Development of Antiricin Single Domain Antibodies Toward Detection

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Anal. Chem. 2008, 80, 9604–9611

Development of Antiricin Single Domain Antibodies Toward Detection and Therapeutic Reagents George P. Anderson,† Jinny L. Liu,† Martha L. Hale,‡ Rachael D. Bernstein,§ Martin Moore,† Marla D. Swain,† and Ellen R. Goldman*,† Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375, Integrated Toxicology Division, USAMRIID, 1425 Porter Street, Fort Detrick, Fredrick, Maryland 21702, and Nova Research Inc, 1900 Elkin Street Suite 230, Alexandria, Virginia 22308-2406 Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.6 ng/mL was obtained. One sdAb pair in particular was found to be highly specific for ricin. While polyclonal antibodies cross react strongly with RCA120, the sdAb pair had minimal cross reactivity. In addition, the binders were found to be thermal stable, regaining their ricin binding activity following heating to 85 °C for an hour. Cycles of thermally induced unfolding of the sdAb and their subsequent refolding upon cooling was monitored by circular dichroism. As several of the sdAb were observed to bind to ricin’s A chain, cell free translation assays were performed to monitor the ability of the sdAbs to inhibit ricin’s biological activity. One of the sdAb (C8) was particularly effective and blocked ricin’s biological activity with an effectiveness equal to that of a mouse antiricin antibody. These results indicate that antiricin sdAb have great potential for both diagnostic and therapeutic applications. Ricin is a high priority threat agent because of its wide availability, ease of purification, and high morbidity.1 Ricin, the only toxin to exist naturally in large quantities, is obtained from the castor bean plant, Ricinus communis, and is a byproduct of castor oil production. Worldwide, one million tons of castor beans are processed annually for castor oil production with the waste containing 5% ricin by weight. The toxin is quite stable and isolation is simple and cheap. The whole process can be scaled down to a “kitchen” process, thus making the development of both sensitive reagents for ricin detection and ricin therapeutics of high importance. Several bio-assays have been used for the detection of ricin including electochemiluminescence, surface plasmon resonance, * To whom correspondence should be addressed. E-mail: ellen.goldman@ nrl.navy.mil. Fax: 202-767-9594. † Naval Research Laboratory. ‡ USAMRIID. § Nova Research Inc. (1) Audi, J.; Belson, M.; Patel, M.; Schier, J.; Osterloh, J. JAMA 2005, 294, 2342–2351.

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fiber optic, as well as hand-held and array based immunoassays.2-7 Most are immunoassays that rely on recognition elements of monoclonal or polyclonal antibodies (IgG) derived from mice, rabbits, goats, or sheep. Functional IgG from most mammals is a 160 kDa protein composed of two heavy and two light chains connected by disulfide bonds. Each chain is composed of one variable domain plus either one constant domain for the light chain or three constant domains for the heavy chain. A variable domain from a light chain (VL) combines with a variable domain from a heavy chain (VH) to form one of two antigen-binding sites. The antigen binding surface is composed of six complementaritydetermining regions (CDRs), three residing in each of the VH and VL protein domains. The interaction of these six CDR loops of varying sizes and sequences allows the formation of diversified antigen binding surfaces with the topologies to recognize a wide range of antigenic targets. Although sensitive and specific, conventional IgG are large molecules that have limited stability.8 Various derivatives of the intact IgG molecule have been utilized over the years. Initially, proteolytic fragments were prepared; these Fab or Fab’2 fragments primary benefit was the lack of the Fc region, but they often were even less stable than the parent molecule. Molecular engineering has facilitated the expression of recombinant binding fragments created by linking the VH and VL together to create scFv fragments. Often these constructs were also less stable than intact IgG, and production could be problematic because of limited solubility. A recent alternative to conventional IgG and its derivatives has been the development of single domain antibodies (sdAb) derived from members of the Camelidae family, such as llamas. These animals have IgG subclasses (IgG2 and IgG3) that possess unique structural arrangement consisting of only two heavy chains (Figure 1).9,10 The heavy chains, lacking the first constant domain, fail to pair with light chains and yield an antibody with a molecular (2) Garber, E. A. E.; O’Brien, T. W. J. AOAC Int. 2008, 91, 376–382. (3) Feltis, B. N.; Sexton, B. A.; Glenn, F. L.; Best, M. J.; Wilkins, M.; Davis, T. J. Biosens. Bioelectron. 2008, 23, 1131–1136. (4) Fulton, R. E.; Thompson, H. G. J. Immunoassay Immunochem. 2007, 28, 227–241. (5) Huelseweh, B.; Ehricht, R.; Marschall, H. J. Proteomics 2006, 6, 2972– 2981. (6) Anderson, G. P.; Lingerfelt, B. M.; Taitt, C. R. Sensor Lett. 2004, 2, 18–24. (7) Taitt, C. R.; Golden, J. P.; Shubin, Y. S.; Shriver-Lake, L. C.; Sapsford, K. E.; Rasooly, A.; Ligler, F. S. Microb. Ecol. 2004, 47, 175–185. (8) Vermeer, A. W. P.; Norde, W. Biophys. J. 2000, 78, 394–404. (9) Hamers-Casterman, C.; Atarhouch, T.; Muyldermans, S.; Robinson, G.; Hamers, C.; Songa, E. B.; Bendahman, N.; Hamers, R. Nature 1993, 363, 446–448. 10.1021/ac8019398 CCC: $40.75  2008 American Chemical Society Published on Web 11/19/2008

Figure 1. Cartoon of the three llama antibody subtypes, conventional IgG (IgG1), heavy chain only sub types (IgG2 and IgG3), and the single domain antibody cloned binding derivative. Variable domains have been colored red and blue for the conventional variable heavy and light chains, respectively, and purple for the variable domain from the heavy chain only antibodies.

weight of ∼110 kDa. The heavy chain antibody’s two antigenbinding sites are each formed by only a single variable domain, giving binding interaction with only three CDRs as opposed to six in the whole IgG. Also, the structure of this domain is altered by the replacement of select surface amino acids that increase its hydrophilicity and stability compensating for the lack of a light chain. The variable regions from camelid heavy chain antibodies can be expressed recombinantly as sdAb.10 The binding site formed from these single domains has been described as being high affinity, highly stable, and able to refold properly after denaturation.11-13 We have developed ricin binding sdAb derived from immunized llamas and evaluated them in terms of those purported properties. In addition, as ricin’s toxicity is mediated by its enzymatic activity, which causes the ADP ribosylation of ribosomes that leads to protein synthesis inhibition, we evaluated the ability of the sdAb to neutralize ricin in a cell free translation assay. These sdAb reagents have the potential to provide both improved detection reagents, as well as having the potential to be highly effective therapeutics against ricin intoxication. MATERIALS AND METHODS Reagents. Ricin, ricin A chain, ricin B chain, R. communis Agglutinin (RCA120), and goat antiricin were purchased from Vector (Burlingame, CA). Ricin toxoid was from Toxin Technologies (Sarasota, FL). The rabbit antiricin was the kind gift of Dr. Jill Czarnecki (Naval Medical Research Center, Silver Spring, MD). Llama immunizations of two animals were through triple J farms (Bellingham, WA). PhycoLink Streptavidin-R-Phycoerythrin PJ31S (SA-PE) was purchased from Prozyme (San Leandro, CA). Phosphate buffered saline (PBS), Tween 20, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO). The Anti-M13 antibody was purchased from GE Healthcare (Piscataway, NJ). SdAb Library Construction. White blood cells were isolated from 100 mL of llama blood, and total RNA was extracted using (10) Ghahroudi, M. A.; Desmyter, A.; Wyns, L.; Hamers, R.; Muyldermans, S. FEBS Lett. 1997, 414, 521–526. (11) Perez, J. M. J.; Renisio, J. G.; Prompers, J. J.; van Platerink, C. J.; Cambillau, C.; Darbon, H.; Frenken, L. G. J. Biochemistry 2001, 40, 74–83. (12) Dumoulin, M.; Conrath, K.; Van Meirhaeghe, A.; Meersman, F.; Heremans, K.; Frenken, L. G. J.; Muyldermans, S.; Wyns, L.; Matagne, A. Protein Sci. 2002, 11, 500–515. (13) Verheesen, P.; ten Haaft, M. R.; Lindner, N.; Verrips, C. T.; de Haard, J. J. W. Biochim. Biophys. Acta 2003, 1624, 21–28.

Trizol (Invitrogen, Carlsbad, CA). RNA was used in an oligo-dT primed reverse transcription reaction, and PCR amplification of the heavy domain antibodies was performed using flanking primers as described previously,10,14 with resulting sdAb genes being cloned into phage display vector pecan21.14 Panning. Selection was carried out as previously described14 using ricin as target. We performed monoclonal phage enzyme linked immunosorbent assay (ELISA) after each of three rounds of panning to identify individual positive clones. Selected positive clones were sequenced to identify unique sdAb genes. Sequences were aligned using the Multalin program.15 SdAb Protein Production. Unique sdAb clones were subcloned from the phage display sdAb-fusion vector to a soluble sdAb expression vector and constructs were transformed into Escherichia coli Rosetta (Novagen, Madison, WI) for protein production. As described previously,14 the sdAb proteins were isolated from the periplasmic compartment of 500 mL scale shake flask cultures by osmotic shocking, immobilized metal affinity chromatography (IMAC), and gel filtration on a Superdex G75 column (GE-Healthcare). Proteins were quantified using microbicinchoninic acid (BCA) assay (Pierce, Rockford, IL) and stored at 4 °C prior to analysis. Preparation of Luminex Reagents and Assay Protocols. Luminex (Austin, TX) carboxylated microspheres were crosslinked to a variety of proteins using the two-step carbodiimide coupling protocol provided by the manufacturer. The signal for Luminex experiments is reported as the median fluorescence intensity of at least 100 separate microspheres. Direct binding assays were performed essentially as described previously.16,17 To determine relative Kd, biotinylated (Bt)- sdAb were serially diluted in a 96-well microtiter plate (60 µL/well). To each well a mixture of protein coated microspheres, including spheres coated with ricin, was added (5 µL/well). The microspheres and Bt-sdAb were allowed to incubate for at least 30 min at room temperature. Then 5 µL/well (10 mg/L) of SA-PE was added and incubated for an additional 30 min prior to measuring using the Luminex 100 flow analyzer. (14) Goldman, E. R.; Anderson, G. P.; Liu, J. L.; Delehanty, J. B.; Sherwood, L. J.; Osborn, L. E.; Cummins, L. B.; Hayhurst, A. Anal. Chem. 2006, 78, 8245–8255. (15) Corpet, F. Nucleic Acids Res. 1988, 16, 10881–10890. (16) Anderson, G. P.; Ortiz-Vera, Y. A.; Hayhurst, A.; Czarnecki, J.; Dabbs, J.; Vo, B. H.; Goldman, E. R. Botulinum J. 2008, 1, 100–115. (17) Anderson, G. P.; Matney, R.; Liu, J. L.; Hayhurst, A.; Goldman, E. R. Biotechniques 2007, 43, 806–811.

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For the thermal stability testing, 30 µL of the sdAb (10 mg/L) or IgGs (100 mg/L) were heated for various times at 85 °C in a thermal cycler (Tetrad 2, MJ Research). After cooling to room temperature, each sdAb or IgG sample was tested (1 or 10 mg/L respectively) for direct binding to toxin-coated microspheres as described for the monoclonal testing. For the sandwich immunoassays, selected antibody-coated microspheres were incubated (30 min, room temperature), in wells of a 1.2 µm multiscreen filter plate (Millipore, Billerica, MA) with different amounts/types of antigens. Antigens were removed by filtration, and selected biotinylated antiricin antibodies (10 mg/ L) were then added. After 30 min of incubation (room temperature) the excess antibody was removed by filtration and then SAPE (2.5 mg/L) was added and the plate incubated at room temperature in the dark for 30 min. Binding was then evaluated using the Luminex instrument. Sandwich immunoassays using ricin-spiked food matrixes were performed in a similar manner using sdAb immobilized on magnetic microspheres. Instead of using filter plates, microspheres were washed in standard microtiter plates, since the microspheres were immobilized by a magnet (Qiagen, Valencia, CA) while the liquid was pipetted from the plate. Circular Dichroism (CD). The sdAb to be measured was diluted to 65 µg/mL using deionized water, resulting in approximately a 5% PBS buffer. The CD was measured on a JASCO 720 using a 1 cm water-jacketed quartz cuvette. The temperature was adjusted using a Neslab rte111 programmable water bath. The temperature was raised from 25 to 85 °C over a 1 h time period and held at 85 °C for 5 min before the temperature was rapidly cooled to 25 °C. CD spectra were acquired every 5 min during the heating cycle from 260 to 195 nm with a data pitch of 0.1 nm. Cell-Free Translation Assay. Ricin biological activity was determined using a microtiter cell-free translation assay that detects luciferase translation from luciferase m-RNA.18 Dilutions of ricin are included in each assay for generation of a standard curve. Briefly, using phosphate buffered saline (PBS), antibodies were diluted in a 96 well microtiter plate. A constant amount of ricin (100 ng/mL final concentration) was added to the antibody dilutions, the plate left on a microplate shaker for 15 min (25 °C), and then 5 µL transferred to a v-shaped microtiter plate. The rabbit reticulocyte lysate, RNasin, amino acids complete, and luciferase m-RNA (Promega, Madison, WI) were mixed together and then 25 µL added to each well. The plates were incubated (37 °C) for 90 min and then 5 µL of the translation lysate transferred to a black microtiter plate (Sigma Chemicals, St. Louis, MO). After the addition of the assay buffer, containing the luciferin substrate (Promega, Inc.), luminescence was measured as counts per second (CPS) on a Victor multiplate reader (Perkin-Elmer Wallac, Boston, MA). Data was expressed as the percent of control [% control ) (CPS treated/CPS control) × 100]. In Vitro Cell Cytotoxicity Assay. The mouse thyoma EL4 cell line, (ATCC, Manasas, VA) was maintained in RPMI-1640 medium supplemented with 5% fetal calf serum. Immediately prior to the assay, cells were pelleted (600 × g, 10 min, 4 °C) and resuspended to 2 × 106 cells/mL in RPMI-1640 medium. Using RPMI-1640 medium, sdAb were diluted (50 µL/well) in a 96 well (18) Hale, M. L. Pharmacol. Toxicol. 2001, 88, 255–260.

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microtiter plate. Ricin was diluted to yield a final concentration of 200 ng/mL and added (50 µL/well) to the antibody dilutions. After the plates had been left on a shaker for 15 min, cells (100 µL) were added to each well. The plates were incubated (37 °C, 5% CO2) overnight (approximately 17 h) after which 20 µL Alamar Blue(Trek Diagnostics, Cleveland, OH) was added to each well and the plate incubated for 4 additional hours. Dilutions of ricin were included in each assay and used to generate a standard curve. Fluorescence (560 nm excitation and 590 nm emission) was measured on a Safire2 Microplate reader (Tecan Group Ltd., Research Triangle, N.C). The average ± SD of three wells was reported as percentage (%) untreated control cells ) [(fluorescence measurement of treated cells/fluorescence measurement of untreated control cells) × 100]. RESULTS AND DISCUSSION Library Construction. We constructed a library of sdAb displayed on phage derived using mRNA obtained from peripheral blood lymphocytes purified from llamas immunized with ricin toxoid. Before starting library construction the polyclonal response was evaluated, and the polyclonal IgG was subfractionated into both conventional and heavy chain antibody fractions to ensure there was a population of high titer heavy chain antibodies that recognized ricin.16 Heavy chain antibody subclasses purified from ricin toxoid immune llama serum exhibited binding toward intact ricin as well as purified A-chain and B-chain fragments. This gave us confidence in the ability to select useful antiricin sdAb from a library constructed from the immunized animals. Our constructed phage display library contained 5.0 × 107 members. Selection of Ricin Binders. Individual phage clones were examined by monoclonal phage ELISA after the first, second, and third rounds of ricin selection, and strong binders were identified after each round. Out of 196 colonies screened, 41 clones were chosen for sequencing based on both their overall signal from the monoclonal phage ELISA, as well as the ratio of signal from each clone on ricin versus signal on the irrelevant protein BSA. We found 24 unique sdAb that fit into 9 families sharing essentially identical CDRs (Supporting Information, Figure S-1). We chose 16 representative clones, spanning each of the nine families, to examine more closely in terms of specificity, affinity, thermal stability, and to screen for overlapping epitopes. Luminex Evaluation of Binders. We utilized the Luminex platform, a specialized flow cytometer for performing multiplexed fluoroimmunoassays on the surface of coded microspheres, to evaluate binding specificity, affinity, and to separate clones that bind non-overlapping epitopes. As the Luminex binding assays can be done in a homogeneous format, affinity can be determined from the equilibrium binding curves to ricin coated microspheres generated by plotting the signal versus sdAb concentration.14,19,20 All the isolated binders showed higher signal on ricin versus irrelevant toxins such as cholera toxin and staphylococcal enterotoxin B (figure 2). Several families, however, showed high signal on the irrelevant proteins. Potentially the less specific binders could be used in a sandwich assay paired with a specific sdAb. All the clones that showed nonspecific binding appeared to bind (19) Seideman, J.; Peritt, D. J. Immunol. Methods 2002, 267, 165–171. (20) Waterboer, T.; Sehr, P.; Michael, K. M.; Franceschi, S.; Nieland, J. D.; Joos, T. O.; Templin, M. F.; Pawlita, M. Clin. Chem. 2005, 51, 1845–1853.

Figure 2. Specific binding of selected sdAb to cognate antigen. C8, F11, and H1 (panels A, B, and C) are shown as representatives of the many isolated sdAb that were highly specific for ricin. B5H (panel D) is representative of the minority of isolated sdAb, it binds preferentially to ricin’s B chain and does not show high specificity. Homogeneous direct binding assays were performed incubating sdAb with microspheres coated with ricin, ricin A chain, ricin B chain, staphylococcal enterotoxin B (SEB), bovine serum albumin (BSA), and cholera toxin (CTX). Binding constants were determined by fitting the ricin-binding curves using the standard binding equation: y ) (Bmax)x/(Kd + x).

preferentially to the ricin B chain. The specific binders either showed preference for the ricin A chain or gave signal on intact ricin but not on direct binding to either of the two ricin subunits. The Kd, of the 16 characterized clones, as determined by fitting the equilibrium ricin binding data, ranged 0.04 nM to 500 nM. Representative sdAb from each of the families were immobilized on luminex microspheres and evaluated as capture reagents in sandwich immunoassays. Using these microspheres we found only four of the families functioned well as capture reagents (Supporting Information, Figure S-2). The method used for microsphere conjugation did not orient the sdAb, and some clones may immobilize preferentially in an unfavorable orientation. Additional experiments may be conducted in the future with those apparently less useful capture sdAb, by utilizing their engineered His tag to coat Ni modified Luminex microspheres (Qiagen). Preliminary studies using these modified microspheres found results comparable to covalently attached sdAb; however, poor performing capture sdAb have yet to be evaluated using this method. Currently, our experiments have focused on the best performing clones. In the case of a non-repetitive protein target such as ricin the development of sandwich assays requires the use of pairs of sdAb that bind non-overlapping epitopes. To screen for sdAb with nonoverlapping epitopes, we examined the best three sdAb microsphere sets in sandwich assays in combination with tracers from each of the sdAb families (Figure 3). In this way we determined that the sdAb recognize at least three distinct epitopes on ricin.

We examined thermal stability of seven of the ricin binding sdAb. Aliquots of each representative were heated to 85 °C, along with aliquots of conventional antibody for periods of time up to an hour; after cooling, the proteins were assayed for performance in direct binding experiments. Figure 4 shows the results of these experiments. Both rabbit and goat antiricin lost all activity after the first 10 min of heating. Clones C8, D1, D12, F11, H1, and H3 retained the majority of their binding ability on heat treatment. Only clone C3 was found to lose activity during the heating process; indicative of the fact the while most sdAb display thermal stability individual clones may not.21 In addition to retaining their binding ability, all of the sdAb retained their selectivity; binding to irrelevant targets did not increase after heat treatment (Supporting Information, Figure S-3). We selected clones F11, C8, and H1 to examine them more closely; these three clones have Kds of 0.41 nM, 0.35 nM, and 1.2 nM respectively. These clones bind to non-overlapping epitopes and can be used in any combination as capture-tracer pairs for the detection of ricin. To further explore the specificity of these three sdAbs, we examined their binding to ricin that had been added to different food matrixes. Using microspheres coated with F11, H1, or rabbit antiricin, we analyzed ricin spiked samples prepared using a variety of foods including: canned corn, tuna, green beans, mushrooms, tomatoes, tomato juice, and milk. These samples (21) Beekwilder, J.; van Houwelingen, A.; van Beckhoven, J.; Speksnijder, A. Eur. J. Plant Pathol. 2008, 121, 477–485.

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Figure 3. Sandwich assays using rabbit antiricin, and sdAb C8, F11, and H1 captures paired with seven different sdAb reporters. All the sdAb reporters worked with the rabbit-antiricin coated microspheres. H3 and D12 give no signal when combined with C8 capture. D1 does not work with the F11 capture, and C3 does not work paired with H1.

Figure 4. Binding ability of soluble sdAb protein preparations after heat exposure. The sdAb and conventional antibodies were heated to 85 °C for various periods of time, cooled, and assayed at concentrations of 1 µg/mL and 10 µg/mL respectively.

were prepared as described by Sapsford et al.,22 except that the ricin concentrations were added following centrifugation, thereby avoiding the confounding effects of target adsorption to particulate matter. In almost all cases the detection of ricin in food matrixes using the sdAb pairs was similar to the detection in buffer (Figure 5, and Supporting Information, Figure S-5). The highest signal was achieved using F11 as a capture paired with C8 as the tracer. (22) Sapsford, K. E.; Taitt, C. R.; Loo, N.; Ligler, F. S. Appl. Environ. Microbiol. 2005, 71, 5590–5592.

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This combination, as can be seen in Figure 5, reliably gave ricin detection at a level of 1.6 ng/mL. Successful detection in complex matrixes is a strong indicator of the veracity of the antiricin sdAb specificity. In addition, because ricin is a potential threat agent, the ability for sensitive detection of ricin in complex matrixes such as food is important.2 The specificity of the F11-C8 pair was further confirmed by challenging the assay with the lectin from Limulus polyphemus (not shown), Concanvalin A (not shown), and R. communis Agglutinin RCA120 (Figure 6). No cross reactivity was observed for the first two lectins; however, RCA120 is clearly more of a challenge as it shares at least 80% sequence homology to ricin.23 The top left panel shows the response of various capture antibodies to ricin when using a biotinylated polyclonal Llama IgG1 antiricin tracer. The top right panel shows the detection of ricin when using the same set of capture antibodies and biotinylated sdAb C8 as tracer. Note that when C8 is used as both the capture and tracer, very little signal results, confirming the presence of a single unique epitope on ricin for C8. The bottom left panel shows the response to various concentrations of RCA120, up to 100 µg/ mL, when using the polyclonal tracer; note that the sdAbs C8 and F11 capture the least RCA120. The panel on the bottom right shows that when using the biotinylated sdAb C8 tracer the amount of cross reactivity toward RCA120 is greatly reduced for all the various capture antibodies. Even at 100 µg/mL of RCA120, the preferred sdAb pair of F11 and C8 has a signal/background response that remains below a ratio of 4 (a conservative limit of detection on the Luminex platform). (23) Roberts, L. M.; Lamb, F. I.; Papping, D. J. C.; Lord, J. M. J. Biol. Chem. 1985, 260, 5682–5686.

Figure 5. Examples of ricin detection in food matrixes. Corn (left) and tuna fish (right) water and blends were spiked with ricin and tested. Sandwich assays for the detection of ricin were performed using F11, H1, and conventional antibody (Llama IgG) coated microspheres paired with a C8 reporter. BSA coated microspheres were included as a control. Assays are denoted as follows: Corn water (CW), corn blend (CB), tuna water (TW), and tuna blend (TB). The data is the average of two experiments; error bars represent the average deviation.

Figure 6. Examination of crossreactivity to RCA 120. Top panel shows the response to ricin using either a polyclonal antibody (left) or sdAb (right), while the bottom panels show the response to RCA120. SdAbs F11 and C8 along with monoclonal and polyclonal antiricin antibodies are used as captures. The pairing of F11 coated microspheres with the C8 reporter provides the least cross reactivity of all the pairs tested, giving signal below the limit of detection at even 100 µg/mL RCA120.

Circular Dichroism. CD can be used to give information about the secondary structure of proteins. To examine in more detail the thermal stability of the sdAb, and confirm our functional results, we took sequential far UV CD spectra of C8, F11, and H1 while slowly heating the sample to 85 °C and again after the samples had cooled to room temperature. While the spectra of these sdAb varied (not shown), all three sdAb were observed to denature on heating and then refold once cooled to room

temperature. The sdAb C8 CD melting curves, and recovery after heat cycling, shown in Figure 7 are typical of what we observed for the other sdAb. On the basis of how the CD signal at 231 and 208 nm changed with temperature, we estimated a melting temperature of 65 °C for C8; H1 and F11 also had melting temperatures around 65 °C. The three clones examined by CD all showed the ability to refold after two cycles of heat denaturation; however, the magnitude of the peaks decreased somewhat Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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Figure 7. CD of single domain antibody C8. Left panel shows the elipticity change of sdAb C8 at 231 and 208 nm as the sample was heated. The right panel shows the CD spectra of the initial C8 sample, the heated sample, after cooling to room temperature, at 85 °C in a second round of heating, and the final cooled sample. Although the spectra of other sdAb differ in their details they show similar behavior on heating and cooling.

Figure 8. Percent ricin inhibition in cell free translation assays. Ricin (100 ng/mL) was mixed with varying concentrations of sdAb or a mouse antiricin IgG and then added to a rabbit reticulocyte lysate with luciferase mRNA. Translation of luciferase was detected by measuring luminescence after the addition of a luciferin buffer. Values are calculated as % control activity [(cps treated sample/cps control sample) × 100]. Results represent the average (SD for three wells.

on each cycle as can be seen in Figure 7. This is in agreement with what has been observed with other sdAb, where the incomplete recovery of the CD spectra was attributed to factors such as aggregation and proteolysis at elevated temperatures.11 Conventional antibodies always, and their cloned variable domains (scFv) often, denature irreversibly on similar heat treatment.24-26 Although some small-molecule binding sdAb have been reported to bind their antigens at high temperature,27,28 this may in part be due to antigen induced folding of the sdAb.29 (24) Young, N. M.; MacKenzie, C. R.; Narang, S. A.; Oomen, R. P.; Baenziger, J. E. FEBS Lett. 1995 377, 135-139. (25) Wirtz, P.; Steipe, B. Protein Sci. 1999, 8, 2245–2250. (26) Pedroso, I.; Irun, M. P.; Machicado, C.; Sancho, J. Biochemistry 2002, 41, 9873–9884. (27) van der Linden, R. H. J.; Frenken, L. G. J.; de Geus, B.; Harmsen, M. M.; Ruuls, R. C.; Stok, W.; de Ron, L.; Wilson, S.; Davis, P.; Verrips, C. T. Biochim. Biophys. Acta 1999, 1431, 37–46. (28) Ladenson, R. C.; Crimmins, D. L.; Landt, Y.; Ladenson, J. H. Anal. Chem. 2006, 78, 4501–4508. (29) Dolk, E.; van Vliet, C.; Perez, J. M. J.; Vriend, G.; Darbon, H.; Ferrat, G.; Cambillau, C.; Frenken, L. G. J.; Verrips, T. Proteins 2005, 59, 555–564.

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Figure 9. Percent inhibition of ricin-mediated cellular cytotoxicity. Antibodies were serially diluted 2-fold and mixed with ricin prior to incubation with EL-4 cells. Cytotoxicity was determined using Alamar Blue as a vital stain. Fluorescence (560 nm excitation and 590 nm emission) was measured and the average (SD of three wells was reported as percentage (%) untreated control cells ) [(fluorescence measurement of treated cells/fluorescence measurement of untreated control cells) × 100]

Ricin Inhibition Assays. Antiricin sdAb C8, F11, and H1, along with mouse antiricin IgG were tested for their ability to inhibit ricin activity in a cell free translation assay (Figure 8). We found that C8, the most effective of the sdAb, was able to neutralize 100% of ricin activity at 40 µg/mL. Even at sdAb concentrations of 0.6 µg/mL, 20% of ricin activity was neutralized versus untreated sample. The mouse antiricin showed ∼20% neutralization at 5 µg/mL, and none at 1.3 µg/mL. Although the C8 sdAb was able to neutralize ricin at a lower concentration in terms of µg/mL than the whole anti-ricin IgG, it is important to remember that sdAb are about one tenth the size of conventional antibodies; on a molar basis the sdAb and the conventional antibody show approximately equivalent neutralization. Neutralization of ricin biological activity is similar in a cell based assay (Figure 9). The fact that from this first small set of selected binders we have obtained such an effective sdAb bodes well for their use as antitoxin therapeutics. Although the F11 and H1 sdAb did not perform as well as C8, each did show some inhibition of ricin.

Our results confirm and build upon the recent demonstration of a recombinant human single-domain antibody, developed by computer-aided design, which bound ricin A chain and showed promising inhibition of ricin activity.30 Their construct was based on the human variable heavy domain, which lacking the changes to key residues that allow camelid sdAb to be produced as soluble monomeric fragments, had limited solubility. Improved therapeutic reagents may be able to be constructed by making multimeric sdAb structures,31,32 linking combinations of naturally occurring sdAb such as C8 with H1 and/or F11 or linking sdAb with “camelized” traditional VH domains. CONCLUSIONS We successfully selected sdAb against ricin toxin that were obtained from libraries derived from immunized llamas. Isolated sdAb were specific for ricin, functional in sandwich immunoassays as tracer and capture reagents, able to retain antigen binding ability after heating, and able to neutralize the action of ricin (30) Wang, S. T.; Feng, J. N.; Guo, J. W.; Guo, L. M.; Li, Y.; Sun, Y. X.; Qin, W. S.; Hu, M. R.; Han, G. C.; Shen, B. F. Mol. Immunol. 2006, 43, 1912– 1919. (31) Conrath, K. E.; Lauwereys, M.; Wyns, L.; Muyldermans, S. J. Biol. Chem. 2001, 276, 7346–7350. (32) Simmons, D. P.; Abrege, F. A.; Krishnan, U. V.; Proll, D. F.; Streltsov, V. A.; Doughty, L.; Hattarki, M. K.; Nuttall, S. D. J. Immunol. Methods 2006, 315, 171–184.

biological activity. Although binders providing the sensitive detection of Marburg virus have been isolated from a semisynthetic naı¨ve llama library,33 the ricin binders isolated from our immune derived library yielded better toxin detection than those we previously isolated from a semisynthetic naı¨ve library.14,15 Whether derived from immune or naı¨ve libraries, the properties of these small binding fragments have potential to provide improved diagnostic, detection, and therapeutic reagents. ACKNOWLEDGMENT This work was supported by JSTO-CBD/DTRA. The opinions expressed here are those of the authors and do not represent those of the U.S. Navy, the U.S. Department of Defense, or the U.S. government. SUPPORTING INFORMATION AVAILABLE Further details are given in Figures S-1 through S-5. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review September 12, 2008. Accepted October 23, 2008. AC8019398 (33) Sherwood, L. J.; Osborn, L. E.; Carrion, R., Jr.; Patterson, J. L.; Hayhurst, A. J. Infect. Dis. 2007, 196 (2), S213–219.

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