Molecular Recognition in Antibody Engineering. Studies on recombinant and phage displayed antibodies

Ann-Christin Malmborg Hager

Research output: ThesisDoctoral Thesis (compilation)


In vitro generation of antibody fragments of desired specificity and affinity plays an important role since it permits the generation of reagents which may be valuable for diagnostic and therapeutic applications. Furthermore, the kinetic parameters for an antibody-antigen interaction, rather than the affinity, has shown to correlate with biological functions like virus neutralization. This points at the importance of being able to evaluate kinetic parameters. In this thesis we have evaluated the BIAcore biosensor for measuring affinity and kinetic constants and pointed at events where precautions need to be taken. The recently launched ORIGEN Analyser was, furthermore, evaluated for measuring affinity constants and a protocol for measuring dissociation rate constants was developed. The ability to select antibodies from phage displayed antibody libraries based on their kinetic parameters would be valuable. Two approaches in this direction were taken. The first was to use BIAcore biosensor for selection. By collecting elution fractions of an injected phage displayed antibody library, it could be shown that the time of dissociation is proportional to the dissociation rate constant. The second approach was to develop a modified protocol for SAP selection. SAP (selection and amplification of phage) links specific interaction to phage infection, by displaying the antibody fragment on a non-infectious phage and the antigen, fused to protein3, is added free in solution. Thus, only specific phages are allowed to infect and are amplified. We showed that addition of competing antigen to the interaction between antigen-protein3 fusion protein and a model phage displayed antibody library favoured low dissociation rate constants. Furthermore, a reduction in interaction time favoured high association rate constants. Thus, it was, for the first time, possible to select antibody fragments based on association rate. The last part of the thesis presents a new concept, which integrates phage and bacterial display in an attempt to physically link the genetic information of specifically interacting antibody-antigen molecules. This was performed by expressing peptide antigens on the surface of E. coli F pilus. The peptides were expressed as a fusion to pilin, the building block of pilus and encoded by the traA gene, which completely blocked protein3-mediated wild type phage infection. However, when a phage displayed scFv antibody was allowed to interact with pilus displayed peptide, we obtained a bacterial infection mediated by the specific antibody-antigen interaction. Thus, specific interaction on a protein-ligand level could be genetically rescued as a cellular linkage of the involved genes.This principle could have the potential to allow for screening of specific molecular interactions by crossing one phage and one bacterial library.
Original languageEnglish
Awarding Institution
  • Department of Immunotechnology
  • [unknown], [unknown], Supervisor, External person
Award date1996 Sep 20
ISBN (Print)91-628-2146-6
Publication statusPublished - 1996

Bibliographical note

Defence details

Date: 1996-09-20
Time: 10:30
Place: Chemical Centre, K:C

External reviewer(s)

Name: Uhlén, Mathias
Title: Prof.
Affiliation: [unknown]


Subject classification (UKÄ)

  • Immunology in the medical area


  • transplantation
  • affinity
  • antibody
  • kinetic
  • Immunologi
  • serologi
  • serology
  • Immunology
  • Bioteknik
  • Biotechnology
  • library
  • selection
  • surface display
  • phage display
  • association rate
  • dissociation rate


Dive into the research topics of 'Molecular Recognition in Antibody Engineering. Studies on recombinant and phage displayed antibodies'. Together they form a unique fingerprint.

Cite this