Enrichment of a single clone from a high diversity library of phage-displayed antibodies by panning with Anopheles gambiae (Diptera: Culicidae) midgut homogenate.
A high diversity library of recombinant human antibodies was selected on complex antigen mixtures from midguts of female Anopheles gambiae Giles. The library of phage-displayed single chain variable region fragment constructs, derived from beta-lymphocyte mRNA of naïve human donors, was repeatedly selected and reamplified on the insoluble fraction of midgut homogenates. Five rounds of panning yielded only one midgut-specific clone, which predominated the resulting antibody panel. In A. gambiae, the epitope was found throughout the tissues of females but was absent from the midgut of males. The cognate antigen proved to be detergent soluble but very sensitive to denaturation and could not be isolated or identified by Western blot of native electrophoresis gels or by immunoprecipitation. Nevertheless, immunohistology revealed that this sex-specific epitope is associated with the lumenal side of the midgut. Severe bottlenecking may limit the utility of phage display selection from naïve libraries for generating diverse panels of antibodies against complex mixtures of antigens from insect tissues. These results suggest that the selection of sufficiently diverse antibody panels, from which mosquitocidal or malaria transmission-blocking antibodies can be isolated, may require improved selection methods or specifically enriched pre-immunized libraries.
Killeen
G F
GF
Department of Tropical Medicine, Tulane School of Public Health and Tropical Medicine, 1430 Tulane Avenue SL29A, New Orleans, LA 70112, USA. Gerry.Killeen@unibas.ch
Foy
B D
BD
Frohn
R H
RH
Impoinvil
D
D
Williams
A
A
Beier
J C
JC
D000818:Animals; D000852:Anopheles / Q000276:immunology*; D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification; D007150:Immunohistochemistry / Q000662:veterinary; D008297:Male; D019151:Peptide Library; D012737:Sex Factors
D000906:Antibodies; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library