Abstract
Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology.
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References
Krakauer T (2013) Update on staphylococcal superantigen-induced signaling pathways and therapeutic interventions. Toxins 5:1629–1654
Miethke T, Wahl C, Heeg K, Echtenacher B, Krammer PH et al (1992) T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J Exp Med 175:91–98
Dellabona P, Peccoud J, Kappler J, Marrack P, Benoist C et al (1990) Superantigens interact with MHC class II molecules outside of the antigen groove. Cell 62:1115–1121
Seth A, Stern LJ, Ottenhoff TH, Engel I, Owen MJ et al (1994) Binary and ternary complexes between T-cell receptor, class II MHC and superantigen in vitro. Nature 369:324–327
Proft T, Fraser JD (2003) Bacterial superantigens. Clin Exp Immunol 133:299–306
Singh PK, Agrawal R, Kamboj DV, Gupta G, Boopathi M et al (2010) Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B. Appl Environ Microbiol 76:8184–8191
Fast DJ, Schlievert PM, Nelson RD (1989) Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production. Infect Immun 57:291–294
Neumann B, Engelhardt B, Wagner H, Holzmann B (1997) Induction of acute inflammatory lung injury by staphylococcal enterotoxin B. J Immunol 158:1862–1871
Vial T, Descotes J (1995) Immune-mediated side-effects of cytokines in humans. Toxicology 105:31–57
Frame KK, Hu WS (1990) The loss of antibody productivity in continuous culture of hybridoma cells. Biotechnol Bioeng 35:469–476
Kessler N, Bertrand S, Aymard M (1993) Stability of a murine hybridoma is dependent on the clonal line and culture media. In Vitro Cell Dev Biol 29A:203–207
Ozturk SS, Palsson BO (1990) Loss of antibody productivity during long-term cultivation of a hybridoma cell line in low serum and serum-free media. Hybridoma 9:167–175
Skerra A, Pluckthun A (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240:1038–1041
Ho M, Nagata S, Pastan I (2006) Isolation of anti-CD22 Fv with high affinity by Fv display on human cells. Proc Natl Acad Sci U S A 103:9637–9642
Galeffi P, Lombardi A, Pietraforte I, Novelli F, Di Donato M et al (2006) Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems. J Transl Med 4:39
Choo AB, Dunn RD, Broady KW, Raison RL (2002) Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells. Protein Expr Purif 24:338–347
Coia G, Hudson PJ, Irving RA (2001) Protein affinity maturation in vivo using E. coli mutator cells. J Immunol Methods 251:187–193
Irving RA, Kortt AA, Hudson PJ (1996) Affinity maturation of recombinant antibodies using E. coli mutator cells. Immunotechnology 2:127–143
Wang SH, Zhang JB, Zhang ZP, Zhou YF, Yang RF et al (2006) Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis. Anal Chem 78:997–1004
Sato M, Kojima K, Sakuma C, Murakami M, Tamada Y et al (2014) Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay. Sci Rep 4:4080
Kim H, Park S, Lee HK, Chung J (2013) Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification. Exp Mol Med 45:e43
Acknowledgement
We are grateful to the Director, DRDE, Gwalior for providing the research facilities. We thank Bruce Rottmann (Wayne State University) for critical reading and editing of this chapter.
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Singh, P.K., Agrawal, R., Kamboj, D.V., Singh, L. (2016). Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology. In: Brosnahan, A. (eds) Superantigens. Methods in Molecular Biology, vol 1396. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3344-0_17
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DOI: https://doi.org/10.1007/978-1-4939-3344-0_17
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