as shown in fig1 and 2 , a preferred immunoassay 10 of the present invention , comprises a sample well 12 , a sample pad 14 , a conjugate label pad 16 , a membrane or detection strip 18 , an absorbent pad 20 , an adhesive layer 22 , a plastic backing 24 , and a plastic housing 40 . sample pad 14 overlaps with conjugate label pad 16 to form a first overlap zone 26 . conjugate label pad 16 and membrane 18 form a second overlay zone 28 . membrane 18 and absorbent pad 20 form a third overlap zone 30 . a sample ( not shown ) can be placed in sample well 12 and filters down through the sample pad 14 and then through a conjugate label pad 16 containing a conjugate label , e . g ., protein a conjugated with colloidal gold . the gold particles , preferably about 45 nm in size , serve as an indicator dye . the conjugate label binds to igg antibodies in the sample to form a complex , and the complex then migrates along the membrane 18 . anthrax pa - specific antibodies bind to the recombinant pa antigen that is immobilized in a discreet location 32 on membrane 18 . formation of this double antigen - antibody complex causes the indicator dye to precipitate and form a detectable colored red line ( illustrated as “ t ” in fig1 ), indicating a positive result that anthrax pa - specific antibodies are present in the sample . fig2 is an illustration of a test strip from millipore corporation ( see mansfield , m . ( 1999 ) a short guide : developing immunochromatographic test strips , millipore corporation , bedford , mass .) that has been modified in accordance with the present invention . an assay is recorded as positive when a distinct band of the pa antigen ( t on the assay 10 in fig1 ) appears in addition to the control band . a protein a control line will also form whether the pa antigen line is visible or not , indicating the test is functioning properly . a negative test results when only the control ( c ) band appears in the membrane window . preferably , each lateral - flow device is individually packaged in a plastic - lined foil pouch with a desiccant pad to ensure stability . these testing devices can be stored long - term at room temperature with no loss of activity . the testing devices comprise 0 . 72 and 1 . 8 μg rpa protein immobilized on nitrocellulose strips as the test indicator ( t ) and protein a as the control band ( c ). the amount of membrane - bound antigen is important . it is necessary to have excess binding capacity to capture all of the desired igg , yet not too great to cause diffusion and band broadening . experience with saliva sample testing has shown that 4 drops , as added by bulb - pipette , work best for obtaining optimum results . this amount provides sufficient testing volume , and subsequent capillary flow pressure , to ensure optimum membrane flow rate as the sample migrates the entire length of the membrane . calibration of several plastic , bulb - pipette medicine droppers that are used to add sample to the device indicates that each drop consists of about 40 μl volume . therefore 4 drops or 160 μl sample volume will be used for each test . four drops of unstimulated saliva followed by 3 drops of saliva dilution buffer as chase solution also appears to facilitate sample flow by breaking up the mucins . test results using pbs serial dilutions spiked with goat anti - ava igg1 polyclonal antibody ( bioport , corporation ) showed a positive result for pa - specific igg antibodies at dilutions ranging from 1 : 250 through 1 : 32 , 000 ( v / v ). this indicates that the testing device and method described above will specifically detect anti - pa igg antibodies with a sensitivity of at least 5 ng protein . saliva specimens that are undiluted and diluted 1 : 1 to 5 : 1 ( saliva : buffer , v / v ) with phosphate - buffered saline ( pbs ) containing 10 mm ethylenediamine tetraacetic acid ( edta ) and 0 . 1 % sodium azide were tested to study sample viscosity due to mucin content and methods to overcome testing difficulties . saliva dilutions up to 5 parts saliva to one part buffer show marked improvement in membrane flow properties and produce a clear signal at the protein a control line . the time for this test is about 5 - 10 minutes , and under 20 minutes . the results can be used to evaluate immunization status of a patient . for example if the sample came from a person who has received an anthrax vaccine , then the positive result will indicate that the appropriate immune response was elicited from that person , particularly if there are no other indications or symptoms of anthrax in the person . if the sample tests negative , and the sample came from a person who has received an anthrax vaccine , then the negative result will indicate that the person has not been properly immunized . if the sample tests positive , and the sample came from a person who has not received an anthrax vaccine , then the positive result will indicate that the person has been exposed to anthrax , and treatment against anthrax should begin immediately . if the sample tests negative , and the sample came from a person who has not received an anthrax vaccine , then the negative result will indicate that the person has not been exposed to anthrax , particularly if there are no other indications or symptoms of anthrax in the patient . membrane 18 can comprise any suitable material , e . g ., a uniform - sized ( 10 × 500 mm ) nitrocellulose membrane ( millipore ™ xa3j072100 ). conjugate label pad 16 can contain any suitable marker , e . g ., dried colloidal gold - labeled protein a as marker ( see fig1 and 2 ) and be placed at one end of membrane 18 . an absorption pad 20 is located at the opposite end of the membrane 18 and serves to draw the sample , e . g ., saliva , along the membrane 18 by capillary action . a plastic backing 24 provides support for the adhesive layer 22 and membrane 18 , and the combination can be cut into individual test strips ( e . g ., 5 × 60 mm ) and fitted into a plastic housing . a round sample application well 12 is positioned directly above the sample pad 14 , and a rectangular detection window 34 is located above the nitrocellulose membrane 18 . 1 . develop a rapid , in vitro method to detect anthrax anti - pa antibodies in whole saliva and / or oral fluid including oral mucosal transudate and gingival crevicular fluid . 2 . optimize reaction conditions to enhance limits of detection , including sample preparation , preventing proteolysis , antigen form and secondary antibody conjugates to amplify the visual signal . 3 . determine assay sensitivity and specificity and refine test method parameters . 4 . evaluate assay performance for detection of anthrax anti - pa antibodies in comparison to enzyme - linked immunosorbent assay ( elisa ) in a small group of immunized military personnel , with final assay refinement . 5 . conduct parallel serum and saliva elisa assays in a large subject population to validate the test method . 6 . analyze data to determine if saliva and / or oral fluid including oral mucosal transudate and gingival crevicular fluid is a viable substitute for serum in immunoassays to evaluate anthrax immune status , and prepare lateral flow device for distribution . a significant strength of this proposal is that rapid diagnostics have now attained sufficient sensitivity to offer a compact , in vitro test method as a viable alternative to instrumental assays . in fact a number of immunochromatographic tests are now commercially available to confirm presence of a variety of infectious diseases , drugs of abuse , allergies and sexually transmitted diseases among others . mansfield , m . ( 1999 ) a short guide : developing immunochromatographic test strips , millipore corporation , bedford , mass . although these assays employ serum or urine as the tested body fluids , in accordance with the present invention , oral body fluids can be used . saliva antibody content mirrors that in serum . saliva consists of over 99 % water with less than 1 % solid constituents including albumins , globulins , enzymes and mucins . salivary mucin is a high molecular weight glycoprotein and is principally responsible for the viscous , lubricating nature of the fluid . saliva has not been as rigorously studied as other body fluids , and data characterizing salivary antibody makeup and concentration are not as common as for serum . however , it is known that saliva contains the same antibody classes as serum in lower , but measurable quantities . based on the available literature , it is expected that about a 900 - fold lower igg level is in whole saliva compared with that in serum . total igg levels in serum have been reported as 1250 mg / 100 ml , while that for whole saliva is 1 . 4 mg / 100 ml . roitt , i . m ., and lehner , t . ( 1980 ) chapter 11 : oral immunity . in immunology of oral diseases p . 307 , blackwell scientific publications , boston , mass . in an average saliva sample of 5 ml , 0 . 07 mg or 70 μg igg would be present . to enhance detection , assay amplification procedures can be studied to improve the ability to measure specific salivary igg antibodies . serum and saliva elisa analyses of anti - pa specific and total igg will allow for normalization of saliva values and obtain comparative data . saliva contains only a trace of , if any , protease enzymes as the bulk of protein digestion begins in the stomach . however , bacteria present in saliva can secrete proteases with some degradative effect on salivary proteins . see orten , j . m ., and neuhaus , o . w . ( 1975 ) chapter 13 : nutrition : digestion , absorption , and energy metabolism . in patient biochemistry pp . 442 - 443 , the c . v . mosby company , st . louis , mo . the protease effect on salivary antibodies can be studied and a protease inhibitor additive can be developed to counteract this potential degradation . as previously noted , the possible threat of personnel exposure to biological warfare agents and the protection of united states armed forces in this event has been a primary concern for the department of defense . the inexpensiveness , accessibility and relative ease of handling anthrax bacteria make this an especially troublesome weapon and a relevant threat to military personnel . anthrax immunizations are required for deployed service members and are an important component of our integrated force health protection program . the present invention provides a method and device to preserve and maintain the health of united states military personnel in support of military operations . another aspect of the present invention is to help monitor a vaccination program and provide methods and devices to monitor vaccine effectiveness . the present invention provides a test method to quickly assess vaccine efficacy and measure antibody response over the course of the vaccination senes . the in vitro salivary diagnostic test of the present invention provides a significant step forward in field - test capability and in making rapid assessment of immune status , including such assessment at remote - site operations . the present invention can be used as a screening test for all immunized military personnel and provide clinicians with a valuable diagnostic tool to monitor anthrax vaccination progress . this salivary diagnostic tool also offers a considerable range of options for verification of vaccine effectiveness , confirming exposure to a number of infectious diseases , and tracking disease progression by monitoring individual antigen level and antibody response . these applications are possible when a specific antigen of the disease and its corresponding antibody are known . the design and rationale for this study rest on the importance of using saliva as the test medium to measure anthrax vaccine effectiveness . this rapid immunoassay avoids venipuncture and the necessity of having trained corpsmen to collect samples for measurement of anti - pa antibodies . sample collection is non - invasive with no perceivable discomfort or risk to the individual . a positive confirmation of anthrax antibodies is readily observed within 5 - 10 minutes by the appearance of the test band . the assay is straight - forward with no other chemicals or reagents needed to perform the test . the following sections describe the experimental approach designed to address three specific aims : ( 1 ) to optimize the assay of the present invention by signal amplification methods to achieve the most sensitive results ; ( 2 ) to determine assay specificity by studying false positive and false negative results in the small pilot study ; and ( 3 ) to validate the assay with parallel serum and saliva elisa measures of antibody levels in a large subject population . assay sensitivity can be developed by testing serial dilutions of commercially available anti - pa and anti - ava antibodies with the lateral - flow device of the present invention . antibody standards can be prepared in saliva and in buffered saline to study the possibility of mucin interference with the assay . antigen selection and antibody detection methods are critical to maximize assay sensitivity and specificity as indicated by a colorimetric marker . factors investigated for optimization of assay conditions include sample preparation , antigen form , various conjugated secondary antibody markers to amplify detection and methods to facilitate antibody capture by membrane - bound antigen . testing and evaluation can lead to a determination of assay specificity to reduce false positive and false negative responses from a range of prepared standards . positive and negative specimen results in the rapid test method can be compared with results of elisa for identical samples . elisa test conditions can be optimized for saliva using appropriate antigen and antibody detection systems to give enhanced responses for specific and total igg . based on these results , various aspects of the assay can be refined . a testing protocol can be developed , including large - scale specimen collection and testing at an established anthrax immunization program site . in a further phase in the development of the present invention , serum and saliva samples can be collected from a large number of military personnel and tested in parallel elisa studies to compare antibody titers and to validate the salivary diagnostic method with quantitative and statistically supported data . the sensitivity and specificity of the assay of the present invention can be evaluated by making serial dilutions of a solution spiked with available polyclonal igg antibodies specific for pa toxin protein . bioport corporation &# 39 ; s goat anti - ava igg1 , 4 . 3 mg / ml in 50 mm 4 -( 2 - hydroxyethyl )- 1 - piperazineethanesulfonic acid ( hepes ) buffer , ph 7 . 0 , can be made to 1 μg / μl concentration in pbs . serial dilutions of this polyclonal antibody can be prepared in pbs , ph 7 . 4 , at dilutions ranging from 1 : 250 to 1 : 64 , 000 . each dilution can be prepared by taking a 1 ml aliquot of the previous solution and diluting 1 : 1 with fresh pbs . the complete progression of antibody dilutions can be 1 : 250 , 1 : 500 , 1 : 1000 , 1 : 2000 , 1 : 4000 , 1 : 8000 , 1 : 16 , 000 , 1 : 32 , 000 and 1 : 64 , 000 . these dilutions can be tested with the assay 10 of the present invention to verify positive reaction results for pa - specific antibodies at least through 1 : 16 , 000 , or about 10 ng protein , and evaluated for dose - response reactions in the indicator test line . test results indicated detection of goat anti - ava igg1 antibody to less than 5 ng . the same dilutions of goat anti - ava igg1 antibody can be made in saliva to test whether mucin interferes with the assay . dilutions of 1 : 500 , 1 : 2000 and 1 : 8000 can also be made by adding the appropriate amount of antibody to saliva that is already diluted 1 : 1 with pbs containing 10 mm edta and 0 . 1 % sodium azide . from the initial 1 : 500 standard , subsequent 1 : 4 dilutions of each saliva solution can be made with pbs . the edta has been shown to minimize mucin viscosity and thereby facilitate saliva testing . this example confirmed that the edta buffer does not affect the specific igg antibody nor does it interfere with antigen - antibody binding . also , in duplicate solutions of these same three saliva antibody dilutions , 1 : 500 , 1 : 2000 and 1 : 8000 , sigma ™ protease inhibitor cocktail can be added 1 : 20 immediately after the solution is made and tested against the spiked saliva sample with no protease inhibitor . these side - by - side samples can be tested at + 15 minutes , 60 minutes , 3 hours and the following day to examine any possible degradation effects of oral bacterial enzymes on the anti - pa antibody . these solutions can be tested with the diagnostic assay and elisa to 1 ) identify limits of detection and 2 ) develop a quantitative standard curve in which dose - response relationships from the elisa assays can be used to evaluate the assay method . a goal of the present invention is to assess anthrax vaccine immune status . protective antigen is the antigen of choice for assay development , supported by a large body of literature indicating it is the antigenic component of the anthrax toxin . for anthrax , the entire antigen - antibody specificity issue then distills down to a single protein and the igg antibodies made against pa , the main component of the ava vaccine . also , various techniques have been employed in other laboratories to enhance expression of pa and thereby increase the relative antibody titer produced against it . techniques to amplify visual detection of antigen - antibody binding can be investigated . preparation of a dendrimer or polymer tree , immobilized on the nitrocellulose with pa protein or reactive peptides bound to branches , may increase accessibility of the antigen and enhance antibody capture . another method of signal amplification to study is using different secondary antibody - conjugate detection systems to find the most sensitive procedure for this assay . an example of one conjugate label is protein a conjugated with colloidal gold to provide a visual detection of specific antibody binding . however , if necessary , the enzymes alkaline phosphatase , horseradish peroxidase and biotin - streptavidin conjugated to a secondary antibody may be used to offer more sensitive probes that will enhance the detection of antigen - antibody complex formation . false positive and false negative tests can be studied to determine specificity of the assay . this determination can be made using salivary standards of known dilutions of goat anti - ava antibody and samples collected from subject volunteers in the pilot study ( to ensure a sufficient number of both positive and negative trials ). reproducibility of test results for these salivary standards and samples will determine the rate of false positives or negatives . results using the immunoassay method and kit of the present invention can be compared with elisa values obtained for the same samples or specimens . specificity determinations can be conducted with 3 repetitions of each standard or sample and elisa comparative analysis . a false negative is defined as a test that only shows the control ( c ) band when other assay test repetitions of this same sample indicate positive with both the control and test bands clearly visible . a false negative determination can be supported by positive elisa results of this same sample indicating the presence of anti - pa antibody above negative control values . conversely , a false positive is defined as visual detection of both test and control bands , but further test repetitions of this sample indicate negative and / or elisa results for this sample are less than those obtained from the lowest detectable anti - pa antibody dilution . preliminary specificity studies were initiated with saliva samples collected from five study volunteers , a , b , c , d , and e . three of the five volunteers , a , b , and c , had not received any of the ava series , and the saliva samples from these three volunteers all tested negative for anthrax igg antibodies using the assay of the present invention . thus , the present invention confirmed that these three volunteers had not been immunized for anthrax . the other two volunteers , d and e , had received at least one of the ava series . volunteer d had received 4 of the ava series with the last injection occurring about 36 months prior to the test . the saliva sample from volunteer d tested negative for anthrax igg antibodies using the assay of the present invention . volunteer e had received three of the ava series , with the last injection occurring about 19 months prior to the test . the saliva sample for volunteer e indicated a positive test in two different dilutions of saliva with dilution buffer . however , when this test was repeated 1½ - 2 hours later , the same diluted saliva sample , and a second dilution made in fresh dilution buffer , indicated negative . although the saliva sample from volunteer e was placed on ice immediately after collection , it was concluded that protease activity of the saliva may have degraded the anti - pa antibody in the 1 - 2 hour interim between the first and second test . further tests can be conducted by adding signa ™ protease inhibitor cocktail 1 : 10 or 1 : 20 to saliva samples just after collection and the test repeated for the same individual . elisa analysis can also be performed for these saliva samples to verify antibody presence . it is also possible to use stimulated saliva samples in which protease activity , if present , should be minimized during collection ( as described below ). saliva samples can be collected from study volunteers in three ways : volunteers initially can provide unstimulated saliva by allowing available saliva to drip into a small polypropylene collection cup over 3 - 5 minutes . according to a preferred embodiment a polypropylene collection cup or container may be used . it is within the scope of the invention to use a pliable material for the container as described in column three of u . s . pat . no . 5 , 922 , 614 , which is expressly incorporated herein by reference thereto . volunteers can be given a single piece of sugarless peppermint chewing gum and asked to chew for 30 seconds before collecting samples of stimulated saliva . typically , volunteer subjects provide sample volumes of about 2 - 4 ml for unstimulated saliva , while a volume of 5 - 8 ml is most common after chewing a piece of gum . alternatively , an absorbent element may be rubbed along the gum line for a short period of time , preferably up to thirty seconds , then the absorbent element may be held in place along the gums for a longer period of time , preferably up to two minutes . u . s . pat . no . 5 , 830 , 410 , which is expressly incorporated herein by reference thereto , more fully describes this method in columns six and seven and also describes a representative collection device in fig1 a and 1b . the pliable material collection cup or container may be placed around the oral - fluid - saturated absorbent element and deformed or squeezed to extract the oral fluid . each of these samples can be placed on ice immediately after collection to ensure stability . to preserve saliva samples for storage and biological assay in accordance with the present invention , a mycobacteriocidal protease inhibitor solution may sometimes be appropriate . for example , a general protease inhibitor cocktail ( premade sigma ™ protease inhibitor cocktail p2714 ) can be added to each sample 1 : 20 to prevent protein degradation from oral bacterial enzymes . these samples can then be returned to the laboratory for testing . a protease inhibitor cocktail can be provided as 100 × lyophilized powder . the protease inhibitor cocktail can be reconstituted to 10 × with barnstead still quality water [ 10 ml ]. at these working concentrations , the protease inhibitor solution can be diluted 10 × for proteolytic inhibition . saliva specimens can be tested immediately for presence of anti - pa antibodies . samples can then be placed into graduated conical tubes , their volume recorded , and finally placed into frozen storage at − 80 ° c . to await elisa comparative testing for anti - pa igg determinations . serum and saliva specimens can be collected from a large population of volunteers ( phase ii of the study ) at distant clinics or medical treatment facilities with an established anthrax vaccination program . collection of these test samples can be in cooperation with trained medical personnel . saliva samples can be collected in small polypropylene collection cups as previously described , and transferred into graduated , conical plastic tubes for frozen storage and shipment . the tubes provided for saliva shipment can have protease inhibitor preweighed into each tube to give about a 1 : 20 concentration in an average sample volume of 5 ml . blood samples can be drawn by trained corpsmen or other medical staff , centrifuged to precipitate red blood cells , and the supernatant collected in graduated tubes for immediate frozen storage . only sequential sample numbers will identify collected samples , with volunteers &# 39 ; social security numbers and other pertinent data recorded in the database of study participants . in accordance with the protection of patient subjects testing protocol , all personal identifying information will be destroyed at the conclusion of the study . assay lateral flow of the present invention and elisa assays can be performed with serum and saliva samples in a double - blind controlled study so that the results of the two assays with each sample type may not be matched against each other until the data are collated and analyzed . the present standard for measuring antibody titer is elisa , a very reliable , but relatively time - consuming and labor - intensive process . highly trained laboratory personnel usually perform these determinations , and the instrumentation required is relatively sophisticated and expensive . while elisa has been the laboratory standard for measuring antibody response , it is more problematic to perform this diagnostic test in forward - deployed regions where personnel are at greater potential risk for anthrax toxin exposure . development of a rapid , economical test procedure that can be performed by minimally trained personnel will provide a field test option to verify protective immunization . serum elisa procedures are well established in the literature and in this laboratory . procedures for saliva elisa analyses must be developed and optimized for anti - pa igg antibody detection . saliva testing procedures previously conducted in this laboratory involve saliva samples diluted 1 : 5 with 1 / 10 × saliva dilution buffer ( pbs with 10 mm edta and 0 . 1 % azide ). about 350 ng sample of rpa , ( reported binding capacity of immulon 4 microtiter plates ), can be used to coat the 96 - well polystyrene plates ( immulon 4 , dynatech ™ incorporated ), which are incubated for 1 hour at 37 ° c ., followed by blocking with 1 % bovine serum albumin ( bsa ) and 0 . 5 % tween 20tm in pbs . specific igg anti - pa antibody derived from a saliva sample diluted 1 : 5 with 1 / 10 × saliva dilution buffer can be incubated with the antigen at 37 ° c . for a minimum of 60 minutes . the plate can be developed with goat anti - patient igg1 ( estimated 1 : 500 dilution ) secondary antibody conjugated with alkaline phosphatase for substrate detection . a sample of 85 % purified recombinant pa in tris buffer ( tris ( hydroxymethyl ) aminomethane ) was provided by bioport corporation , lansing , mich ., manufacturer of the u . s . anthrax vaccine . briefly , the recombinant protein is made by transforming avirulent bacillus anthracis bh441 cells ( pxo1 − and pxo2 − ) with a pa expression vector containing the pag gene . this shuttle vector then directs recombinant pa antigen over - expression using the replicating mechanisms of escherichia coli host bacteria . the recombinant protein is purified from the culture supernatant using phenyl sepharose , a hydrophobic interaction column , followed by q sepharose , anion - exchange chromatography . the final product is then dialyzed into tris buffer at ph 7 . 2 . upon receipt , the purity of the rpa protein ( 83 kda ) was verified by gel electrophoresis and immunoblotting with monoclonal anti - pa igg1 and polyclonal anti - ava igg1 antibodies . a phs study protocol has been written and submitted for approval by the command institutional review board ( irb ) to obtain authorization to recruit volunteer subjects for the pilot study . in accordance with this protocol , local area active duty personnel and reservists who have had at least one of the ava injections can be recruited for participation in phase i according to the procedures outlined in the phs protocol . potential study volunteers can be contacted through the naval hospital great lakes immunization clinic , the naval reserve center , great lakes , and the armed forces dental research facility , great lakes . also , a brief notice of the study and request for volunteers can be published in general health and fitness mailings to area active duty and reserve personnel , and in the naval training center , great lakes bulletin , a weekly base newspaper . the local immunization clinic begins the ava vaccine series for personnel who have received orders for deployment to foreign areas considered to be at greatest risk for possible anthrax exposure . phase i of the salivary diagnostic study has received approval from the irb , guided by appropriate federal regulations at 45 cfr 46 and other requirements governing the use of patient subjects in research protocols . at least one study assistant can review the testing procedures and purpose of the study with individual volunteers , obtain signed permission to collect a sample on an informed consent form , and obtain authorization to take personal data as it relates directly to the study in a signed privacy act statement . as testing progresses for the pilot study , a phs protocol can be written and submitted for review by the command irb and the review committee of our collaborating laboratory ( ies ) to obtain authorization to recruit volunteer subjects for phase ii of this study . study volunteers can be asked how many of the six ava injections they have received and how long it has been since their last immunization . this information will be recorded in the subject database to compare with observed results . study volunteers can be grouped , if possible , based upon the number of ava injections they have completed . individual medical records can be reviewed only to confirm self - declared immunization history and any medical conditions that could potentially impact the outcome of the diagnostic test . a statistician can consult with the principal investigator on experimental design to ensure appropriate open and blind controls are incorporated into the study . the mechanisms of the assay system can be developed so that the assay can provide sensitivity and specificity of at least 90 % based on final tests of 150 negative and 150 positive samples . using serum and saliva samples from subjects with and without ava immunizations , the saliva test method can be optimized so that , compared to gold - standard serum methods ( elisa ), the assay of the present invention can have sensitivity and specificity of at least 80 % that of serum methods using 90 % one - tailed confidence limits . some preliminary work has been done with pa peptide fragments that may form some of the antigenic sites within the 735 amino acid toxin ( excluding the 29 amino acid n - terminal signal sequence that is cleaved upon activation ). specific peptide fragments were selected for study based on demonstrated antigenic reactions to anti - pa antibodies in the literature and on very hydrophilic values obtained through application of the data to the hopp - woods plot . hopp , t . p ., and woods , k . r . ( 1981 ) prediction of protein antigenic determinants from amino acid sequences . proc natl acad sci usa 78 , 3824 - 3828 . little and others described antigenic reactions of a series of polyclonal and monoclonal antibodies to three specific pa amino acid sequences : ( 1 ) in the furin - sensitive ( protease ) region ; ( 2 ) the ef / lf factor binding region ; and ( 3 ) the c - terminal membrane - binding domain . little , s . f ., novak , j . m ., lowe , j . r ., leppla , s . h ., singh , y ., klimpel , k . r ., lidgerding , b . c ., and friedlander , a . m . ( 1996 ) characterization of lethal factor binding and cell receptor binding domains of protective antigen of bacillus anthracis using monoclonal antibodies . microbiology 142 , 707 - 715 . antibody binding to each of these antigenic regions neutralized toxin activity by preventing pa activation or membrane recognition and binding of pa to the host cell . the rationale for using small peptide fragments is that it may be possible to better define the antigenic sites of pa critical to anthrax toxicity . a more complete understanding of pa binding domains and formation of the antigen - antibody complex will help elucidate mechanisms of toxin action and its inhibition . in preliminary findings , five pa peptide fragments of 3000 approximate mass have been identified that may have antigenic activity to anti - pa antibodies . of these peptides , two have proven to be antigenic by dot blot procedures . these active fragments are : a 26 - mer of amino acids # 576 - 601 that is a part of the ef / lf binding site of pa ; and a 29 - mer , amino acids # 707 - 735 at the c - terminal end where the cell membrane receptor recognition and binding site is located . further study may further define the binding properties of these synthetic antigenic reaction sites . the term patient used herein includes humans , as well as animals . thus , the present invention can be used for diagnostics for veterinary tests . obviously , many modifications and variations of the present invention are possible in light of the above teaching . it is therefore to be understood that , within the scope of the appended claims , the invention may be practiced otherwise than as specifically described . the principles described above can be readily modified or adapted for various applications without departing from the generic concept , and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the enclosed embodiments . it is to be understood that the terminology and phraseology herein is for the purpose of description and not of limitation .