J Periodontal Implant Sci.  2011 Apr;41(2):54-59.

Identification of mono- or poly-specific monoclonal antibody to Porphyromonas gingivalis heat-shock protein 60

• Choi J
• Lee SY
• Kim K
• Choi BK
• Kim MJ

Abstract

PURPOSE
The aim of this study was to define the immunoreactive specificity of Porphyromonas gingivalis (P. gingivalis) heat shock protein (HSP) 60 in periodontitis and atherosclerosis.
METHODS
In an attempt to define the cross-reactive bacterial heat-shock protein with human self-antigen at molecular level, we have introduced a novel strategy for cloning hybridoma producing anti-P. gingivalis HSP 60 which is polyreactive to bacterial HSPs or to the human homolog.
RESULTS
Five cross-reactive clones were obtained which recognized the #19 peptide (TLVVNRLRGSLKICAVKAPG) among 37 synthetic peptides (20-mer, 5 amino acids overlapping) spanning the whole molecule of P. gingivalis HSP 60. We have also established three anti-P. gingivalis HSP 60 monoclonal antibodies demonstrating mono-specificity. These clones recognized the #29 peptide (TVPGGGTTYIRAIAALEGLK).
CONCLUSIONS
Peptide #19 and #29 of P. gingivalis HSP 60 might be important immunoreactive epitopes in the immunopathogenic mechanism of bacterial antigen-triggered autoimmune diseases.

Keyword

Antibodies; Periodontitis; Porphyromonas gingivalis

MeSH Terms

Amino Acids
Antibodies
Antibodies, Monoclonal
Autoimmune Diseases
Chaperonin 60
Clone Cells
Cloning, Organism
Epitopes
Heat-Shock Proteins
Humans
Hybridomas
Peptides
Periodontitis
Porphyromonas
Porphyromonas gingivalis
Sensitivity and Specificity
Amino Acids
Antibodies
Antibodies, Monoclonal
Chaperonin 60
Epitopes
Heat-Shock Proteins
Peptides

Figure

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  Figure 1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis profile of purified recombinant Porphyromonas gingivalis HSP60 (A) and one (clone JC2) of purified anti-P. gingivalis HSP60 monoclonal IgG antibody demonstrating the heavy and the light chain, respectively (B). MW: molecular weight marker, HSP: heat shock protein.

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  Figure 2 Reactive pattern of cross-reactive (A: clones JC5-JC9) and mono-reactive (B: clones JC1-JC3) or anti-Porphyromonas gingivalis heat shock protein 60 monoclonal antibodies with cognate antigen or with human counterpart demonstrated by immunoblot analysis.

• 

  Figure 3 Reactive pattern of poly-reactive (A: clones JC 5-JC9) and mono-reactive (B: clones JC1-JC3) anti-Porphyromonas gingivalis heat shock protein (HSP)60 monoclonal antibodies with cognate HSP antigen or with HSP's of five periodontopathogenic bacteria as demonstrated by immunoblot analysis. Pg: P. gingivalis HSP60, Aa: Actinobacillus actinomycetemcomitans, Pi: Prevotella intermedia, Fn: Fusobacterium nucleatum, Sm: Streptococcus mutans, Td: Treponema denticola.

• 

  Figure 4 Poly-reactive monoclonal antibody (A: clones JC5-JC9) recognized #19 peptide among 37 synthetic peptides spanning a whole molecule of Porphyromonas gingivalis heat shock protein (HSP)60 as well as human HSP60, while mono-reactive monoclonal antibody (B: clones JC1-JC3) recognized #29 peptide among the same synthetic peptides without responding to human HSP60, as evidenced by dot immunoblot analyses. Hu: human HSP60, Pg: P. gingivalis HSP60.

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  Figure 5 In addition to reactivity with peptide #19 (Hu#19) from self-antigen (human heat shock protein 60), inter-species cross-reactivity of the five monoclonal antibodies (clones JC5-JC9) with #19 synthetic peptides from Porphyromonas gingivalis (Pg#19), Mycobacterium tuberculosis (Myco#19) and Chlamydia pneumoniae (Chla#19), respectively, is demonstrated by dot immunoblot analysis. Note that none of them recognized #19 peptide from M. tuberculosis.

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