Landini M P, Lazzarotto T, Maine G T, Ripalti A, Flanders R
Institute of Microbiology, University of Bologna, Italy.
J Clin Microbiol. 1995 Oct;33(10):2535-42. doi: 10.1128/jcm.33.10.2535-2542.1995.
Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.
由于抗原组成以及缺乏抗原标准化，人巨细胞病毒（HCMV）特异性抗体的血清学检测结果差异很大。经分子生物学方法制备的、由单一特征明确的病毒蛋白或其部分组成的抗原物质，已被证明是改善血清学诊断的有前景的工具。我们构建了一种包含ppUL32（p150）两个区域和ppUL44（p52）一半的重组蛋白，并将这种三抗原融合蛋白与包含两个ppUL32片段的双抗原融合蛋白以及包含ppUL44一半的单抗原融合蛋白的免疫球蛋白M（IgM）反应性进行了比较。我们还构建并测试了另外两种单抗原融合蛋白，分别包含大部分ppUL80a和一部分ppUL83。通过重组酶免疫测定法（rec - EIA）和市售酶免疫测定法，对来自免疫功能正常和免疫抑制不同组别的700多份血清样本进行了HCMV IgM检测。还进行了蛋白质印迹法（免疫印迹）以及（针对免疫抑制个体）通过免疫荧光和多形核白细胞PCR进行的抗原血症检测。所获得的结果表明：（i）三抗原融合蛋白可以替代单个蛋白；（ii）在抗CMV IgM的血清学检测中，三抗原融合蛋白不能单独用于替代病毒或感染细胞；（iii）添加包含ppUL83和ppUL80a部分的融合蛋白对于形成能够替代病毒用于检测HCMV特异性IgM的抗原混合物至关重要；（iv）rec - EIA非常特异，并且比市售酶免疫测定法更灵敏，所获得的结果与蛋白质印迹法一致；（v）rec - EIA能够可靠地用于检测不同组活动性HCMV感染患者中的HCMV特异性IgM。
