Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points.

When panned with a transient heat denaturation approach against target enzymes, a human V(H) (antibody heavy chain variable domain) phage display library yielded V(H)s with composite characteristics of binding, non-aggregation and reversible thermal unfolding. Moreover, selection was characterized by enrichment for V(H)s with (i) an even number of disulfide forming Cys residues in complementarity-determining region (CDR) 1 and CDR3 and (ii) acidic isoelectric points. This parallels naturally occurring camelid and shark single-domain antibodies (sdAbs) which are also characterized by (i) solubility and reversible unfolding, (ii) a high occurrence of disulfide forming Cys in their CDRs, particularly, in CDR1 and CDR3 and (iii) acidic V(H)s as inferred here by a pI distribution analysis, reported here, of pools of human and camelid V(H) and V(H)H (camelid heavy chain antibody V(H)) sequences. Our results, reinforced by previous observations by others, suggest that protein acidification may yet be another mechanism nature has devised to create functional sdAbs and that this concept along with the inclusion of inter-CDR disulfide linkages may be applied to human V(H) domains/libraries for non-aggregation optimization. In addition, calculation of theoretical pIs of V(H)s selected by panning may be used for rapid and precise identification of non-aggregating V(H)s.

Prototype single step lateral flow technology for detection of avian influenza virus and chicken antibody to avian influenza virus.

A rapid and effective lateral flow assay (LFA) for detection of avian influenza virus (AIV) was developed. For antigen capture, the assay used monoclonal antibody specific for a conserved nuclear protein (NP) epitope, immobilized on a cellulose acetate matrix, in conjunction with a second NP monoclonal antibody chemically linked to either coloured latex beads or colloidal gold particles contained in a sample pad for detection. Virus sample added to the sample pad flowed into the trapping antibody to form a visible band as well as a second, control band further along the acetate strip. The control band consisted of recombinant protein A/G, also immobilized on the matrix. A second LFA for detection of chicken antibody to AIV was developed where NP antigen was immobilized on the matrix with recombinant protein A/G immobilized as a control band. Latex beads or colloidal gold particles to which monoclonal anti-chicken antibody was attached, were used as the indicator system.

Optimal design features of camelized human single-domain antibody libraries.

We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.

Thermodynamic analysis of monoclonal antibody binding to duplex DNA.

A technique based on fluorescence polarization (anisotropy) was used to measure the binding of antibodies to DNA under a variety of conditions. Fluorescein-labeled duplexes of 20 bp in length were employed as the standard because they are stable even at low ionic strength yet sufficiently short so that both arms of an IgG cannot bind to the same duplex. IgG Jel 274 binds duplexes in preference to single-stranded DNA; in 80 mM NaCl Kobs for (dG)20.(dC)20 is 4.1x10(7) M-1 compared with 6.4x10(5) M-1 for d(A5C10A5). There is little sequence specificity, but the interaction is very dependent on ionic strength. From plots of log Kobs against log[Na+] it was deduced that five or six ion pairs are involved in complex formation. At low ionic strength,Kobs is independent of temperature and complex formation is entropy driven with DeltaH degrees obs and DeltaC degrees p,obs both zero. In contrast, in 80 mM NaCl DeltaC degrees p,obs is -630 and -580 cal mol-1K-1 for [d(TG)]10.[d(CA)]10 and (dG)20.(dC)20 respectively. IgG Jel 241 also binds more tightly to duplexes than single-stranded DNA, but sequence preferences were apparent. The values for Kobs to [d(AT)]20 and [d(GC)]20 are 2.7x10(8) and 1.3x10(8) M-1 respectively compared with 5.7x10(6) M-1 for both (dA)20. (dT)20 and (dG)20.(dC)20. As with Jel 274, the binding of Jel 241 is very dependent on ionic strength and four or five ionic bonds are involved in complex formation with all the duplex DNAs which were tested. DeltaC degrees p,obs for Jel 241 binding to [d(AT)]20 was negative (-87 cal mol-1K-1) in 80 mM NaCl but was zero at high ionic strength (130 mM NaCl). Therefore, for duplex-specific DNA binding antibodies DeltaC degrees p,obs is dependent on [Na+] and a large negative value does not correlate with sequence-specific interactions.

Sequence and structure specific antibodies from phage display libraries.

A large combinatorial phage display library was panned against five nucleic acid antigens, calf thymus DNA, poly[d(GC)], poly[d(AT)], poly(dA) x poly(dT) and poly(rA) x poly(dT). After the third and fourth rounds of panning, many positive clones were selected against poly[d(GC)], poly(dA) x poly(dT) and poly(rA) x poly(dT). The specificity of these antibodies was tested by both direct and competitive solid phase radioimmune assays. All the clones derived from panning with poly[d(GC)] were non-specific and bound to all nucleic acids. The poly(rA) x poly(dT) derived clones were specific for single-stranded nucleic acids, with some sequence preferences, and the poly(dA) x poly(dT) derived clones showed considerable specificity for this antigen. The sequences of these phage-derived antibodies showed no similarities with DNA-binding antibodies from other sources. Even after six rounds of panning no positive clones were detected which bound to poly[d(AT)] and after seven rounds only two were derived from panning with calf thymus DNA. Therefore, sequence- and structure specific antibodies can be recovered from phage display libraries but not all sequences may be represented in the repertoire.

Characterization of four nucleic acid-binding single-chain Fv fragments by direct and competitive solid-phase radioimmunoassays.

The heavy- and light-chain variable region genes of four different nucleic acid-binding antibodies (Jel 274 and Jel 72 (specific for duplex DNA), Jel 103 (specific for poly(rI)), and Jel 318 (specific for DNA triplexes)) were cloned. Single-chain Fv fragments (scFv) in which the heavy and light chains were joined by a linker were constructed by polymerase chain reaction. The linker of 21 amino acids also served as a tag since it consisted of a repeating heptapeptide that was recognized by a specific antipeptide antibody. scFv were expressed in the cytoplasm of Escherichia coli as inclusion bodies. After purification and renaturation, a cross-linking assay was used to demonstrate that > 90% of scFv were in the form of monomers. The specificity of scFv was analyzed by both direct and competitive solid-phase radioimmunoassays. scFv.103 retained its specificity for poly(rI), whereas the other three scFv still bound the original antigen, but subtle changes in the overall specificity were noted. Thus, in some cases, the conformation of the binding site may be different in the context of an scFv compared with the original IgG.