Enrichment of a single clone from a high diversity library of phage-displayed antibodies by panning with Anopheles gambiae (Diptera: Culicidae) midgut homogenate

G. F. Killeen, B. D. Foy, R. H. Frohn, D. Impoinvil, A. Williams, John C Beier

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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 β-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 immuno-precipitation. 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.

Original languageEnglish
Pages (from-to)31-37
Number of pages7
JournalBulletin of Entomological Research
Volume93
Issue number1
DOIs
StatePublished - Feb 1 2003
Externally publishedYes

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DNA libraries
Anopheles gambiae
midgut
Culicidae
clones
antibodies
antigens
epitopes
selection methods
denaturation
detergents
bacteriophages
malaria
gel electrophoresis
Western blotting
lymphocytes
insects
gender
tissues

ASJC Scopus subject areas

  • Insect Science

Cite this

Enrichment of a single clone from a high diversity library of phage-displayed antibodies by panning with Anopheles gambiae (Diptera : Culicidae) midgut homogenate. / Killeen, G. F.; Foy, B. D.; Frohn, R. H.; Impoinvil, D.; Williams, A.; Beier, John C.

In: Bulletin of Entomological Research, Vol. 93, No. 1, 01.02.2003, p. 31-37.

Research output: Contribution to journalArticle

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