To obtain thermostable immunoreagents specific for the spore form of Bacillus anthracis two llamas were immunized with a combination of six different recombinant proteins. These proteins BclA, gerQ, SODA1, SOD15, BxpB and the protein p5303 have all been shown as components of the B. anthracis spore and could potentially serve as targets for the detection of spores in multiplexed biosensors. Peripheral blood lymphocytes were used to construct a phage display library from which single domain antibodies (sdAbs) targeting each of the proteins were isolated. Unique sdAbs exhibiting nanomolar or better affinities for the recombinant proteins were obtained and most of the isolated sdAbs retained their ability to bind antigen after cycles of heating as determined by enzyme linked immunosorbent assay (ELISA). SdAbs targeting the BclA and gerQ proteins were able to successfully detect bacterial spores, whether broken or intact, using a direct ELISA; the sdAbs were specific, showing binding only to B. anthracis spores and not to other Bacillus species. Additionally, SODA1 and p5303 binding sdAbs detected spores in sandwich assays serving as both captures and tracers. Used in combination, sdAbs targeting B. anthracis proteins could be integrated into emerging biosensors to improve specificity in multiplex assays.
References
[1]
Inglesby, T.V. Anthrax as a biological weapon, 2002: Updated recommendations for management. JAMA 2002, 287, 2236–2252, doi:10.1001/jama.287.17.2236.
[2]
Schmid, G.; Kaufmann, A. Anthrax in europe: It’s epidemiology, clinical characteristics, and role in bioterrorism. Clin. Microbiol. Infect. 2002, 8, 479–488, doi:10.1046/j.1469-0691.2002.00500.x.
[3]
Drobniewski, F. Bacillus cereus and related species. Clin. Microbiol. Rev. 1993, 6, 324–338.
[4]
Taitt, C.R.; Malanoski, A.P.; Lin, B.; Stenger, D.A.; Ligler, F.S.; Kusterbeck, A.W.; Anderson, G.P.; Harmon, S.E.; Shriver-Lake, L.C.; Pollack, S.K.; et al. Discrimination between biothreat agents and a near neighbor species using a resequencing array. FEMS Immunol. Med. Microbiol. 2008, 54, 356–364, doi:10.1111/j.1574-695X.2008.00486.x.
[5]
Ellerbrok, H.; Nattermann, H.; Ozel, M.; Beutin, L.; Appel, B.; Pauli, G. Rapid and sensitive identification of pathogenic and apathogenic bacillus anthracis by real-time pcr. FEMS Microbiol. Lett. 2002, 214, 51–59, doi:10.1111/j.1574-6968.2002.tb11324.x.
[6]
Qi, Y.; Patra, G.; Liang, X.; Williams, L.E.; Rose, S.; Redkar, R.J.; DelVecchio, V.G. Utilization of the rpob gene as a specific chromosomal marker for real-time pcr detection of bacillus anthracis. Appl. Environ. Microbiol. 2001, 67, 3720–3727, doi:10.1128/AEM.67.8.3720-3727.2001.
Ghahroudi, M.A.; Desmyter, A.; Wyns, L.; Hamers, R.; Muyldermans, S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997, 414, 521–526, doi:10.1016/S0014-5793(97)01062-4.
[9]
Vu, K.B.; Ghahroudi, M.A.; Wyns, L.; Muyldermans, S. Comparison of llama vh sequences from conventional and heavy chain antibodies. Mol. Immunol. 1997, 34, 1121–1131, doi:10.1016/S0161-5890(97)00146-6.
[10]
Greenberg, A.S.; Avila, D.; Hughes, M.; Hughes, A.; McKineey, E.C.; Flajnik, M.F. A new antigen recepetor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 1995, 374, 168–173.
[11]
Nuttall, S.D.; Krishnan, U.V.; Hattarki, M.; Gori, R.D.; Irving, R.A.; Hudson, P.J. Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries. Mol. Immunol. 2001, 38, 313–326, doi:10.1016/S0161-5890(01)00057-8.
[12]
Eyer, L.; Hruska, K. Single-domain antibody fragments derived from heavy-chain antibodies: A review. Vet. Med.-Czech 2012, 57, 439–513.
[13]
Wesolowski, J.; Alzogaray, V.; Reyelt, J.; Unger, M.; Juarez, K.; Urrutia, M.; Cauerhff, A.; Danquah, W.; Rissiek, B.; Scheuplein, F.; et al. Single domain antibodies: Promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009, 198, 157–174, doi:10.1007/s00430-009-0116-7.
[14]
Dumoulin, M.; Conrath, K.; Van Meirhaeghe, A.; Meersman, F.; Heremans, K.; Frenken, L.G.J.; Muyldermans, S.; Wyns, L.; Matagne, A. Single-domain antibody fragments with high conformational stability. Protein Sci. 2002, 11, 500–515.
[15]
Walper, S.A.; Anderson, G.P.; Brozozog Lee, P.A.; Glaven, R.H.; Liu, J.L.; Bernstein, R.D.; Zabetakis, D.; Johnson, L.; Czarnecki, J.M.; Goldman, E.R. Rugged single domain antibody detection elements for bacillus anthracis spores and vegetative cells. PLoS One 2012, 7, e32801.
[16]
Love, T.; Redmond, C.; Mayers, C. Real time detection of anthrax spores using highly specific anti-ea1 recombinant antibodies produced by competitive panning. J. Immunol. Methods 2008, 334, 1–10, doi:10.1016/j.jim.2007.12.022.
[17]
Williams, D.D.; Turnbough, C.L. Surface layer protein ea1 is not a component of bacillus anthracis spores but is a persistent contaminant in spore preparations. J. Bacteriol. 2003, 186, 566–569, doi:10.1128/JB.186.2.566-569.2004.
[18]
Cybulski, R.J., Jr.; Sanz, P.; McDaniel, D.; Darnell, S.; Bull, R.L.; O’Brien, A.D. Recombinant bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen. Vaccine 2008, 26, 4927–4939, doi:10.1016/j.vaccine.2008.07.015.
[19]
Cybulski, R.J., Jr.; Sanz, P.; Alem, F.; Stibitz, S.; Bull, R.L.; O'Brien, A.D. Four superoxide dismutases contribute to bacillus anthracis virulence and provide spores with redundant protection from oxidative stress. Infect. Immun. 2008, 77, 274–285.
[20]
Ghahroudi, M.A.; Desmyter, A.; Wyns, L.; Hamers, R.; Muyldermans, S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997, 414, 521–526, doi:10.1016/S0014-5793(97)01062-4.
[21]
Anderson, G.; Ortiz-Vera, Y.A.; Hayhurst, A.; Czarnecki, J.; Dabbs, J.; Vo, B.; Goldman, E. Evaluation of llama anti-botulinum toxin heavy chain antibody. Botulinum J. 2008, 1, 100–116, doi:10.1504/TBJ.2008.018953.
[22]
Vandeventer, P.E.; Weigel, K.M.; Salazar, J.; Erwin, B.; Irvine, B.; Doebler, R.; Nadim, A.; Cangelosi, G.A.; Niemz, A. Mechanical disruption of lysis-resistant bacterial cells by use of a miniature, low-power, disposable device. J. Clin. Microbiol. 2011, 49, 2533–2539, doi:10.1128/JCM.02171-10.
[23]
Nuttall, S.D.; Wilkins, M.L.; Streltsov, V.A.; Pontes-Braz, L.; Dolezal, O.; Tran, H.; Liu, C.-Q. Isolation, kinetic analysis, and structural characterization of an antibody targeting the bacillus anthracis major spore surface protein bcla. Proteins 2011, 79, 1306–1317, doi:10.1002/prot.22971.
[24]
Walper, S.A.; Brozozog Lee, P.A.; Goldman, E.R.; Anderson, G.P. Comparison of single domain antibody immobilization strategies evaluated by surface plasmon resonance. J. Immunol. Methods 2013, 388, 68–77, doi:10.1016/j.jim.2012.11.014.
[25]
Goldman, E.R.; Anderson, G.; Conway, J.O.; Sherwood, L.J.; Fech, M.; Vo, B.; Liu, J.L.; Hayhurst, A. Thermostable llama single domain antibodies for detection of botulinum a neurotoxin complex. Anal. Chem. 2008, 80, 8583–8591, doi:10.1021/ac8014774.
[26]
Griffiths, A.; Malmqvist, M.; Marks, J.; Bye, J.; Embleton, M.J.; McCafferty, J.; Baier, M.; Holliger, K.P.; Gorick, B.; Hughes-Jones, N.; et al. Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 1993, 12, 725–734.
[27]
Clackson, T.; Lowman, H.B. Phage Display; Oxford University Press: New York, NY, USA, 2004; p. 331.
[28]
Anderson, G.P.; Legler, P.M.; Zabetakis, D.; Goldman, E.R. Comparison of immunoreactivity of staphylococcal enterotoxin b mutants for use as toxin surrogates. Anal. Chem. 2012, 84, 5198–5203, doi:10.1021/ac300864j.
