Construction of dimeric F(ab) useful in blood group serology

Abstract

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.

Alteration of amino acid residues at the L‐chain N‐terminus and in complementarity‐determining region 3 increases affinity of a recombinant F(ab) for the human N blood group antigen

“…All of these F(ab) were seen to be highly purified when analyzed by SDS‐PAGE and formed H‐chain Fd fragment‐L‐chain heterodimers (Fig. 2 and data not shown; also see Czerwinski et al 8 ).…”

“…The pComb3H vectors 8 containing the L‐chain cDNA and the H‐chain Fd fragment cDNA of N92, NNA7, or the resulting mutants (see below) were each digested with Nhe I and Spe I to remove the gene III fragment and then religated with T4 ligase. In addition to removing the gene III fragment, this manipulation coupled the hexahistadyl affinity tag to the 3′ end of the H‐chain Fd fragment.…”

“…After dialysis against 20 mmol per L Tris, 150 mmol per L NaCl, pH 8.0, followed by concentration with an ultrafiltration system (Micro‐Prodicon, Spectrum, Houston, TX), the protein content was measured by the bicinchoninic acid protein assay (Pierce, Rockford, IL). The purity of the protein preparations was confirmed by SDS‐PAGE with and without 2‐mercaptoethanol, as described previously 8 . The activity of the purified proteins was measured by both hemagglutination and ELISA (see below).…”

“…Nevertheless, few studies have examined the humoral immune response to glycopeptide antigens, where both the underlying peptide sequence and the attached carbohydrates are important for antigen recognition 2‐4 . To this end, we have focused on antibodies recognizing the human M and N glycopeptide blood group antigens 5‐13 . Antibodies to these antigens are clinically relevant in the settings of hemolytic transfusion reactions and HDN 14‐17 .…”

MNS Blood Group System

“…Stable dimers of F(ab) fragments with anti -M and -N specifi cities directly agglutinated red cells at concentrations similar to those of corresponding IgG antibodies [487] . Comparison of high -and low -affi nity recombinant F(ab) fragments with N specifi city and site -directed mutagenesis experiments demonstrated that L -chain amino acid sequences, and particularly Gly91 in complementaritydetermining region 3 (CDR3), were important for determining high affi nity [488] .…”