title: Construction of dimeric F(ab) useful in blood group serology.
abstract: BACKGROUND
  When expressed in Escherichia coli, recombinant F(ab) contain a heavy-chain Fd fragment and a complete light-chain fragment. Because these F(ab) are monovalent, their avidity is significantly lower than that of a corresponding bivalent IgG antibody. In addition, when monovalent F(ab) are used in hemagglutination assays, antiglobulin reagents are required. Therefore, it would be useful to develop a system that expresses recombinant bivalent F(ab) in E. coli.
  
  
  METHODS
  Three modified vectors were constructed. Each contained cDNA sequences encoding a peptide linked to the C terminus of a heavy-chain CH1 region: an IgG1 hinge region (Hinge), a leucine zipper (Zip), or a peptide containing the Hinge and Zip sequences in tandem (HingeZip). The vectors were used to express two cloned F(ab) recognizing human antigens M and N: NNA7 (anti-N) and 425/2B (anti-M). The recombinant proteins were expressed in E. coli and were purified and evaluated by ELISA and hemagglutination.
  
  
  RESULTS
  By gel filtration chromatography, 35, 90, and 70 percent of the purified F(ab) expressing the Hinge, Zip, and HingeZip tails, respectively, were dimers. By ELISA, the avidity of F(ab) containing the Zip or HingeZip tails was six to eight times higher than that of the corresponding monovalent F(ab). In addition, the dimeric F(ab) directly agglutinated RBCs in concentrations similar to those of corresponding bivalent IgG antibodies.
  
  
  CONCLUSIONS
  An introduction of dimer-inducing peptides allowed the isolation of bacterially produced, bivalent F(ab). This approach could be useful for obtaining inexpensive, serologic reagents that may replace or complement conventional MoAbs produced by mammalian tissue culture methods.
authors.affiliation: Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland. czerwins@immuno.iitd.pan.wroc.pl
authors.forename: Marcin
authors.initials: M
authors.lastname: Czerwinski
authors.forename: Anna
authors.initials: A
authors.lastname: Krop-Watorek
authors.forename: Elwira
authors.initials: E
authors.lastname: Lisowska
authors.forename: Steven L
authors.initials: SL
authors.lastname: Spitalnik
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001788:Blood Grouping and Crossmatching; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006386:Hemagglutination Tests; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D011994:Recombinant Proteins