Construction of dimeric F(ab) useful in blood group serology.
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.
Czerwinski
Marcin
M
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland. czerwins@immuno.iitd.pan.wroc.pl
Krop-Watorek
Anna
A
Lisowska
Elwira
E
Spitalnik
Steven L
SL
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
D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D011994:Recombinant Proteins