Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System

Abstract

Staphylococcal enterotoxins (SEs) are the second most common causal agents of food poisoning throughout the world. Staphylococcal enterotoxin B (SEB) is one of the most potent and a listed biological warfare agent. Therefore, its quick, accurate and sensitive detection is of paramount importance. But availability of sensitive and specific antibodies against SEB is the major bottleneck in the development of an immunodetection system. Therefore, in the present study seb gene was cloned and expressed in a heterologous host resulting in a yield of 92 mg pure toxin per litre of culture broth after Ni-NTA affinity purification. Antibodies raised against the recombinant toxin did not cross react with related enterotoxins and organisms that can gain access in the food. Further, a sandwich ELISA was developed to detect SEB after extraction from artificially spiked food samples like milk, orange juice, skim milk and khoya. The sandwich ELISA was able to detect SEB in the range of 0.25 to 0.49 ng/ml or g of food. The detection system developed in the present study is at least as specific and sensitive as other commercially available kits which use monoclonal antibodies.

Development and Evaluation of Simple Dot–Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food

“…Although, numerous reports have been published in the recent past for detection and quantification of SEs in food, most of them are using methodologies which require costly equipment and sophisticated laboratory set up. Several assays reported in recent past include, a double-antibody sandwich ELISA [10] with sensitivity of 32 and 64 pg ml -1 SEA in buffer and milk respectively; a monoclonal antibody-based sandwich ELISA [11] with sensitivity of 0.0282 ng ml -1 SEA; hydrogelbased microarray biochips [14] with detection limit of 0.5 ng ml -1 SEA in milk; a sandwich ELISA [15] for staphylococcal enterotoxin B (SEB) with the sensitivity of 0.25-0.45 ng ml -1 in different foods. Several kits for detection and quantification of classical SEs are also available commercially viz., RIDASCREEN (R-Biopharm GmbH, Germany), Transia (Transia-Diffchamb S.A. Lyon, France), VIDAS Staph enterotoxin 2 (SET2) and TECRA (3M Science, USA).…”

Preliminary Extraction and Identification of the 44.5 kDa Outer Membrane Proteins Isolated from Bovine Fusobacterium necrophorum (AB)

“…Following the removal of the normal cells at 4°C with a low-speed centrifugation step (1,000 rpm, 30 min), the remaining supernatant (1,000 rpm, 4°C, 30 min) containing ruptured cells was centrifuged at 15,000 rpm for 30 min. The sediment (15,000 rpm, 4°C, 30 min) obtained was suspended in 20 mL of 0.5 % sodium lauryl sarkosinate (SLS, Yi Ji Industrial Co., Ltd. Shanghai, China) aqueous solution, and the suspension was gently stirred at 25°C for 30 min to solubilize the inner membrane proteins [12]. The suspension solution was then centrifuged at 15,000 rpm for 30 min and the sediment collected.…”

Therapy Decision Support Based on Recommender System Methods

“…While most works related to CDSS deal with diagnosis decision support [2, 3], predicting patient condition [4–8], or determining drug interaction [9], data-driven CDSS for therapy decision support are rare to date. This fact can be partially attributed to traditional data-mining and machine-learning techniques, which have limitations in case of missing and inhomogeneous data and often show an undesired black-box behavior.…”