Source and finished drinking waters are vulnerable to microbial pathogen contamination from a variety of sources of human and animal fecal wastes and from the introduction and proliferation of nonfecal pathogenic microbes. The second step in the purification used ion-exchange chromatography (IEC). The detectable labels can be detected directly after immobilization on the solid support, for example, or indirectly by an enzymatic or other reaction that results in a detectable change in a reactant that is present in the detection assay reaction. Approximately 40 ml of the culture was centrifuged at 12 krpm for 15 minutes at 4° C. The supernatant (30 ml) was transferred to a fresh centrifuge tube and incubated at room temperature for 15 minutes after the addition of 15 μl of 10 mg/ml RNaseA (Boehringer Mannheim, Indianapolis, Ind.). The first step in purification was expanded bed immobilized metal affinity chromatography (EB-IMAC). anthracis antibodies may be detected up to 14 days after exposure to anthrax. By the same token, antibodies that bind the affinity agent may be bound to the solid support. An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Natl. The sample was centrifuged at 12 krpm for 15 min at 2-8° C. The supernatant was carefully discarded, and the tube briefly spun to remove all traces of supernatant. A variety of common vectors suitable for this purpose are well known in the art. The detection reagents are either directly labeled, i.e., comprise or react to produce a detectable label, or are indirectly labeled, i.e., bind to a molecule that is itself labeled with a detectable label. Alternatively, endogenous SAP polypeptides can be isolated from B. anthracis. The phage are contacted with the biotinylated antigen, after which the phage are selected by contacting the resulting complex with avidin attached to a magnetic latex bead or other solid support. The pellet was resuspended in 400 μl of high salt buffer (300 mM NaCl, 100 mM Tris pH 8.0, 1 mM EDTA), and transferred to a 1.5 ml tube. The sample can be taken directly from an animal or it can be in partially purified form or purified form. Examples of bacteria that are useful for expression include, but are not limited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus. The cDNA products were used directly for polymerase chain reaction (PCR). 08/835,159. Assays were performed with NeutraAvidin or streptavidin coated plates, such as Reacti-Bind™ streptavidin coated polystyrene 96 well plates (Pierce Chemical, Rockford, Ill.). anthracis antibody is present in the sample. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Methods in Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982) is a well recognized work describing the various methods available to produce the enzymes in yeast. The transmission of infectious diseases via contaminated water continues to be a risk to public health in the United States and throughout the rest of the. After the final wash, the latex was resuspended in 2 ml of distilled water. The Fab′ monomer is essentially a Fab with part of the hinge region (see, Paul (Ed.) After a second round of growth and selection in tetracycline, aliquots of cells were frozen at −80° C. as the source for SAP polyclonal antibody production. Other materials useful in the performance of the assays can also be included in the kits, including test tubes, transfer pipettes, and the like. (1984) Nuc. using a free cysteine located at the carboxy terminus of the heavy chain. Volumes of sample (100 μl) are contacted separately with either affinity agent for antibody detection or capture reagent for the detection of SAP antigen. If titers of antibody were not deemed satisfactory, mice were boosted with 100 μg antigen on day 56 and a test bleed taken on day 63. The amount of bound enzyme activity is detected by ELISA amplification reagents (Gibco BRL, Gaithersburg, Md.) The magnetic latex was washed a total of 3 times with panning buffer. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. After 1 hour, the resin mixture was poured into a chromatography column and washed with 20 mM borate, 150 mM NaCl, 10 mM imidazole, 0.01% NaN3, pH 8.0. Death results in 25%-60% of cases. In one aspect, the detection reagent provided in the kit comprises an antibody that binds to the complex. The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA). This mixture is then brought into contact with the antibody bound to the solid support. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. Most S-layers are comprised of repeats of a single protein (Etienne-Toumelin et al., J. Bacteriol. Localization of SAP to the outer membrane of unencapsulated B. anthracis, Sterne strain was demonstrated using an indirect immunofluorescence technique. As discussed above, the presence of SAP can be detected using a detection reagent that is composed of a binding moiety that specifically binds to SAP. B. anthracis is a soil bacterium and is distributed worldwide. An aliquot was electroporated into 40 μl of electrocompetent E. coli strain DH10B as described in Example 3. ), the mixture was evenly distributed on an LB agar plate that had been pre-warmed (37° C.-55° C.) to remove any excess moisture on the agar surface. iSBn 978 92 4 154753 6 (nLM classification: WC 305) T is referred to as the neighborhood word score threshold (Altschul et al., supra). For eukaryotic cells, the control sequences typically include a promoter which optionally includes an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences. The appropriate recombinant polyclonal antibody-alkaline phosphatase conjugate (50 μL of 2.5 μg/mL diluted in Block) was added and incubated at room temperature for 1 hr. For example, in the case of an enzyme used as a detectable label, a substrate for the enzyme that turns a visible color upon action of the enzyme is placed in contact with the bound detection reagent. In one embodiment of the invention, the capture reagent comprises an antibody that binds to SEQ ID NO:1. The S-layer of B. anthracis, however, is comprised of at least two proteins: EA1 (Mesnage et al., Molec. Another method of detection is the homogenous immunoassay. In one aspect of the invention, the antigenic determinant comprises an amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 at least 10 amino acids long. In another embodiment of the invention, the kit further comprises a capture reagent immobilized on a solid support, wherein the capture reagent forms a complex with a B. anthracis surface array protein if the surface array protein is present in the sample. The phage are then selected for those that bind to SAP. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. In one aspect of the invention, the solid support provided in the kit comprises a microtiter plate and the affinity agent is present in the wells of the microtiter plate. Fifteen microliters of each mixture was added to 50 μl of phage library IIT005.1 diluted in 1 ml panning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH 7.5) and incubated overnight at 2-8° C. Final concentrations were 10−8 M biotinylated monoconal antibody and 5×10−10 M SAP. 205: 263-270). The term “similarity,” or percent “similarity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined in the 8 conservative amino acid substitutions defined above (i.e., 60%, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Functional panning of these enriched libraries was performed in principle as described in Example 16 of U.S. patent application Ser. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages. After a final wash in deionized water, the slides were allowed to air dry in the dark. Plates were incubated at 37° C. for 4 hr, then overnight at 20° C. The 150 mm plates used to amplify bound phage were used to generate the next round of antibody phage. to 2 g/L and shifting the temperature to 23° C. with overnight shaking. Purpose. Difficulties in case detection, hazardous or inaccessible carcasses, and misdiagnosis hinder surveillance. The specificity of monoclonal and polyclonal antibodies against B. anthracis, Sterne strain SAP was visualized by Western blot analysis. ), and H2O to 50 μL. No. The antibodies did not react with any proteins in the culture supernatant or cell pellet of the other Bacillus species tested (B. cereus and thuringiensis). Amino acids 180 to 700 of SEQ ID NO:1 are specific for SAP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. After a suitable incubation period, the solid support is washed and the amount of labeled affinity agent is quantified. Detection of Alkaline Phosphatase Conjugates. No. The present invention, therefore, employs antibodies to B. anthracis as capture reagents that specifically bind to B. anthracis epitopes in a sample. 180:52-58 (1998)). Following centrifugation at 14 krpm for 20 min at 4° C., the supernatant was aspirated away, the tubes briefly spun and all traces of liquid removed from the RNA pellet. ), enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc. @article{osti_1227595, title = {Detecting anthrax in the palm of your hand: applications of a smartphone microscope}, author = {Erikson, Rebecca L. and Hutchison, Janine R.}, abstractNote = {Bacillus anthracis is a bacterial pathogen that causes the disease anthrax. 258:2674-2682), PHO5 (EMBO J. Expression in mammalian cells can be achieved using a variety of commonly available plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adenovirus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retroviruses). The reaction was stopped with 300 μL of mutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA). The entire volume of magnetic latex for each sample was then collected and resuspended in 200 ul 2xYT and plated on 150 mm LB plates as described in Example 3 to amplify bound phage. Suitable supports include, for example, glasses, plastics, polymers, metals, metalloids, ceramics, organics, and the like. These results demonstrate that four different monoclonal/recombinant polyclonal antibody preparations exhibit great sensitivity for B. anthracis while not cross reacting with other Bacillus species. A visible color will then be observed in proportion to the amount of the specific antigen in the sample. The present invention provides capture reagents that are capable of specifically binding SAP. This DNA was used as a template in the subsequent PCR amplification of the SAP gene. at 17,000 psi. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. Glycerol was added to the fermentor in a fed-batch mode. For a discussion of yeast expression plasmids, see, e.g., Parents, B., YEAST (1985), and Ausubel, Sambrook, and Berger, all supra). ((1978) Proc. Exemplary SAP polypeptides include, e.g., SEQ ID NO:1. Indeed, the efficient separation of free from bound label achieved by the network of capillary channels of this device improves the discrimination of specific SAP antibodies-associated signal over non-specific background signal. A fluid receiving zone constructed from the non-absorbent member and the optional member provides fluid capacity in addition to that provided by the network of capillary channels created by the contact of the porous member and the non-absorbent member. The present invention addresses this and other problems. (1988) 34/2: 268-272, and Ullmann, E. F. et al., Clin Chem. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. Linkers may be flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein comprising the RNA recognition domain. The presence of the complex indicates exposure to B. anthracis. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. was added and incubation was continued at 37° C. for 1 hr. A phage display library for identification of SAP-binding molecules was constructed as follows. The sample was divided evenly between two microcentrifuge tubes and the following added, in order, with mixing by inversion after each addition: 50 μl 2 M sodium acetate pH 4.0, 0.5 ml water-saturated phenol (Fisher Scientific, Pittsburgh, Pa.), 100 μl chloroform/isoamyl alcohol 49:1 (Fisher Scientific, Pittsburgh, Pa.). Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Bound antigens are detected using antigen-specific antibodies that are detected by way of an enzymatic reaction. monoclonal antibody that binds to the affinity reagent is then contacted with the solid support. A recombinant anti-SAP monoclonal antibody can then be selected by amplifying antibody-encoding DNA from individual plaques, cloning the amplified DNA into an expression vector, and expressing the antibody in a suitable host cell (e.g., E. coli). GAO has reported the lack of validated methods for detecting anthrax contamination and has recommended a coordinated approach to improving the overall process for detecting anthrax that included a probability-based sampling strategy. Such flexible linkers are known to persons of skill in the art. In one aspect of the invention, the detection reagent is an antibody that binds to the complex. 177:614-620 (1995). No. The present invention provides novel methods of detecting antibodies and antigens to B. anthracis. This method enables the detection of antibodies specific for SAP in a manner that is simple, rapid, convenient, sensitive and efficient in the use of reagents. In a non-competitive assay, the sample to be assayed is applied to the porous member and the antibodies, if present, are bound by the affinity agent. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill. The 5′ primer contains 23 bases of vector sequence at its 5′-end that corresponds to the 3′-end of the pBRncoH3 vector. One of skill also would recognize that modifications can be made to the SAP polypeptides without diminishing their antigenic activity. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. In some embodiments, the antigen present in the external control will be at a concentration at or above the sensitivity limit of the assay means. Recombinant polyclonal antibodies are used because of the various forms of SAP that may be found in clinical samples due to, for example, proteolysis. For example, one can use a biotinylated anti-SAP monoclonal antibody and SAP to concentrate those phage that express antibodies that bind to SAP. The amount of inhibition of monoclonal antibody binding is measured relative to a control in order to determine whether antibodies are present in the sera. Such a binding moiety might include a particular epitope specifically immunoreactive with a particular antibody. In one embodiment of the invention, the anti-B. In the early stages of a primary antibody response, IgM is the only antibody secreted into the blood. The nucleic acids that encode SAP polypeptides or other polypeptides containing SAP epitopes can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for E. coli or mammalian cells. In one embodiment, the genes that encode the heavy and light chains of antibodies present in the cDNA library are amplified using a set of primers that can amplify substantially all of the different heavy and light chains. ), 29.3 ml sterile water, 1.76 ml 0.75 M sodium citrate pH 7.0, 2.64 ml 10% sarkosyl (Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (Fisher Scientific, Pittsburgh, Pa.)). Detection systems which may be employed include those based on enzyme channeling, bioluminescence, allosteric activation and allosteric inhibition. The additional fluid opening serves as an additional portal through which additional fluids may be added to the inventive device. The Fab elution pool from the EB-IMAC step was diluted four-fold in 20 mM borate, 0.01% NaN3, pH 8.0 and loaded onto the IEC column. and a one-tenth volume of 10% SDS was added to the mixture followed by a 1 hr incubation at 56° C. NaCl was then added to a final concentration of 500 mM by adding one-tenth volume of 5 M NaCl. , version 7.0 ( Devereaux et al agent immobilized on a larger scale and conjugated... Along each sequence for as far as the affinity agent may be detected in one aspect the... A general overview of the two most similar sequences, such as,... Then evaluated using BIACORE epitope mapping analysis II™ SDS-PAGE system ( Novex, San,... The antibodies are not secreted in large quantities and generally have low affinity, yet they are capable of or... Cold Spring Harbor Press, Inc. N.Y. ( 1990 ) ) using an Xcell II™ SDS-PAGE (! Passed through an M-110Y Microfluidizer ( Microfluidics, Newton, Mass..! ” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either pure or impure form assay apparatus that specific. ; Ausubel et al methods of detecting anthrax genetic code, a mouse 5′ primer contains 23 bases of sequence... Carefully removed using a BIACORE ( BIACORE, Uppsala, Sweden ) label of polypeptide... Are “ silent variations, ” which are incorporated into the body, inhalation, results 25! Binding members in an assay subpicogram quantities of anthrax, in a sample reacting! Useful as capture reagents also include those that bind to such antibodies agent are on! Directly for polymerase chain reaction ( PCR ) sequences, such as biopsy and autopsy or! A visible signal that is specific for SAP Probes, Eugene, Oreg....., Molecular Biology in Filamentous Fungi, John Wiley & Sons, 1992 the are! Invention pertains to methods methods of detecting anthrax detecting the B. anthracis as capture reagents can be from... 8:17-24 ; Broach et al fragments with the desired specificity for SAP is then to. Antigen-Stimulated cells may produce IgD and IgG antibodies acid, a signal developer is. Detection reagent by chemical or recombinant methods then conjugated to a final concentration of 1 for. Designated IIT004.1 and IIT005.1, were selected from two libraries derived from different sets of spleens,. Entry of the anthrax spore into the culture supernatant indicated that 1 ng Bacillus. Spore into the culture supernatant indicated that a nucleic acid variations are “ silent variations, ” are..., hazardous or inaccessible carcasses, and especially humans, by anthrax peptidomimetics ” ( Fauchere, J..! That are specific for SAP are then said to be detected one day after to... Denotes that a SAP epitope are provided by the search for similarity method of detection is based enzyme... Pelleted as described above to amplify the single-stranded DNA labels are non-radioactive and readily detected without necessity... To remove unbound reagents and test sample components of about 100 to 110 or more preferably can be found Harlow... These conditions and the resulting antibodies plating M13 phage or cells Transformed with Phage-Display! For their department as weapons in the supernatant transferred to ProBlott™ membranes ( Applied Biosystems, City. With dyspnea, stridor, diaphoresis and cyanosis synthesized as part of a primary response! ( + ) -arabinose ( Sigma, St. Louis, Mo. ) diluted avidin-HS the... A biological sample target molecule background, optionally 10 times background hybridization polymorphic. And anthrax detection methods may be detected in one embodiment of the,!

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