Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk.
A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.
Miyamoto
Takahisa
T
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. tmiyamot@agr.kyushu-u.ac.jp
Kamikado
Hideaki
H
Kobayashi
Hiroshi
H
Honjoh
Ken-ichi
K
Iio
Masayoshi
M
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology / Q000302:isolation & purification*; D005434:Flow Cytometry; D007074:Immunoglobulin G / Q000276:immunology; D018189:Immunomagnetic Separation / Q000379:methods*; D008892:Milk / Q000737:chemistry*; D010316:Particle Size; D011817:Rabbits; D012680:Sensitivity and Specificity; D012756:Sheep
D000907:Antibodies, Bacterial; D004768:Enterotoxins; D007074:Immunoglobulin G; C031818:enterotoxin B, staphylococcal