Melting temperatures of L1 binding sdAbs and mAbs.

<p>ND  =  not determined.</p><p>* Fluorescent melt assays performed in triplicate on at least 2 days. The triplicate measurements were typically identical within less than 0.2°C, and always within 0.6°C, while samples measured from different protein lots or on different days agreed within a degree. ** Most of the CD assays were performed on at least two different days. Values determined by CD agreed within a degree.</p><p>Melting temperatures of L1 binding sdAbs and mAbs.</p

Refolding of sdAb.

<p>Circular dichroism was used to monitor the secondary structure of purified sdAbs over a temperature range of 25–95°C. Representative plots are shown of clones H7 and H2 typifying the distinctly different refolding properties. Temperature is expressed in °C and the change in ellipticity is in milidegrees. CD experiments were performed in duplicate for most of the sdAbs; the observed Tm values agreed within a degree, and the refolding trends were the same among the replicate measurements.</p

Melting temperatures of vaccinia binding sdAbs.

<p>* Fluorescent melt assays performed in triplicate; the measurements were typically identical within less than 0.2°C, and always within 0.6°C.</p><p>Melting temperatures of vaccinia binding sdAbs.</p

Specificity of L1 binding sdAbs.

<p>Direct binding assays were used to demonstrate target specificity for L1 binding antigens. Antigen was immobilized to magnetic beads and biotinylated (bt)- sdAbs (noted at the top of each graph) served as the tracers. Independent direct binding assays were performed different days; a representative data set is shown. The MAGPIX assays typically measures binding from at least 50 beads and the error of the mean fluorescence intensity (MFI) is ≤5%.</p

Amino acid alignments of vaccinia virus binding sdAbs.

<p>The representatives from each family that were chosen for further characterization are presented. Red denotes positions where the amino acid is 90% conserved in the compared sequences, blue indicates low consensus (50%) and black are not conserved. The three CDRs are underlined.</p

Binding kinetics for sdAbs targeting the L1 antigen^*.

<p>* From binding to at least 3 L1-coated spots, reported as average (standard deviation).</p><p>Binding kinetics for sdAbs targeting the L1 antigen^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106263#nt101" target="_blank">*</a>}.</p

Increased melting temperature of L1-G2+.

<p>Fluorescence based melting assay showing melting of L1-G2 in blue and the mutant with the added disulfide, L1-G2+, in black. Melting temperature is determined from a plot of the first derivative of the melt curve. The measurements were performed in triplicate on two different days and showed identical results; for clarity only one replicate is shown.</p

Sequences of L1 binding sdAb.

<p>The alignment shows the sequences of L1 binders isolated in sandwich-style selection. Three families, based on homology within the CDR regions were isolated. CDR regions are underlined. Red denotes positions where the amino acid is 90% conserved in the compared sequences, blue indicates low consensus (50%) and black are not conserved. The most dominant family (L1-H7 family) showed some variation in amino acid sequences of the framework regions that showed an impact of binding affinities and thermal stability. Arrows mark the positions of the cysteine substitutions in the L1-G2+ construct.</p

Recombinant vaccinia virus proteins and antibodies from BEI resources.

<p>Recombinant vaccinia virus proteins and antibodies from BEI resources.</p

Thermal stability assay.

<p>Following a one hour incubation at the defined temperature optical density at 280 nm was measured to assess protein aggregation (upper panel). Samples G2 (blue triangles) and G2+ (gray circles) were measured in duplicate, and showed the same solubility behavior in each replicate. Since all of the mAbs behaved similarly they are all represented by the same symbol (red upside-down triangle). Binding activity following incubation was measured using a direct binding assay with the L1 antigen directly immobilized to four rows of a sensor chip for SPR analysis (lower panel). The mAbs have the same color and symbol but are differentiated by line type: mAb419 with short dashes, mAb418 with long dashes, and mAb417 with a solid line.</p