An improved procedure for the generation of recombinant single-chain Fv antibody fragments reacting with human CD13 on intact cells

Matthias Peipp, Nicola Simon, Alexandra Loichinger, Wolfgang Baum, Kerstin Mahr, Susan J. Zunino, Georg H. Fey

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

A procedure was developed to generate recombinant single chain Fv (scFv) antibody fragments reacting with the extracellular domain of human cell surface antigen CD13 (hCD13; aminopeptidase N) on intact cells. Membrane fractions prepared from a stably transfected hCD13-positive murine NIH/3T3 cell line were used to immunize BALB/c mice, with the intention that hCD13 would be the major immunogenic molecule recognized by the immune system. Spleen RNA from the immunized mice served to generate a combinatorial scFv phage display library. The library was adsorbed against non-transfected NIH/3T3 or Sf21 insect cells to eliminate nonrelevant binders. The supernatant was then used for panning with either hCD13-transfected Sf21 insect cells or a hCD13-expressing human leukemia-derived cell line. Therefore, the key concepts of the procedure were the presentation of hCD13 as the sole human antigen on murine NIH/3T3 cells and a screening strategy where hCD13 was the major common antigen of the material used for immunization and panning. Two different hCD13-reactive phages were isolated and the soluble scFvs were expressed in E. coli and purified. The two scFvs, anti-hCD13-1 and anti-hCD13-3, differed at four amino acid positions in their VH regions and both had high affinities for hCD13 as determined by surface plasmon resonance (KD=7 and 33×10-10 M, respectively). Both efficiently recognized hCD13 on intact cells. Therefore, the procedure allowed the production of high affinity scFvs reacting with a desired antigen in its native conformation without requiring extensive purification of the antigen and should be useful for the preparation of scFvs against other conformation-sensitive cell-surface antigens.

Original languageEnglish (US)
Pages (from-to)161-176
Number of pages16
JournalJournal of Immunological Methods
Volume251
Issue number1-2
DOIs
StatePublished - May 1 2001
Externally publishedYes

Keywords

  • Aminopeptidase N
  • CD13
  • Leukocyte surface antigens
  • Mixed lineage leukemia
  • Phage display
  • Recombinant antibodies
  • Single chain Fv-fragments

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

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