Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk

被引:30
作者
Scotcher, Miles C. [1 ]
Cheng, Luisa W. [1 ]
Stanker, Larry H. [1 ]
机构
[1] ARS, Western Reg Res Ctr, USDA, Albany, CA USA
基金
美国农业部;
关键词
MONOCLONAL-ANTIBODIES; DODECYL-SULFATE; SEQUENCE; BINDING; PROTEIN; REGION; IDENTIFICATION; OUTBREAK; TETANUS;
D O I
10.1371/journal.pone.0011047
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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. Methods and Findings: 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.
引用
收藏
页数:10
相关论文
共 27 条
[1]   Botulinum toxin as a biological weapon - Medical and public health management [J].
Arnon, SS ;
Schechter, R ;
Inglesby, TV ;
Henderson, DA ;
Bartlett, JG ;
Ascher, MS ;
Eitzen, E ;
Fine, AD ;
Hauer, J ;
Layton, M ;
Lillibridge, S ;
Osterholm, MT ;
O'Toole, T ;
Parker, G ;
Perl, TM ;
Russell, PK ;
Swerdlow, DL ;
Tonat, K .
JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, 2001, 285 (08) :1059-1070
[2]  
BHUYAN AK, 2009, MECH SDS INDUCED PRO
[3]   BOTULINUM NEUROTOXIN-A SELECTIVELY CLEAVES THE SYNAPTIC PROTEIN SNAP-25 [J].
BLASI, J ;
CHAPMAN, ER ;
LINK, E ;
BINZ, T ;
YAMASAKI, S ;
DECAMILLI, P ;
SUDHOF, TC ;
NIEMANN, H ;
JAHN, R .
NATURE, 1993, 365 (6442) :160-163
[4]   NUCLEOTIDE-SEQUENCE OF THE GENE CODING FOR CLOSTRIDIUM-BOTULINUM (CLOSTRIDIUM-ARGENTINENSE) TYPE-G NEUROTOXIN - GENEALOGICAL COMPARISON WITH OTHER CLOSTRIDIAL NEUROTOXINS [J].
CAMPBELL, K ;
COLLINS, MD ;
EAST, AK .
BIOCHIMICA ET BIOPHYSICA ACTA, 1993, 1216 (03) :487-491
[5]  
CDC, 1998, Botulism in the United States, 1899-1996 Handbook for Epidemiologists, Clinicians, and Laboratory Workers
[6]   Antibody Protection against Botulinum Neurotoxin Intoxication in Mice [J].
Cheng, Luisa W. ;
Stanker, Larry H. ;
Henderson, Thomas D., II ;
Lou, Jianlong ;
Marks, James D. .
INFECTION AND IMMUNITY, 2009, 77 (10) :4305-4313
[7]   Botulism: Cause, Effects, Diagnosis, Clinical and Laboratory Identification, and Treatment Modalities [J].
Dembek, Zygmunt F. ;
Smith, Leonard A. ;
Rusnak, Janice M. .
DISASTER MEDICINE AND PUBLIC HEALTH PREPAREDNESS, 2007, 1 (02) :122-134
[8]   TOXIGENIC CLOSTRIDIA [J].
HATHEWAY, CL .
CLINICAL MICROBIOLOGY REVIEWS, 1990, 3 (01) :66-98
[9]   MOST POISONOUS POISON [J].
LAMANNA, C .
SCIENCE, 1959, 130 (3378) :763-772
[10]   Conserved sequence and structure association motifs in antibody-protein and antibody-hapten complexes [J].
Livesay, DR ;
Subramaniam, S .
PROTEIN ENGINEERING DESIGN & SELECTION, 2004, 17 (05) :463-472