Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin

Sheila Nathan, Hongbin Li, Rahmah Mohamed, Mohammed Noor Embi

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 108 clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.

Original languageEnglish
Pages (from-to)45-53
Number of pages9
JournalJournal of Biochemistry, Molecular Biology and Biophysics
Volume6
Issue number1
DOIs
Publication statusPublished - Feb 2002

Fingerprint

Burkholderia pseudomallei
Exotoxins
Bacteriophages
Display devices
Antibodies
Clone Cells
Light
Single-Chain Antibodies
Antibody Affinity
Polymerase chain reaction
Hybridomas
Digestion
Immunoglobulin G
Genes
Enzyme-Linked Immunosorbent Assay
Polymerase Chain Reaction
Enzymes

Keywords

  • Antibody
  • Exotoxin
  • Gel electrophoresis
  • Hybridoma
  • Immunotherapy
  • Reverse transcription

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics
  • Biophysics

Cite this

Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin. / Nathan, Sheila; Li, Hongbin; Mohamed, Rahmah; Embi, Mohammed Noor.

In: Journal of Biochemistry, Molecular Biology and Biophysics, Vol. 6, No. 1, 02.2002, p. 45-53.

Research output: Contribution to journalArticle

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