Nano-Immunodetection and Quantification of Mycobacteria in Metalworking Fluids

The accurate detection and enumeration of mycobacteria in metalworking fluids (MWFs) is imperative from an environmental protection and occupational health perspective. We report here on a comparison of the labeling efficiency of nano-immunomagnetic particles (NIMP) and free antibody (FAb) to detect mycobacteria in semisynthetic MWF by using both traditional visualization analysis and cluster analysisaided visualization analysis (CAAVA). The NIMP labeling method involved coating nanometer-scale magnetic particles with Protein A, and oriented conjugation of polyclonal antibodies specific to Mycobacterium spp. The FAb labeling method is modified from the traditional immunofluorescence (IF) method for more efficient detection of mycobacteria in a model MWF. The labeling efficiency of NIMP and FAb were 7.2 4.6 and 16.3 5.5%, and the specificity 85.0 6.1 and 88.1 10.5%, respectively, based on traditional visualization analysis. Based on CAAVA analysis, the labeling efficiency of NIMP and FAb increased to 12.4 1.6 and 20.5 3.9%, and the specificity to 97.8 3.2 and 98.5 2.5%, respectively. A linear relationship of FCM counts and seeded concentrations was observed over four orders of magnitude (R2 0.99) in pure and ternary cultures. The results strongly support the applicability of either FAb or NIMP coupled with CAAVA and flow cytometry for rapid detection and enumeration of mycobacteria in complex matrices.