Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal anti-D: the same framework 1 residues are required for both reactivities.
BACKGROUND
The heavy-chain V4-34 germline gene segment is mandatory for pathologic cold-reacting autoantibodies with anti-I/i specificity (cold agglutinins) and is also preferentially used by monoclonal immunoglobulin M alloantibodies against D and other Rh antigens. The use of the V4-34 segment by monoclonal anti-D has previously been shown to also confer anti-I/i reactivity (cold agglutinin activity), which has implications for the use of such antibodies for Rh blood typing. V4-34 framework 1 (FR1) sequence is believed to be critical for cold agglutinin activity of cold agglutinins.


METHODS
The aim of this investigation was to use site-directed mutagenesis of a recombinant V4-34-encoded anti-D to determine the contribution of V4-34 FR1 sequence to anti-D activity and whether mutational modifications in the FR1 region could separately alter anti-D and anti-i activities.


RESULTS
The results show that amino acid changes in V4-34 FR1 at W7, A23, and Y25 have a profound effect on anti-D activity as well as on anti-i activity. It was not possible to substantially reduce or remove anti-i activity without reducing anti-D activity to a comparable extent.


CONCLUSIONS
The same nonpolar hydrophobic amino acids in FR1 are critical for maintaining both anti-D and anti-i activity. It is proposed that these residues influence the conformation of the antigen-binding site.
Thorpe
Susan J
SJ
Biotherapeutics Group and Technology Development and Infrastructure Group, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK. sthorpe@nibsc.ac.uk
Ball
Christine
C
Fox
Bernard
B
Thompson
Keith M
KM
Thorpe
Robin
R
Bristow
Adrian
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D000918:Antibody Specificity; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003450:Cryoglobulins / Q000276:immunology; D015870:Gene Expression / Q000276:immunology; D018095:Germ-Line Mutation; D006801:Humans; D006825:Hybridomas; D007048:I Blood-Group System / Q000276:immunology*; D007075:Immunoglobulin M / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D012204:Rh-Hr Blood-Group System / Q000276:immunology*
D000911:Antibodies, Monoclonal; D003450:Cryoglobulins; D007048:I Blood-Group System; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; C029443:cold agglutinins