Use of a new functional dual coating (FDC) assay to measure low toxin levels in serum and food samples following an outbreak of human botulism

Abstract

Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. The active dichain neurotoxin molecule is composed of a heavy chain (H-chain) of~100 kDa with the carboxy-terminal end consisting of a receptor-binding (H C ) domain, while the amino-terminal (H N ) domain is linked by a critical disulfide bond to a light chain (L-chain) of~50 kDa. Although the mouse bioassay (MBA) is traditionally used to confirm the presence of toxin in serum or food, its sensitivity is insufficient to detect low toxin levels in approximately 30 to 60 % of botulism patients. A novel FDC (functional dual coating) microtitre plate immuno-biochemical assay, which quantifies botulinum toxicity by measuring the H C domain linked with L-chain endopeptidase activity, was modified to allow human serum (lysed or unlysed) to be tested without interference from the matrix, with toxin detection down to 0.03 mouse LD 50 per ml serum or 0.13 pg ml -1 using just 100 ml of clinical samples. The assay was specific for type A toxin and could additionally be applied to whole blood and food samples. Low levels of 1 to 2 mouse LD 50 per ml serum of type A toxin were quantified for the first time using the modified FDC assay in two severely intoxicated UK patients who required mechanical ventilation and antitoxin. Toxin levels in recovered food sample extracts were also detected and one MBAnegative sample was found to contain 0.32 LD 50 per ml extract. The FDC assay provides a real alternative for public health laboratories to unambiguously confirm all cases of type A botulism and, due to its sensitivity, a promising new tool in toxin pharmacokinetic studies.

Targeted localized use of therapeutic antibodies: a review of non-systemic, topical and oral applications

“…Following immunization, animals capable of producing large amounts of potent antibody at minimal cost, such as sheep, goats, cows, camels, horses and chickens (Abbott et al, 2013;Berghman et al, 2005;Carlander et al, 2000;Kovacs-Nolan & Mine, 2012;Landon & Smith, 2003;Meddeb-Mouelhi et al, 2003;Schade et al, 2005;Stevens et al, 2013), may therefore be particularly suited for plasma, serum, colostrum, milk or egg production. Any residual unwanted taste or color, which generally results from a variable hemoglobin contamination within some batches of crude serum or plasma preparations for oral use, may be easily removed by applying skimmed milk, as described previously (Jones & Marks, 2013). Existing monoclonal antibodies may perhaps be utilized via this route of administration.…”

Toxic Phytochemicals and Their Potential Risks for Human Cancer

“…In spite of these bans, safrole can still be purchased on the Internet as a primary ingredient in sassafras oil or brown camphor oil and instructions as to its synthesis from methylenedioxy compounds are also readily available. In addition, safrole is a precursor in the synthesis of the insecticide synergist piperonyl butoxide, which when added to insecticide mixtures, such as pyrethrin, pyrethroid, and carbamate insecticides, increases their potency (66–68). Many common insecticides and pesticides for home and garden use, mosquito and termite control, veterinary use, and human clothing and bedding use contain piperonyl butoxide, which is classified as a possible human carcinogen by the EPA (69).…”

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

“…Endopeptidase assays usually detect the LC activity only. Therefore in different approaches HC-specific antibodies or alternatively receptor binding have been applied for toxin extraction and combined with detection of cleaved substrates [ 42 , 43 , 44 ]. Independent of the aforementioned approaches different cell-based assays have been developed which are currently in use for precise potency determination of highly pure pharmacological preparations and for BoNT inhibitor screening [ 45 , 46 ].…”