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Immunodetection of the recombinant GroEL by the Nanobody NbBruc02

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Abstract

Brucella has a great impact on health and economy in Syria, thus much effort is being placed on the development of diagnostics and vaccines. In this context, a wide Nanobody “immune” library was previously established, from which several Brucella-specific binders were isolated. One of these camel genetically engineered heavy-chain antibody fragments was referred to as NbBruc02. The precise antigen of NbBruc02 was presumed to be, according to proteomic approaches, the Brucella heat shock protein of 60 kDa (HSP-60). HSP-60, or alternatively named GroEL, is an interesting Brucella immunodominant antigen with important roles in the parasite life cycle, mainly adhesion and penetration during the infection of macrophages. In the present work, the capacity of NbBruc02 to filtrate the native GroEL from Brucella total extract was tested by immunochromatography approach. The interaction between NbBruc02 and its antigen was further confirmed using recombinant GroEL from Brucella. Interestingly, NbBruc02 was able to immunodetect the native as well as the denatured forms of the rGroEL in ELISA and immunoblotting, respectively. In agreement with previously reported data, NbBruc02 was able only to detect the denatured Yersinia rGroEL. Using surface plasmon resonance (SPR) biosensor, NbBruc02 showed a strong interaction, with nanomolar affinity (K D = ~10−8 M), with the native rGroEL of Brucella and not of Yersinia. Because the casual conformational changes in the GroEL 3D structure make the base of its function, NbBruc02 by its ability to recognize a “conformational epitope,” could open wide perspectives to study the role of GroEL in Brucella physiology.

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Abbreviations

AEC:

3-amino-9-ethylcarbazole

FPLC:

Fast protein liquid chromatography

HCAb:

Heavy-chain antibody

HSP-60:

Heat shock protein of 60 kDa

IPTG:

Isopropyl β-d-thiogalactoside

LPS:

Lipopolysaccharide

Ni-NTA:

Nickel-charged nitrilotriacetic acid

SPR:

Surface plasmon resonance

TMB:

3,3′,5,5′-tetramethylbenzidine

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Acknowledgments

The authors would like to thank the Director General of the Atomic Energy Commission of Syria and the head of the Molecular Biology and Biotechnology department for their continuous support throughout this work.

Conflict of interest

The authors report no potential conflicts of interest in this work.

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Authors and Affiliations

  1. Department of Plant Biology, Faculty of Science, Damascus University, Damascus, Syria

    Lubna Abo Assali

  2. Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), Damascus, Syria

    Ayman Al-Mariri & Abdul Qader Abbady

  3. Department of Environmental Science, Faculty of Science, Damascus University, Damascus, Syria

    Ebtisam Hamad

Authors
  1. Lubna Abo Assali
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  2. Ayman Al-Mariri
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  3. Ebtisam Hamad
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  4. Abdul Qader Abbady
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Correspondence to Abdul Qader Abbady.

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Abo Assali, L., Al-Mariri, A., Hamad, E. et al. Immunodetection of the recombinant GroEL by the Nanobody NbBruc02. World J Microbiol Biotechnol 28, 2987–2995 (2012). https://doi.org/10.1007/s11274-012-1109-z

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  • DOI: https://doi.org/10.1007/s11274-012-1109-z

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