Bhunia A K, Steele P J, Westbrook D G, Bly L A, Maloney T P, Johnson M G
Department of Food Science, University of Arkansas Biotechnology Center, Fayetteville 72703.
Microb Pathog. 1994 Feb;16(2):99-110. doi: 10.1006/mpat.1994.1011.
An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10(8) cfu/ml into a suspension of Ped-2E9 (10(6)/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test. Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 microns filter.
开发了一种使用骨髓瘤细胞和杂交淋巴细胞的体外细胞培养检测方法，该方法在短短6小时内就能检测出致病性李斯特菌菌株。三种不同的杂交瘤细胞系，即小鼠Ped-2E9和EM-7G1以及人RI.37和小鼠骨髓瘤NS1细胞，在对有毒力的李斯特菌属的反应中表现出同样的敏感性。从食品和临床来源收集的单核细胞增生李斯特菌以及其他李斯特菌属，以10(8) cfu/ml的浓度接种到Ped-2E9（10(6)/ml）的悬浮液中。通过台盼蓝排斥试验测定，致病性李斯特菌属在4小时内杀死了80%的杂交瘤细胞。相反，所有非致病性李斯特菌属都没有杀死杂交瘤细胞。通过台盼蓝染色和91-97%的乳酸脱氢酶（LDH）释放测定，暴露于三株单核细胞增生李斯特菌菌株的Ped-2E9细胞在6小时内显示出96-97.5%的死亡。RI.37细胞也显示出类似的结果。单核细胞增生李斯特菌与杂交瘤细胞的感染复数（MOE）为100:1或10:1时，分别在4小时或6小时内杀死了约80%的杂交瘤细胞。单核细胞增生李斯特菌与杂交瘤细胞的体外毒力检测与免疫受损小鼠模型相比具有优势，结果在6小时内得出，而不是3天。暴露2或4小时后，未从Ped-2E9杂交瘤细胞中回收细胞内的单核细胞增生李斯特菌和无害李斯特菌。然而，在落射荧光显微镜下观察到单核细胞增生李斯特菌和无害李斯特菌细胞都附着在Ped-2E9细胞表面。溶血素阳性的单核细胞增生李斯特菌与杂交瘤细胞的直接接触是导致死亡的关键，因为当杂交瘤细胞通过0.45微米的滤膜与生长的细菌分离时，它们没有被杀死。
