Development of a sandwich ELISA for the detection of Listeria spp. using specific flagella antibodies.
Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x 10(5) cells/0.1 ml at 22 degrees C. and 1 x 10(6) cells/0.1 ml at 30 degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 10(4) cells/0.1 ml at 22 degrees C and 30 degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.
Kim
Seong-Hee
SH
National Veterinary Research and Quarantine Service, Ministry of Agriculture and Forestry, Anyang 430-824, Korea.
Park
Min-Keun
MK
Kim
Jin-Young
JY
Chuong
Pham Duc
PD
Lee
Yong-Soon
YS
Yoon
Byoung-Su
BS
Hwang
Kyu-Kye
KK
Lim
Yoon-Kyu
YK
D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry*; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005407:Flagella / Q000235:genetics*; D005516:Food Microbiology; D007136:Immunoglobulins / Q000032:analysis; D008087:Listeria / Q000145:classification* / Q000276:immunology / Q000302:isolation & purification*; D008460:Meat / Q000382:microbiology; D008892:Milk / Q000382:microbiology; D012680:Sensitivity and Specificity; D013552:Swine
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; C016512:IgY; D007136:Immunoglobulins