• close up view of gate and parade ground of Fort Clatsop

    Lewis and Clark

    National Historical Park OR,WA

Immunological Analysis of Musket Ball (Bioarch)

Process Of Discovery
Immunological Analysis of a Musket Ball from the Fort Clastop Site, Oregon

Bioarch, Inc.
59 Glenpatrick Crescent
Cochrane, AB TOL 0W3
Canada

November 6, 1996

BACKGROUND

Scientific research carried out by reputable scientists in North America and Europe in the past 10 years clearly show that organic residues recovered from lithics, ceramics, coprolites and soils, can be identified through he use of chemical and molecular biological techniques. Although these techniques are used with confidence in the 'hard sciences', their application to archaeology is relatively new and, as such, there are still problems areas that need to be resolved (Thomas 1993). However, it is clear that data obtained by the use of these modes of analysis can provide unique insight into the evolution of animals and humans, prehistoric environments, prehistoric diet and subsistence, and tool function, information that cannot be obtained by other means.

Although questions concerning the preservation and viability of ancient protein materials have recently been made (Eisele 1995, Fiedel 1996) evidence shows that proteins are extremely hardy molecules. Proteins have been recovered from shells of planktonic foraminifera dating between 2 and 4Ka BP (Robbins and Brew, 1990), from dinosaur bones (Miller and Wyckoff 1968) and dinosaur eggs (Voss-Foucart, 1968), from frozen mammoth dated ca. 40,000 BP (Prager et al. 1980), and in 1500-year-old bones (Cattaneo et al. 1992). Although proteins may not be preserved in their tertiary form, linear epitopes are generally preserved which can be identified by Western blot and other immunological methods (Abass et al. 1994). Given the viability of proteins under the conditions discussed there is a high probability that artifacts used in hunting, butchering, plant collection and processing will also retain adequate amounts of detectable protein.

In forensic work stains are obtained from a variety of sources - clothing, metal, plaster, cement etc. Moreover, criminals frequently attempt to remove bloodstains by a variety of methods such as laundering, scrubbing with bleach, etc. yet, such degraded samples are still identified by immunological methods (Lee and De Forest 1976; Milgrom and Campbell 1964; Shinomiya et al. 1978, among others). It is only in very recent years that immunological analysis has been replaced by DNA testing in crime labs. Forensic wildlife laboratories use immunological techniques in their investigation of hunting violations and illegal trade, often from contaminated evidence (Bartlett and Davidson 1992; Guglich et al. 1994; Mardini 1984; McClymont et al. 1982; among others). Immunological methods are also used to test the purity of food products such as canned luncheon meat and sausage, products which have also undergone considerable degradation (Ashoor et al. 1988; Berger et al. 1988; King 1984). The age of stains does not preclude obtaining positive results (Gaensslen 1983:225).

Immunological methods have been used to identify plant and animal residues on flaked and groundstone lithic artifacts (Hyland et al. 1990; Kooyman et al. 1992; Newman 1990; Yohe et al. 1991) and in Chumash paint pigment (Scott et al. 1996). Plant and animal residues on ceramic artifacts have been identified by their amino acid sequences (Broderick 1979) and by analysis of lipid and fatty acids (Fredericksen 1988; Heron et al. 1991; Bonfield and Heron 1995), while serological methods have been used to determine blood groups in skeletal and soft tissue remains (Heglar 1972; Lee et al. 1989) and in the detection of hemoglobin from 4500-year-old bones (Ascenzi et at. 1985). Human leukocyte antigen (HLA) and deoxyribonucleic acid (DNA) determinations made on human and animal skeletal and soft tissue remains have demonstrated genetic relationships and molecular evolutionary distances (Hansen and Gurtler 1983; Lowenstein 1985, 1986; Paabo 1985, 1986, 1989; Paabo et al. 1989). Recent studies have shown that it is possible to detect DNA in ancient wheat and radish seeds (Brown et al. 1995; O'Donoghue et al. 1995), providing the potential for evolutionary studies of plant domesticates.

MATERIALS AND METHODS

The method of analysis used in this laboratory is cross-over electrophoresis (CIEP). Minor adaptations to the original method were made following procedures used by the Royal Canadian Mounted Police Serology Laboratory, Ottawa (1983) and the Centre of Forensic Sciences (Toronto). Although this test is not as sensitive as RIA, it has a long history of use in forensic laboratories, does not require expensive equipment, is reasonably rapid and lends itself to the processing of multiple samples (Culliford 1964). In this test the antigen and antibody are driven together by an electrophoretic force instead of simple diffusion as in the Ouchterlony test. The test is performed in agarose gels with a pH of 8.6. Paired wells, approximately 1.5 mm. in diameter are punched in the agarose gel 5 mm. apart. The antigen (unknown extract) is placed in the cathodic well of the pair and the antiserum in the anodic one. The gel is placed in an electrophoresis tank containing a barbital buffer, pH 8.6, and triple thicknesses of filter paper are used as wicks to connect the ends of the slides with the buffer. The application of an electrical current, set at a constant 100v, moves the two reactants towards each other. If the unknown sample contains protein corresponding to the species antiserum against which it is being tested, an extended lattice forms as a result of cross-linking, and a precipitate forms where they reach equivalence concentrations between the two wells. Weak positive reactions, common in archaeological samples, are more readily observed if the gel is dried and stained with a protein stain, such as Coomassie Blue. Appropriate positive and negative controls, prepared in 5% ammonia solution, are run with each gel. These are: positive - blood of species being tested for e.g., deer blood for deer antiserum and negative - blood of species in which antiserum is raised e.g., rabbit if raised in that animal. Duplicate testing is carried out on all positive results.

A musket ball recovered from the Fort Clatsop site near Astoria, Oregon, was submitted for potential identification of animal protein residues by immunological analysis. Possible residue was removed from the artifact using a 5% ammonium hydroxide solution. This has been shown to be the most effective extractant for old and denatured bloodstains and does not interfere with subsequent testing (Dorrill and Whitehead 1979; Kind and Cleevely 1969). The artifact was placed in a shallow plastic dish and 4.0 mL of 5% ammonia solution applied directly to it. Initial disaggregation was carried out by floating the dish and contents in an ultrasonic cleaning bath for two to three minutes. Extraction was continued by placing the boat and contents on a rotating mixer for thirty minutes. The resulting ammonia solution was removed with a pipette and placed in a plastic vial. The sample was concentrated by lyophilization then reconstituted by the addition of 200ul of sterile phosphate-buffered-saline (PBS). Initial testing was carried out against pre-immune serum (i.e., serum from a non-immunized animal). A positive result against pre-immune serum could arise from non-specific protein interaction not based on the immunological specificity of the antibody (i.e., nonspecific precipitation), however, a negative reaction was obtained. Complete testing of the sample was continued against the antisera shown in Table 1.

Antisera obtained from commercial sources are developed specifically for use in forensic medicine and, when necessary, these sera are solid phase absorbed to eliminate species cross- reactivity. However, these antisera recognize epitopes shared by closely related species and will often identify other species within the individual family. The relationship of animal antisera used to potential prey species identified is shown in Table 3.

Table 1: Animal antisera used in analysis.

ANTISERA TO:
SOURCE
bear
Organon Teknika
bovine
Organon Teknika
cat
Organon Teknika
chicken
Organon Teknika
deer
Organon Teknika
dog
Organon Teknika
guinea-pig
Organon Teknika
human
Organon Teknika
rabbit
Organon Teknika
rat
Organon Teknika
sheep
Organon Teknika
elk
University of Calgary

Table 3: Relationship of animals to antisera used in analysis.

ANTISERA
MOST PROBABLE SPECIES
bear Black, grizzly
bovine Bison, cow
cat Bobcat, lynx, mountain lion, cat
chicken Chicken, turkey, quail, grouse, pheasant
deer Deer (all species), elk, moose, caribou, pronghorn
dog Coyote, wolf, fox, dog
guinea-pig Porcupine, squirrel, beaver, guinea-pig
human Human, monkey
rabbit Rabbit, hare, pika
rat Mouse (all species), rat (all species)
sheep Sheep, goat

RESULTS

A weak positive reaction to human antiserum was obtained on this artifact. This reaction is probably due to the presence of recent human saliva on the artifact as noted by the excavator. No other positive results were obtained in this analysis. The absence of identifiable proteins on artifact may be due to poor preservation of protein or that it was used on species other than those encompassed by the antisera. It is also possible that the artifact was not utilized.

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Did You Know?

Archeologist

Archeological excavations have been conducted at Fort Clatsop in 1996,1997,1998 and 2005. So far, a few items that may be from the expedition, have been found: a sharpened stick, a brass bead, a glass bead, and a lead rifle ball.