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Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library.

Abstract：

:Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V(HH)s all belonging to the V(HH) subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V(HH)s persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K(D)s were 3 and 254 muM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a nai;ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a V(HH) nai;ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.

journal_name

J Immunol Methods

journal_title

Journal of immunological methods

authors

Yau KY,Groves MA,Li S,Sheedy C,Lee H,Tanha J,MacKenzie CR,Jermutus L,Hall JC

doi

10.1016/j.jim.2003.07.011

keywords:

subject

Has Abstract

pub_date

2003-10-01 00:00:00

pages

161-75

issue

1-2

eissn

0022-1759

issn

1872-7905

pii

S0022175903002680

journal_volume

281

pub_type

杂志文章

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Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library.