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Porphyromonas gingivalis PAD Antibody Case Study
Case Study: The Venables Group

The Venables group, based at the Kennedy Institute of Rheumatology, enlisted the assistance of Cambridge Research Biochemicals to generate a custom polyclonal antibody directed to a recombinant protein, Porphyromonas gingivalis PAD known as PPAD, where PAD stands for peptidylarginine deiminases. PADs are known to catalyse the modification of peptidyl-arginine to peptidyl-citrulline and these modifying enzymes are rarely found in bacteria. 

Immunoblotting and ELISA data

Image (above right): (A) Correlation of antibody detection by ELISA and immunoblotting. Representative immunoblot positive and negative of serum reacting to PPAD (PPAD-GST 89 kDa) and RgpB (RgpB-His 47kDa) Validation of the ELISA analysis relative to the immunoblotting results (B+C). The 27 sera (10 control, 7 peridontitis and 10 rheumatoid arthritis) were divided into two groups based on presence (+) or absence (−) of IgG antibodies to (B) PPAD or (C) RgpB as determined by immunoblotting.

The red line indicates the ELISA mean U/ml value for the serum groups. The Mann–Whitney U test was used to calculate p values for the difference between the arbitury units for the anti-PPAD/anti RgpB blot positive (+) serum group and blot negative (−) serum group (** p<0.05 and ***p<0.01).Using this antibody, the group have shown that the localisation of PPAD in P. gingivalis is exclusively on the cell surface.

The antibody was also shown not to cross react with human PAD2 and PAD4 and has been used to detect PPAD in oral epithelial cell cultures infected with P. gingivalis. Rheumatoid arthritis (RA) is characterised by the presence of disease-specific antibodies to citrullinated protein antigens (ACPA). PPAD has previously been shown to citrullinate both human and bacterial proteins and recombinant GST-tagged PPAD is capable of autocitrullination and therefore PPAD could stimulate an anti-citrulline antibody response in RA susceptible patients.

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Peptide Synthesis by Cambridge Research Biochemicals

To investigate the peptidyl citrulline-specific antibody response to PPAD itself, 13 cyclic peptides spanning the PPAD molecule were synthesised by Cambridge Research Biochemicals, encompassing all the arginine bases in PPAD, where the arginines were substituted by citrulline.

The group used these peptides to gauge the antibody response to these targets in patients with RA. In two of these peptides, there was a 40% increase in positivity, indicating that these targets could be used for screening for anti-PPAD antibodies in future epidemiological studies.

The group concluded that the immune response to both citrullinated PPAD and citrullinated PPAD peptides suggests that as a bacterial protein, it could be a target for RA therapy in ACPA positive patients.

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Testimonial: The Venables Group

"Our lab has used Cambridge Research Biochemicals for the synthesis of several custom peptides, with and without modifications, and also for the production of polyclonal antibodies. We have been very happy with their service and products. The whole process, from the initial point of contact to final product, including scientific advice and processing, has been highly professional and friendly as well as skilled and of high quality. We keep going back for more!".

Elena Lugli, Anna Montgomery and Patrick Venables- The Venables Group, The Kennedy Instititute of Rheumatology, Oxford, UK.

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