Levine M, Miller F C
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
J Clin Microbiol. 1991 Dec;29(12):2809-16. doi: 10.1128/jcm.29.12.2809-2816.1991.
Inflammatory periodontal diseases are provoked by bacteria which adhere to teeth at the gingival margin and form plaques containing toxins detectable by their effect on mammalian cells in culture. The aim of this study was to make toxin-neutralizing monoclonal antibodies and determine whether they detect antigen in specific oral bacteria. Bacterial plaque was collected from teeth and homogenized, and the fluid phase (plaque extract) was boiled or first fractionated over Sephacryl S-300. Hybridomas from immunized mice secreted immunoglobulin M (IgM) antibodies which reacted to plaque antigens. Neutralization was detected by an increase in the growth of HL60 cells which were exposed to plaque toxins in the presence of IgM from hybridoma culture or ascitic fluids. However, the neutralization was obvious only when the plaque toxins reduced growth by 50% or less. Plaque toxin preparations were found to contain proteases which hydrolyzed all of the IgM in ascitic fluids within 24 h. Replenishing the IgM daily preserved protection compared with protection from IgM from other hybridomas or saline only. The decrease in the specific activity of plaque proteins caused by replenishing one such antibody (3hE5) was 2.5-fold compared with activity with unreplenished 3hE5, 3.8-fold compared with activity with saline only, and 10.7-fold compared with activity with replenished, unrelated antibody. The neutralizing IgM detected an array of 14,000- to 22,000-molecular-weight antigens. The native toxins may be aggregates of these antigens, or the array may indicate fragments of an undetected, larger antigen or a common, nonpeptide adduct. Only 0.5 to 0.8% of the bacteria from sites with periodontitis and grown on blood agar contained antigen. One group of reactive bacteria was identified as Actinomyces odontolyticus serotype I. Other isolates were identified as Staphylococcus epidermidis, but antigen disappeared from the these isolates within 6 weeks of subculture. Epitope-containing antigens were also found in streptococcal and Eikenella isolates, and it is likely that the antigens from only some of these bacteria are toxic.
炎症性牙周疾病是由附着在牙龈边缘牙齿上的细菌引发的，这些细菌会形成含有毒素的菌斑，通过其对培养中的哺乳动物细胞的作用可检测到这些毒素。本研究的目的是制备毒素中和单克隆抗体，并确定它们是否能检测特定口腔细菌中的抗原。从牙齿上收集细菌菌斑并进行匀浆处理，液相（菌斑提取物）经过煮沸或先在Sephacryl S - 300上进行分级分离。免疫小鼠产生的杂交瘤分泌与菌斑抗原发生反应的免疫球蛋白M（IgM）抗体。通过在杂交瘤培养物或腹水的IgM存在下，检测暴露于菌斑毒素的HL60细胞生长的增加来检测中和作用。然而，只有当菌斑毒素使生长降低50%或更低时，中和作用才明显。发现菌斑毒素制剂含有蛋白酶，这些蛋白酶在24小时内会水解腹水中所有的IgM。与来自其他杂交瘤的IgM或仅用生理盐水相比，每天补充IgM可保持保护作用。补充一种这样的抗体（3hE5）导致菌斑蛋白比未补充的3hE5的活性降低2.5倍，比仅用生理盐水的活性降低3.8倍，比补充的无关抗体的活性降低10.7倍。中和性IgM检测到一系列分子量为14000至22000的抗原。天然毒素可能是这些抗原的聚集体，或者该系列可能表明未检测到的较大抗原的片段或常见的非肽加合物。在牙周炎部位且在血琼脂上生长的细菌中，只有0.5%至0.8%含有抗原。一组反应性细菌被鉴定为溶牙放线菌血清型I。其他分离株被鉴定为表皮葡萄球菌，但在传代培养6周内这些分离株中的抗原消失。在链球菌和艾肯菌分离株中也发现了含表位的抗原，并且可能只有这些细菌中的一些细菌的抗原具有毒性。
