Development of immunodetection system for botulinum neurotoxin serotype E

Abstract

Background & objectives:Botulism, a potentially fatal paralytic illness, is caused by the botulinum neurotoxins (BoNTs) secreted by Clostridium botulinum. It is an obligate anaerobic, Gram-positive, spore-forming bacterium. BoNTs are classified into seven serotypes based on the serological properties. Among these seven serotypes, A, B, E and, rarely, F are responsible for human botulism. The present study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA)-based detection system for the detection of BoNT/E.Methods:The synthetic gene coding the light chain of BoNT serotype E (BoNT/E LC) was constructed using the polymerase chain reaction primer overlapping method, cloned into pQE30UA vector and then transformed into Escherichia coli M15 host cells. Recombinant protein expression was optimized using different concentrations of isopropyl-β-D-1-thiogalactopyranoside (IPTG), different temperature and the rBoNT/E LC protein was purified in native conditions using affinity column chromatography. The purified recombinant protein was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and further confirmed by western blot and matrix-assisted laser desorption ionization-tandem time-of-flight (MALDI-TOF). Polyclonal antibodies were generated against rBoNT/E LC using Freund's adjuvant in BALB/c mice and rabbit. Sandwich ELISA was optimized for the detection of rBoNT/E LC and native crude BoNT/E, and food matrix interference was tested. The developed antibodies were further evaluated for their specificity/cross-reactivity with BoNT serotypes and other bacterial toxins.Results:BoNT/E LC was successfully cloned, and the maximum expression was achieved in 16 h of post-induction using 0.5 mM IPTG concentration at 25°C. Polyclonal antibodies were generated in BALB/c mice and rabbit and the antibody titre was raised up to 128,000 after the 2nd booster dose. The developed polyclonal antibodies were highly specific and sensitive with a detection limit about 50 ng/ml for rBoNT/E LC and 2.5×103 MLD50 of native crude BoNT/E at a dilution of 1:3000 of mouse (capturing) and rabbit (revealing) antibodies. Further, different liquid, semisolid and solid food matrices were tested, and rBoNT/E LC was detected in almost all food samples, but different levels of interference were detected in different food matrices.Interpretation & conclusions:There is no immune detection system available commercially in India to detect botulism. The developed system might be useful for the detection of botulinum toxin in food and clinical samples. Further work is in progress.

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ELISA Based Detection of Botulinum Neurotoxin Type F in Red Meat and Canned Fish

“…Hence, determination of detection limit in various foods will help in investigation of the food borne outbreaks. BoNTs was determined in different food matrices with LOD of 5 ng/ml for type A and E and 10 ng/ml type B and F using optical immunoassay 24 Digoxigenin labelled antibodies further increased the limit of detection of different BoNTs in the range of 60 -176 pg/ml 17,20 .…”

“…The cut-off value for the developed method was calculated using mean specific OD + 3SD of the blank. The concentration of the rBoNT/F LC protein at which the absorbance value was two times the cut-off value (A 450 nm using the well having no antigen) was considered to be the ELISA endpoint (detection limit) 17 .…”

“…This method is very sensitive and specific, but this assay require a huge number of animals. Hence, it is necessary to replace the mouse bioassay with similarly sensitive another assays [16][17][18] .…”

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