Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins.
Rapid ultrasensitive detection of gastrointestinal pathogens presents a great interest for medical diagnostics and epidemiologic services. Though conventional immunochemical and polymerase chain reaction (PCR)-based methods are sensitive enough for many applications, they usually require several hours for assay, whereas as sensitive but more rapid methods are needed in many practical cases. Here, we report a new microarray-based analytical technique for simultaneous detection of five bacterial toxins: the cholera toxin, the E. coli heat-labile toxin, and three S. aureus toxins (the enterotoxins A and B and the toxic shock syndrome toxin). The assay involves three major steps: electrophoretic collection of toxins on an antibody microarray, labeling of captured antigens with secondary biotinylated antibodies, and detection of biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shear-flow. All the stages are performed in a single flow cell allowing application of electric and magnetic fields as well as optical detection of microarray-bound beads. Replacement of diffusion with a forced transport at all the recognition steps allows one to dramatically decrease both the limit of detection (LOD) and the assay time. We demonstrate here that application of this "active" assay technique to the detection of bacterial toxins in water samples from natural sources and in food samples (milk and meat extracts) allowed one to perform the assay in less than 10 min and to decrease the LOD to 0.1-1 pg/mL for water and to 1 pg/mL for food samples.
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, Russia. shlyapnikov@online.stack.net
Yuri M
YM
Shlyapnikov
Elena A
EA
Shlyapnikova
Maria A
MA
Simonova
Anna O
AO
Shepelyakovskaya
Fedor A
FA
Brovko
Ravilya L
RL
Komaleva
Eugene V
EV
Grishin
Victor N
VN
Morozov
D000818:Animals; D055910:Antibodies, Immobilized / Q000276:immunology; D001427:Bacterial Toxins / Q000032:analysis* / Q000276:immunology; D002772:Cholera Toxin / Q000032:analysis / Q000276:immunology; D004768:Enterotoxins / Q000032:analysis* / Q000276:immunology; D004867:Equipment Design; D004926:Escherichia coli / Q000276:immunology / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation; D057230:Limit of Detection; D060526:Magnetic Fields; D008460:Meat / Q000382:microbiology; D008892:Milk / Q000382:microbiology; D040081:Protein Array Analysis / Q000295:instrumentation*; D013211:Staphylococcus aureus / Q000276:immunology / Q000302:isolation & purification*; D014734:Vibrio cholerae / Q000276:immunology / Q000302:isolation & purification*; D014871:Water Microbiology
D055910:Antibodies, Immobilized; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli