Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

<div><p>Single domain antibodies (sdAbs) are the recombinantly-expressed variable domain from camelid (or shark) heavy chain only antibodies and provide rugged recognition elements. Many sdAbs possess excellent affinity and specificity; most refold and are able to bind antigen after thermal denaturation. The sdAb A3, specific for the toxin Staphylococcal enterotoxin B (SEB), shows both sub-nanomolar affinity for its cognate antigen (0.14 nM) and an unusually high melting point of 85°C. Understanding the source of sdAb A3’s high melting temperature could provide a route for engineering improved melting temperatures into other sdAbs. The goal of this work was to determine how much of sdAb A3’s stability is derived from its complementarity determining regions (CDRs) versus its framework. Towards answering this question we constructed a series of CDR swap mutants in which the CDRs from unrelated sdAbs were integrated into A3’s framework and where A3’s CDRs were integrated into the framework of the other sdAbs. All three CDRs from A3 were moved to the frameworks of sdAb D1 (a ricin binder that melts at 50°C) and the anti-ricin sdAb C8 (melting point of 60°C). Similarly, the CDRs from sdAb D1 and sdAb C8 were moved to the sdAb A3 framework. In addition individual CDRs of sdAb A3 and sdAb D1 were swapped. Melting temperature and binding ability were assessed for each of the CDR-exchange mutants. This work showed that CDR2 plays a critical role in sdAb A3’s binding and stability. Overall, results from the CDR swaps indicate CDR interactions play a major role in the protein stability.</p> </div

Primary structure and sequence of sdAbs used in this study.

<p>A) The overall primary structure of sdAbs is shown schematically with alternating framework and CDRs. Melting temperature for the wildtype sdAbs is given in parentheses next to the name. The framework regions are grouped together above the schematic while the CDRs are shown below. The percent identity of sdAb D1 and sdAb C8 toward sdAb A3 is shown for each region. B) Construct identifications are shown schematically for all hybrid antibodies in this study. Regions are color coded for clarity. Observed melting point is shown as a bar graph. Detailed measurements are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077678#pone-0077678-t001" target="_blank">Table 1</a>.</p

CDR swaps lower affinity but may increase or decrease melting temperature.

<p>Melting temperature for sdAbs A3, D1 and C8, as well as full CDR swaps, are plotted against affinity. For each data point the framework origin is indicated before the dot while the CDR origin is indicated after the dot. The affinity shown is that towards the target specified for the CDR origin antibody.</p

Comparison of Immunoreactivity of Staphylococcal Enterotoxin B Mutants for Use as Toxin Surrogates

The development and testing of detection methodologies for biothreat agents are by their very nature complicated by the necessity to handle hazardous materials. Toxoids prepared by thermal or chemical inactivation are often used in place of the native toxin; however, the process of detoxification can decrease the agent’s ability to be detected at similar concentrations. One method to overcome this limitation is the use of toxin mutants which have altered amino acid sequences sufficient to abrogate or greatly reduce their toxic activity. While this method of toxoid preparation is much more controlled, there is still no guarantee that the resulting product will be equal in detectability to the native toxin. In this work, we have evaluated the utility of two recombinantly expressed Staphylococcal Enterotoxin B (SEB) mutants, a single point mutant (Y89A), and a mutant with three amino acids changed (L45R, Y89A, Y94A), to act as surrogates for SEB in immunoassays. We evaluated the affinity of a number of anti-SEB monoclonal antibodies (mAb) and an anti-SEB single domain antibody (sdAb) for SEB and its surrogates. One of the mAb’s affinity was decreased by a factor of 3000 for the triple mutant, and another mAb’s affinity for the triple mutant was decreased by 11-fold while the others bound the mutants nearly as well as they did the native toxin. MAGPIX sandwich immunoassays were used to evaluate the ability of all combinations of the recognition reagents to detect the SEB mutants in comparison to SEB and a chemically inactivated SEB. These results show that recombinant mutants of SEB can serve as much more useful surrogates for this hazardous material relative to the chemically inactivated toxin; however, even the point mutant impacted limits of detection, illustrating the need to evaluate the utility of toxin mutants on a case-by-case basis depending on the immunoreagents being employed

Square Wave Voltammetry of TNT at Gold Electrodes Modified with Self-Assembled Monolayers Containing Aromatic Structures

<div><p>Square wave voltammetry for the reduction of 2,4,6-trinitrotoluene (TNT) was measured in 100 mM potassium phosphate buffer (pH 8) at gold electrodes modified with self-assembled monolayers (SAMs) containing either an alkane thiol or aromatic ring thiol structures. At 15 Hz, the electrochemical sensitivity (µA/ppm) was similar for all SAMs tested. However, at 60 Hz, the SAMs containing aromatic structures had a greater sensitivity than the alkane thiol SAM. In fact, the alkane thiol SAM had a decrease in sensitivity at the higher frequency. When comparing the electrochemical response between simulations and experimental data, a general trend was observed in which most of the SAMs had similar heterogeneous rate constants within experimental error for the reduction of TNT. This most likely describes a rate limiting step for the reduction of TNT. However, in the case of the alkane SAM at higher frequency, the decrease in sensitivity suggests that the rate limiting step in this case may be electron tunneling through the SAM. Our results show that SAMs containing aromatic rings increased the sensitivity for the reduction of TNT when higher frequencies were employed and at the same time suppressed the electrochemical reduction of dissolved oxygen.</p></div

MOESM1 of Selection, characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein

Additional file 1: Figure S1. Biopanning against EBOV GP. The titer of eluted phage was determined at the end of each round of biopanning. Figure S2. Thirty-four potential GP-binding sdAb sequences selected from the second and third rounds of panning against EBOV GP. Sequences were divided into families based on similarity of CDR sequences. Figure S3. Representative measurements of binding kinetics and KD using SPR biosensor. Figure S4. Table of IMGT number and corresponding amino acid at each position for clones EBOV-GP-A8, EBOV-GP–H7 and EBOV-GP-G6. Figure S5. Competition data assessing the ability of the sdAbs to bind GP that was pre-bound with EBOV-GP-A8. Figure S6. Western blotting analysis of GP and VLP binding specificity and cross reactivity for EBOV-GP-G6-neg+-GSKKK

Electrochemical simulation output^a.

<p><i>E</i> = formal redox potential, <i>k</i> = the standard heterogeneous rate constant.</p>a<p>Each peak was modeled as a 2 electron transfer step. Other parameters are listed in the text.</p><p>Electrochemical simulation output^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115966#nt106" target="_blank">a</a>}.</p

Experimental details.

<p><b>A.</b> Chemical structures for biphenyl-4-thiol (Biphenyl), 4-(phenylethynyl)benzenethiol (OPE) and undecane-1-thiol (C11) used to from SAMs on gold electrodes. <b>B.</b> The electrochemical setup.</p

Electrochemical experimental data for the reduction of TNT^a.

<p>1st E_pc = the first cathodic peak potential, 2nd E_pc = the second cathodic peak potential, 1^st I_pc = the first cathodic peak current.</p>a<p>Measured in air saturated water buffered at pH 8 with 100 mM potassium phosphate.</p>b<p>Limit of detection calculated at S/N = 3.</p><p>Electrochemical experimental data for the reduction of TNT^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115966#nt103" target="_blank">a</a>}.</p

Characteristics of SAMs on gold^a.

a<p>average of three electrodes.</p><p>Characteristics of SAMs on gold^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115966#nt101" target="_blank">a</a>}.</p