Detection of botulinum neurotoxin serotype B at sub mouse LD(50) levels by a sandwich immunoassay and its application to toxin detection in milk.
BACKGROUND
Botulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H). We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.


RESULTS
Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5-0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.


CONCLUSIONS
We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk.
Scotcher
Miles C
MC
Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, California, USA.
Cheng
Luisa W
LW
Stanker
Larry H
LH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001905:Botulinum Toxins / Q000032:analysis* / Q000276:immunology / Q000633:toxicity; D019274:Botulinum Toxins, Type A; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007928:Lethal Dose 50; D051379:Mice; D008892:Milk / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A