A molecular approach for isolating high-affinity Fab fragments that are useful in blood group serology.
BACKGROUND
Multiple mouse hybridoma antibodies recognize the antigens of the MNS blood group system. The Fab fragments of several of these antibodies were expressed on bacteriophage and as soluble proteins. The parental N92 anti-N IgG monoclonal antibody (parental N92 MoAb), but not its monovalent, soluble Fab fragment (N92 Fab fragment), agglutinated antigen-positive red cells by an antiglobulin method. Light-chain shuffling was used to isolate mutant N92 Fab fragments with higher affinity that would function by agglutination.


METHODS
Light-chain cDNA libraries, constructed from mice immunized with N-type glycophorin A, were inserted into a recombinant pComb3H vector containing the N92 Fd fragment. The N92 Fd fragment:light-chain libraries were panned on N-type glycophorin A or NN red cells, and antigen-binding clones were isolated. Purified parental N92 MoAb and the Fab fragments were evaluated by enzyme-linked immunosorbent assay and agglutination.


RESULTS
The novel NNA7, C1, and G11 Fab fragments all bound to N-type glycophorin A with higher affinity than did the N92 Fab fragment. The affinity of the library-derived clones was equivalent to that of the parental N92 MoAb. Although their fine specificity differed slightly from the parental N92 MoAb, the clones functioned equivalently by agglutination using an antiglobulin method.


CONCLUSIONS
Light-chain shuffling allowed the isolation of bacterially produced, high-affinity, soluble, monovalent recombinant anti-N Fab fragments that functioned well by agglutination. This approach is useful in obtaining inexpensive serologic reagents that may replace conventional MoAbs produced by tissue culture methods.
Czerwinski
M
M
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland.
Krop-Watorek
A
A
Siegel
D L
DL
Spitalnik
S L
SL
D000371:Agglutination; D000818:Animals; D015723:Gene Library; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008951:MNSs Blood-Group System; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000302:isolation & purification
D007140:Immunoglobulin Fab Fragments; D008951:MNSs Blood-Group System; D011994:Recombinant Proteins