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Applied and Environmental Microbiology, April 2004, p. 2098-2104, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2098-2104.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Immunodetection of the Bacteriocin Lacticin RM: Analysis of the Influence of Temperature and Tween 80 on Its Expression and Activity

Tomer Keren, Merav Yarmus, Galia Halevy, and Roni Shapira^*

Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Received 1 September 2003/ Accepted 7 January 2004

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 µg ml^–1 for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 µg ml^–1 at 37, 30, 20, 15, 10, and 4°C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.

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* Corresponding author. Mailing address: Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. Phone: 972-8-9489818. Fax: 972-8-9476189. E-mail: shapira{at}agri.huji.ac.il.

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Applied and Environmental Microbiology, April 2004, p. 2098-2104, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2098-2104.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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