Immunodetection of the bacteriocin lacticin RM: analysis of the influence of temperature and Tween 80 on its expression and activity.
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 micro g 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 micro g ml(-1) at 37, 30, 20, 15, 10, and 4 degrees 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.
Keren
Tomer
T
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Yarmus
Merav
M
Halevy
Galia
G
Shapira
Roni
R
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D000918:Antibody Specificity; D001430:Bacteriocins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western / Q000706:statistics & numerical data; D004797:Enzyme-Linked Immunosorbent Assay / Q000706:statistics & numerical data; D015151:Immunoblotting / Q000706:statistics & numerical data; D013294:Lactococcus lactis / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011136:Polysorbates; D011817:Rabbits; D012680:Sensitivity and Specificity; D013696:Temperature
D000907:Antibodies, Bacterial; D001430:Bacteriocins; D011136:Polysorbates; C408784:lacticin RM