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. 2010 Jul;2(3):85-93.

Fingerprint-like Analysis of "Nanoantibody" Selection by Phage Display Using Two Helper Phage Variants

S V Tillib  1 , T I Ivanova, L A Vasilev
Affiliations

Fingerprint-like Analysis of "Nanoantibody" Selection by Phage Display Using Two Helper Phage Variants

S V Tillib et al. Acta Naturae. 2010 Jul.

Abstract

This paper discusses the selection of mini-antibody (nanoantibody, nanobody® or single domain antibody) sequences of desired specificity by phage display-based method using a generated library of antigen-binding domains of special heavy-chain only antibodies (single-stranded antibodies) of immunized camel. A comprehensive comparison of the efficiency of parallel selection procedures was performed by using the traditional (M13KO7) and modified (with N-terminal deletion in the surface gIII protein) helper phages. These two methods are partly complementary, and by using them in parallel one can significantly improve the selection efficiency. Parallel restriction analysis (fingerprinting) of PCR-amplified cloned sequences coding for mini-antibodies (HMR-analysis) is proposed for identifying individual clones, as a replacement to sequencing (to a certain extent). Using this method, unique data were collected on the selection of mini-antibody variants with the required specificity at various stages of a multi-stage selection procedure. It has been shown that different sequences coding for mini-antibodies are selected in different ways, and that, if this feature is not taken into account, some mini-antibody variants may be lost.

Keywords: fingerprinting; helper phage; immunisation; phage display; recombinant mini-antibody.

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Figures

Fig. 1
Fig. 1
HMR analysis data (HMR fingerprints) of clones from the original library of cloned mini-antibody nucleotide sequences. Ninety-four clones were selected and analysed. Eighty-five different fingerprints were identified with only 9 of them twice as repeats in this set (marked as 2x). The majority of fingerprints correspond to clones with PstI-NotI insertions of adapted nanoantibody sequences. The smaller part of fingerprints (marked by the letter N) corresponds to clones with NcoI-NotI insertions of adapted nanoantibody sequences into the plasmid vector. Fingerprints found in both sub-libraries are underlined. Each HMR fingerprint is an electrophoretogram consisting of three gel lanes with separated DNA fragments obtained after parallel treatment of the PCR product (an amplified nanoantibody sequence) with one of three restrictases, HinfI (H), MspI (M) or RsaI (R), and the forth lane with marker DNA (the sizes of the marker DNA fragments are shown at the bottom of the figure).
Fig. 2
Fig. 2
HMR fingerprints of the selected nanoantibody clones that bind in ELISA to immobilised recombinant anthrax lethal factor. These clones are designated as “alf” with a number.
Fig. 3
Fig. 3
HMR fingerprints of the selected nanoantibody clones that bind in ELISA to immobilised Rabies virus. These clones are designated as “aRv” with a number.
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