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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Imaging of Lactobacillus brevis single cells and microcolonies without a microscope by an ultrasensitive chemiluminescent enzyme immunoassay with a photon-counting television camera.
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Imaging of Lactobacillus brevis single cells and microcolonies without a microscope by an ultrasensitive chemiluminescent enzyme immunoassay with a photon-counting television camera.

机译：通过具有光子计数电视摄像机的超灵敏化学发光酶免疫测定法无需显微镜即可对短乳杆菌单细胞和微菌落进行成像。

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An ultrasensitive chemiluminescent enzyme immunoassay (CLEIA) was developed for the rapid detection and quantification of Lactobacillus brevis contaminants in beer and pitching yeast (Saccharomyces cerevisiae slurry collected for reinoculation). L. brevis cells trapped on a 47-mm nucleopore membrane (0.4-micron pore size) were reacted with a peroxidase-labelled Lactobacillus group E antibody and then subjected to an enhanced CLEIA analysis with 4-iodophenol as the enhancer. The combination of a nucleopore membrane with low background characteristics that enables the antigen-antibody reaction to proceed through the pores of the membrane and a labelled antibody prepared by the maleimide hinge method with minimal nonspecific binding characteristics was essential to minimize background in the detection of single cells. An ultrahigh sensitive charge-coupled device (CCD) camera equipped with a fiber optics image intensifier permitted the imaging of single cells. A clear correlation existed between the number of luminescent spots observed and the plate count [y (CLEIA) = 0.990x (plate count) + 15.9, where n = 7, r = 0.993, and P < 0.001]. Microscopic observation confirmed that the luminescent spots were produced by single cells. This assay could be used to detect approximately 20 L. brevis cells in 633 ml of beer within 4 h. Our ultrasensitive CLEIA could also be used to detect microcolonies approximately 20 microns in diameter which had formed on a membrane after 15 to 18 h of incubation. This method, which we called the microcolony immunoluminescence (MIL) method, increased the signal-to-noise ratio dramatically. The MIL method could be used to detect a 10(0) level of L. brevis contamination in 633 ml of beer and a 1/10(8) level of L. brevis contamination in pitching yeast within 1 day (15 to 18 h to form microcolonies and 2 h for CLEIA).
机译：开发了一种超灵敏的化学发光酶免疫法（CLEIA），用于快速检测和定量啤酒和沥青酵母（收集的酿酒酵母浆液用于再接种）中的短乳杆菌污染物。使捕获在47毫米核孔膜（孔径为0.4微米）上的短乳杆菌细胞与过氧化物酶标记的乳杆菌E组抗体反应，然后以4-碘苯酚为增强剂进行增强的CLEIA分析。具有低本底特性的核孔膜（使抗原抗体反应能够通过该膜的孔进行）与通过马来酰亚胺铰链法制备的具有最小非特异性结合特性的标记抗体的结合对于将检测单个底物的本底最小化至关重要。细胞。配备光纤图像增强器的超高灵敏度电荷耦合器件（CCD）相机允许对单个细胞进行成像。观察到的亮点数量与板数之间存在明显的相关性[y（CLEIA）= 0.990x（板数）+ 15.9，其中n = 7，r = 0.993，P <0.001]。显微镜观察证实，发光点是由单个细胞产生的。该测定法可用于在4小时内检测633 ml啤酒中的大约20 L. brevis细胞。我们的超灵敏CLEIA也可用于检测孵育15至18小时后在膜上形成的直径约20微米的微菌落。这种方法，我们称为微菌落免疫荧光法（MIL），可显着提高信噪比。 MIL方法可用于检测633毫升啤酒中10（0）的短乳杆菌污染和1天（15至18 h至15h的发酵酵母中）发酵酵母中1/10（8）的短乳杆菌污染。形成小菌落，CLEIA为2小时）。

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