O-polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry
O-polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry
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DOI：
10.1128/cdli.5.6.862-870.1998
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发表时间：
1998-11-01
期刊：
影响因子：
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通讯作者：
Zygmunt, MS
中科院分区：
文献类型：
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作者：
Cloeckaert, A;Weynants, V;Zygmunt, MS
根据与A、M单特异性多克隆血清的玻片凝集反应，将光滑布鲁氏菌分为A(+)M(-)、A(-)M(+)和A(+)M(+)三种血清型。所涉及的表位位于光滑脂多糖(S-脂多糖)的O-多糖(O-PS)部分，它代表布鲁氏菌表面暴露最多的抗原结构。通过使用单抗，已经报道了O-PS上的一些表位特异性：A、M以及A和M优势菌株所共有的表位，这些表位被称为公共(C)表位。根据酶联免疫吸附试验(ELISA)对A和M显性布鲁氏菌的相对单抗结合力以及与小肠结肠炎耶尔森菌O：9的交叉反应，将后者进一步细分为五个表位特异性：C(M>；A)、C(M=A)、C/Y(M>；A)、C/Y(M=A)和C/Y(A>；M)。在本研究中，我们通过分析与整个布鲁氏菌细胞结合的单抗水平和流式细胞术分析，研究了这些表位在包括活疫苗株在内的所有布鲁氏菌种和生物群的代表表面的出现情况。在ELISA法中，单抗结合水平与先前定义的表位特异性和每个布鲁氏菌种、生物群或菌株的多克隆血清所定义的血清型有很好的相关性。然而，与C(M=A)和C(M>；A)表位结合的单抗与猪链球菌生物群2株结合不显著，与猪链球菌生物群3和新红假单胞菌结合的滴度比与其他布鲁氏菌株低。部分流式细胞仪检测结果与酶联免疫吸附试验检测结果相矛盾。事实上，弓形细胞术表明，所有的O-PS表位，包括A表位和M表位，都不同程度地被布鲁氏菌共享。然而，根据MAb结合强度，它们显示出高度的O-PS异质性。因此，弓形细胞仪法对单抗特异性和布鲁氏菌血清型的细分不如用ELISA法明显。而在ELISA法中，A表位的单抗与小肠结肠炎耶尔森菌O：9的结合不明显，而在流式细胞术中，该单抗与小肠结肠炎耶尔森菌O：9有很强的结合。针对C(M=A)表位的两个单抗中的一个也与猪链球菌生物型2菌株结合在一个低但显著的水平上。然而，与在EL ISA中一样，在流式细胞术中，针对C(M>；A)表位的单抗根本不能与猪链球菌生物型2菌株结合。流式细胞术提供了有关单抗特异性的新信息，并可能进一步解释某些单抗对小鼠光滑布鲁氏菌感染的被动保护能力的某些方面，如本研究表明，布鲁氏菌株显然完全缺乏一个特定的C O-PS表位(例如，猪布鲁氏菌生物型2缺乏C[M>；A]表位)，这为获得缺乏诊断O-PS表位的疫苗株提供了可能性，这将进一步有助于解决区分感染动物和接种动物的问题，这仍然是布鲁氏菌研究的主要目标。
Smooth Brucella strains are classified into three serotypes, i.e., A(+)M(-), A(-)M(+), and A(+)M(+), according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by bow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by Bow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice, As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e,g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.