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 journalArticle

23 Citations (Scopus)

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

Fingerprint

Single-Chain Antibodies
Sf9 Cells
Antigens
NIH 3T3 Cells
Surface Antigens
Bacteriophages
Libraries
Insects
CD13 Antigens
Cell Line
Surface Plasmon Resonance
Immune System
Immunization
Leukemia
Spleen
RNA
Escherichia coli
Amino Acids
Membranes

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

An improved procedure for the generation of recombinant single-chain Fv antibody fragments reacting with human CD13 on intact cells. / Peipp, Matthias; Simon, Nicola; Loichinger, Alexandra; Baum, Wolfgang; Mahr, Kerstin; Zunino, Susan J.; Fey, Georg H.

In: Journal of Immunological Methods, Vol. 251, No. 1-2, 01.05.2001, p. 161-176.

Research output: Contribution to journalArticle

Peipp, Matthias ; Simon, Nicola ; Loichinger, Alexandra ; Baum, Wolfgang ; Mahr, Kerstin ; Zunino, Susan J. ; Fey, Georg H. / An improved procedure for the generation of recombinant single-chain Fv antibody fragments reacting with human CD13 on intact cells. In: Journal of Immunological Methods. 2001 ; Vol. 251, No. 1-2. pp. 161-176.
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AU - Zunino, Susan J.

AU - Fey, Georg H.

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