Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae
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Abstract. Darniati, Setiyaningsih S, Agungpriyono DR, Handharyani E. 2021. Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae. Biodiversitas 22: 99-105. The aim of the research was to produce monospecific antibodies against Klebsiella pneumoniae serotype K1 and K2. Klebsiella pneumonia isolates were recovered from pneumonic lungs of Aceh cattle. The bacteria were identified by rpoB (RNA polymerase ? subunit) PCR amplification specific for K. pneumoniae. The presence of capsule cluster gene magA and k2A was used to characterize capsular serotype K1 and K2, respectively. Antisera were produced using serial immunization of New Zealand White rabbits with K. pneumoniae serotypes K1 and K2. The presence of antibodies was determined by using immunodiffusion test and purification was performed by immunoaffinity purification method. K. pneumoniae antibodies began to appear on the 14th day after the first injection and progressively intensified after the 1st and 2nd booster. The cross-reactivity between the antigen was eliminated by absorbing the antisera with the opposite antigen and several serotypes of non-K1/K2. After purification, serum protein concentrations were substantially decreased from 58.48 µg/µL and 53.99 µg/µL to 2.38 µg / µL and 3.72 µg / µL for K1 and K2, respectively). The SDS-PAGE analysis showed two bands with molecular weight at 54 kDa and 25 kDa representing the heavy and light chains of immunoglobulin G, respectively. Purified polyclonal antiseras against K. pneumoniae serotypes K1 and K2 showed high affinity and specificity for homologous antigens in immunodiffusion and direct agglutination tests, and immunohistochemical staining.
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References
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Ascoli CA, Aggeler B. 2018. Overlooked benefits of using polyclonal antibodies. BioTechniques. 65: 127–136. doi:10.2144/BTN-2018-0065
Awate S, Babiuk LA, Mutwiri G. 2013. Mechanisms of action of adjuvants. Frontiers in Immunology. 4: 1-10. doi:10.3389/fimmu.2013.00114
Bronte V, Pittet MJ. 2013. The spleen in local and systemic regulation of immunity. Immunity. 39: 806–818. doi: 10.1016/j.immuni.2013.10.010
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Danielsson D, Kronvall G. 1974. Slide agglutination method for the serological identification of Neisseria gonorrhoeae with anti-gonococcal antibodies adsorbed to protein A-containing Staphylococci. Applied Microbiology. 27: 368-374
Desjardins P, Hansen JB, Allen M. 2010 Microvolume protein concentration determination using the NanoDrop 2000c Spectrophotometer. Journal of Visualized Experiments. 33: 1-4. doi:10.3791/1610
Dong D, Liu W, Li H, Wang Y, Li X, Zou D, Yang Z, Huang S, Zhou D, Huang L, Yuan J. 2015. Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China. Frontiers in Microbiology. 6: 1–6. doi:10.3389/fmicb.2015.00519
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Liu S, Li S, Zhang Y, Wang Y, Zhu Y, Wang B, Chen ZN. 2017. Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography. Molecular Medicine Reports. 15: 4035–4040. doi:10.3892/mmr.2017.6523.
M’lan-Britoh A, Meité S, Boni C, Zaba F, Koffi KS, Guessennd N, Kakou NS, Faye- Kette H. Dosso M. 2017. First molecular investigation of capsular serotyping and hypervirulent (hvlp) of K. Pneumoniae in University Hospital Center of Yopougon Cote D'ivoire. African Journal of Clinical and Experimental Microbiology. 19: 70-75
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Martin RM, Bachman MA. 2018. Colonization, infection, and the accessory genome of Klebsiella pneumoniae. front. Cell. Infect. Microbiol. 8: 1-15. doi:10.3389/fcimb.2018.00004.
Matsumoto H, Haniu H, Komori N. 2019. Determination of protein molecular weights on SDS-PAGE. In: Kurien BT, Scofield RH (eds) Methods in Molecular Biology. Humana Press Inc. United states.
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Okwor EC, Eze DC, Okonkwo KE, Ibu JO. 2011. Comparative evaluation of agar gel precipitation test (AGPT) and indirect haemagglutination test (IHA) for the detection of antibodies against infectious bursal disease (IBD) virus in village chickens. African Journal of Biotechnology. 10: 16024–16027. doi:10.5897/AJB11.2006.
Opoku-Temeng C, Kobayashi SD, DeLeo FR. 2019. Klebsiella pneumoniae capsule polysaccharide as a target for therapeutics and vaccines. J. Comput. Struct. Biotechnol. 17: 1360–1366. doi: 10.1016/j.csbj.2019.09.011
Parija S, Biswas R. 2011. A rapid slide agglutination test for the diagnosis of neurocysticercosis in the rural health set up. Tropical Parasitology. 1: 94-98. doi:10.4103/2229-5070.86942.
Patterson K, Olsen B, Thomas, C, Norn D, Tam M, Elkins C. 2002. Development of a rapid immunodiagnostic test for Haemophilus ducreyi. Journal of Clinical Microbiology. 40: 3694–3702. doi:10.1128/JCM.40.10.3694-3702.2002.
Pohanka M. 2009. Monoclonal and polyclonal antibodies production - preparation of potent biorecognition element. Journal of Applied Biomedicine. 7: 115–121.
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Seo R. Kudo D, Gu Y, Yano H, Aoyagi T, Omura T, Irino S, Kaku M, Kushimoto S, 2016. Invasive liver abscess syndrome caused by Klebsiella pneumoniae with definite K2 serotyping in Japan: a case report. Surg. Case Reports 2: 1–5. doi:10.1186/s40792-016-0201-2. doi:10.1186/s40792-016-0201-2.
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Ascoli CA, Aggeler B. 2018. Overlooked benefits of using polyclonal antibodies. BioTechniques. 65: 127–136. doi:10.2144/BTN-2018-0065
Awate S, Babiuk LA, Mutwiri G. 2013. Mechanisms of action of adjuvants. Frontiers in Immunology. 4: 1-10. doi:10.3389/fimmu.2013.00114
Bronte V, Pittet MJ. 2013. The spleen in local and systemic regulation of immunity. Immunity. 39: 806–818. doi: 10.1016/j.immuni.2013.10.010
Chander Y, Ramakrishnan MA, Jindal N, Hanson K, Goyal SM. 2011. Differentiation of Klebsiella pneumoniae and K. oxytoca by multiplex polymerase chain reaction. International Journal of Applied Research in Veterinary Medicine. 9: 138.
Chuang Y, Fang C, Lai S, Chang S, Wang J. 2006. Genetic determinants of capsular serotype K1 of Klebsiella pneumoniae causing primary pyogenic liver abscess. The Journal of Infectious Diseases. 193: 645–654. doi:10.1086/499968
Danielsson D, Kronvall G. 1974. Slide agglutination method for the serological identification of Neisseria gonorrhoeae with anti-gonococcal antibodies adsorbed to protein A-containing Staphylococci. Applied Microbiology. 27: 368-374
Desjardins P, Hansen JB, Allen M. 2010 Microvolume protein concentration determination using the NanoDrop 2000c Spectrophotometer. Journal of Visualized Experiments. 33: 1-4. doi:10.3791/1610
Dong D, Liu W, Li H, Wang Y, Li X, Zou D, Yang Z, Huang S, Zhou D, Huang L, Yuan J. 2015. Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China. Frontiers in Microbiology. 6: 1–6. doi:10.3389/fmicb.2015.00519
Doud MS, Grimes-Zeppegno R, Molina E, Miller N, Balachandar D, Schneper L, Schneper L, Poppiti R, Mathe K. 2009. A k2A-positive Klebsiella pneumoniae causes liver and brain abscess in a Saint Kitt’s man. International Journal of Medical Sciences. 6: 301–304. doi:10.7150/ijms.6.301
Duraiyan J, Govindarajan R, Kaliyapan K, Palanisamy M. 2012. Applications of immunohistochemistry. Journal of Pharmacy and Bioallied Sciences. 4: S307–S309. doi:10.4103/0975-7406.100281
Effah CY, Sun T, Liu S, Wu Y. 2020. Klebsiella pneumoniae: An increasing threat to public health. Annals of Clinical Microbiology and Antimicrobials. 19: 1–9.
Gameel AA, El Sanousi SM, Al Nawawi F, Al Shazly MO. 1991. Association of Klebsiella organisms with pulmonary lesions in sheep. Revue d’élevage et de Médecine Vétérinaire Des Pays Tropicaux. 44: 161–164.
Guachalla LM, Stojkovic K, Hartl K, Kaszowska M, Kumar Y, Wahl B, Paprotka T, Nagy E, Lukasiewicz J, Lukasiewicz J, Nagy G, Szijártó V. 2017. Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae. Sci. Rep. 7: 1–13. doi:10.1038/s41598-017-06682-2
He Y, Guo X, Xiang S, Li J, Li X, Xiang H, Chen D, Chen J. 2016. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae. Antonie van Leeuwenhoek. 109: 1029–1040.
Hellman J, Zanzot EM, Loiselle PM, Amato SF, Black KM, Ge Y, Kurnick JT, Warren HS. 1997. Antiserum against Escherichia coli J5 Contains Antibodies Reactive with Outer Membrane Proteins of Heterologous Gram?Negative Bacteria. The Journal of Infectious Diseases. 176: 1260–1268. doi:10.1086/514121.
Hober S, Nord K, Linhult M. 2007. Protein A chromatography for antibody purification. Journal of Chromatography B. 848: 40–47. doi: 10.1016/j.jchromb.2006.09.030.
Jang S, Wheeler L, Carey RB, Jensen B, Crandall CM, Schrader KN, Jessup DC, Kathleen G, Frances MD. 2010. Pleuritis and suppurative pneumonia associated with a hypermucoviscosity phenotype of Klebsiella pneumoniae in California sea lions (Zalophus californianus). Veterinary Microbiology. 141: 174–177.
Jun JB. 2018. Klebsiella pneumoniae liver abscess. Infect. Chemother. 50: 210–218. doi:10.3947/ic.2018.50.3.210.
Li L, Yu T, Ma Y, Yang Z, Wang W, Song X, She Y, Guo T, Kong J, Wang M, Xu H. 2019. The genetic structures of an extensively drug resistant (XDR) Klebsiella pneumoniae and its plasmids. Frontiers in Cellular and Infection Microbiology. 9: 1-11. doi:10.3389/fcimb.2018.00446.
Lin ZW, Zheng JX, Bai B, Xu GJ, Lin FJ, Chen Z, Sun X, Qu D, Yu ZJ, Deng QW. 2020. Characteristics of hypervirulent Klebsiella pneumoniae: Does low expression of rmpA contribute to the absence of hypervirulence? Front. Microbiol. 11: 1-11. doi:10.3389/fmicb.2020.00436.
Liu S, Li S, Zhang Y, Wang Y, Zhu Y, Wang B, Chen ZN. 2017. Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography. Molecular Medicine Reports. 15: 4035–4040. doi:10.3892/mmr.2017.6523.
M’lan-Britoh A, Meité S, Boni C, Zaba F, Koffi KS, Guessennd N, Kakou NS, Faye- Kette H. Dosso M. 2017. First molecular investigation of capsular serotyping and hypervirulent (hvlp) of K. Pneumoniae in University Hospital Center of Yopougon Cote D'ivoire. African Journal of Clinical and Experimental Microbiology. 19: 70-75
Majidi, Amirkhiz. 2007. Production and purification of polyclonal antibody against bovine immunoglobulins in rabbits. African Journal of Biotechnology. 6: 1369–1372. doi:10.4314/ajcemv19i1.10.
Martin RM, Bachman MA. 2018. Colonization, infection, and the accessory genome of Klebsiella pneumoniae. front. Cell. Infect. Microbiol. 8: 1-15. doi:10.3389/fcimb.2018.00004.
Matsumoto H, Haniu H, Komori N. 2019. Determination of protein molecular weights on SDS-PAGE. In: Kurien BT, Scofield RH (eds) Methods in Molecular Biology. Humana Press Inc. United states.
Nandin IS, Fong C, Deantonio C, Torreno-Pina JA, Pecetta S, Maldonado P, Gasparrini F, Ordovas-Montanes J, Kazer SW, Kjaer, Svend, Borley DW, Nair U, Coleman JA, Lingwood D, Shalek AK, Meffre E, Poignard P, Burton DR, Batista FD. 2017. Novel in vitro booster vaccination to rapidly generate antigen-specific human monoclonal antibodies. Journal of Experimental Medicine. 214: 2471–2490. doi:10.1084/jem.20170633.
Nutt SL, Hodgkin PD, Tarlinton DM, Corcoran LM. 2015. The generation of antibody-secreting plasma cells. Nature Reviews Immunology. 15: 160–171. doi:10.1038/nri3795.
Okwor EC, Eze DC, Okonkwo KE, Ibu JO. 2011. Comparative evaluation of agar gel precipitation test (AGPT) and indirect haemagglutination test (IHA) for the detection of antibodies against infectious bursal disease (IBD) virus in village chickens. African Journal of Biotechnology. 10: 16024–16027. doi:10.5897/AJB11.2006.
Opoku-Temeng C, Kobayashi SD, DeLeo FR. 2019. Klebsiella pneumoniae capsule polysaccharide as a target for therapeutics and vaccines. J. Comput. Struct. Biotechnol. 17: 1360–1366. doi: 10.1016/j.csbj.2019.09.011
Parija S, Biswas R. 2011. A rapid slide agglutination test for the diagnosis of neurocysticercosis in the rural health set up. Tropical Parasitology. 1: 94-98. doi:10.4103/2229-5070.86942.
Patterson K, Olsen B, Thomas, C, Norn D, Tam M, Elkins C. 2002. Development of a rapid immunodiagnostic test for Haemophilus ducreyi. Journal of Clinical Microbiology. 40: 3694–3702. doi:10.1128/JCM.40.10.3694-3702.2002.
Pohanka M. 2009. Monoclonal and polyclonal antibodies production - preparation of potent biorecognition element. Journal of Applied Biomedicine. 7: 115–121.
Priyanka A, Akshatha K, Deekshit VK, Prarthana J, Akhila DS. 2020. Klebsiella pneumoniae infections and antimicrobial drug resistance. In: Sidharda B, Dyavaiah,M, Syed A (eds) Model organisms for microbial pathogenesis, biofilm formation and antimicrobial drug discovery. Springer, Singapore.
Remya P, Shanthi M, Sekar U. 2019. Characterisation of virulence genes associated with pathogenicity in Klebsiella pneumoniae. Indian Journal of Medical Microbiology. 37: 210-218. doi: 10.4103/ijmm.IJMM_19_157.
Ribeiro MG, Motta RG, Paes AC, Allendorf SD, Salerno T, Siqueira AK, Fernandes MC, Lara GHB. 2008. Peracute bovine mastitis caused by Klebsiella pneumoniae. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia. 60: 485–488. doi:10.1590/S0102-09352008000200031.
Rodrigo G, Gruvegård M, Van Alstine J. 2015. Antibody Fragments and Their Purification by Protein L affinity Chromatography. Antibodies. 4: 259–277. doi:10.3390/antib4030259
Russo TA, Olson R, Fang CT, Stoesser N, Miller M, MacDonald U, Hutson A, Barker JH, La Hoz RM, Johnson JR, Backer M, Bajwa R, Catanzaro AT, Crook D, De Almeida K, Fierer J, Greenberg DE, Klevay M, Patel P, Ratner A, Wang JT, Zola J. 2018. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae. Journal of Clinical Microbiology. 56: 1-12. doi:10.1128/JCM.00776-18.
Sadeghi S, Aghebati-Maleki L, Nozari S, Majidi J. 2018. A methodological approach for production and purification of polyclonal antibody against dog IgG. Veterinary Research Forum. 9: 13–18.
Sallusto F, Lanzavecchia A, Araki K, Ahmed R. 2010. From vaccines to memory and back. Immunity. 33: 451–463. doi: 10.1016/j.immuni.2010.10.008.
Sarba EJ, Kudama K, Dandecha M, Megersa L, Borena BM, Gebremdhin EZ. 2020. Prevalence, organ distribution and antimicrobial susceptibility profile of Salmonella isolated from chickens purchased from markets in selected districts of West Shoa, Ethiopia. Ethiopian Veterinary Journal. 24: 73–89. doi:10.4314/evjv24i2.5
Schroeder Jr HW, Cavacini L. 2010. Structure and function of immunoglobulins. The Journal of Allergy and Clinical Immunology. 125: S41–S52. doi: 10.1016/j.jaci.2009.09.046
Seo R. Kudo D, Gu Y, Yano H, Aoyagi T, Omura T, Irino S, Kaku M, Kushimoto S, 2016. Invasive liver abscess syndrome caused by Klebsiella pneumoniae with definite K2 serotyping in Japan: a case report. Surg. Case Reports 2: 1–5. doi:10.1186/s40792-016-0201-2. doi:10.1186/s40792-016-0201-2.
Siu LK, Yeh KM, Lin JC, Fung CP, Chang FY. 2012. Klebsiella pneumoniae liver abscess: a new invasive syndrome. The Lancet Infectious Diseases. 12: 881–887.
Snider GL. 2000. Clinical Relevance Summary. Chest. 117: 244S-246S. doi:10.1378/chest1175suppl_1.244s.
Sorden SD, Harms PA, Nawagitgul P, Cavanaugh D, Paul PS. 1999. Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue. Journal of Veterinary Diagnostic Investigation. 11: 528–530. doi:10.1177/104063879901100607.
Talib N, Salam F, Sulaiman Y. 2018. Development of polyclonal antibody against clenbuterol for immunoassay application. Molecules. 23: 789. doi:10.3390/molecules23040789.
Weber J, Peng H, Rader C. 2017. From rabbit antibody repertoires to rabbit monoclonal antibodies. Experimental and Molecular Medicine. 49: 1-12. doi:10.1038/emm.2017.23.
Whitton JL, Slifka MK, Liu F, Nussbaum AK, Whitmire JK. 2004. The Regulation and Maturation of Antiviral Immune Responses. Advances in Virus Research. 63: 181–238. doi:10.1016/S0065-3527(04)63003-X.
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