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 2^nd booster dose. The developed polyclonal antibodies were highly specific and sensitive with a detection limit about 50 ng/ml for rBoNT/E LC and 2.5×10^[3] 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.

Keywords: Biowarfare agents - botulinum neurotoxins - botulism - polyclonal antibody - recombinant protein.

Fig. 1

PCR amplification of botulinum neurotoxin serotype E light chain gene. Lane 1, molecular weight marker (bp); lanes 2-4, Botulinum neurotoxin serotype E light chain gene.

Fig. 2

SDS-PAGE for protein expression of Botulinum neurotoxin serotype E light chain clone (A) induced at 20°C; lane 1, protein molecular weight marker (kDa); lane 2, Escherichia coli M15 host cells; lane 3, uninduced clone; lanes 4-6, induced with 0.25, 0.5, 0.75 mM isopropyl-β-D-1-thiogalactopyranoside, (B) induced at 25°C; lane 1, protein molecular weight marker (kDa); lanes 2,4,6, uninduced clone; lane 3,5,7, induced with 0.25, 0.5, 0.75 mM isopropyl-β-D-1-thiogalactopyranoside. Circle indicates 50 kDa protein. SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Fig. 3

(A) SDS-PAGE. Lane 1, protein molecular weight marker (kDa); lanes 2 to 10, purified protein. (B) Western blot. Lanes 1 to 3, purified protein; lane 4, protein molecular weight marker (kDa). Circle indicates Botulinum neurotoxin (50 kDa) protein.

Fig. 4

Antibody titre raised against rBoNT/E light chain-soluble protein in mice (intramuscular route) and rabbit (intramuscular route). Optical density values represented are mean±standard deviation of triplicate determinations. Circle indicates antibody titre raised up to a serum dilution of 1:128000 in both mice and rabbit.

Fig. 5

Minimum detection limit of botulinum neurotoxin type serotype E by sandwich ELISA. OD values represented are mean±standard deviation of triplicate determinations. Horizontal line indicates the detection limit (absorbance value two times of the cut-off value).