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Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus Aureus Enterotoxin B

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Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus Aureus Enterotoxin B

Russell R Graef et al. BMC Biotechnol.

Abstract

Background: Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays.

Results: Here we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL.

Conclusion: The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies.

Figures

Figure 1
Figure 1
ELISA results of direct binding of llama plasma to SEB toxin and toxoid coated wells. This data verifies the presence of SEB toxin and toxoid binding IgG in the immunized llama plasma.
Figure 2
Figure 2
Amino acid sequence and western blot of sdAb A3. A) Predicted amino acid sequence of the A3 anti-SEB sdAb. CDRs are highlighted. B) Western Blot of purified sdAb A3 using anti-His-HRP antibody. Benchmark ladder.
Figure 3
Figure 3
Evaluation of specificity by Luminex fluid array direct binding assays: Bt-sdAb A3 (top panel), Bt- llama anti-SEB IgG1 (middle panel), and Bt-IgG2 (bottom panel) subclass fraction. Result show A3 is highly specific for SEB.
Figure 4
Figure 4
Evaluation of thermal stability by binding activity. SdAb A3 at 10 μg/ml and antibodies (llama anti-SEB IgG1, RA-SEB, and anti-SEB MAb 3b2a) at 100 μg/ml were heated at 85°C. Aliquots were removed and tested. The samples were diluted 10-fold and tested for their antigen binding ability in a Luminex direct binding assay.
Figure 5
Figure 5
Evaluation of A3's thermal stability by monitoring secondary structure by CD. A3 was heated to 85°C and then cooled to 25°C repeatedly: A) CD spectra of A3 (blue line) and upon heating to 85°C (1st cycle, pink; 2nd cycle, orange) and upon cooling to 25°C (1st cycle, green line; 2nd cycle, red line). Alternatively, secondary structure can be monitored and Tm determined by tracking the ellipticity change at single wavelength 202 nm: B) 4 heat cycles to 95°C, first cycle blue, second cycle green, third cycle red, and fourth cycle black; C) 4 cooling cycles color coded as in panel B.
Figure 6
Figure 6
Differential Scanning Calorimetry of the sdAb A3. Antibody melting was observed in PBS with a protein concentration of 1.6 mg/ml. Heating rate was 1°C per minute. Melting temperature was about 82.6°C and refolding upon cooling was not observed.
Figure 7
Figure 7
The Tm of A3 deviates from a normal distribution. A total of 12 SdAb for which the melting temperatures have been determined are presented on a normal probability plot. The Y-axis values are specified by the probability distribution function for an assumed normal distribution. The straight line is a robust linear regression of the data and all data points will be near the line if the assumption is correct. The Tm for A3 can be seen to fit poorly.
Figure 8
Figure 8
Comparison of CD spectra and intrinsic fluorescence intensity of sdAb A3 to llama IgG1 taken at 5°C intervals from 25°C to 85°C. SdAb A3 a) CD spectra b) fluorescence intensity. Llama anti SEB IgG1 c) CD spectra d) fluorescence intensity. Results indicate that intrinsic fluorescence changes fail to correlate with the sdAb's loss of ellipticity upon heating, while intrinsic fluorescence does correlate with ellipticity for the polyclonal IgG.
Figure 9
Figure 9
SPR binding profiles for sdAb A3. Panel A shows five concentrations of SEB (30, 10, 3.3, 1.1, and 0.37 nM) flowed for 3 minutes over immobilized sdAb A3, followed by 15 minutes dissociaiton. Data shown was corrected by subtraction of interspot data and the buffer only response. Panel B shows a similar experiment performed on a NeutrAvidin coated chip with Bt-sdAb A3 immobilized. Panel C shows sdAb A3 (30, 10, 3.3, 1.1, and 0.37 nm binding to a surface coated with SEB.
Figure 10
Figure 10
SEB detection using Luminex sandwich assays. Three different anti-SEB antibody coated bead sets plus a negative control were used in each assay performed as described in the methods. Top panel used Bt-sdAb-A3 (1 μg/mL), middle panel used Bt-MAb 3b2a (10 μg/mL), and bottom panel Bt-Llama anti-SEB (10 μg/mL), respectively as the detector antibody, followed by SA-PE (5 μg/mL) to fluorescently label the immuno-complex prior to measurement on the Luminex 100.

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