Analyzing ancient microbial proteins
Detection of microbial antigens in ancient specimens can be achieved using several techniques including immunochromatographic detection, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analysis (31,34, 37-40). While the latter technique detects and visualizes microbial antigens in fixed, unaltered tissues (30, 41-43), the former techniques necessitate the destruction of tissues for extracting microbial antigens prior to analysis (31,34,37,38,44).Immunochemistry
Immunohistochemistry combines morphology and immunochemical detection of antigenic determinants in tissues (39,45). Detection of microbial antigens can be done using immunofluorescence analysis of frozen tissues and immunoperoxidase applied to formalin-fixed, paraffin-embedded tissues (39). Immunohistochemistry is low cost and easy to perform (46); avoiding in-laboratory contamination and the visualization of ancient microbe tissues (30,39,41,42,46) are also possible using this technique. However, the degradation of ancient antigenic protein and the limited number of specific bacterial antibodies available limit the application of immunohistochemistry (39). Immunohistochemistry has been used for the detection of intact smallpox virus particles in an Italian mummy from the 16th century by incubating the skin with anti-vaccinia virus antiserum followed by protein A-gold and electronmicroscopy(15).Immunohistochemistry was also used to detect the syphilis bacterium T. pallidum in the mummy of Maria d'Aragona (1503–1568) recovered from an abbey in Naples, Italy 16, as well as the Rocky Mountain spotted fever bacterium Rickettsia rickettsii in different tissues collected from a patient who died in 1901 with a false diagnosis of typhus, providing an example of retrospective diagnosis of closely related diseases (41). Immunodetection of Tropheryma whipplei in intestinal tissues from Dr. Whipple's patient in 1907, Whipple's first patient with Whipple's disease, yielded the observation that the lamina propria of the small intestine was filled with foamy macrophages and markedly dilated lacteals, causing the villi to appear widened and blunted by immunostaining (42). As for parasites, immunohistochemistry demonstrated taeniasis in a stomach cystic lesion through the use of serum collected from a Taenia solium–infected individual as a primary antibody and anti-human IgG as secondary antibody (30).Immunochromatography and ELISA
The detection of ancient microbial antigens can be further accomplished using either immunochromatography or ELISA, both of which require the destruction of ancient tissues for protein extraction (31,34,37,38,44). For immunochromatography, protocols use 50 mg ancient bone material or 7.5 mg ancient dental pulp reconstituted in 200 mL sterile saline solution that is subjected to three freeze/thaw cycles (1 min per cycle), sonication for 15 min, a fourth freeze/thaw cycle, a 24-h incubation at 4°C to solubilize the remaining antigens, centrifugation at 2400× g at room temperature, and incorporation of the supernatant in the dipstick assay (34). In another work, the circulating anodic antigen, a proteoglycan with a strong negative charge at neutral pH (47), was extracted by homogenizing 150 mg mummified tissue with phosphate-buffered saline at 0°C, sonicating the sample mixture, and centrifuging at 25,000× g for 20 min. The supernatant was then mixed with trichloroacetic acid solution (5%–7%) and centrifuged at 25,000× g for 20 min. After dialysis of the supernatant for 24 h againstdistilled water (4°C), extracted protein was used for ELISA (40). Immunochromatography has been applied to the detection of the plague bacterium Y. pestis–specific F1 antigen in several burial sites in Europe (24,34,37,46,48). Using a rapid diagnostic test, Y. pestis was demonstrated in four Benedictine nuns and two priests in two burial sites in France, Poitiers and La Chaize-le-Vicomte, dating between 1578 and 1632 (37). ELISA and immunohistochemical analysis also identified Y. pestis in ancient plague victims in Venice (San Leonardo in Fossa Mala, 14th century) and Genoa (Bastione dell'Acquasola, 14th century), Italy, and in one individual in Briançon, France (17th century) (46). In that work, four individuals from the same period were collected from graves not related to the epidemics and were used as negative controls (46). ELISA has also been used for the detection of circulating schistosome antigen in Egyptian mummies (44). Cheek and colon samples from a mummy (1198–1150 BC), in which Schistosoma hematobium eggs were found, and shin tissue from a late predynastic (3200 BC) mummy were used. Gut tissues from Atacama (2000 BC) mummies were used as negative controls (44). Likewise, the pig tapeworm T. solium was detected in the stomach of a 20-year-old female Egyptian mummy who lived in the late Ptolemaic period (second to first centuries BC) (30). Because pigs are the unique reservoir for T. solium, this finding suggests that swine farming was concurrent with the Hellenistic Egypt period (30). Likewise, the detection of the Plasmodium falciparum histidine-rich protein-2 antigen (PfHRP-2) demonstrated the presence of malaria in naturally desiccated 1450- to 5200-year-old mummies from Egypt and Nubia (32). The authors suggested that the stability of PfHRP-2 over several thousand years indicated the potential value of paleoimmunological diagnosis in investigating the distribution of such an important disease as malaria during prehistoric and even Paleolithic times (32). The authors used gut tissue from unembalmed, desiccated mummies that were >3000 years old from the site of Camarones in the Atacama Desert near Arica, northern Chile, as a negative control (32). Recently, P. falciparum was detected using qualitative-double antibody immunoassays to analyze the skeletons of four members of the Medici family from 16th century Italy (38,49). In our laboratory, we now use ELISA to detect Y. pestis in dental pulp collected from plague victims from Venice, Italy, (14th to 16th centuries) and Poitiers, France, (16th to 18th centuries); dental pulp collected from 10 individuals without evidence of infection was used as a negative control (D. Raoult, unpublished data).