Trimer forming interactions.

<div><p>(a) A stereo view of the composite β-sheet: One of the β-sheets of the V_H consists of five β-strands and it joins together with identical sheets from the symmetry related molecules to form a composite β-sheet with a 3-fold symmetry. The outer strand (Tyr H100 to Ser H112) is the longest, with a significant bend which separates it into two segments. The two segments interact with separate molecules joining the three sheets together. The residues Tyr H100, Gln H105 and Ser H112 are labeled as 100, 105 and 112 respectively. The location of the CDR H3 loop is labeled as H3. </p> <p>(b) Interactions of the sulfate moiety: The sulfur atom and one of the oxygen atoms sits on the crystallographic 3-fold axis. The oxygen atom on the three fold interacts with three Gln residues (Gln H77 of the three V_H) related by symmetry. The other oxygen atoms interact with Ser residues (Ser H23 of the three V_H). (c) A view of the electron density of the His-tag residues. This diagram depicts the 2Fo-Fc electron density map of the His-tag residues. The contours are drawn at 1.2 σ level. (d) Ni-coordination: The Ni ²⁺ ion sits on the crystallographic 3-fold symmetry axis coordinating with 6 Histidine residues in octahedral geometry. Each of the symmetry related chains contributes two histidine residues for coordination. The bound nickel is shown as a sphere and histidine residues are represented as stick models. The three symmetry related molecules are shown in green, cyan and pink. </p></div

A comparison of the free form of scfv6H4 with the METH bound form.

<div><p>(a) A stereo-view depicting the superposition of the Cα atoms of the free form (pink) with the METH complex (purple). The largest deviations are all in the CDR loops. The METH is shown in green. The CDR H1, CDR H2 and CDR H3 are labeled as H1, H2 and H3. Likewise, CDR L1, CDR L2 and CDR L3 are labeled as L1, L2 and L3. Residues Gly H26, Asp H27, Ser H97 and Met H100B are labeled as 26, 27, 97 and 100B. The light chains are shown in lighter colors. </p> <p>(b) <b>Trimer formation</b>. Left panel: A lateral of a view cartoon representation of the trimer. The three molecules assemble around the 3-fold axis to give it a light bulb-like shape. The Ni²⁺ ion is shown as a sphere (in the stem region on the top) and the sulfate moiety (towards the bottom of the sphere) is shown as a CPK model. Right panel: A view from the top along the 3-fold axis. The three molecules are labeled as Mol-A (red), Mol-B (green) and Mol-C (blue). The light chains are shown in lighter colors. </p></div

AMP and p-OH-METH binding.

<p>(a) Electron density of the ligands: Left panel: A view showing the electron density (2Fo-Fc) of AMP. Right panel: A 2Fo-Fc map of p-OH-METH. The contours are drawn at 1.2 σ level. (b) A comparison of the binding modes of the two metabolites with METH. Left panel: A superposition of AMP (pink) with METH (blue). The antibody binds both AMP and METH in a similar manner, but there is a shift in the position of the nitrogen atom which forms hydrogen bonds with Glu H101 of the V_H chain and His H89 of the V_L chain. The hydrogen bond distances indicate that in the AMP structure they are much weaker. Right panel: p-OH-METH: The geometry of the binding pocket is the same in the METH structure and the hydrogen bonds are of comparable strength as in the METH structure. </p

Simple Radiometric Method for Accurately Quantitating Epitope Densities of Hapten–Protein Conjugates with Sulfhydryl Linkages

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses ¹⁴C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten–BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (<i>r</i>² = 0.98, <i>y</i> = 0.98<i>x</i> + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten–protein conjugations, and choosing the most effective epitope densities for conjugate vaccines

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

<div><p>Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10¹² vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH <i>in vivo</i>. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.</p></div

A comparison of METH or AMP brain and serum concentrations over time, after a 0.56 mg/kg <i>ip</i> injection of METH, between AAV-scFv6H4, AAV-scFv7F9, and a saline control at day 21 post AAV8 administration.

<p>Mice treated with either AAV-scFv6H4 or AAV-scFv7F9 showed significantly lower brain METH concentrations (a) and significantly higher serum concentrations of METH (b) than the saline-treated mice (*, p < 0.05; #, p < 0.001). There was also a significant decrease in AMP brain concentrations (c) in the AAV-scFv treated groups compared to control mice but no difference in serum AMP concentrations (d). Points are shown as mean ± SEM (n = 3–4 per group).</p

Comparison of METH and scFv molar concentrations over time.

<p>On day 0, mice were injected with PBS (control treatment), AAV-scFv6H4, or AAV-scFv7F9. Serum scFv6H4 and 7F9 concentrations were measured via ELISA on days 8 and 15. On day 22, mice were treated with METH as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200060#pone.0200060.g006" target="_blank">Fig 6</a>. Both serum scFv and METH concentrations were measured. Mice treated with either AAV-scFv6H4 or AAV-scFv7F9 showed significantly higher serum concentrations of METH than the control mice (*, p < 0.05; #, p < 0.001). Data points are mean ± SEM (n = 3–4 per group).</p

Comparison of IC₅₀ values for METH between culture-produced scFv and expressed AAV-scFv.

<p>A competitive binding assay was performed with each of the variants. The IC₅₀ (in nM) for both scFv and AAV-scFv variants was estimated at 50% ³H-METH bound (dotted line). Individual data points are shown as the mean ± SEM (n = 8 per group).</p

Schematic of the prototype scFv design.

<p>V_H, variable heavy region; V_L, variable light region; Linker, 15 amino acid linker; His6, 6-histidine tag for purification and identification; FLAG, FLAG tag for identification; HMM38, a secretory signal sequence. The HMM38 at the 5’ end of the sequences is cleaved during secretion at the site indicated (triangle).</p