ABSTRACT
Bacillus cereus is a spore-forming ubiquitous bacterium notable as a food poisoning agent. Detection of B. cereus spores using selective media is laborious and non-specific. Herein, the quantitative detection of B. cereus spores was investigated with commercial antibodies and published aptamer sequences. Several detection reagents were screened for affinity to Bacillus collagen-like protein A (BclA), an abundant exosporium glycoprotein. Sensitivity and selectivity toward B. cereus spores were tested using immunoassays and multi-analyte profiling (xMAP). A recombinant antibody developed in llama against BclA protein showed B. cereus spore selectivity and sensitivity between 102 and 105 spores/mL using xMAP. DNA aptamer sequences demonstrated sensitivity from 103 to 107 spores/mL and no cross-reaction to B. megaterium and B. subtilis. Selectivity for B. cereus spores was also demonstrated in a mixture of several diverse microorganisms and within a food sample with no compromise of sensitivity. As proof of concept for multiplexed measurement of human pathogens, B. cereus and three other microorganisms, E. coli, P. aeruginosa, and S. cerevisiae, were simultaneously detected using xMAP. These data support the development of a rapid, sensitive, and selective system for quantitation of B. cereus spores and multiplexed monitoring of human pathogens in complex matrices.
ABSTRACT
Thiol 2-mercaptoethanol (2-ME) has been reported to enhance growth in lymphocytes by various investigators. Some have used 50 µM for growing hybridomas in vitro. Concentrations of 50 and 5 µM in 5% FBS supplemented D-MEM were tested to determine their effects on the growth of 5 monoclonal antibody secreting mouse B cell hybridomas and the myeloma Sp2/O-Ag14. Viability after 24 and 48 h exposure was determined by Trypan blue exclusion. Analysis by one-way ANOVA confirmed that 50 µM 2-ME has a significant negative impact (p<0.05) on hybridoma as well as on myeloma growth, whereas no significant difference (p>0.05) between the control and the 5 µM treatment group was observed after 48 h. Also, no significant difference (p>0.05) in the mortality rates between the control and the treatment groups was found. When combined with the observed protracted doubling time in the 50 µM treatment group, these results indicate that the impact of 2-ME is due to inhibition of cell division. The degree of inhibition was observed to vary between the different hybridomas as well as the myeloma. Although the impact of 2-ME on mitosis has been demonstrated in organisms such as the ciliated protozoan Tetrahymena pyriformis, the yeast Saccharomycess cerevisiae, and the egg of the echinoid the sand dollar Dendraster excentricus, this work demonstrates for the first time that 2-ME impedes the growth of mouse B cell hybridomas. We conclude that adding 2-ME to mouse B cell hybridoma growth media may not be beneficial.
