Patent Application: US-6103602-A

Abstract:
an assay method and kit is disclosed for detecting the presence of at least one predesignated , target antibody to a mycobacterium in a sample selected from one or more patient bodily fluids . the method comprises the following steps : contacting the sample of one or more patient bodily fluids with at least one mycobacterium antigen on a lateral - flow assay membrane to bind to the target antibody in the sample ; previously , simultaneously or subsequently to step , binding the at least one mycobacterium antigen with a conjugated label producing a detectable signal ; and detecting the signal whereby the presence of the target antibody is determined in the sample by the intensity or presence of the signal . the method can further comprise the step of evaluating immunization status of the patient from whom the sample came by comparing the signal or lack thereof with immunizations previously received by the patient and in comparison to a known standard control . in a preferred embodiment , the mycobacterium antigen specifically binds to mycobacterium tuberculosis specific antibodies . preferably , the immunoassay of the present invention comprises a lateral - flow assay comprising a membrane , a conjugated label pad , and at least one mycobacterium antigen bound to the membrane . in a preferred embodiment , the at least one mycobacterium antigen is selected from the group consisting of 38 kda and 16 kda antigens .

Description:
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 lateral - flow assay 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 body fluid specimen ( 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 having a diameter size in the range of about 20 - 55 nm , and more preferably in the range of about 40 - 45 nm , serve as an indicator dye . the conjugate label binds to antibodies in the sample to form a complex , and the complex then migrates along the membrane 18 . in a preferred embodiment , mtb specific antibodies bind to a mixture of at least two antigens that are immobilized in a discrete location 32 on membrane 18 . formation of this antigen mixture - antibody complex causes the indicator dye to precipitate and form a detectable colored red line ( illustrated as “ t 1 ” in fig1 ), indicating a positive result that mtb 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 . in a preferred embodiment , the mixture of at least two antigens comprises two antigen proteins , the 38 kda ( also known as antigen 5 , antigen 78 , pab , and phos ) and the 16 kda antigens . recombinant proteins of the 38 kda , 16 kda , esat - 6 , or cfp - 10 may also be used instead of mtb culture products . an assay is recorded as positive when a distinct band of the antigen mixture ( t 1 on the assay 10 in fig1 ) appears in addition to the control band . a control line ( c ) of protein a will also form whether the antigen mixture line is colored 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 ( more than 6 months ) at room temperature with no loss of activity . the testing device can comprise a mixture of 0 . 72 or 1 . 8 μg recombinant 38 kda and 16 kda antigen proteins and / or other antigens such as esat - 6 , mpt - 63 , cfp - 10 , tb23 hyt6 , f29 . 47 , 21 - 2h3 , mpt40 and others , immobilized on nitrocellulose strips as the test indicator ( t 1 ) and protein a as the control band ( c ). since the 38 kda and the 16 kda antigen proteins are only found in mtb , a positive test is a highly specific indicator for mtb exposure . in a preferred embodiment , the assay 10 of the present invention can have a single test stripe ( t 1 ). as shown in fig1 and 2 , the assay 10 of the present invention can further comprise a second test stripe t 2 , which can be located between t 1 and the control stripe c . in a preferred embodiment , the second test stripe t 2 can comprise an antigen mixture of shared common mycobacterial antigens ( such as p32 of m . bovis , 65 or 64 kda bcg antigen , mpb57 or bcg - a , lam , and others ), and stripe t 1 can be comprised of 16 kda protein only as a measure of latent tb . while two test stripes t 1 and t 2 are shown in fig1 and 2 , as noted above , assay 10 can have a single test stripe , as shown in fig3 . in accordance with the present invention , assay 10 can comprise more than two stripes to test for the presence of additional targeted antibodies in a sample selected from one or more patient bodily fluids . experience with saliva sample testing has shown that 4 drops of saliva and / or oral mucosal transudate and gingival crevicular fluid mixed with 2 drops of buffer , 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 - 50 μl volume . therefore , 4 drops or 160 - 200 μl sample volumes can be used for each test . four drops ( 160 - 200 μl ) of stimulated saliva mixed with 2 drops ( 80 - 100 μl ) of protease inhibitor / edta dilution buffer also appears to facilitate sample flow . in a preferred embodiment , 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 tb 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 tb in the person . if the sample tests negative , and the sample came from a person who has received a tb 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 a tb vaccine , then the positive result will indicate that the person has been exposed to mtb . if the sample tests negative , and the sample came from a person who has not received a tb vaccine , then the negative result will indicate that the person has not been exposed to mtb , particularly if there are no other indications or symptoms of tb 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 and in fluid communication with the sample pad 14 , and a rectangular detection window 34 is located above the nitrocellulose membrane 18 . thus , the present invention provides a rapid , simple , immunoassay test ( two versions 1 = non - invasive using saliva , and 2 = invasive using diluted serum ) for tuberculosis . the present invention provides a rapid test method for simultaneously determining the presence of antibodies in saliva ( or serum ) to mycobacterium tuberculosis ( mtb ) and other mycobacteria . the method is fast ( usually 5 minutes or less ) and technically easy to perform . maekura et al . ( 3 ) noted that the antibody titers to a mycobacterial antigen declined to normal levels as a result of antituberculosis chemotherapy . therefore , an additional objective of the invention is to monitor prognosis following conventional mtb antibiotic therapy . the rapid lateral flow devices of the present invention comprise a combination of striped antigens that will simultaneously determine exposure to both specific ( mtb and m . bovis ) and general mycobacterial antigens . the present invention provides a salivary diagnostic method and assay to supplant and / or complement invasive procedures such as the ppd skin test and their corresponding slow and labor intensive methods . using saliva or sera in accordance with the immunoassay of the present invention would aid in the diagnosis of tuberculosis . using saliva as a source of specific antibodies directed at tb - related antigens is a new and unique technological approach in the diagnosis of tb . the method of the present invention preferably tests either saliva specimens or diluted serum . saliva is preferred as the antibody source since whole saliva is the most easily collected of available specimens and generally mirrors serum antibody contents . pre - filtered or unfiltered saliva may be used . if patient serum is used , a loopful ( 5 μl ) can be diluted with 6 drops ( 240 - 300 μl ) of a diluent and will be aliquoted and mixed with the diluent . the diluted sample can be added dropwise to a lateral - flow device using a transfer pipette . either a single test stripe or at least two antigen test stripes in addition to a positive control stripe can be present in the device . salivary samples and serum can be collected from patients known to be infected with mtb according to positive culture results , af staining and radiographic results . the lateral flow device test concentrations are optimized by setting sensitivity thresholds using positive clinical samples and known negative controls . a patient sample can be added to the sample well . a pad containing protein a colloidal gold comes in contact with the flowing sample . as the samples flow from the application spot , the first test stripe can contain a single antigen or a mixture of two or more antigens ( e . g ., the 38 kda plus the 16 kda antigens )( recombinant proteins may be used instead of mtb culture products ). since these antigens are only found in m . tuberculosis , a positive result is highly specific for mtb exposure . the second stripe ( if present ) encountered by the flowing sample will contain either a single antigen or a mixture of non - specific mycobacterial antigens . in other words , the second stripe can comprise a shared mycobacterial antigen common to all mycobacteria or a mixture of such antigens . it is believed that the position of the two test stripes is important . if the second stripe is positive , it indicates that the patient has been exposed to any mycobacteria ( including mtb ) or has sero - converted in response to ppd skin tests . the patient sample antibodies first bind by their fragment crystallizable ( fc ) portion to the protein - a colloidal gold label reagent . a positive result is visualized as a red stripe , which indicates where an antigen antibody reaction has occurred and has complexed along with the protein - a - colloidal gold label ( or other suitable label ). if the second stripe only is positive , the patient has been exposed to a non - mtb mycobacterium . if both test stripes are positive , or only the first test stripe is positive , then the patient has been exposed or is still infected with mtb . the positive control ( c ) must be positive ( red stripe ) or the test is invalid . this control is accomplished by striping a material that will react with patient sample antibodies as they flow across the c position . results from lateral flow saliva and serum tests are usually available in one to five minutes , and less than twenty minutes , including that from an internal positive control . the method can further comprise the step of evaluating immunization status of the patient from whom the sample came by comparing the signal or lack thereof with immunizations previously received by the patient and in comparison to a known standard control . the assay of the present invention is a rapid , sensitive and specific screening device . it can be suitable for field and shipboard use by ancillary medical personnel . no electrical equipment is required to perform the method of the present invention . 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 ]. table of working concentrations in protease inhibitor solution edta 10 mm aebsf 20 mm bestatin 1300 μm e - 64 14 μm leupeptin 10 μm aprotinin 3 μm at these working concentrations , the protease inhibitor solution can be diluted 10 × for proteolytic inhibition . 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 . 1 . wilkinson r . j ., haslov , k ., rappuoli , r ., giovannoni , f ., narayanan , p . r ., desai , c . r ., vordermeier , h . m ., paulsen , j ., pasvol , g ., ivanyi , j ., and singh , m . evaluation of the recombinant 38 - 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