用于诊断卡氏肺孢子虫肺炎的抗人卡氏肺孢子虫单克隆抗体的制备与特性鉴定

Production and characterization of monoclonal antibodies to human Pneumocystis carinii for the diagnosis of P. carinii pneumonia.

作者信息

Nato F, Contini C, Zamora-Zavala C, Viscardi P, Delia S, Mojon M, Mazie J C

机构信息

Hybridolab, Département des Biotechnologies, Institut Pasteur, Paris, France.

出版信息

Eur J Med. 1992 Jun;1(3):132-8.

PMID:1341432

Abstract

OBJECTIVES

Monoclonal antibodies against human Pneumocystis carinii were produced by fusion of myeloma cells (X63-AG8.653) with splenocytes from Biozzi mice that had been immunized against P. carinii cysts isolated from infected human lung. The aim of this study was to evaluate the usefulness of these monoclonal antibodies for the diagnosis of P. carinii pneumonia by indirect immunofluorescence in comparison with a modified silver stain method and commercial kits.

METHODS

One hundred fifty-seven specimens from 87 patients, infected or non-infected with human immunodeficiency virus, were examined for the presence of P. carinii. Specimens were either induced sputum samples or bronchoalveolar lavage fluids. Indirect immunofluorescence was performed with six stable clones obtained by limiting dilution. Four of the monoclonal antibodies were IgG1 isotypes, one was an IgG3 and one was an IgM. Their isoelectric points varied from 6.5 to 8.3. Tests were also performed with silver methenamine staining and with two commercially available monoclonal antibodies (Monofluo kit from Diagnostics Pasteur and MAb from Dako).

RESULTS

The 6 antibodies all recognized P. carinii cysts in indirect immunofluorescence. No cross reactivity was observed with yeast or host cells. P. carinii antigens could not be identified with western immunoblotting suggesting that the monoclonal antibodies recognized native antigens. This result was confirmed by dot blot analysis. Spots were observed with native but not with denatured antigens. Inhibition studies showed that these 6 antibodies recognized the same or overlapping sites. The sensitivities of detection of P. carinii in sputum were 87% by silver stain and from 93.5 to 96.7% by immunofluorescence. The sensitivities of detection in bronchoalveolar lavage were 67.3% by silver stain and from 75.7% to 76.8% by immunofluorescence.

CONCLUSION

Immunofluorescence was more sensitive than silver staining and the best results were obtained with E5-8 and A8-13 monoclonal antibodies and with Monofluo kit.

摘要

目的

通过骨髓瘤细胞（X63-AG8.653）与经感染人肺分离的卡氏肺孢子虫囊肿免疫的Biozzi小鼠脾细胞融合，制备抗人卡氏肺孢子虫的单克隆抗体。本研究的目的是通过间接免疫荧光法，与改良银染法和商业试剂盒比较，评估这些单克隆抗体在诊断卡氏肺孢子虫肺炎中的实用性。

方法

对87例感染或未感染人类免疫缺陷病毒的患者的157份标本进行卡氏肺孢子虫检测。标本为诱导痰标本或支气管肺泡灌洗液。用通过有限稀释获得的6个稳定克隆进行间接免疫荧光检测。其中4种单克隆抗体为IgG1亚型，1种为IgG3，1种为IgM。它们的等电点在6.5至8.3之间。还用亚甲胺银染色和两种市售单克隆抗体（来自巴斯德诊断公司的Monofluo试剂盒和来自达科公司的单克隆抗体）进行检测。

结果

6种抗体在间接免疫荧光中均能识别卡氏肺孢子虫囊肿。未观察到与酵母或宿主细胞的交叉反应。用免疫印迹法无法鉴定卡氏肺孢子虫抗原，提示单克隆抗体识别的是天然抗原。斑点印迹分析证实了这一结果。观察到天然抗原出现斑点，而变性抗原未出现斑点。抑制研究表明，这6种抗体识别相同或重叠的位点。痰中卡氏肺孢子虫检测的灵敏度，银染法为87%，免疫荧光法为93.5%至96.7%。支气管肺泡灌洗中检测的灵敏度，银染法为67.3%，免疫荧光法为75.7%至76.8%。