Characterization of a Broadly Specific LPS-Neutralizing Single Domain Antibody

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  • V. Yadav Department of Veterinary Microbiology and ICAR Centre of Advanced Faculty Training, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India
  • A. Singh Department of Veterinary Microbiology and ICAR Centre of Advanced Faculty Training, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India
  • J. Rawat Department of Veterinary Microbiology and ICAR Centre of Advanced Faculty Training, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India

Klíčová slova:

Endotoxin, single domain antibody, SPR, MALDI-TOF, dAb-LPS co-crystallisation

Abstrakt

Gram negative endotoxaemia or sepsis remains a lethal challenge to humans and animals, globally. Single domain antibodies (dAbs) have emerged as therapeutic biologics for challenging antigens such as lipopolysachharide (LPS). The present study characterized a broadly specific LPS-neutralising dAb produced from phage display library from LPS-immunized Indian desert camel, for its sequence validation with mass spectrometry. Also, the dAb clone 26 was subjected to co-crystallisation with LPS, extracted from E. coli O6 (ATCC 25922). The mass spectrometry of dAb clone 26 was performed upon in-gel trypsin digestion, MALDI-TOF analysis of the derived peptides and sequence identification on Mascot protein identification. The co-crystallization grid screening was performed with affinity purified dAb clone 26 in complex with LPS in a hanging drop vapor diffusion set up with pH and various precipitants i.e., ammonium sulphate, PEG3350, 6000 and 8000 as variables. The sequence of dAb clone 26 was validated with the sequence coverage of 35% through mass spectrometry. The study led to further validation of dAb clone 26 for developing it as a therapeutic candidate for endotoxemia.  Keywords: Endotoxin, single domain antibody, MALDI-TOF, dAb-LPS co-crystallizationCite this Article Yadav V, Singh A, Rawat J. Characterization of a Broadly Specific LPS-Neutralizing Single Domain Antibody. Research & Reviews: Journal of Immunology. 2017; 7(1): 1–8p.

Stažení

Publikováno

2017-03-04

Číslo

Vol 7 No 1 (2017)

Sekce

Research Articles

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