Once again the Marseille research group is pushing the bounds of plague detection. This time their target is looking for a more sensitive method of detecting non-nucleic acid biomolecules from Yersinia pestis, ‘the plague’. We have now moved into an era where PCR is being used in the mechanics of testing, rather than amplifying the ultimate target of the test.
The immuno-PCR (iPCR) method is outlined in the figure. The selective component of the assay is the mouse polyclonal anti-Yersinia pestis antibody. Polyclonal antibodies are the products of several different B lymphocytes reacting to the same antigen, protein in this case. This means the antibodies in preparation will bind to different parts (epitopes) of the same protein. This should be an advantage in working with badly degraded material.
In iPCR the selective reagent is the non-human antibody generated against the microbial target. The second antibody is against the non-human antibody (mouse, rabbit, etc) and carries biotin as a marker. These biotinylated antibodies are a very common and widely available immunology reagent. The third antibody is against the biotin and carries a reporter sequence of DNA. Quantitative real-time PCR is then done on the reporter DNA sequence attached to the antibody. This amplified reporter DNA can easily measure very tiny amounts of DNA. The iPCR system utilizes advances in PCR technology to measure specific protein levels.
Malou et al (2012) took known positive and negative teeth, and subjected them to iPCR, PCR and the standard ELISA antibody assay. They first determined the detection limit for ELISA and iPCR, and set a threshold for a positive iPCR result with 10 known negative teeth (5 ancient and 5 modern). They then coded and randomized 34 historically known positive teeth, the 10 negative teeth (5 ancient and 5 modern) and two blanks for testing with ELISA, PCR, and iPCR. The results and how they compare are shown in the diagram below. The ELISA only picked up four teeth with one being a known negative resulting in a sensitivity of 9% and specificity of 90%. Of the three ELISA true positives, two were from a 17th century site at Lariey and one from a 6th century site at Sens, both in France. For the iPCR, 14 of 34 exceeded the threshold and were considered positive for a 41% sensitivity with a 100% specificity (all positives were historically positive, no false positives). These teeth came from Lariey, Bourges, Sens, Bondy, and Venice with a date range from the 6th to 17th century. Traditional PCR identified 10 out of 34 historically positive teeth fora sensitivity of 29% with 8 of 10 also being identified by iPCR.
Immuno-PCR compares well with the efficiency and specificity of standard PCR for Yersinia pestis DNA and is more sensitive than the standard ELISA antibody assay. A standard ELISA produced the worst results. Although not quantitative, it would have been interesting if they had also done the Rapid Diagnostic Dipstick Test (RDT), another antibody based protein detection method, that has been used in several studies. They are suggesting that iPCR be added to traditional PCR as a method of further confirmation of Yersinia pestis at a site. Immuno-PCR can be done on the same teeth as traditional PCR and should be easily doable with the expertise and equipment in labs that conduct traditional aDNA PCR. By identifying both aDNA and protein, the confirmation of Yersinia pestis in the ancient remains should become quite strong.
Malou, N., Tran, T., Nappez, C., Signoli, M., Le Forestier, C., Castex, D., Drancourt, M., & Raoult, D. (2012). Immuno-PCR – A New Tool for Paleomicrobiology: The Plague Paradigm PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031744