Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.

Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, California 94110, USA.
Nature Biotechnology (Impact Factor: 39.08). 02/2007; 25(1):107-16. DOI: 10.1038/nbt1269
Source: PubMed

ABSTRACT Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.

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    ABSTRACT: Botulinum neurotoxin (BoNT) causes the disease known as botulism, which can be lethal. Rapid determination of exposure to BoNT is an important public health goal. Our laboratory has developed Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT. Here, we demonstrate that this method is very sensitive, detecting as little as 0.5 mouse LD<sub align="right"> 50 </sub> of BoNT/A and as little as 0.05 mouse LD<sub align="right"> 50 </sub> of BoNT/B, /E, and /F spiked into human serum samples. Additionally, the ability to further differentiate BoNT as the subtype of BoNT/A spiked into milk using toxin proteomics and mass spectrometry has been demonstrated. This method does not require DNA and can be performed on the same sample as that used for Endopep-MS analysis. The combination of these techniques, all performed on the same sample, provides a sensitive and selective analysis of BoNT isolated from a food or clinical sample and measures the toxin's activity.
    The Botulinum J 01/2012; 2(2):119 - 134.
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    ABSTRACT: Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg mL(-1) (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg mL(-1) (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg mL(-1) (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation <20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little cross-reactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.
    The Analyst 08/2014; · 3.91 Impact Factor
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    ABSTRACT: Botulinum neurotoxins (BoNTs) are some of the most potent biological toxins to humans. They are synthesized by the gram-positive, spore-forming bacterium Clostridium botulinum. BoNT is secreted from the bacterium as a ~150 kDa polypeptide which is cleaved by bacterial or host proteases into a ~50 kDa light chain and a ~100 kDa heavy chain disulfide-linked protein. The light chain of the toxin contains the catalytic domain that blocks acetylcholine release from neurons and results in flaccid muscle paralysis. Four serotypes of this pathogen have thus far been associated with human foodborne contamination. Due to their potent toxicity, botulinum neurotoxins pose bioterrorism concerns and are listed as select agents. Ironically, BoNTs, also known as BOTOX®, are used quite liberally among the population for cosmetic reasons. Less well known are the many medical uses of BoNT, such as treatment for strabismus, cervical distonia, and an ever-increasing list of medical ailments. In this review, BoNTs will be explored from perspectives as human poisons or medicines. New technologies to identify and neutralize the effects of the toxin will also be presented and discussed. A brief historical perspective on human botulism The history of botulism is an interesting story of a double-edged sword, in this case a bacterial toxin associated with contaminated foods, its use as a biological weapon, and ironically its use as intervention for a number of medical problems and even for cosmetic purposes. A detailed description of human botulism was reported as late as the eighteen century when an outbreak in south western German spurred then District medical officer Justinus Kerner (1786-1862) to write and publish the first detailed and accurate description of disease symptoms [1,2]. He collected data over a five-year period (1817 through 1822) and suggested the cause of disease to be a biological poison. He coined the phrase "sausage poison" and "fatty poison" to describe botulism since it was often associated with improperly handled meat. In 1895 another outbreak in the small Belgian village of Ellezelles advanced understanding of the disease by identifying the associated pathogen as Clostridium botulinum. In the 1970s, Drs. Alan B. Scott and Edward J. Schantz pioneered the use of botulinum toxins for medical treatment when they carried out a number of ground-breaking animal studies and demonstrating that the toxin may be effective for treatment of strabismus. In 1989, the US Food and Drug Administration approved the use of BOTOX for cosmetic purposes. Research continues to better delineate properties of the toxin, its mechanism of action, and its potential use in human medicine [1,2].

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