Identifying the cause of past epidemics answers on the specific sensing of pathogens in inhumed persons, alleged paleomicrobiology, an emerging field of research which gained from technological progresss in microbiology. For about 15 old ages, the sensing, designation and word picture of bugs in ancient environmental and human specimens emerged on the footing of ancient DNA analyses. Restrictions in ancient DNA elaboration and taint led to research alternate methods of sensing and word picture of non-DNA biomolecules including mycolic acids for observing ancient mycobacteriums and proteins. Besides, chemoimmunology, immuno-chromatography, enzyme linked immunosorbent check and immuno-PCR have been developed for the specific sensing of bugs in ancient homo and environmental specimens. As for antediluvian DNA, rigorous protocols have to be enforced in order to guarantee the genuineness of informations. We herein reexamine that the plural analysis of non-DNA biomolecules from ancient bugs, opens new chances for the designation of ancient bugs and new locales to assist deciding the contentions about the cause of some historical pandemics and to analyze the co-evolution of bugs with hosts.
Paleomicrobiology, the quest for bugs in ancient environmental and human remains, is an emerging field of research which gained from technological progresss in microbiology. The most abundant principal of research used molecular biological science techniques, aimed to observe and to analyze ancient microbic DNA ( Drancourt and Raoult, 2005 ) . This DNA-based attack is prone to taint of the ancient stuff by environmental DNA, including the aDNA antecedently PCR-amplified in the research lab ( Green et al. , 2009 ) which may take to false positive consequences. Besides, the natural decaying of DNA ensuing from chemical alterations and from its atomization, along with the presence of ill characterised PCR inhibitors in antediluvian specimens, may restrict the molecular sensing of microbic DNA in some antediluvian specimens ( Paabo, 1989 ) . A theoretical bound of the D- and L-enantiomers ( D/L ) of aspartic acid ratio & amp ; gt ; 0.08 has been calculated, beyond which ancient Deoxyribonucleic acid could be non-detectable ( Poinar et al. , 1996 ) . For illustration, it was shown that ancient treponema DNA was non preserved in human bone specimens dating dating back to the 9th -19th centuries ( Bouwman and Brown, 2005 ) . For these grounds, construing the consequences of some initial surveies has been questioned ( Spencer and Howe, 2004 ) and rigorous regulations of experimental procedures and reading of informations have been enforced in order to guarantee the genuineness of informations ( Drancourt and Raoult, 2005 ) .
Bibliography indicates a displacement in the methods used in paleomicrobiology from aDNA analysis forwards non-DNA biomolecule analyses ( Figure XX ) . [ NY, DECRIRE LE RESULTAT DE VOTRE RECHERCHE BIBLIOMETRIQUE ] . In fact, methods based on the immunodetection of microbic antigens have been early developed in paleomicrobiology ( Fornaciari and Marchetti, 1986 ; Fornaciari et al. , 1989 ; Dumler, 1991 ; Dumler et al. , 2003 ; Bruschi et al. , 2006 ; Cerutti et al. , 2007 ; Bianucci et al. , 2008b ; Bianucci et al. , 2009 ; Fornaciari et al. , 2010a ) and these attacks are now in the model of extended presentation that ancient proteins at big, including works and animate being proteins could prevail across geological times ( Schweitzer et al. , 2007 ; Asara et al. , 2007a ; Asara et al. , 2007b ; Organ et al. , 2008 ; Schweitzer et al. , 2009 ; Travis2010 ) . Several surveies further indicated that proteins may break resist than aDNA to taphonomic decaying ( Nielsen-Marsh et al. , 2005 ; Organ et al. , 2008 ; Heaton et al. , 2009 ) .
These observations opened new positions for the sensing and word picture of microbic non-DNA biomolecules in antediluvian remains. Two lines of methods have been developed, i.e. the immunodetection taking to the sensing of pathogen specific antigens and the analytical techniques taking to analyse a set of pathogen specific biomolecules. Methods and first consequences on such non-DNA microbic biomolecules sensing in antediluvian remains are herein reviewed. Analysed documents have been retrieved through Pubmed and Medline databases utilizing the undermentioned combined keywords: antediluvian, past, virus, parasite, bacteriums, infection, pathogen, proteome, proteomics, DNA taint, paleomicrobiology, quantification protein, immunodetection, immunological check, late serology, mass spectroscopy, MALDI-TOF, mycolic acid, dental mush. Mentions from a old reappraisal ( Drancourt and Raoult, 2005 ) every bit good as mentions from selected documents were besides reviewed. We searched within the database of Journal of Archaeological Science ( Journal home page: hypertext transfer protocol: //www.elsevier.com/locate/jas ) and the database of American Journal of Physical Anthropology ( Journal home page: hypertext transfer protocol: //onlinelibrary.wiley.com/journal/10.1002/ ( ISSN ) 1096-8644 ) . The selected documents were evaluated by the standards for strength and quality of grounds based on antecedently publication ( Drancourt and Raoult, 2005 ) ( Box 1 ) ( Table 1 ) .
Ancient proteins for paleomicrobiology.
The techniques. After a seminal survey proposing that ancient proteins retained some unity and antigenic specificity ( Smith and Wilson, 1990 ) , the presentation that proteins could be detected in some insects preserved in gold ( Poinar et al. , 1996 ) paved the manner towards the analysis of non-DNA, ancient biomolecules ( Heaton et al. , 2009 ) . Paleopathologists have used a assortment of methods to show the presence of microbic antigens and Igs in archaeological tissues ( Willcox, 2002 ) . A first group of methods purposes to the sensing and quantification of antigenic antigenic determinants of selected microbic proteins extracted from ancient specimens. Indeed, microbic antigens can be extracted from antediluvian specimens and their presence can be detected by several techniques including immunochromatographic sensing, enzyme-linked immunosorbent check ( ELISA ) , and XXXXXXX [ NY, LA IL FAUT DETAILLER LES METHODES D’EXTRACTION ET LES TECHNIQUES COMME VOUS L’AVEZ FAIT POUR BIANUCCI ] . As for immunochromatographic sensing, a protocol indicated that 50 milligram of bone stuff or 7.5 milligram of ancient dental mush were reconstituted in 200 milliliter of unfertile saline solution and subjected to three freeze/thaw rhythms ( 1 min each rhythm ) ; sonication for 15 min, a 4th freeze/thaw rhythm ; 24 – hours incubation at 4 & A ; deg ; C in order to solubilise the staying antigens ; centrifugation at 5,000 revolutions per minute et room temperature and so they could utilize the supernatant with the dipstick check ( Bianucci et al. , 2009 ) . In this survey, the output of protein extraction was non reported and how to stock the protein for the following utilizing? ? ? ? . The capacity of immunodetection to follow infinitesimal measures of microbic stuffs could be farther increased by the application of the immuno-PCR, a technique uniting the advantages of immunodetection with the logarithmic, PCR-based elaboration of the signal ( Sano et al. , 1992 ) . Immuno-PCR was foremost presented as a technique in which a specific antibody-DNA conjugate is used to observe antigen ( Sano et al. , 1992 ) . Consequently, immuno-PCR has a sensitiveness grater than any bing antigen sensing system because of the tremandous elaboration capableness and specificity of PCR ( Sano et al. , 1992 ) . For illustration, we are developing quantitative immuno-PCR for the sensing of the pestilence agent Yersinia plague in the dental mush of past pestilence ‘s victims ( D. Raoult, unpublished information ) .
A derived method purposes to the sensing of Igs in ancient specimens. Indeed, it was demonstrated that Igs could be extracted from ancient bone tissues ( Torres et al. , 2002 ) [ NY, DETAILLER LA TECHNIQUE ; EST-CE UN DES PROTOCOLES PRESENTES PLUS HAUT? SI OUI? LE COPIER/COLLER ] . It was further demonstrated that in these Igs could retain their antibody belongings of adhering to a specific antigen, therefore establishing alleged late serology ( Kolman et al. , 1999 ) . In this attack, Igs extracted from ancient bone specimens are made responding with a pathogen-specific antigen ( Kolman et al. , 1999 ) .
A 3rd group of methods utilizations immunohistochemistry for observing microbic antigens and even full microbic cells without extraction straight in ancient tissues [ NY, DETAILLER UN PEU LES TECHNIQUES: IMMUNOPEROXYDASE ET MICROSCOPIE OPTIQUE ; IMMUNOGOLD ET MICROSCOPIE ELECTRONIQUE ] .
Immunodetection techniques may nevertheless give false negative consequences since antigenic proteins may be extracted from ancient tissues in concentration below that noticeable by techniques. This major challenge may explicate the few publications in paleomicrobiology refering this method ( Lepidi, 2008 ) . Besides, faithlessly positive consequence may ensue from cross-reactivity between primary antibody and non-specific antigen in ancient samples. For illustration, the initial claim of sensing of hemoglobin in 4,500-year-old human castanetss ( Ascenzi et al. , 1985 ) was farther shown to be a false consequence due to cross-reactivity of the primary antibody with non-haemoglobin bone proteins ( Lendaro et al. , 1991 ) ; former survey deficiency appropriate negative controls, such as indistinguishable tissue in non-related, persons ( Fornaciari et al. , 2010b ) . In the experiments of immunodetection, measuring the specificity of the primary antibody is important for the accurate reading of the information. As for antediluvian pathogens, the primary antibody has to be specific for the targeted pathogen and therefore non cross-react with other micro-organisms and non cross-react with non-pathogen constituents of the tissue under analysis. This can be demonstrated by proving a aggregation of relevant beings prior to the trial and by the incorporation of the appropriate negative controls into the trial. At last, the secondary antibody has to hold no cross-reactivity with the tissue under analysis, as demonstrated by another negative control dwelling in the incubation with non-infected, ancient tissue. In contrast, immunological sensing methods are easy to execute as they require a few specific equipments, for illustration ELISA technique or immunohistochemistry technique ( Cerutti et al. , 2007 ) . Besides, it is possible to place microbic cells in antediluvian, mummified and fixed tissues ( Dumler et al. , 2003 ; Fornaciari and Marchetti, 1986 ) . A 3rd advantage of the immunological sensing is the capacity to accurately observe a pathogen from even closely related beings, depending on the specificity of the primary antibody. This fact has been illustrated by the late diagnosing of fatal Rocky Mountain spotted febrility in the remains of patient died in 1901 with an erroneous diagnosing of typhus due to Rickettsia prowazekii ( Dumler, 1991 ) . A last advantage is its capacity to observe infinitesimal measures of antigenic stuff. Immunodetection could therefore complement that of aDNA, by supplying an independent presentation with a low hazard of cross-contamination ; and by supplying extra informations in the instance of false-negative aDNA-based sensing.
Although several surveies reported the usage of the matrix-assisted XXXX ( MALDI-TOF MS ) analysis of ancient carnal proteins or peptides, these techniques have been used merely one time for the sensing of ancient microbic proteins ( Hollemeyer et al. , 2008 ) . The application of mass spectroscopy in the field of archeology and evolutionary biological science is the work of Hollemeyer et Al. 2008 which identified species from 5300-year-old Tyrolean ma, besides called iceman or Oetzi ( Hollemeyer et al. , 2008 ) . With the successful obtain of mass informations from single hairs in their survey, these writers suggested that mass spectroscopy constitutes a powerful analytical method for the rapid and high-throughput word picture of antediluvian samples ( Hollemeyer et al. , 2008 ) . It is nevertheless notable that the accurate designation of ancient animate beings is conducive to the survey of possible reservoirs of antediluvian pathogens as illustrated by the rapid designation of mammals which could hold been ancient reservoirs for ancient Bartonella organisms [ PAPIER DE DANG SUR LES CHATS ; PAPIER MD + HENRI DE LUMLEY SUR B. QUINTANA ] . [ NY, IL FAUT QUE NOUS REPARLIONS DE CETTE PARTIE ] . We so developed a simple and efficient protocol to pull out entire ancient proteins from ancient dental mush specimens for the MALDI-TOF MS analysis. After incubating the dental mush with 1 milliliters of 500 millimeter EDTA, pH 8.0 with agitation at room temperature for 24 hours, the specimen is so sonicated five times for 1 min each and centrifuged at 17,900 tens g for 40 min at room temperature. The specimen is so dialysied overnight in 2 L of a solution incorporating 50 mM Tris-Hcl, pH 8.0, and 150 millimeter NaCl. After proper extraction, protein concentration can be determined utilizing the Bradford protein quantification protocol ( Bradford, 1976 ) . With dental mush of eight persons from four entombment sites dating between 700 and 8,500 old ages ago, we obtained the protein concentrations of 0.28 ± 0.12 g/l yielded by Bradford protein quantification protocol ( Figure 1 ) . We stocked the extract protein at -80 & A ; deg ; C for the hereafter utilizing. In our experiments, the stock of the extract protein from ancient specimens at -80 & A ; deg ; C yielded for one twelvemonth consistent MALDI-TOF MS profiles and we have obtained an placing MALDI-TOF peptidic profile from a two 8,500-year-old human alveolar consonant mush specimens, including two deciduous dentitions collected in two different Neolithic kids in Syria ( 6500 BC ) ( T-N-N. Tran and M. Drancourt, unpublished information ) . However, when using the same ways to ancient microbic proteins, there are some troubles because of low concentration of microbic proteins in entire infusion or ancient proteins. For illustration, MALDI-TOF MS comparing of dental mush peptides profiling between a negative control group and antediluvian dentitions collected from pestilence victims failed to happen the different between two groups. A recent publication analysed ancient mycobacterial proteins in antediluvian remain was achieved by utilizing matrix-assisted optical maser desorption/ionization tandem time-of-flight mass spectroscopy ( MALDI TOF/TOF MS ) ( Boros-Major et al. , 2010 ) . Just as for microbic protein analyses, an advantage of analytical techniques of protein is less capable to false positives or taint by comparing with PCR techniques ( Schweitzer, 2004 ) .
Protein-based sensing of antediluvian pathogens. Immunodetection has been largely applied to fastidious beings as illustrated by the immunohistochemical sensing of the syphilis bacteria Treponema globus pallidus in the ma of Maria d’Aragona ( 1503-68 ) recovered from an abbey in Naples, Italy ( Fornaciari et al. , 1989 ) . Further presentation was achieved in a 200-year-old skeleton [ NY, IL FAUT EN DIRE UN PEU PLUS ] ( Kolman et al. , 1999 ) . [ NY, QU’EST-CE QUE CES TRAVAUX NOUS APPRENNENT? ] . The same attack has been used to observe the emerging bacteria Tropheryma whipplei responsible for alleged Whipple ‘s disease, in the enteric tissues preserved from the autopsy index instance described by Dr. Whipple in 1907 ; legion intracellular bacteriums were observed and a bulk of the foaming macrophages identified in the widened lamina propria stained intensely ( Dumler et al. , 2003 ) . This work confirmed that the disease such as described by Dr Whipple was so due to T. whipplei ( La Scola 2001 ) . Likewise, an immunoperoxydase trial detected Rickettsia rickettsii, the Rocky Mountain spotted fever bacteria but non Rickettsia prowazekii, the typhus bacteria, in different tissues collected from a patient who died in 1901 with the diagnosing of typhus ( Dumler, 1991 ) . This work illustrated the capacity of these techniques to supply an accurate, retrospective diagnosing of an deathly infection in forensic instances. As for antediluvian viruses, the immunostaining of tegument incubated with anti-vaccinia-virus antiserum followed by protein-A/gold and negatron microscopy has been successfully employed in the survey of the sensing of integral smallpox virus atoms in an Italian ma dated from the 16th century ( Fornaciari and Marchetti, 1986 ) . This consequence demonstrated that the antigenic construction of the viral atoms is good preserved in mas tissues ( Fornaciari and Marchetti, 1986 ) .
Detection of the pestilence bacteria Yersinia pestis-specific F1 antigen has been helpful to show that several burial sites in Europe were plague sites including the Black Death sites. ( Pusch et al. , 2004 ; Bianucci et al. , 2008b ; Bianucci et al. , 2009 ) . Using a rapid diagnostic trial for pestilence ( RDT ) , pestilence was demonstrated in four Benedictine nuns and two priests in two burial sites in France, Poitiers and La Chaize-le-Vicomte, between 1578 and 1632 ( Bianucci et al. , 2008c ) . ELISA and immunohistochemical analysis was besides used to place Y. plague in ancient pestilence victims in Venice ( San Leonardo in Fossa Mala, XIV century ) and Genoa ( Bastione dell’Acquasola, XIV century ) , Italy in add-on with a topic suspected Y. plague infection from Brian & A ; ccedil ; on ( France, XVII century ) ( Cerutti et al. , 2007 ) . They used four specimens from the same period deducing from Gravess non related to epidemics for negative controls ( Cerutti et al. , 2007 ) . ELISA has been besides used for the sensing of go arounding schistosome antigen in Egyptian mas ( Deelder et al. , 1990 ) . Cheek and colon from a ma ( 1198 – 1150 BC ) in which Schistosoma haematobium eggs had been found and shin tissue from a late predynastic ( 3200 BC ) were used ; negative control samples were gut tissue from mas from the Atacama ( 2000 BC ) ( Deelder et al. , 1990 ) . Likewise, the sensing of the histidine-rich protein-2 antigen ( PfHRP-2 ) derived from Plasmodium falciparum for the designation of malaria in samples from of course desiccated 1,450 -5200-year-old mas from Egypt and Nubia, stands as a peculiarly successful usage of a double antibody immunoassay in palaeopathology ( Miller et al. , 1994 ) . These writers suggested that the stableness over several thousand old ages of PfHRP-2 indicated the possible value of paleo-immunological diagnosing in look intoing the distribution of such an of import disease as malaria during prehistoric and even Palaeolithic times ( Miller et al. , 1994 ) . These writers used gut tissue from unembalmed, dried-out mas & A ; gt ; 3,000 old ages old from the site of Camarones in the Atacama desert near Arica, northern Chile as negative control ( Miller et al. , 1994 ) . A recent illustration of sensing past pathogens via immunological technique is the usage of two different qualitative-double antibody immunochemical assaies for examine the presence of Plasmodium falciparum antediluvian proteins in the skeletal remains of four members of the Medici household, Italia 16th century ( Fornaciari et al. , 2010a ; Fornaciari et al. , 2010b ) . Because of hog is the alone reservoir for T. solium ; the determination of this parasite suggested that swine agriculture was current in Hellenistic Egypt, as supported by other archaeological groundss confirmed an ancient instance of cysticercosis in an Egyptian ma of 20 year-old adult female about who lived in the late Ptolemaic period ( 2nd to first centuries B.C. ) ( Bruschi et al. , 2006 ) [ NY, IL FAUT REPRENDRE CET EXEMPLE, IL N’EST PAS COMPLET ] .
Other non-DNA biomolecules for paleomicrobiology.
The techniques. The sensing of mycolic acids and mycocerosic acids, two constituents of the TB bacteria Mycobacterium TB cell wall, has been used as a alternate to observe this pathogen in ancient human skeletons [ REF ; Kremer et al. , 2000 ) . Because of these lipoids are peculiarly robust, they appeared as ideal biomarkers in complementing the sensing of M. TB Deoxyribonucleic acid for the diagnosing of ancient TB ( Redman et al. , 2009 ) . With mycolic avec biomarkers, there are some advantages for the sensing of M. TB. First, it can avoid the job of taint with DNA/PCR technique. Second, this non-DNA biomolecule is robust and the technique is sensitive for sensing M. TB in antediluvian remains ( Donoghue et al. , 2010 ) Indeed, mycolic acids have been detected in ancient homo remains by utilizing high public presentation liquid chromatography ( HPLC ) and mass spectroscopy ( Gernaey et al. , 2001 ; Donoghue, 2008 ; Mark et al. , 2010 ) . These attacks are sensitive plenty to observe the molecules straight without elaboration ( Donoghue, 2008 ) . In a limited figure of surveies, these biomarkers have been used in complement to the sensing of M. TB DNA ( Donoghue et al. , 2009 ) . [ NY, IL FAUT DETAILLER UN PEU ] .
Mycolic acid-based sensing of ancient TB. Human TB caused by Mycobacterium TB, is thought to hold emerged in Africa at the terminal of the Neolithic Period with farther development towards M. TB complex beings with specialised ecological niches ( Gagneux and Small, 2007 ) . For illustration, the findings of M. TB DNA the Karkur calcified pleura ( AD 600 ) by PCR were confirmed by parallel HPLC-detection of mycolic acids ( Donoghue et al. , 1998 ) , set uping the first sensing of M. TB in a non-mummified tissue other than bone ( Donoghue et al. , 1998 ) . After proving two biomarkers for sensing M. TB in the ribs of skeletons 1000-year-old derived from Addingham, West Yorkshire, from an early medieval ( Anglo-Saxon ) graveyard, Gernaey et Al. 2001 concluded that mycolic acids seemed more dependable to name ancient TB than IS6110 and this biomarker could be of value in following paleomicrobiology of TB back into antiquity ( Gernaey et al. , 2001 ) . Another survey detected M. TB DNA and used HPLC as an independent method of verification by straight observing mycolic acids in the ribs, arm castanetss ( grownup ) and long castanetss ( baby ) from human skeletons at the site of Atlit-Yam, 10 kilometer South of Haifa, calibrated carbon 14 day of the months range from 9250-8160 old ages BP, bespeaking a day of the month during the last stage of the Pre-Pottery Neolithic C period ( Hershkovitz et al. , 2008 ) . Mycocerosic acid has been detected to follow M. TB in the ribs from 49 persons of the 19th and twentieth century Coimbra Identified skeletal aggregation, half with documental informations bespeaking TB as a cause of decease ( Redman et al. , 2009 ) . They have found mycocerosic acids in archeological remains for the first clip and they showed that mycocerosic acid are valuable biomarkers for the diagnosing of ancient TB ( Redman et al. , 2009 ) . Besides, ancient mycolic acids from archeological bone samples ( 1,400-year-old ) have been detected by MALDI-TOF-MS ( Mark et al. , 2010 ) . Their informations suggested that the MALDI-TOF-MS has possible as a rapid and consistent technique for the sensing and designation of ancient mycobacterial infections ( Mark et al. , 2010 ) .
The first publication of the application MALDI-TOF technique in the field of paleomicrobiology is the work of Mark et Al. 2010 ( Mark et al. , 2010 ) . In fact, these writers analyzed five anthropological bone samples from different archeological periods ( from 600 AD to 1600 AD ) in which four samples were investigated for Deoxyribonucleic acid of Mycobacterium TB composite and the trial was positive in all instances ( Mark et al. , 2010 ) . After mycolic acerb extraction from these castanetss, they used MALDI-TOF mass spectroscopy to obtain the spectra and they compared with the spectra obtained from negative control which was non-infected, healthy bone samples from the processed graveyards ( Mark et al. , 2010 ) . [ NY, IL FAUT REFERENCER ET COMMENTER LA RECENTE CRITIQUE DE CE TRAVAIL ++ ] .
Probationary recommendations for utilizing non-DNA biomolecules in paleomicrobiology.
As for the analysis of ancient microbic Deoxyribonucleic acid, the common types of sample preserved in archeological records and that may incorporate ancient proteins, are bone tissues ( Brandt et al. , 2002 ; Schweitzer et al. , 2007 ; Asara et al. , 2007a ; Asara et al. , 2007b ; Organ et al. , 2008 ; Schweitzer et al. , 2009 ) . Another beginning of ancient protein might come from mummified tissues. In the instance of mummified tissues, an necropsy revealed a package of linen patchs, enfolding a hollow muscular organ, identified as the tummy was used to diagnosis the cysticercosis in an Egyptian ma dating back to the late Ptolemaic period ( 2nd to first centuries B.C. ) ( Bruschi et al. , 2006 ) . Other beginnings for ancient protein might be stuffs such as hair ( Hollemeyer et al. , 2008 ) , teeth ( Bianucci et al. , 2009 ) , skin tissues ( Fornaciari and Marchetti, 1986 ) and other mummified tissues: musculus ( Bianucci et al. , 2008a ) , encephalon, lung ( Miller et al. , 1994 ) . However, the debasement of ancient proteins is of import and it is the challenge for this sphere ( Willcox, 2002 ) . In our lab, we use dental mush as stuffs of pick for the survey in paleomicrobiology ( Figure 2 ) .
4.2. Principles for obtaining dependable informations in immunodetection
Principles for obtaining dependable informations in analytical techniques
Due to the trouble of obtaining ancient microbic Deoxyribonucleic acid and the critical inquiry of taint by utilizing PCR, it is non ever possible to use the PCR/sequencing technique to find the cause of past infections. For illustration, in the survey investigated the cause of Black Death, the writers used PCR method and the dipstick check ( F1 antigen ) for the seventeenth century skeletal remains, their consequences suggested that in 83 % of samples the PCR reactions started from less templet molecules than is needed for positive designation ( Pusch et al. , 2004 ) . Even if sensing and word picture of DNA is the most widely used attack for the survey of antediluvian pathogens ( Drancourt and Raoult, 2008 ) , for the infective diseases that kill quickly leave no hints in dentitions, bone, or coprolites, which is debatable ( Cunha and Cunha, 2008 ) in Deoxyribonucleic acid technique. The surveies of past infection via the manner of supermolecules can be a complement method to decide the jobs in the yesteryear. As for immunodetection technique and late serology, these techniques can merely reply one inquiry at a clip. However, a various immunodetection technique named auto-immunochemistry has been developed for the diagnosing of modern infective diseases ( Lepidi, 2008 ) . The other trouble in paleoproteomic is the reserve of ancient protein ; it is the same trouble when we investigate about the antediluvian samples. Immunodetection of infective agent in antediluvian specimens have proven that ancient proteins can retain their unity and antigenic speficity and it may open the new door beyond of DNA for paleomicrobiology. But, until now there are fewer surveies refering the sensing of past pathogens by proteomic methods than surveies by DNA methods in the same field. Lepidi gave one account about this restriction that may be due to the antigenic determiners in ancient tissues are frequently impaired or destroyed ( Lepidi, 2008 ) . Since it has been shown that antibodies could last for centuries, this method could potentially be applied to ancient specimens, in order to show the presence of micro-organism. This technique relies on the immunodetection of a pathogen by utilizing the patient ‘s serum. This attack could be sometimes applied to ancient diseases specimens.