ASTROBIOLOGY Volume 5, Number 1, 2005 © Mary Ann Liebert, Inc.
Research Paper Experimental Support for an Immunological Approach to the Search for Life on Other Planets MARY HIGBY SCHWEITZER,1 JENNIFER WITTMEYER,1 RECEP AVCI,2 and SETH PINCUS3,4
ABSTRACT We propose a three-phase approach to test for evidence of life in extraterrestrial samples. The approach capitalizes on the flexibility, sensitivity, and specificity of antibody–antigen interactions. Data are presented to support the first phase, in which various extraction protocols are compared for efficiency, and in which a preliminary suite of antibodies are tested against various antigens. The antigens and antibodies were chosen on the basis of criteria designed to optimize the detection of extraterrestrial biomarkers unique to living or once-living organisms. Key Words: Antibody reactivity—Extraction methods—Exobiology-Astrobiology. Astrobiology 5, 30–47.
for the following reasons: First, antibodies are designed to primarily recognize structure, not sequence, thereby allowing the detection of overall similarities and avoiding the problems inherent in using methods that require previous knowledge or a priori assumptions of specific sequence data (e.g., as does polymerase chain reaction analyses). (Note that although structure is dependent upon primary sequence, sufficient differences in sequence will result in alterations in structure.) Second, antibodies are extremely well characterized, and a large database exists for AbAg interactions among terrestrial biomolecules (over 58,000 references listed in the Web of Science database at http://isi6.isiknowledge.com/ portal.cgi). Third, antibodies can be targeted to
for extraterrestrial life must be sensitive, specific, repeatable, and able to distinguish endogenous from contaminating compounds as well as biotic and abiotic substances. Life detection instruments must be compact, lightweight, and capable of producing quantifiable data that can be transmitted to Earth for analyses. A number of instruments and/or techniques to search for life on other planets have been proposed, each with inherent strengths and weaknesses that make them appropriate for the task. We have chosen to capitalize on the flexibility, sensitivity, and specificity of antibody–antigen (Ab-Ag) interactions ECHNIQUES TO DETECT EVIDENCE
of Marine, Earth and Atmospheric Sciences, North Carolina State University, Raleigh, North Carolina. of Physics, Montana State University, Bozeman, Montana. 3Department of Microbiology Louisiana State University Health Sciences Center, New Orleans, Louisiana. 4Research Institute for Children, Children’s Hospital, New Orleans, Louisiana. 2Department
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highly conserved epitopes of proteins that may be widely distributed among taxa, allowing for the recognition of members of very large groups (i.e., Archaea) and maximizing the possibility of positive identification of biomarkers (Bergendahl et al., 2003). Alternatively, antibodies can be specific enough to distinguish between amino acid isoforms, possibly allowing for the distinction between terrestrial and non-terrestrial origins of molecular markers (Hofstetter et al., 1999). Antibodies can be designed to differentiate between compounds produced exclusively by living organisms (e.g., heme, hopanes) and organic compounds produced abiotically (e.g., polyaromatic hydrocarbons), thereby reducing the chances of false-positives. Fourth, a single molecule or organism may contain thousands of epitopes, each capable of being recognized by different antibodies (Edwards et al., 2003). Use of polyclonal antisera can optimize the chances of recognizing a foreign antigen that may contain some, but not all, of the epitopes found on organisms. Fifth, when a sample from Mars is retrieved and returned to Earth for additional detailed analyses, Ab-Ag interactions will facilitate the purification and concentration of small amounts of antigen from large volumes of test sample for further characterization (e.g., Cuatrecasas et al., 1968; Wofsy and Burr, 1969). Sixth, because of the exquisite flexibility and virtually unlimited nature of Ab-Ag interactions, it is possible to make antibodies against epitopes that do not occur naturally on Earth but could conceivably be incorporated by other life forms (Hofstetter et al., 1999). For example, while non-synthetic proteins consist of L-enantiomers of amino acids (Bada, 1985), antibodies can be raised against peptides consisting of D-enantiomers (Hofstetter et al., 2002; Vandenabeele-Trambouze et al., 2002), to test whether life on other planets exhibits varied chemical “handedness.” (Some organisms possess isomerase enzymes that, after synthesis, will racemize some amino acids to the D-isomer.) Seventh, monoclonal antibodies can be raised against specific peptide regions, or, alternatively, phage-displayed antibodies can be employed to recognize antigens contained in martian samples and may provide an eventual means for yielding amino acid sequence data for phylogenetic comparisons with Earth taxa. Finally, Ab-Ag interactions can be extremely sensitive, with antibodies capable of detecting nanogram to picogram or even femtomolar amounts of material (Lueking et al., 1999),
thus optimizing the chances for success if extraterrestrial molecules are low in concentration and/or abundance. Additionally, for organisms difficult to culture or with extremely slow replication/metabolic rates (e.g., Gordon et al., 2000; Siegert et al., 2001), consistent with hypotheses regarding organisms on Mars, antibody reactivity may be the most efficient means of detection. We propose a three-phase approach to the search for extraterrestrial life, and in this paper present data that represent our progress toward accomplishing the first phase. Phase 1 involves defining and designing systematic experimental approaches to the identification of extraterrestrial biomarkers. This phase includes optimizing extraction techniques for the highest yield of biological/organic material from extraterrestrial samples, identifying and characterizing likely target molecules and raising antibodies against them, and testing antibodies for titer, specificity, and cross-reactivity with various antigens. Phase 1 also includes optimizing Ab-Ag detection to attain the maximum number of tests in a minimum of steps, time, space, and weight, and incorporating a sensitive detection system reliable under martian conditions. Employing newly developed protein microarray technology (Lueking et al., 1999; Templin et al., 2002) will allow thousands of separate experiments to be conducted within 1 cm2, thus conserving space and weight without compromising data collection. Finally, Phase 1 involves testing the technology and assays in terrestrial environments analogous to conditions existing on Mars. Phase 2 involves remote, automated testing carried out on the surface of Mars. Adopting existing bioassays to the martian environment is far from trivial. Well-tested methods yielding predictable results on Earth will need to be adapted for Mars Lander applications. Crucial to the success of this project is selection of an appropriate landing and sampling site, which may be at odds with engineering constraints given that areas most amenable to life may be those least amenable to a successful landing. Therefore, site selection may require a compromise between optimal landing safety and optimal success for life detection. Advanced robotics designed for fully automated data acquisition will be required to collect quantifiable data for interplanetary transmission. All data must be compared with those obtained from Earth-based experiments using the same pa-
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rameters for accurate interpretation of extraterrestrial data. Data generated in this manner will provide a baseline with which to compare results generated in Phase 3. Phase 3 is directed toward analyses of martian samples returned to Earth. Engineering constraints will greatly limit sample return; therefore data from phase 2 will be critical for selecting return samples. Phase 2 results will be verified in these samples through multiple analytical methods not feasible for a remote mission, such as immunoblot analyses. Sample return will also allow in situ testing, using methods such as immunohistochemistry and/or atomic force microscopy, which would verify endogeneity and rule out contamination. Finally, petrographic analyses of sediments from the target areas may support biochemical evidence for life, either directly, in the form of microbes trapped in the sediments, or indirectly, with identifiable traces such as mineralized biofilms (Toporski et al., 2002). Here we present Phase 1 data, a comparison of the efficacy of various extraction protocols, and the results of testing of a preliminary suite of antibodies made against selected antigens selected unique to living or once-living organisms.
MATERIALS AND METHODS Extraction protocols To adequately test extraterrestrial samples for the presence of biomarkers, the samples must be extracted in a manner that maximizes the recovery of low concentrations of organic matter. We have compared the efficacy of several methods of chemical extraction outlined in the literature by applying them to samples of identical mass and composition and then measuring total resulting product by bicinchoninic acid (BCA) protein detection (Pierce, Rockford, IL) and specific antibody interaction using enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and immunoblotting.
Potting soil extractions We chose to compare methodologies using commercial potting soil, as it contains a complex mixture of organic components, yet is more similar to soil/sediment samples targeted for ex-
traterrestrial investigations than cultured organisms or tissues would be. Identical 5-g aliquots were subjected to the methods outlined in Table 1. Soil components were difficult to saturate completely; therefore potting soil extractions were extended to 7 days to ensure maximum exposure of all components to extracting buffers. After extraction, samples were centrifuged at 3,000 rpm (1,925 g) for 15 min, and the supernatant was collected and dialyzed against distilled H2O in Pierce 3,500 molecular weight cutoff (MWCO) cassettes (Slide-A-lyzer) for 96 h with daily changes. The dialysate was centrifuged to remove remaining particulate matter, lyophilized to dryness, and reconstituted in sterile phosphatebuffered saline (PBS). Samples were tested for protein content (BCA, Pierce) and used as antigen in ELISAs to test for cross-reactivity with various polyclonal antisera.
Sediment extraction Methods 3 [guanidine thiocyanate (GuSCN)] and 4 [guanidine hydrochloric acid (GuHCl)] in Table 1 were applied to two sediment samples very low in apparent organic content to further compare buffer efficacy in recovering proteinaceous/antigenic material, and to test assay sensitivity. Calcite-cemented sediments surrounding fossil material previously shown to contain organic compounds (Schweitzer et al., 1997a,b) and Patagonian (Argentina) sandstone sediments, high in oxidized iron and surrounding dinosaur eggs containing embryonic soft tissues (Chiappe et al., 1998), were selected because the fossils found within them demonstrated exceptional preservation, possibly mediated by microbes, the molecular remains of which may be preserved in the entombing sediments. These extracts were subjected to BCA assays for protein concentrations and ELISAs for immunoreactivity as described.
Spiked sediment extraction To test the efficiency of extraction methods specifically for molecular DNA, degraded DNA, or its component nucleotides in sediments, separate tubes with 0.5 g of the above sediments were mixed with 3 mg (in solution) of the following: deoxynucleotide monophosphate (dNMP) mix (AMP, GMP, CMP, and UMP) conjugated to bovine serum albumin (BSA) (0.75 mg each of
IMMUNOLOGICAL APPROACH TO SEARCH FOR LIFE
TABLE 1. EXTRACTION METHODS FOR REMOVAL OF PROTEIN AND DNA IN FOSSIL BONE, MICROBIAL SAMPLES, AND SEDIMENTS Extraction buffer
1. Morse Buffer (D.E. Morse, personal communication) 2. Merriwether reducing buffer (Merriwether et al., 1994) 3. GuSCN (Hoss and Paago, 1993) 4. GuHCl EDTA (Tuross and Stathoplos, 1993) 5. CelLytic B-II 6. PBS (saline) 7. GuHCl
General extraction procedure: The outersurface of mammoth bone samples was cleaned with a sterile dremel tool. Microbial samples were frozen at 80°C and lyophilized at time of collection. all samples, including fossil bone, sediment, and microbial samples (Cyanobacteria/spp. and Archaea) were ground to powder using a sterile mortar and pestle. The ground powder was washed with 0.5 M NaCl multiple times (to remove any surface ions or contaminants) and rinsed with sterile water multiple times. Following these preparatory steps, samples were treated with one of the described extraction methods. After extraction, all samples were dialyzed against water and lyophilized to completion. Powdered samples were demineralized with sterile 0.5 M EDTA, rinsed with sterile distilled H2O, and centrifuged to pellet. The pellet was partially solubilized in 5 ml of buffer containing 8 M urea, 0.01 M Tris/HCl (pH 7.2), and 10 mM -mercaptoethanol per 0.5 g of powdered bone, and stirred at 4°C for 48 h. Powdered samples (0.5 g) were vortex-mixed with 5 ml of buffer containing 10 mM Tris/HCl, 10 mM EDTA, 10 mg/ml of dithiothreitol, 0.5 mg/ml of proteinase K (Sigma), and 0.1% SDS, and incubated at 60°C for 24 h and then at 28°C for 72 h. Five milliliters of buffer containing 10 M GuSCN, 0.2 M EDTA (pH 8.0), 0.1 M Tris/HCl (pH 6.4), and 1.3 g/100 ml of octylglucosidase was added to 0.5 g of pelleted sample and extracted at 60°C for 24 h. Five milliliters of a buffer containing 4 M GuHCl, 0.5 M Tris, and 0.5 M EDTA (pH 7.4) was added to 0.5 g of powdered sample, vortex-mixed, and incubated at 60°C with occasional agitation for 24 h. Five milliliters of bacterial cell lysis extraction reatent in 20 mM Tris-Cl (pH 7.5) (Sigma) was incubated to 1 g of washed, powdered sample with shaking at 28°C for 24 h. Phosphate buffer (1 mM Na2HPO4 7 H2O, 10 mM NaH2PO4 H2O, 120 mM NaCl, pH 6.8) was added to preweighed material, sonicated on ice for three 15-s pulses, and then incubated at 28°C for 24 h. Five milliliters of a buffer containing 4 M GuHCl and 0.5 M Tris was added to 0.5 g of cleaned, powdered sample, vortex-mixed, and incubated at 60°C with occasional agitation for 24 h.
AMP-BSA, GMP-BSA, CMP-BSA, and UMPBSA); salmon sperm DNA (Sigma Chemical Co., St. Louis, MO) diluted in enzyme buffer (100 mM sodium acetate, pH 5.0, and 5 mM MgCl2); and digested DNA (incubated at 0.05 g of DNase; Sigma)/g of DNA for 2 h at 37°C. Sediments were incubated in these solutions for 2 h at 37°C and then lyophilized, weighed, and extracted with GuHCl (Method 7, Table 1). These samples are referred to as “spiked sediments.” Individual controls were performed using the extraction buffers and enzyme buffers either alone or added to sediments not incubated with molecular solutions, but dialyzed and lyophilized in the same manner as the spiked sediments. Lyophilized extracts were reconstituted in PBS buffer and used for ELISA cross-reactivity assays with polyclonal antibodies to dNMP-human immunoglobulin G (HgG) and monoclonal antibodies to DNA.
Fossilized bone extraction We also compared the efficiency of extraction buffers in removing intact or naturally degraded protein material, using Pleistocene mammoth bone fragments. Skull bone fragments were taken from an adult specimen of Mammuthus cf M. columbi (MOR 604) collected in eastern Montana (Hill and Schweitzer, 1999; Schweitzer et al., 2002). Samples were prepared as described in Table 1, then centrifuged to pellet the bone powders, and extracted using Methods 1–4 (Table 1). For all samples, control tubes containing only extraction buffers were treated in an identical manner. Samples and controls were centrifuged after extraction as previously described, and the supernatant was dialyzed against distilled H2O in 2,000 MWCO dialysis tubing (Spectra/Por, Spectrum Laboratories, Inc., Rancho Dominguez, CA)
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for 7 days with daily changes. The dialysate was lyophilized to dryness and reconstituted in 1 ml of sterile distilled H2O. Protein concentration was determined using a micro-BCA assay (Pierce). Reconstituted samples were used as antigen in ELISA and immunoblot assays against mammoth antibodies (Schweitzer et al., 2002).
Rio Tinto microbial fauna The Rio Tinto river system has been suggested as a possible analogue environment for early Mars (Fernandez-Remolar et al., 2003). The river is a naturally acidic [pH 1.7–2.5 (Amaral-Zettler et al., 2003; Fernandez-Remolar et al., 2003)] cold water system flowing over pyrite beds. Over time, the acidic waters act to solubilize iron that is then quickly oxidized. Evidence suggests that iron-rich and highly oxidized martian sediments may, at some point in its evolution, have supported flowing surface water conditions (Fairen et al., 2003; Lunine et al., 2003); therefore organisms adapted to the acidic and iron-rich conditions of the Rio Tinto may be good analogues for similar organisms once or currently existing on Mars. Various samples were collected from different regions of the river that varied in pH and hydrodynamics, and placed on ice until extraction. Sample specimens are further described in Table 2. Samples were centrifuged, and the resulting pellets were subjected to extraction using Method 4 (Table 1). After dialysis against sterile distilled H2O, extracts were lyophilized and then reconstituted in PBS for use in ELISA cross-reactivity studies.
Immunogen descriptions and antibody preparation An immunogen is a substance used to immunize a host in order to raise specific antibodies. An antigen, on the other hand, is a substance to which antibodies react. An immunogen can also be an antigen. Taxa representing Archaea and cyanobacteria were used to generate antisera for this study. Colonies of colorless, filamentous Hydrogenbacter acidophilus and green, mat-forming Cyanidium spp. were collected from Nymph Creek sampling sites in Yellowstone National Park (courtesy of J. Henson, Montana State University, Bozeman, MT). Samples were separated from surrounding sediments and debris and then freeze-dried. Four milligrams of powdered, freeze-dried organisms were reconstituted in 1 ml of PBS, ultrasonicated for eight 10-s bursts (Method 6, Table 1), and used to immunize rabbits. Rabbits were injected with immunogen in Freund’s complete adjuvant and then boosted twice at 2-week intervals with immunogen in Freund’s incomplete adjuvant. Antisera were collected 2 weeks after the second boost and tested for reactivity by ELISA and immunoblot against the immunogen and other antigens. Sediment material from the same sites at Nymph Creek were extracted by Method 4 (Table 1), dialyzed, lyophilized, and then reconstituted in PBS and used to measure reactivity of antisera to dead organismal components within the sediments. Two taxa of cyanobacteria, Nostoc spp. and Phormidium spp. (Hawes and Howard-William,
TABLE 2. DESCRIPTION OF SPECIMENS AND SITE PARAMETERS FROM SAMPLES COLLECTED FROM THE RIO TINTO RIVER SYSTEM, SPAIN Specimen number Description 2A-1, 2A-2
Colorless, filamentous (morphologically similar to Hydrogenbacter spp.) Filamentous green algae
Soft, yellow, laminated mounds
Green algae and cyanobacterial growth
Parameters Site 2, left arm of stream
Deep red waters pH 1.0
Site M, exposed rock near source Site L, wall of dam forming pond Site 3, right arm of stream Site 3, right arm, out of water flow Site 5, 5 m below area of convergence of left and center arm
Clear water, low oxygen, pH 1.0 Deep red waters, pH 1.5–2.0 pH 3.0 No visible growth Thin film on rocks in fast moving water
IMMUNOLOGICAL APPROACH TO SEARCH FOR LIFE
1998; McKnight et al., 1998), were collected from the McMurdo Dry Valley lakes in Antarctica and freeze-dried immediately after collection (courtesy of J. Priscu, Montana State University). The McMurdo Dry Valley ecosystem has also been proposed as a terrestrial analogue for martian environments (MacClune et al., 2003); hence the organisms adapted to those environments are suitable for antibody generation. Samples were extracted by Method 4 and used at 100 g/ml of immunogen in Freund’s complete adjuvant per rabbit, followed by two boosts of immunogen/Freund’s incomplete adjuvant 2 weeks apart. Sera were collected 2 weeks after each boost and tested for specificity and cross-reactivity by ELISA and immunoblot. Because all terrestrial life contains nucleic acids, we also made antibodies to a mixture of nucleotides, the building blocks of nucleic acid polymers. Nucleotides are composed of a pentose sugar, phosphate, and one of four nitrogenous bases (adenine, thymine, guanine, or cytosine), collectively labeled nucleotide triphosphates. Precursor molecules are nucleotide monophosphates (NMPs). To obtain nucleotide antibodies, two rabbits were injected with a mixture of 50 g each of AMP-HgG, CMP-HgG, UMP-HgG, and GMP-HgG in Hunter’s adjuvant, followed by four boosts at 3–4-week intervals. Sera were collected 2 weeks after the fourth boost and tested for reactivity. Monoclonal antibodies to DNA (cell line 2C10) were generously provided by D. Stollar (Tufts University, Boston, MA). Finally, antibodies were raised to purified cytochrome c, a heme-based biomolecule necessary for cellular respiration as part of the electron transport chain. The prosthetic heme group that is at the core of this molecule is highly resistant to degradation (Huseby and Ocampo, 1997), and its presence is easily confirmed using varied techniques, which makes it an ideal target for the search for either extinct or extant extraterrestrial life, assuming a common biochemistry. The molecule is widely distributed among all aerobic taxa, identified in Archaea and Eubacteria, as well as eukaryotes, including human (Grossman and Lomax, 1997). Although there is variation in cytochromes across taxa, the motifs binding the heme are quite conserved (Zamocky, 2004). For extraterrestrial experiments, more than one source of cytochrome should be used as immunogen for antibody elicitation. For this study, Saccharomyces cerevisiae cytochrome c (catalogue
number C-2436, Sigma) was injected into rabbits at 250 g/ml emulsified in Freund’s complete adjuvant, followed by two boosts of 250 g/ml in Freund’s incomplete adjuvant to produce polyclonal antisera. Sera were collected 2 weeks after each boost for screening by ELISA. Sera from all animals used in this study were taken prior to immunization and used as negative controls (referred to as pre-immune sera). Two rabbits were injected for each immunogen. Additional boosts with immunogen and subsequent test bleeds were performed when necessary to increase antibody titer.
Immunologic assays ELISA parameters. ELISAs were performed to detect protein, inhibition, and cross-reactivity of various antisera. Microtiter 96-well polystyrene plates (Immulon 2HB, Dynatech, Chantilly, VA) were coated with extracted or pure antigen diluted to varying concentrations in sterile PBS. Plates were incubated for 4–6 h at 25°C or 12 h at 4°C and then blocked with PBS containing 1% BSA, 0.1% Tween 20, and 0.01% thimerosal (BSA blocking buffer) or with PBS containing 1% dried milk, 0.1% Tween 20, and 0.01% thimerosal (Blotto) for 6–12 h. Immune and pre-immune antisera were diluted in either of these buffers and incubated with antigen for 4–6 h at 25°C or overnight at 4°C. The antisera were removed, and the plates were washed multiple times in a wash buffer consisting of PBS containing 0.1% Tween 20. Alkaline phosphatase-conjugated secondary antibody [goat anti-rabbit IgG (H L), and goat anti-mouse IgG (H L), Zymed, San Francisco, CA] was diluted 1:1,000, 1:2,000, or 1:3,000 in either BSA blocking buffer or Blotto and incubated as above. Following washing, the colorimetric substrate, p-nitrophenyl phosphate (Sigma), was diluted to 0.5 mg/ml in 9.8% diethanolamine containing 0.5 mM MgCl2, and absorbance was measured at 405 nm over various time points, using a SpectraMax plus 384 plate reader and Softmax Pro software (Molecular Devices, Sunnyvale, CA). Cross-reactivity and inhibition studies. To demonstrate that the antisera we produced were sufficiently specific to identify and differentiate biomarkers and/or organisms, ELISAs were conducted on multiple antigens as described above, using polyclonal sera produced in rabbits. To test
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the range of cross-reactivity, antisera were incubated with both the antigens used to produce them (immunogens) and with antigens other than those against which they were raised, and relative reactivities were noted. Both specificity and cross-reactivity were also tested by inhibition. Antisera were incubated prior to testing with both immunogens and antigens in known concentrations to block the binding sites of the antibodies, and then reactivity tested by incubating with immunogens and antigens as described above. The degree of inhibition is directly correlated to the similarity between the test antigen and the immunogen used to generate the antibodies. For example, lyophilized extracts of Cyanidium and Nostoc were added at 0, 10, 100, or 1,000 g/ml concentrations to anti-Cyanidium antisera diluted 1:600 in PBS and incubated at 28°C for 2 h. This allowed the antigen to bind reactive sites on antibodies, inhibiting them from binding to antigen. The more similar these antigens were to Cyanidium, the more effective the inhibition, and the ELISA will measure less reactivity to antigen after inhibition. Immunoblot parameters. To confirm ELISA results, antigens extracted as described were electrophoretically separated on a 12% SDS-PAGE gel in duplicate, and blotted to 0.22-m (pore size) nitrocellulose membranes (Nitropure, G.E. Osmonics, Inc., West Borough, MA). The membranes were incubated in Blotto to prevent nonTABLE 3.
Morse (1) Gu/SCN (3)
Gu/HCL EDTA (4)
RESULTS Extraction methods For all samples tested, extraction buffers resulting in highest protein concentrations and greatest reactivity to test antisera were GuHCl and GuSCN (Tables 3 and 4 and Fig. 1). Guanidine is a powerful protein denaturant, facilitating solubilization of many proteins otherwise insoluble in their native states, so these results are not surprising. Table 3 illustrates the results of BCA assays performed on potting soil extracted by the various methods described. Based upon colorimetric change and absorbance at 562 nm, the same starting material and parameters resulted in clear differences in recovered protein. This was further supported by antibody reactivity, using
PROTEIN CONCENTRATION OBTAINED FROM VARIOUS EXTRACTION METHODS ON DIFFERENT SAMPLES, USING THE BCA PROTEIN ASSAY (PIERCE), WITH A MINIMUM DETECTION LIMIT OF 6 G/ML
Extraction buffer (method of extraction from Table 1)
Cell lysis (5)
specific binding of antibodies and then with either test or pre-immune serum diluted in Blotto for 5 h at 28°C. Following three washes in PBS/Tween to remove unbound antibodies, membranes were incubated with horseradish peroxidase-conjugated secondary antibody [goat anti-rabbit IgG (H L), Zymed] diluted 1:3,500 in Blotto. After washing to remove unbound secondary, bound antibodies were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce) and recorded on x-ray film (Hyperfilm, Amersham, Piscataway, NJ).
Protein concentration (mg/ml)
Potting soil Control Potting soil Argentine sediments Calcite sediments Control Potting soil Argentine sediments Calcite sediments Control Potting soil Control Potting soil Control
2.500 No protein 4.600 0.042 0.046 No protein 6.600 0.030 0.335 No protein 0.192 No protein 2.100 No protein
Sample starting weights: potting soil, 5 g/extraction; Argentine sediments, 6 g/extraction; and calcite sediments, 10 g/extraction. The Merriwether reducing extraction (Method 2) was not included, as proteinase K, a component of this buffer, interferes with the assay.
IMMUNOLOGICAL APPROACH TO SEARCH FOR LIFE TABLE 4.
EXTRACTED MAMMOTH BONE MATERIAL ANALYZED FOR PROTEIN CONCENTRATION USING PROTEIN ASSAY (PIERCE), WITH A MINIMUM DETECTION LIMIT OF 0.5 G/ML
Extraction buffer (method of extraction) Morse (1) GuSCN (3) GuHCL EDTA (4)
Protein concentration (g/ml)
Mammoth fossil Control Mammoth fossil Control Mammoth fossil Control
39.83 No protein 189.63 No protein 190.17 No protein
Material obtained using Merriwether reducing extraction (Method 2) was not measured for protein content as proteinase K interferes with this assay.
antibodies generated against various microorganisms to test against the extracted material (Fig. 1). For all extraction methods, purified antiserum to Cyanidium had the highest cross-reaction to potting soil extracts. Antisera to Phormidium and Hydrogenbacter also demonstrated cross-reactivity with potting soil extracts, while antisera to Nostoc and dNMP showed only slight reactivity to potting soil extracts. The results, shown in Fig. 1, indicated that with the same amount and source of starting material (potting soil), antibody, and experimental parameters, soils extracted by GuHCl and/or GuSCN demonstrated far greater
reactivity to antisera by ELISA, supporting the efficiency of these two buffers for recovery of antigenic material from heterogeneous samples. The efficacy of these two extraction methods was also evaluated when applied to antibodies developed against mammoth bone. More antigenic material was recovered from the fossil samples extracted by the two guanidine-based buffers (Methods 3 and 4) than when other buffers were employed (Fig. 2). Background values with fossil material were high because of variations in experimental factors, such as time of incubation of the colorimetric substrate in the wells and the
FIG. 1. Comparative efficiency of extraction buffers, measured by reactivity of polyclonal antisera raised against Yellowstone (Cyanidium spp. and Hydrogenbacter spp) and Antarctic (Nostoc spp. and Phormidium spp.) extremophiles and of a mix of dNMPs with various extractions of identical aliquots of potting soil. Numbers in parentheses refer to extraction methods described in Tables 1 and 3. All antisera were diluted 1:400 in Blotto. Alkaline phosphatase-conjugated secondary goat anti-rabbit antibodies were diluted 1:2,000 in Blotto. Error bars indicate standard deviations from the mean.
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FIG. 2. Comparison of extraction efficiency for fossil mammoth bone monitored by ELISA antibody cross-reactivity. Numbers in parentheses refer to extraction methods described in Tables 1 and 3. Mammoth fossil extracts were diluted 1:100 in PBS buffer and coated onto 96-well plates. Test sera were produced using rabbits immunized with mammoth bone extracted with GuSCN buffer (Method 3). Anti-mammoth serum was diluted 1:500 in Blotto. Secondary goat anti-rabbit antibody was diluted 1:1,000 in Blotto. Error bars designate the standard deviation from the mean.
temperature of the room at the time. To detect any minimal binding that would be demonstrated with the control buffers, incubation time was exaggerated, which led to higher background. These results were confirmed by the resulting protein concentration detected by micro-BCA (Table 4) and immunoblot (data not shown), which demonstrates differential reactivity of the antisera to bone extracted by each of the first four methods shown in Table 1. Data from potting soil extractions and fossil extractions clearly showed that of the different extraction methods tested, guanidine-containing buffers were consistently more efficient in recovering organic material from all types of samples, and employing either one maximizes the chances for recovering organic components from extraterrestrial samples.
Immunological assays Figure 3A demonstrates cross-reactivity of polyclonal antiserum raised against whole-cell extract of Cyanidium spp. with antigens derived from six microbial taxa (Cyanidium, Hydrogenbacter, Phormidium, Nostoc spp., and two samples of Rio Tinto organisms). As expected, reactivity was strongest with the immunogen (optical density value was saturated after 20 min), but relative dif-
ferences in binding to other antigens was noted. Significant cross-reactivity to Hydrogenbacter spp., Phormidium spp., Nostoc spp., and, surprisingly, lithified bacterial overgrowths from Spain’s Rio Tinto river system (L1-A) indicated the presence of similar epitopes in the antigen extracts. Immunoblot results confirmed these shared epitopes. As shown in Fig. 3B, as expected, the strongest reactivity was observed against Cyanidium extract (lane 2). In addition, there was some cross-reactivity with Hydrogenbacter (lane 3), Nymph Creek sediments (lane 5), Phormidium (lane 6), and Nostoc (lane 7), with bands in each lane similar in molecular size and mobility to bands from Cyanidium extract (arrows), while other bands were unique to each sample. This indicates that while some epitopes, perhaps microbial membrane components, are shared among microbial taxa, antibodies bind in patterns sufficiently distinct to allow for the differentiation of extracts. The relatively broad crossreactivity can be used to identify biomarkers in extraterrestrial samples that are similar, but not identical, to Earth forms. It should be noted, however, that it is possible for molecules of different sizes to express similar epitopes, so this method should not be used in isolation to characterize molecular components. Antisera generated against Yellowstone extremophiles (Cyanidium spp.) were used to mea-
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1. 2. 3. 4. 5. 6. 7. 8. 9.
1 mg/ml Argentina sediment Gu/HCI extract 200 mg/ml Oyanidium PBS sonicated 50 mg/ml Hydrogentracter PBS sonicated Molecular weight markers (wide range, Sigma) 500 mg/ml Yellowstone sediment Gu/HCI extract 1 mg/ml Phormidium Gu/HCI extract 1 mg/ml Nostoc Gu/HCI extract 2 mg/ml ss DNA dH2O used to dilute samples
C FIG. 3. A: Antibodies to Cyanidium spp. were used to test specificity and relative cross-reactivity across samples, monitored by ELISA. Cyanidium and Hydrogenbacter antigens were extracted in PBS (Method 6), while all other antigens were extracted with GuHCl (Method 4), and 1 g of each sample diluted in PBS was plated as antigen. Secondary goat anti-rabbit antibody was diluted 1:2,000 in Blotto. Cyanidium/anti-Cyanidium reached saturation and was read at a 20-min time point. All other data were collected at t 40 min. Error bars designate the standard deviation from the mean. B: Immunoblot showing samples incubated with pre-immune (a) and anti-Cyanidium test serum (b). ss, singlestranded; dH2O, distilled water. Twenty-five microliters of sample was mixed with a reducing sample buffer for electrophoretic separation at 120 V, 80 mA and then electrophoretically transferred to nitrocellulose membrane at 30 V, 80 mA. Pre-immune and test sera were diluted 1:800 in Blotto, while horseradish peroxidase-conjugated secondary antibodies were used at 1:3,500 dilution in Blotto. C: Anti-Cyanidium sera were tested in an ELISA against 1 g of various sediment samples extracted using Method 4 (Table 1) to test both antibody sensitivity and cross-reactivity. Secondary goat anti-rabbit antibody was diluted 1:2,000 in Blotto. Error bars designate the standard deviation from the mean.
sure sensitivity and determine whether antibodies were capable of (1) recognizing molecular remnants of immunogens in soil/sediment extracts not containing visible organisms and (2) recognizing other organisms retained in sediments from other localities. Because samples collected for testing for biomolecular markers would likely be drawn from similar samples on Mars or other extraterrestrial surfaces, this level of sensitivity would be critical. In Fig. 3C it can be seen that antiserum reacted significantly above background levels with sediments obtained from nonsubmerged regions adjacent to where Cyanidium spp. were collected. This reactivity supports the hypothesis that antibodies can be used to detect dormant or dead or very low levels of organism. Significant cross-reactivity was observed between antibodies to Cyanidium spp., extracts of potting soil, and Rio Tinto sediment samples (Fig. 3C), indicating the possibility that organisms in these samples share molecular components and may be phylogenetically related. This is not unrealistic as Cyanidium spp. are widely distributed, with a high degree of genetic diversity and tolerance for environmental extremes (e.g., Ciniglia et al., 2004). In contrast, sediment samples taken from Cretaceous fossil sites (Argentine sediments and calcite sediments, Fig. 3C) did not show significant reactivity to antibodies used in this study. Low reactivity with this antiserum is consistent with low protein levels measured by BCA (Table 3), despite preliminary evidence for microbial fossil components in these sediments (authors’ manuscript in preparation). Low protein levels (Table 3) and low cross-reactivity with antisera (Fig. 3C) may indicate that microbial epitopes within the Cretaceous fossil samples were either not preserved or were preserved at levels below detectability by the parameters employed here. Alternatively, the antibodies employed may not have been sufficiently similar to antigens in these sediments to elicit binding. Antibody binding can be inhibited or blocked by incubation of the antiserum with immunogen or antigen. The effectiveness of inhibition is directly correlated to the degree of structural similarity between the antigen used to inhibit and the immunogen used to produce the antibody. Inhibition studies can therefore be used to infer regions of similarity in multiple antigens (Bayard and Lottspeich, 2001). A polyclonal antiserum contains antibodies to many different epitopes on
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a complex antigen. When examining related antigens for cross-reactivity, some of these epitopes are shared, while others are specific to only one organism. Figure 4 demonstrates that antibodies raised against Cyanidium spp. bound most strongly to its immunogen, but antibodies also reacted to varying degrees with other microbial antigens and sediment material. When immunogen (Cyanidium spp.) was incubated with antisera, antibody binding was inversely correlated to inhibitor concentration (Fig. 4A), although this correlation was nonlinear. It is entirely expected that blocking with the homologous antigen (Cyanidium-Cyanidium, Fig. 4A) will demonstrate much greater inhibition than when a heterologous antigen is used. Antibody binding to other antigens also decreased with increasing concentrations of inhibitor, but the decrease was less dramatic. Because Nostoc shared only some epitopes with Cyanidium, it did not inhibit all antibodies in the serum (Fig. 4B), which allowed remaining unblocked antibodies to bind to Cyanidium antigen and decreased antibody binding to Cyanidium only slightly. However, binding of anti-Cyanidium antibodies to Nostoc antigen was greatly reduced when the sera was inhibited with Nostoc antigen. Inhibition with Nostoc does not block the antibodies in the polyclonal sera that bind Hydrogenbacter as efficiently. These data demonstrate that an immunological cross-reaction exists between taxa, and support the utility of using polyclonal sera capable of recognizing a broad array of antigens in extraterrestrial exploration. Antisera generated against a cocktail containing equal concentrations of individual nucleotides were effective in recognizing both the mixture and the individual monomers (Fig. 5), but binding was less intense with individual monomers. Significantly, these data show that the NMPs were not equally immunogenic and that differences in immunogenicity may not reflect concentration differences. Strongest reactivity was seen against UMP antigen, and the least reactivity was with CMP. The polyclonal antiserum was also specific, and inhibition studies demonstrated differential binding, depending on the inhibitor used (Fig. 6). For example, antisera generated against the dNMP mix responded with measurable variation to nucleotide monomers when first inhibited with AMP. Binding decreased with increasing concentration of AMP inhibitor most significantly with AMP as antigen,
IMMUNOLOGICAL APPROACH TO SEARCH FOR LIFE
FIG. 4. A: Antibody inhibition studies, monitored by ELISA. Polyclonal antisera raised against Cyanidium spp. were incubated with varying concentrations of Cyanidium antigen to block binding sites and then incubated with various antigens. Secondary goat anti-rabbit antibody was diluted 1:3,000 in BSA block buffer. Error bars designate standard deviation from the mean. B: Nostoc as inhibitor to anti-Cyanidium antibodies. Antiserum was incubated with increasing concentrations of Nostoc antigen and then with various antigens as in A. Secondary goat anti-rabbit antibody was diluted 1:3,000 in BSA blocking buffer. Error bars designate the standard deviation from the mean.
because only antibodies to adenosine phosphates were blocked, and antibodies to other molecules were not affected. These data show that inhibition studies were effective in distinguishing between molecules and have the potential to yield structural information about extraterrestrial unknowns.
Antisera raised against NMPs also showed strong reactivity to extracts of sediment and soil extracts, which supports the presence of these compounds in extracts and demonstrates the efficacy of extraction buffers for nucleotides (Fig. 7A). While relative reactivity was strongest to the immunogen, reactivity greater than two times
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FIG. 5. Polyclonal antiserum raised against a mix of NMPs recognizes individual monomers. Secondary goat anti-rabbit antibody was diluted 1:1,000 in Blotto. Error bars designate standard deviation from the mean.
background was also seen when these antibodies were exposed to extracts of potting soil and Rio Tinto samples, particularly the extracts of lithified overgrowth (L1-A). However, antibodies to nucleotide monomers did not bind significantly over background to PBS-sonicated Cyanidium or Hydrogenbacter spp. (Fig. 7A). This lack of binding may be due to the relative inefficiency of this extraction method, rather than to the lack of antigenic material present in these samples. Interestingly, the anti-nucleotide antibodies did not show significant binding to double-stranded DNA, possibly demonstrating that these anti-dNMP antibodies may not be able to find buried epitopes
FIG. 6. Polyclonal antibodies to NMPs inhibited by incubation with AMP at various concentrations and then tested against individual NMPs as antigens. Secondary goat anti-rabbit antibody was diluted 1:1,000 in Blotto. Error bars designate the standard deviation from the mean.
on undenatured DNA. It should be noted that the high pre-immune values for some samples (potting soil and Spain samples) may be the result of exposure of the test animals to compounds similar in structure to these antigens. Nucleotide antibodies were capable of detecting NMPs in spiked sediments (Fig. 7B). While reactivity was minimal to untreated sediments, both Argentine and calcite sediments spiked with DNA responded strongly to monoclonal antibodies. When sediments were spiked with mixed NMPs in solution, reactivity was stronger with polyclonal anti-NMP antibodies than with monoclonal sera. Finally, while monoclonal antibod-
IMMUNOLOGICAL APPROACH TO SEARCH FOR LIFE
FIG. 7. A: ELISA cross-reactivity assay for anti-NMPs. Antibodies to NMPs are capable of recognizing components in guanidine-based extracts (Method 7) of various soil and sediments. Secondary goat anti-rabbit antibody was diluted at 1:2,000 in Blotto. Error bars designate the standard deviation from the mean. B: Spiked sediment experiment, tested by ELISA with polyclonal anti-dNMP and monoclonal x-DNA antibodies (mAb). Sediments with minimal measurable organic components were spiked with known quantities of DNA, dNMPs, and DNA degraded with DNase enzyme and then extracted by Method 7 (Table 1) to test extraction efficiency for nucleotide retrieval. Secondary goat anti-rabbit and goat anti-mouse antibodies were diluted 1:2,000 in BSA blocking buffer. Error bar designates the standard deviation from the mean.
ies recognized enzyme-degraded DNA, NMP antibodies did not bind above background to degraded DNA. Anti-DNA monoclonal antibodies detected extracted DNA in spiked sediments, which verified that antibody reactivity to this molecule may be used as an indicator of life in sediments where no visible evidence exists and that the extraction procedure (GuHCl) is efficient at recovering nucleotides, molecular DNA, and enzyme-degraded DNA from sediment material in a form recognized by antibodies. Because extraterrestrial biomarkers will likely differ from even broadly distributed terrestrial biomolecules, it is important that antibodies be capable of recognizing “foreign” markers, a characteristic confirmed in Fig. 8. Extracts of Rio Tinto organisms were tested for cross-reactivity with antibodies raised to Cyanidium and Hydrogenbacter spp. from Yellowstone National Park, Phormidium spp. from Antarctica, and cytochrome c. Different antisera reacted with varying strengths to epitopes from these geographically distinct organisms. Antibodies raised to Yellowstone filamentous organisms (Hydrogenbacter) demonstrated higher reactivity than other antisera to the Rio Tinto filamentous (2A) organisms with which they shared similar habitats (low pH)
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and morphologies (colorless and filamentous) (Table 2). Alternatively, antibodies to Cyanidium spp. demonstrated strong cross-reactivity to the mound/mat forming organisms in the less acidic region of the river (Sites 3 and L, see Table 2). However, the cross-reactivity of Phormidium antisera was not as strong (although still two times greater than background) and would indicate fewer epitope similarities between this Antarctic cyanobacterium and the organisms found in the highly acidic Rio Tinto River. Cytochrome c is widely distributed among extant organisms, including members of Archaea and Prokaryota (e.g., Pereira et al., 2004, and references therein). Although all organisms possess a form of this biomarker, it is internal to the membrane-bound mitochondria and may be in low concentrations in whole-cell extracts. Figure 9 demonstrates the strong reactivity observed between antibodies raised against cytochrome c with the cytochrome c immunogen and indicates that antibodies raised against this biomarker are capable of recognizing cytochromes to varying degrees in a variety of samples. Samples demonstrated to have high levels of organic/proteinaceous material by other assays responded with different, but relatively high, reactivity to cy-
FIG. 8. ELISA measuring antibody reactivity to antigens derived from environments that are geographically and temporally distinct from the immunogen used to elicit them. All antisera were diluted 1:1,000 in Blotto. Secondary goat anti-rabbit antibody was diluted 1:2,000 in Blotto. Absorbance readings indicating antibody reactivity were saturated at t 2 h for antisera to Cyanidium and Hydrogenbacter tested against samples from site 3A; therefore these readings cannot be quantified. Error bars designate the standard deviation from the mean.
IMMUNOLOGICAL APPROACH TO SEARCH FOR LIFE
FIG. 9. ELISA measuring differential reactivity of cytochrome c antibodies. Absorbance values for cytochrome c antigen binding with cytochrome c antisera were saturated at t 45 min. Secondary goat anti-rabbit antibody was diluted 1:2,000 in Blotto. Error bars designate the standard deviation from the mean.
tochrome c antiserum. This antiserum responded with varying reactivity to lyophilized extracts of Nostoc spp., potting soil, and both filamentous (2A-2) and acidophilic green algae (5A) Rio Tinto organisms. However, very little reactivity was measured with Yellowstone Cyanidium spp. and Hydrogenbacter spp. extracts, possibly because of inefficiency of the PBS-sonicated extraction method. Likewise, antisera did not react significantly with sediment samples (Argentina sediments and calcite sediments) low in organic content. As noted earlier, high pre-immune response for Spain samples and Phormidium spp. may be due to exposure of the test animals to substances sharing epitopes with these organisms.
DISCUSSION The search for extraterrestrial life is conducted using what is known about life on Earth and models for detecting life with which we are familiar. Though such search strategy assumptions may not be accurate (e.g., may not reflect the range and conditions of life on other planets), we assume that extraterrestrial life will share some aspect of metabolism, chemistry, genetics, or habitat with known life and that such life will be recognizable with methodologies designed to characterize Earth-based life-forms. We also assume that we will be able to differentiate extraterrestrial life from Earth-borne contaminants.
These assumptions constrain the search for extraterrestrial life. We have designed tests to identify target biomolecules that are universally or widely distributed on this planet or are present in organisms inhabiting environments serving as extraterrestrial analogues as good possibilities for extraterrestrial life indicators. If molecules are conserved on this planet across a wide number of taxa, their function must be critical to life processes on Earth and, perhaps, on other planets as well. Target molecules must also be well characterized and identifiable by a number of independent methods. For example, if antibodies demonstrate binding to extracts of martian sediments, then concurrent amino acid analyses should detect amino acids, and spectroscopy should detect absorbance at approximately 280 nm, or circular dichroism studies should detect aromatic functional groups. Target molecules must also be unique to life, and not produced abiotically. Some aliphatic compounds or cyclic hydrocarbons would not necessarily be indicative of life, nor would individual amino acids, so positive identification of these compounds should not be used as the sole evidence for the presence of extraterrestrial life. An ideal target molecule meeting the above criteria is heme, the small chromophore that gives blood its red coloration. One atom of iron binds to the core of the heme group, giving this metallocompound many distinctive features that can be uniquely defined by mass spectroscopy, ul-
traviolet/visible spectroscopy, nuclear magnetic resonance, and other analytical methods, thus allowing independent verification of its presence in extraterrestrial samples. Because this molecule is also a key component of cytochromes, compounds vital to cellular respiration, it is present in virtually every organism on Earth in one form or another. This small molecule is not produced abiotically, but is unique to living or once-living forms. Finally, heme is a modified porphyrin molecule, shown to be stable across geological time (Huseby and Ocampo, 1997). Therefore, if life on Mars utilized metabolic pathways similar to terrestrial life, even if it eventually became extinct, then porphyrin-based molecules may be preserved as evidence of that life. Because we have no way of knowing the molecular structure of life that may have emerged on other planets, it is important to use antisera capable of recognizing multiple epitopes to ensure that the recognition of biomarkers due to overspecificity will not be ruled out. The antisera we have tested are suitable for this purpose, and demonstrate specificity to the organism against which they were raised as well as measurable cross-reactivity to other organisms.
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ACKNOWLEDGMENTS We thank A. Steele and J. Toporski (Carnegie Institute, Washington DC) for hours of discussion and helpful insight; J. Priscu and J. Henson (Montana State University); V. Parro, C. Briones, and D. Fernandez-Remolar (CAB, Madrid); and R. Coria (Museo Carmen Fuentes) for providing samples for analyses, and the reviewers whose comments made this manuscript much stronger.
ABBREVIATIONS Ab-Ag, antibody–antigen; BCA, bicinchoninic acid; BSA, bovine serum albumin; dNMP, deoxynucleotide monophosphate; ELISA, enzymelinked immunosorbent assay; GuSCN, guanidine(aminomethanamidine) thiocyanate; GuHCl, guanidine(aminomethanamidine) hydrochloride; HgG, human immunoglobin G; MWCO, molecular weight cutoff; NMP, nucleotide monophosphate (AMP, TMP, GMP, CMP); PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.
Criteria for acceptance of extraterrestrial life To optimize the chance of life detection while minimizing false-positives it is important to agree upon acceptable evidence for endogenous martian life, extant or extinct. If antibody binding to extracts of martian sediments can be demonstrated, would that be sufficient evidence to make the claim for extant or extinct life on Mars? The need for an assay system with multiple antibodies and the ability to employ independent verification of positive results is obvious. Perhaps most significantly, it will be necessary to detect forward contamination. Microbes contaminating the surfaces of Earth probes may not be easily recognizable as Earth-based molecular forms if they are chemically or structurally altered during space flight. We propose that a positive indicator of extraterrestrial life would be characterized by at least two biomarkers incapable of being produced abiotically, and that the presence of these biomarkers be verified by at least two different methods of analysis to reduce the chance of falsepositives (e.g., immunoreactivity and mass spectroscopy).
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Address reprint requests to: Dr. Mary Higby Schweitzer Department of Marine, Earth and Atmospheric Sciences North Carolina State University 1125 Jordan Hall Box 8208 Raleigh, NC 27695 E-mail: [email protected]