Background: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT) which consist of a family of seven serotypes (A-H) produced by the anaerobic bacterium Clostridium botulinum.BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc) and a 50 kDa light-chain (Lc).F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A) and is used in a sensitive immunoassay for toxin detection.We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody.Methods and Findings: To characterize the binding epitope of F1-40, three complementary experimental approaches were selected.Firstly, recombinant peptide fragments of BoNT/A light-chain were used in Western blots to identify the epitope domains.Secondly, a peptide phage-display library was used to identify the specific amino acid sequences.Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phagedisplay studies were mapped onto the three-dimensional structure in order to visualize the epitope.F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200.The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3.When the three amino acids Q138, P139 and D140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished.Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding. Conclusions:The epitope of F1-40 was localized to a single exposed loop (ß4, ß5) on the Lc of BoNT.Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.
Compost water extracts (compost teas) are gaining popularity among organic growers, largely because of their disease suppressive activity when applied to foliage or soil. Production methods often include addition of supplemental constituents, particularly molasses, to stimulate plant-beneficial microbial populations. We have found that molasses amendments also favor regrowth of human pathogenic bacteria, raising public health concerns about potential contamination of treated crops, particularly produce intended for fresh consumption. Using disease outbreak strains marked with green fluorescent protein (GFP) and spontaneous antibiotic-resistance, we found that regrowth of Salmonella enterica serovar Thompson and Escherichia coli O157:H7 was positively correlated with molasses concentration. For Salmonella, regrowth was also dependent on the type of starter compost material used. Salmonella populations increased from 1 at time 0 to over 1000 CFU ml−1 in dairy manure compost tea with 1% molasses, and from 1 at time 0 to over 350,000 CFU ml−1 in chicken manure compost tea by 72 h. E. coli populations increased from 1 at time 0 to approximately 1000 CFU ml−1 in both types of tea by 72 h. Pathogen regrowth did not occur when molasses was eliminated or kept to 0.2%.
Abstract Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, GalNAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu‐NAcα2‐3Galβ1‐3(NeuNAcα2‐6)GalNAcα1‐ O ‐Ser(Thr) tetrasaccharide or to fixed neuraminidase‐digested cells presenting the Gal‐GalNAc disaccharide. The percentages of Tn‐positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A‐specific monoclonal antibodies showed that the erythrocytes composing the Tn‐negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn‐positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 X 10 7 erythrocytes, suggesting that the frequency of such cells in normal individuals is > 1 X 10 −6 .
ABSTRACT Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms. Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system. Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California. Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water. The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria). The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences. Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue , comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences. Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTES&T Critical Review: Environmental monitoring by immunoassayMartin. Vanderlaan, Bruce E. Watkins, and Larry. StankerCite this: Environ. Sci. Technol. 1988, 22, 3, 247–254Publication Date (Print):March 1, 1988Publication History Published online1 May 2002Published inissue 1 March 1988https://pubs.acs.org/doi/10.1021/es00168a002https://doi.org/10.1021/es00168a002research-articleACS PublicationsRequest reuse permissionsArticle Views403Altmetric-Citations81LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.
Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL-1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for β-amanitin (β-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.
