田间分离株F6）的单克隆抗体（MAb）。针对菌株M62产生了3个杂交瘤克隆，针对菌株F6产生了13个杂交瘤克隆。主要抗体类别为免疫球蛋白M（IgM），不过也获得了IgG2a和IgG2b。针对田间分离株F6产生的3个MAb（5C5、1E10和5H7）不识别4型参考菌株，另一个（6F7）与4型和7型参考菌株均有反应，其余12个MAb仅与同源血清型的参考菌株反应。除一个（6F7）外，MAb识别的所有表位对高碘酸氧化敏感，并且通过免疫斑点法证明，在存在十二烷基硫酸钠和还原条件下，所有表位均耐煮沸。增强化学发光免疫印迹分析显示，10个MAb（3E12、5B8、7C3、6F7、7F5、7E6、5G4、4F1、7E10和4B8）识别出一种梯状条带模式，这与脂多糖（LPS）的O侧链抗原一致，而其余6个MAb（5C5、5H7、1E10、6D11、6B4和5E4）印迹显示为单一条带模式组成的高分子量区域。还检测了MAb对78个田间分离株进行血清分型的适用性。发现先前通过多克隆血清间接血凝法确定的结果与通过MAb酶联免疫吸附测定法获得的结果之间具有高度相关性。根据MAb的不同免疫反应性，分为三组：I组（MAb 3E12、5B8、7C3、6F7和7F5），II组（MAb 7E6、5G4、4F1、7E10和4B8），III组（MAb 5C5、5H7和1E10）。MAb 6D11、6B4和5E4不能归入上述任何一组。确定了胸膜肺炎放线杆菌4型LPS的O抗原上至少6个不同的免疫显性表位。最后，清楚地证明了LPS的O抗原在4型和7型血清型之间交叉反应中的作用。
