Detection of Mycobacterium bovis bacillus Calmette-Guerin using quantum dot immuno-conjugates

*Corresponding author: Mailing address: International Medical Center of Japan, Toyama 1-21-1, Shinjuku-ku, Tokyo 162-8655, Japan. Fax: +81-3-3202-7364, E-mail: tkirikae@ri.imcj.go.jp Luminescent quantum dots (QDs) are a novel and promising class of fluorophores for cellular imaging (1,2). The benefits of QDs include their photostability, high brightness, multi-target labeling with several colors, and single-source excitation for QDs of all colors. We have developed procedures for using QDs to detect mycobacteria in a speciesspecific manner. Mycobacterium bovis BCG strain 172 was obtained from Japan BCG Laboratory, Tokyo, Japan. A green fluorescent protein (GFP) expressing M. bovis BCG, containing plasmid pGFM-11, was supplied by C. Locht, Institut Pasteur de Lille, France. The BCG strains were grown in liquid Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich., USA) supplemented with 10% oleic acid-albumin-dextrose-catalase enrichment (OADC, Difco) and incubated at 37°C. Ten microliters of liquid medium was mounted on a glass coverslip beneath a hole in a plastic petri dish bottom (Matsunami Glass Industry., Ltd., Tokyo, Japan; code. D110100) and were subsequently air dried. Two percent glutaraldehyde in PBS was applied for 1 h at room temperature. After several rinses with PBS, the 1% bovine serum albumin (BSA) in PBS (BSA/ PBS) was applied for 20 min at room temperature to block nonspecific binding. Antiserum obtained from rabbits immunized with heat-killed BCG was applied at a dilution of 1:4000 with BSA/PBS, and the dishes were incubated for 1 h at room temperature. After several rinses with 0.02% Tween 20 in PBS (PBS/Tween 20), Qdot 655 goat F(ab )2 anti-rabbit IgG conjugate (H+L) highly cross-absorbed (antibodies QDconjugate: Quantum Dot Corp., Hayward, Calf., USA) was applied at a dilution of 1:1000 with 1% BSA for 1 h at room temperature. The dishes were then rinsed three times with PBS/ Tween 20, and microscopic examinations were conducted with a confocal laser scanning microscope (LSM 510, Carl Zeiss, Oberkochen, Germany) equipped with a × 100/1.40 oil immersion objective, an HBO 50 illuminator, and an FITC/ Rhodamine dual-band filter set. The results of immunofluorescent staining (A, B), conventional mycobacterial staining (C, D), and Ziehl-Neelsen staining (E, F) are shown in Fig. 1. BCG strains were labeled in red when treated with anti-BCG antibodies (Fig. 1A), whereas Mycobacterium smegmatis (Fig. 1B) was not labeled when treated with anti-BCG antibodies, indicating that these antibodies was specific to M. bovis BCG. As shown by the confocal image in Fig. 2A, the surface of