Patent Application: US-23944405-A

Abstract:
the invention relates to a recombinant dna and polypeptide sequence of an immuno - dominant phage particle associated protein from bartonella bacilliformis . the recombinant protein is easily produced permitting the conduct of more accurate and rapid diagnositic assays for the detection of b . bacilliformis infection with reduced reagent and equipment requirements over that required by currently available methods of diagnosis . the dna and polypeptide sequence is also useful in vaccine preparations against b . bacilliformis .

Description:
we have identified a protein antigen , pap31 , associated with a bacteriophage harbored in bartonella , spp that is an excellent diagnostic laboratory reagent for the laboratory diagnosis of b . bacilliformis infection . the pap31 protein , from the virulent peruvian strain of b . bacilliformis , is found to be a highly expressed antigen in growing cultures of b . bacilliformis . furthermore , the protein is immunologically dominant , making it an ideal antigen for use in enzyme - linked immunosorbent assays ( elisa ) and in other antibody - based assays . the pap31 has been sequenced and a recombinant construct produced thereby permitting easy production , in bulk , with high quality control and allowing the reagent to be stored until required for use . therefore , use of this single protein antigen permits the elisa and western blot - based laboratory methods for the diagnosis of b . bacilliformis that are sensitive and more reliable over currently available procedures . additionally , in addition to its use as a diagnostic reagent , the inventive pap 31 polypeptide reagent is extremely useful either as a subunit vaccine or subunit vaccine component . furthermore , the pap 31 nucleotide sequence is useful as a component in an anti - b . bacilliformis dna vaccine . the recombinant pap31 dna sequence was obtained by sds - page extraction of the american type culture collection ( atcc ) b . bacilliformis strains kc 583 and 584 to form a bacterial cell lysate . lysate proteins were separated by polyacrylamide gel electrophoresis yielding a dominant protein at approximately 36 kda for the kc 583 strain and at 31 kda for the kc 584 strain . dna from these genes was amplified by polymerase chain reaction ( pcr ). under 2 - dimensional ( 2 - d ) gel electrophoresis , 4 to 5 moieties migrate near , but not identically to the pap31 protein . these 2 - d spots were easily discriminated by differences in pi and slight differences in relative molecular weight . pap31 migrated as a band at approximately the same location as the 33 - 31 kda molecular weight standard . strain kc 583 is found to have some repeat units making it slightly larger than the kc 584 strain . n - terminal amino acid sequencing of one spot confirmed the polypeptide as pap31 based on homology with pap31 of b . henselae . in order to obtain b . bacilliformis dna , pcr primers were designed using the kc 583 and 584 dna as templates . the pcr products were subsequently cloned into a pcr2 . 1 vector that was then used to transform escherichia coli . plasmid , containing e . coli colonies , were selected and their dna analyzed and sequenced , using forward and reverse primers from pcr2 . 1 . the dna sequence of pap31 determined by this general strategy is as shown in seq id no . 1 . the polypeptide sequence is as shown in seq id no . 2 . 1 - d and 2 - d gel electrophoresis of whole b . bacilliformis bacterial lysates shows that pap31 represents less than 10 % of total bacterial protein . however , serum from patients , known to be infected with b . bacilliformis , react strongly to pap31 , compared to total protein staining . therefore , the recombinant pap31 polypeptide is an excellent candidate for use as a vaccine or vaccine component against bartonella infection . the pap31 dna construct is also suitable for insertion in an expression system , such as any number of dna expression vectors or viral vectors , as a dna vaccine . expression and purification of the recombinant pap31 polypeptide can be carried out using any of a number of methods and expression systems , including attaching immuno - reactive tags , such as t7 and his , and purifying the expressed product by affinity chromatography . purification of expressed protein is best conducted under denaturing conditions using 2m urea . because pap31 is strongly reactive to serum from b . bacilliformis patients , the recombinant polypeptide or fragments of it are useful in immuno - assays to detect circulating antibody in serum from these patients . the protein would be well suited for use in any antibody - based assay , including elisa and rapid antibody - based assays such as immunochromatographic assays . the recombinant polypeptide is also useful for use in confirmatory western blot analysis . use of these methods , verses that currently employed for b . bacilliformis , this obviates the need for specific detection of bacteria or bacterial proteins greatly enhancing speed of diagnosis and reducing the required infrastructure to support currently available diagnostic methods . currently available methods for the diagnosis of b . bacilliformis are time consuming and require significant infra - structural support . in addition to bacterial isolation and culture from patient samples , the gold - standard immuno - assay currently available for the detection of b . bacilliformis infection is by indirect fluorescence assay ( ifa ). this assay , however , is often not sensitive and can lead to inaccurate results . the assay is also relatively labor and reagent intensive . a typical ifa assay for bartonella diagnosis is conducted by a series of relatively laborious steps including producing b . bacilliformis ( antigen ) by growing the bacteria in vero cell cultures . the antigen is then harvested and irradiated in order to render the material safe . the antigen is drawn up using a hematocrit tube and the material dotted onto to 12 - well slides . twenty - five ( 19 ) microliter dilutions of serum samples , diluted in skim milk diluent , are added to the rows of antigen and incubated for 30 minutes , washed with phosphate buffered saline ( pbs ) and let air - dry . to each well , 25 μl of diluted anti - human fluorescently - labeled antibody is added and incubated at 37 ° c . for 30 minutes , washed with pbs and let air - dry . using a glycerol mounting medium , slides are covered and read within 12 hours under a fluorescent microscope . elisa significantly reduces the labor and time required for ifa diagnosis . use of recombinant pap31 in elisa diagnostic assays is accomplished essentially as follows : 1 . microtiter plates with 96 wells were coated with 0 . 3 μg / well of pap31 and stored in 40 ° c . for 2 days . 2 . plates are washed × 3 with wash buffer ( 0 . 1 % tween - 20 in pbs ). 3 . plates are blocked with 200 μl / well of blocking buffer ( 5 % skim milk in wash buffer ) × 45 minutes and then rinsed three times . 4 . sera is diluted in blocking buffer and 100 μl / well is added and incubated × 2 hours . 5 . plates are washed three times with wash buffer . 6 . plates are then incubated with 100 μl / well of enzyme - labeled ( e . g . peroxidase ) anti - human immunoglobulin for 1 hour . 7 . the plates are washed three times with wash buffer . 8 . substrate is added to the wells and read after 15 to 30 minutes . a standard curve is constructed in conjunction with the above elisa procedure following exposure of the pap31 bound plates to a range of concentrations of a known pap31 - specific antibody . the extent of measured binding of patient serum antibody is compared to a graphic representation of the binding of the pap31 - specific antibody concentrations . an example of elisa results is shown in fig1 . in fig1 , mean and 95 % confidence interval ( ci ) of elisa optical density ( od ) is grouped by ifa results . for the ifa results shown in fig1 , sera with titers greater than 256 were designated as positive . samples labeled as indetermined are those where the sample could not be scored as positive or negative even after repeated testing up to five times by ifa . as shown in fig1 , of the 137 samples tested , 107 were ifa negataive , 29 were ifa positive and one was indetermined . the elisa optical density range ( 95 % confidence interval ( ci )) of the ifa negative samples did not overlap with those of positive ifa samples . the results show that the pap31 reagent incorporated elisa was both highly sensitivity and specific . because the recombinant pap31 is easy to purify , store and able to survive multiple rounds of freeze - thawing cycles without a loss of antigenicity , antibody - based assays using recombinant pap31 will yield more reproducible diagnostic results compared to purified antigen . the inventive polypeptide sequence can also be utilized as capture antigen in rapid antibody - based assays including immunochromographic assays ( 20 ) or other biosensors utilizing metal matrixes ( 21 , 22 ). pap 31 polypeptide is spotted onto a solid matrix strips , such as nitrocellulose . serum from suspected bartonella infected patients is then applied to pads containing anti - human antibody conjugated with colloidal gold or other indicator . the anti - bartonella / anti - human colloidal gold complex migrates up the strip until captured by the bartonella polypeptides . visualization is then made detection of a color change . in assays using metal matrixes for ligand attachment , pap 31 polypeptide as capture antigen is bound to chips or wafers composed of metal thin - films such as silicone . patient serum applied to the chips is then captured by the pap 31 polypeptide . bound antibody is visualized by exposing the serum - exposed chips / wafers to secondary antibody probes . 1 . after sds - page separation of pap31 , the protein is transferred to a solid substrate , such as pvdf membrane ( e . g . 0 . 45 μm pore size ) or nitrocellulose in a buffer consisting of 0 . 025 m tris , 0 . 192 m glycine and 20 % ( vol / vol ) methanol for 1 hour at 400 ma . 2 . unreacted sites are blocked with 5 % milk in tris - borate - saline ( tbs ) with 0 . 2 % tween - 20 overnight . 3 . the membrane is washed twice for 5 minutes each in tbs - tween . 4 . the membrane is then cut into strips . each strip is incubated with a single primary antibody ( sera ) diluted in 5 % ( mass / vol ) milk powder in tbs - tween for 1 hour . 5 . unbound serum are removed by rinsing twice with tbs - tween followed by three rounds of agitation in tbs - tween for five minutes each . 6 . the strips are then incubated with enzyme - labeled anti - human antibody ( e . g . goat anti - human igg ) diluted in 5 % milk powder in tbs - tween for 1 hour on an orbital shaker . 7 . unbound secondary antibody is removed with two rinses in tbs - tween and three successive washes by agitation in tbs - tween for 5 minutes followed by distilled water . 8 . the strips are exposed to substrate and the antibody binding is measured . the assay can be carried out with a strip being used for each serum sample to be tested . a standard curve can be made by conducting the above western blotting procedure following exposure of pap31 bound strips to a range of concentrations of a known pap31 - specific antibody . the extent of measured binding of patient serum antibody is compared to a graphic representation of the binding of the pap31 - specific antibody concentrations that is measured by a number of methods including densitometry . 1 ) groot h , 1951 . human bartonellosis or carrion &# 39 ; s disease . gradwohl r b h , benitez - soto l . felsenfeld o , eds . clinical topical medicine . st . louis , mo . : mosby publishing co ., 615 - 640 . 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( 22 ) jenison , r ., h . la , a . haeberli , r . ostroff and b . polisky . 2001 . silicon - based biosensors for rapid detection of protein or nucleic acid targets . clin . chem . 47 , 1894 . having described the invention , one of skill in the art will appreciate in the appended claims that many modifications and variations of the present invention are possible in light of the above teachings . it is therefore , to be understood that , within the scope of the appended claims , the invention may be practiced otherwise than as specifically described .