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Establishment And Application Of Micro-plate Chemiluminescence Enzyme Immunoassay For The Determination Of Staphylococcal Enterotoxin B

Posted on:2012-12-09Degree:MasterType:Thesis
Country:ChinaCandidate:F LiuFull Text:PDF
GTID:2214330338994604Subject:Immunology
Abstract/Summary:PDF Full Text Request
Staphylococcal enterotoxins (SEs) are bacterial exotoxins secreted by staphylococcus aureus. SEs represent a group of low molecular weight (26 to 30kDa) toxic proteins which can remain biologically active after exposure to both proteolytic enzymes and high temperatures. Seven serologically distinct enterotoxins, SEA, SEB, SEC1, SEC2, SEC3, SED, and SEE, have been identified. Toxic shock syndrome toxin 1 (TSST-1) is known as SEF. Various types of SE have similarities in structure and function. With the development and application of new detection technologies, some new SE and other toxins which are closely related to staphylococcal food poison have been found. Among these toxins, some have been identified with emetic ability, such as SEG,SHE and SEI. Other related toxins (i.e., SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, SEU and SEV) that either lack emetic properties or their potential role in staphylococcal food poisoning has not been confirmed should be designated"staphylococcal enterotoxin–like"(SEl) SAgs.The most prominent property of SE is superantigenicity. Superantigens (SAg) are a group of antigens which can activate 2%20% of naive T cells in T cell repertoire at very low concentrations (110ng/ml) and thus can induce strong immune responses. SAg can be divided into two families, T cell SAg and B cell SAg, according to the target cell type. T cell superantigens can be further divided into TCRαβand TCRγδsuperantigen and SEs belong to TCRαβsuperantigen family. One end of SE molecule binds with the outerα-helix of antigen binding groove of MHC classⅡmolecules on the surface of APC, the other end binds with the variable region of certain TCRβchain on T cells, and thus form the MHCⅡ-SE-TCRVβtriple complex. The MHCⅡ-SE complex and TCR are not bound firmly together and thus the SE molecule can active other naive T cells after it actives one naive T cell. The activated T cells can secrete a variety of cytokines such as IFN-γ, IL-2, CSF and TNF-α, etc., which are closely related to the occurrence and development of some pathological processes.Different strains of staphylococcus aureus could produce different SE and the same strain can produce several types of SE. It has previously shown that different enterotoxins display a high degree of gene sequence similarity in their primary sequence. To fully understand the pathogenic mechanism of various types of enterotoxins, it is critical for us to carry out intensive study on the distribution of different enterotoxins, the relationship between differert types of enterotoxins, and the expression and property of enterotoxin proteins.As one of the major SEs causing food poisoning, SEB is extremely toxic with a half lethal dose (LD50) of about 20 ng/kg and a half effective dose (ED50) of about 0.4 ng/kg. SEB has a stable property and can be prepared easily and can spread in the form of aerosol. According to the Centers for Disease Control and Prevention (CDC) list, SEB belongs to a category B potential biological weapon. The highly sensitive and specific methods of detection of SEB in food, environmental water, and even body fluids are of critical importance in food safety and the war against terrorism.Different assay formats have been developed with the aim of detecting SEB in an ultrasensitive, specific, simple and rapid way. Traditional detection methods, such as reversed passive latex agglutination (RPLA), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence technique are often of poor detection sensitivity and among these the best sensitivity reported was 0.02 ng/mL of SEB in assay buffer. Recently, several useful antigen/antibody-based biosensors for SEB assay have been proposed, such as surface plasmon resonance (SPR) biosensors, resonant acoustic profiling (RAP), biological semiconductors (BSCs), and flow-injection capacitive biosensor. Some of these biosensors for the detection of SEB are highly sensitive, especially the flow-injection capacitive biosensor with the LOD 0.3 pg/mL. However, these methods are mostly used in scientific researches in special professional department and some biosensors are more expensive and not suitable for high-throughput detection.Chemiluminescent immunoassay (CLEIA) has been widely exploited in recent years due to its high sensitivity, wide dynamic range, and suitability for miniaturization. It has been reported that the chemiluminescent reaction often exceeds the sensitivity of radioactivity assays. The basic principle of CLEIA is that the chemiluminescence intensity produced by a chemical reaction is directly proportionate to the amount of analytes present in a sample. One common characteristic of CLEIA is that they all based on antibody-antigen complexes, of which the enzyme-linked immunosorbent assays are highly specific and sensitive. The common enzymes used for chemiluminescence are peroxidase and alkaline phosphatase.As antibodies and enzyme substrates are often key factors that affect the detection level of the CLEIA, we developed a micro-plate CLEIA based on a pair of highly specific mAbs and a sensitive HRP–luminol–H2O2 chemiluminescence system for detecting SEB. The analysis speed is rapid, approximately 2 min per 96-sample plate, which is ideal for clinical practice in hospital where the micro-plate luminometer is easily available and the high-throughput detection is needed.In this study, 20 hybridomas secreting monoclonal antibody (mAb) to SEB were obtained, and the ascites titer and epitope specificities of these mAbs were evaluated using direct and competitive ELISA, respectively. Two mAbs with high titer, high specificity, and different epitope recognized on SEB molecule were chosen to further establish a CLEIA for detecting SEB in a sandwich format. In this pair of mAbs, NO2 was used as capture antibody and NO1 as a detection antibody in CLEIA. The reaction parameters of SEB CLEIA were studied and optimized. This method exhibited high performance with a dynamic range 0.01–5 ng/mL and the measured limit of detection (LOD) 0.01 ng/mL. Intra- and inter-assay coefficient variations were all lower than 13%. For specificity studies, when this method was applied to test SEA, SEC1 and SED, no cross-reactivity was observed. It has been successfully applied to the analysis of SEB in a variety of environmental, biological and humoral matrices such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, and human urine and serum. Thus, we established a micro-plate-CLEIA successfully for detection of SEB with highly sensitive, specific, and simple, based on a pair of highly specific monoclonal antibodies, which has potential applications for quantifying SEB in public health and military reconnaissance.
Keywords/Search Tags:chemiluminescence immunoassay, Staphylococcal enterotoxins, detection
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