Efficient Detection of Amyloida using PMMA-Tag Fused VHH Antibody

Source: AIChE
  • Type:
    Conference Presentation
  • Conference Type:
    AIChE Annual Meeting
  • Presentation Date:
    November 10, 2015
  • Duration:
    30 minutes
  • Skill Level:
    Intermediate
  • PDHs:
    0.50

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VHH domains represent the smallest antigen-binding unit with a molecular size of∼15 kDa in comparison to scFv fragments (30 kDa), Fab fragments (60 tkDa), and whole antibodies (150 kDa) [1]. For this reason, VHH fragments have been considered to have great potential in various industrial applications. Furthermore, the genetically fused PMMA tag to VHH antibody allow the high rate of refolding and immobilization. Recently, Amyloid beta (Aβ) denotes peptides of 36–43 amino acids that are crucially involved in Alzheimer's disease as the main component of the amyloid plaques found in the brains of Alzheimer patients. In the present study, we examined whether PMMA-tag fused VHH antibody could offer the potential for detecting Amyloid β antigen. To do it, the (His)6-tagged recombinant protein expressed as inclusion bodies in Escherichia coli Rosseta (DE3 component cell) were purified by affinity chromatograph on an Histrap Ni 2+-Chealate column using a linear gradient of imidazole. His-tag-VHHs was purified as a monomer, eluting on the range from 0 to 400 mM imidazole under pH 6.0. As a results, the highly purified VHH-PM antibodies showed different recovery rate and antigen-binding activity, according to the different strains of VHH-PM antibodies. The recovery rate of Anti-Aβ-VHH-PM (L1-3) and Anti-Aβ-VHH-PM (61-3) was lower in pH 8.5. However, Anti-Aβ-VHH-PM (61-3) was highly recovered at pH 6.0. Furthermore Anti-Aβ-VHH-PM (L1-3) refolded in pH 8.5 showed high antigen binding activity, compared to that Anti-Aβ-VHH-PM (61-3). It was demonstrated that the optimal condition of refolding in Anti-Aβ-VHH-PM was changeable with pH and strain.

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