Immunological detection of albumin in ancient human cremations using ELISA and monoclonal antibodies

The immunological detection of human albumin in a cremation from the Roman period during a pilot investigation led to studies being carried out to determine (i) whether this protein survived in a significant number of ancient cremations, (ii) its immunological thermostability, and (iii) factors affecting its survival in cremated specimens. All tests were carried out using an extremely sensitive and specific enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibodies. In the first investigation, the skeletal remains of 31 cremated individuals from a variety of sites covering a time span of some 3000 years were examined. In the second investigation, ceramic cups containing small samples of human serum were placed in an oven and exposed to increasingly higher temperatures. In the third investigation, a fragment of human tibial condyle and a bovine vertebra were burnt on small funeral pyres built in a manner similar to those used in ancient times. A full range of control specimens was included within each study. Albumin was detected in eight ancient human cremations (26%): six were from the Spong Saxon cemetery, Norfolk (5th-6th centuries AD), one was from the Luni Roman necropolis, Italy (1st century BC-1st century AD) and one was from the iron age necropolis of the Quattro Fontanili, Veio (7th-9th centuries BC). The results of the serum experiment showed that albumin survived for at least 10 min at 300°C, which corresponded to the serum becoming a crisp black layer. No protein was detected in the modern cremated specimens, suggesting that the lack of surrounding soft tissue had resulted in temperatures within the bones of well above 300°C. The control reactions confirmed that there was no cross-reactivity between human and non-human material and that the assay was unaffected by extraneous factors such as the cremation process itself or soil from subsequent burial. It was concluded that the present studies have unequivocally shown that human albumin survives cremation in a number of archaeological specimens, that this protein has a high degree of thermostability and, because of the likelihood of incomplete combustion and the insulating effects of body tissues, that skeletal elements containing albumin could easily have failed to reach 300°C in many ancient cremations. Cremated bone can now be regarded as a valuable source material for biomolecular investigations of ancient populations.