Patent Application: US-58445103-A

Abstract:
the present invention provides compounds and methods for the detection of anti - leishmanial antibodies in individuals suspected of infection with the protozoan parasite of the genus leishmania , where the infectious agent is an indian strain and similar or closely related to indian leishmania strains . the compounds provided include polypeptides as shown in seq id no : 5 or seq id no : 6 which are useful for the detection of anti - leishmanial antibodies in individuals where the immune responses are elicited against leishmania species of indian strains and similar or closely related to indian leishmania strains , the compounds are also useful as a vaccine and therapeutic agent to prevent and treat leishmaniasis . the present invention further provides a diagnostic kit consisting of antibody raised against polypeptides as shown in seq id no : 5 or seq id no : 6 for detecting leishmanial antigens .

Description:
the present invention provides a recombinant antigen developed from the indian isolates of l . donovani , which is a variant of existing recombinant kinesin related antigen ( rk39 ) for diagnosing vl in india and other geographical areas where l . donovani is the causative agent . this recombinant antigen of indian origin varies upto 60 % in the predicted amino acid sequence at the immunodominant repeat epitope to that of the rk39 . recently , the kinesin related antigen gene has been cloned and characterized from l . chagasi an , american visceralising species by burns et al . ; this led to the development of rk39 antigen , a recombinant protein with 39 amino acid tandem repeats . this above [ burns et al ., 1993 ] antigen from a new world visceralising species l . chagasi is reported not sensitive in some highly endemic regions for vl , sudan and southern europe [ zijlstra , e . e ., 2001 ; jelinek t ., 1999 ] as it is on some other geographical areas , india , bangladesh , nepal etc . india carries the majority of the vl population in the global leishmaniasis burden . so , it is very important to have precise and well characterized tools for the diagnosis and other control measurements in india . the applicants have characterized the kinesin related antigen from two strains namely , a ) mhom / in / dd8 a who reference strain originally obtained from a kala - azar patients from bihar , india and b ) mhom / in / ke16 / 1998 obtained from a 10 year old female kala - azar patient from musafarpur , bihar , india of l . donovani at the sequence level to study , whether the sequence is similar to that one of l . chagasi and to rule out the suspicion that , are some cases are going unnoticed in india as in sudan ? also it becomes very important to know that , are the various ethnic groups responding differently to the rk39 antigen or it is the sequence divergence at the immunodominant region that causes the difference in sensitivity . in this invention we found that , the sequence of kinesin antigen / polypeptide significantly differs from that of the published l . chagasi from which the rk39 was derived . in the present invention we have also raised antibodies against the kinesin antigen / polypeptide and utilized it for the detection of leishmanial antigens . parasites were initially isolated as promastigotes in nnn medium from clinical samples of kala - azar patients and subsequently adapted to grow at 25 ° c . in medium 199 containing 10 % heat inactivated fcs . for routine maintenance , samples of the inoculum containing parasites were introduced aseptically into culture tubes with 4 ml of medium 199 supplemented with 10 % fcs . the tubes were placed in cooled incubator at 25 ° c . and the growth was monitored at regular intervals by microscopy . for mass cultivation of the parasite , samples of inoculum containing parasites were introduced aseptically into 200 ml of m199 containing 10 % fcs in a 500 ml tissue culture flask and incubated in a cooled incubator at 25 ° c . until mid log phase ( 7 - 10 days ). the parasites were then harvested and used for nuclear dna isolation . the parasites in their mid log phase was harvested by centrifuging at 5000 rpm in a refrigerated centrifuge . parasite nuclear dna was isolated following standard protocol with minor modifications [ lu h . g . et al ., 1994 ]. approximately 1 - 5 × 10 9 promastigotes were lysed in 10 volumes of lysis buffer ( nacl , 100 mm , tris - hcl , 10 mm ( ph 8 . 0 ), edta 10 mm , proteinase k / ml 100 μg , sarcosyl 1 . 5 %) at 60 ° c . for 3 hours . the kinetoplast dna networks were sedimented by centrifugation at 27 , 000 × g for 1 hour and resuspended in te buffer ( tris - hcl ( ph 8 . 0 ) 10 mm , edta ( ph 8 . 0 ) 1 mm ). the nuclear dnas were isolated from the supernatants left after sedimentation of the kdnas . these supernatants were incubated overnight for further digestion of proteins at 65 ° c . the nuclear dna was subjected to several cycles of phenol / chloroform extractions by adding equal volume of phenol / chloroform mixture , mixing thoroughly followed by sedimentation by centrifugation at 5000 rpm for 15 minutes . the nuclear dna was precipitated by adding 1 / 10 th the volume of 3m - sodium acetate and 2 volumes of 100 % ethanol mixed well and incubated at − 20 ° c . for 1 hour . the mixture was sedimented by centrifugation at 5000 rpm for 30 minutes at 4 ° c . the pellet was washed with 70 % ethanol , dried and resuspended in te buffer . the concentration and purity of the dna was measured by taking od at 260 / 280 nm . the dna was stored at − 70 ° c . until use . the pcr for the amplification of kinesin related antigen was performed as below using 50 ng of the isolated nuclear dna and using the following primers in various combinations to amplify the overlapping parts in the kinesin gene . the primes were designed based on the sequence data from the genbank for the kinesin gene ( accession no . l07879 ). the available kinesin gene sequence from l . chagasi is 3319 bp in length and has an orf from position 454 with a putative atg starting codon and extends until the last base at position 3319 . this gene has 5 ′ non repeat region from the starting codon at position 2563 and a conserved immunodominant repeat domain from base 2564 to till the end in the 3 ′. the repetitive epitope of 39 amino acid is part of the kinesin gene at the 3 ′ end from the base position 2564 to 3319 . this repetitive epitope has 117 bp tandem repeat at 6 . 5 copies reported and extending upto 3 to 4 kb in the 3 ′ end . the highly conserved short stretches of sequences at various positions were considered for primer designing . the whole 3 . 33 kb gene of the l . chagasi was considered and we attempted to amplify the open reading frame ( orf ) from the position 454 to 3319 bp . the main focus being on the immunodominant tandem repeats of 117 bp size ; however we attempted to amplify and characterize complete orf with as many repeats as possible . three forward and two reverse primers were designed to amplify this gene detailed as follows ; the primer lkf93 ( seq id no : 7 ) is upstream to the predicted starting codon at 455 bp and it covered 362 bp 5 ′ to the starting of orf . the primer lkf1527 ( seq id no : 9 ) is another forward primer , which starts from the position at the base 1527 from the base one i . e . it covers 1073 bases from the beginning of the orf . the third forward primer is lkf 2564 ( seq id no : 10 ) that is intended to amplify the immunodominant tandem repeats in combination with the reverse primer lkr3266 ( seq id no : 11 ). the first reverse primer is lkr 1803 ( seq id no : 8 ), which includes an overlapping region of 276 bp with that of the second primer set with another reverse primer lkr3266 ( seq id no : 11 ). the primer sequences are given in the seq id no : 7 to seq id no : 11 . the first pcr primer set lkf 93 ( seq id no : 7 ) and lkr 1803 ( seq id no : 8 ) amplified a 1 . 71 kb fragment from the position at 93 base at the 5 ′ end of the kinesin gene to the position 1803 bp in the 3 ′ position . the second pcr primer set lkf 1527 ( seq id no : 9 ) and lkr 3266 ( seq id no : 11 ) amplified a 1 . 15 kb fragment with one 117 bp tandem repeat . this pcr amplicons had 276 bp overlapping nucleotides at the 5 ′ end with that of first pcr product amplified . the third pcr primer set lkf 2564 ( seq id no : 10 ) and lkr 3266 ( seq id no : 11 ) amplified the immunodominant 117 tandem repeat region . this third primer set amplified a fragment of size approximately 470 bp corresponding the four 117 bp tandem repeat for mhom / in / ke16 / 1998 and approximately 590 bp size product corresponding five immunodominant tandem repeat for the strain mhom / in / dd8 . all the pcr products were purified as described below and cloned in pgemte cloning vector and sequenced in an automated dna sequencer . by using aerosol free pipette tips and keeping the pre - and post - pcr products separately , amplicons carry over contamination was avoided . all pcr reactions were performed using standard protocols ( sambrook et al ., 1989 ) with a set of negative controls . the tubes were kept in thermal cycler ( mj research , usa ) and incubated at 95 ° c . for 5 minutes followed by 35 cycles of amplification . the amplified pcr products were resolved on agarose gel electrophoresis . the gel was visualized under ultraviolet transilluminator ( uvp ) and photographed using a polaroid camera . the kinesin gene following amplification by pcr was cloned in a ta cloning vector as below . the pcr amplified dna were resolved on agarose gel and the portion containing the band of interest was excised with a sterile scalpel . the dna was eluted from the gel using gel elution kit ( qiagen , germany ) following the manufacturer &# 39 ; s protocol . concentration of eluted dna was measured by absorbance at 260 nm in spectrophotometer . the gel purified pcr product of interest was ligated directly in pgemt - easy vector . in a 1 . 5 ml micro centrifuge tube the following components were added . after mixing gently , the tubes were incubated at 4 ° c . overnight and heated for 10 minutes at 70 ° c . the samples were stored at − 20 ° c . until transformation . the ligated mixture was then transformed by heat sock treatment . the competent cells were prepared by using calcium chloride method [ sambrook et al ., 1989 ]. a single colony of e . coli was inoculated in 5 ml of lb medium and incubated at 37 ° c . overnight with shaking ( 200 rpm ). next day fresh stock of 100 ml lb medium was inoculated with 1 ml of the overnight culture and incubated at 37 ° c . with continuous shaking until the o . d . reached 0 . 6 at 600 nm . the culture was chilled on ice for 30 minutes and the cells were harvested by centrifugation at 4000 g , 4 ° c . for 10 min . the cell pellet was resuspended in 1 / 10 th volume of ice cold filtered sterile 50 mm cacl 2 and kept on ice for 30 min . the cells were pelleted down and finally resuspended in 1 / 25 th volume of ice - cold 50 mm cacl 2 ( with 20 % v / v of autoclaved glycerol ) and either stored at − 70 ° c . in aliquots of 200 μl or used immediately for transformation . approximately 5 μl of ligation mixture was gently mixed with competent cell ( 200 μl ) and incubated in ice for 30 min . after incubation the cells were placed in water bath set at 42 ° c . for 90 seconds ( heat shock ) and immediately transferred to ice . 800 μl of lb medium was added to the cells and kept at 37 ° c . for 90 minutes with shaking ( 150 rpm ). the cells were plated with 16 μl of x - gal and 10 μl of 1m iptg on lb agar plates containing 50 μg / ml of ampicillin . the plates were incubated at 37 ° c . for 12 - 16 hours . the white colonies were selected and checked for the insert . for screening , the plasmid was isolated by rapid boiling method [ holmes and quigley , 1981 ]. the colonies were picked up with sterile toothpick and inoculated in 5 ml of fresh medium . the cultures grown overnight were pelleted and the cells were resuspended in 500 μl of stet buffer ( sucrose 8 % ( w / v ), tris - hcl , ( ph 8 . 0 ), 50 mm , edta na 2 , ( ph 8 . 0 ) 50 mm , triton - x 100 5 % ( w / v ) followed by lysis with 40 μl of lysozyme . the mixture was vortexed well and incubated in a boiling water bath for 90 seconds . the bacterial debris and chromosomal dna were removed by centrifugation at 12000 g for 10 minutes . the supernatant was mixed with 400 μl of isopropanol and 200 μl of 7 . 5 m ammonium acetate . the plasmid dna was pelleted by centrifugation . the dried pellet was resuspended in 100 μl of sterile water , mixed with 50 μl of 7 . 5 m ammonium acetate and incubated on ice for 30 minutes . the samples were centrifuged at 4 ° c . for 10 minutes and plasmid dna was precipitated with ethanol . the pure dna pellet obtained after centrifugation was washed twice with 70 % ethanol dried and dissolved in 50 μl of sterile water . the restriction analysis of the recombinant plasmid was done using appropriate restriction enzyme sites flanking the multiple cloning site of the vectors . the reaction was set as follows : the reaction was incubated at 37 ° c . for 6 - 8 hours and the products were analyzed on 1 . 5 % agarose gel along with standard molecular weight markers . the positive clones containing the insert as detected by the restriction digestion were used for sequencing and preserved as glycerol stock . all the sequencing was done by chain termination method [ sanger et al ., 1977 ] in an automated dna sequencer , abi prism version 7 . 0 . the sequences were analyzed using various software like dnasis , lasergene : edit ; megalign etc ( dna star inc . ), clustal w ( multiple alignment of various sequence files ). all the sequences obtained were aligned to form continuous stretch by using seqman ii ( lasergene package ). along with this , the sequence was searched for homology by using blast [ altschul et al ., 1997 ] option from various websites . the fall - length kinesin sequence obtained after assembling the sequence was presented in the seq id no : 1 for the strain mhom / in / dd8 , it was found to be 3016 bp in length with an open reading frame of 2670 bp . the length of the sequence for the strain mhom / in / ke16 / 1998 was found to be 2937 bp with a long open reading frame of 2577 bp ( seq id no : 2 ). the sequence analysis revealed that the size of the pcr product amplifying the immunodominant region was 563 bp for the strain mhom / in / dd8 ( fig1 ) corresponding to 4 . 8 tandem repeats of 117 bp corresponding to 4 . 8 units of 39 amino acid repeats and 466 bp for the strain mhom / in / ke16 / 1998 ( fig2 ) corresponding to 4 tandem repeats of 117 bp ( 4 units of 39 amino acid repeats ). further , the sequence analysis shows significant variation in both at dna level as well as in the predicted amino acid sequence from that of the l . chagasi sequence . the strain mhom / in / dd8 shows enormous variation from that of l . chagasi as well as from the strain mhom / in / ke16 / 1998 . the blast search for predicted protein sequence of the strain mhom / in / dd8 ( seq id no : 5 ) using non - redundant and patent division of genbank database reveals only up to 38 % identity with that of published l . chagasi and us patent nos . i ) u . s . pat . no . 5 , 411 , 865 and ii ) u . s . pat . no . 5 , 912 , 166 respectively fig3 . the clustal w multiple sequence alignment of the immunodominant repeat region at the nucleotide level for the l . donovani strains mhom / in / dd8 and mhom / in / ke16 / 1998 ( seq id no : 3 and seq id no : 4 ) with that of the immunodominant repeat region of l . chagasi was done and presented in the fig4 . the identities are shadowed dark . the multiple sequence alignment of the immunodominant repeat region at the amino acid level between l . donovani ( seq id no : 5 and seq id no : 6 ) l . chagasi is presented in fig5 . multiple sequence alignment of the immunodominant repeat region at the amino acid level for the l . donovani strain mhom / in / dd8 ( seq id no : 5 ) with that of the immunodominant repeat region of l . chagasi is presented in the fig6 . sequence alignments of the immunodominant repeat region at the amino acid level for the two l . donovani strains mhom / in / dd8 and mhom / in / ke16 / 1998 ( seq id no : 5 and seq id no : 6 ) is presented in fig7 . immunodominant 39 amino acid repeats unit with intra repeat variation for the indian isolates of l . donovani , strains mhom / in / dd8 and mhom / in / ke16 / 1998 ( seq id no : 5 and seq id no : 6 ) are presented in fig8 and fig9 . the sequence analysis reveals that , the amino acids varies enormously having many substitution both conserved and variable at many positions in the 39 amino acid sequence reported for l . chagasi . at least five amino acids for l . donovani are different to l . chagasi at positions 18 , 27 , 29 , 32 and 38 indicated by “*” in the table 1 below , after analyzing the sequences insilico we found a significant variation in the amino acid sequence at the immunodominant region of the strain mhom / in / dd8 with that of the l . chagasi . then , to confirm whether the sequence variation causes any effect on the performance of the antigen to detect anti leishmanial antibodies , we expressed the immunodominant region from the two indian leishmania donovani isolates in e . coli expression vector prset ( invitrogen ) as 6 ×- his tagged protein . we have expressed various combinations of the immunodominant repeat like one , two , three , four , 4 . 8 units and one repeat with the 889 nucleotides upstream to the immunodominant domain . we found that recombinant peptides with four and 4 . 8 repeat units having higher sensitivity and specificity . thus , our work was focused on polypeptides containing 4 . 0 and 4 . 8 repeat units of 39 amino acids . the e . coli expression vector prset c ( invitrogen , netherlands ) was completely digested with psti and ncoi restriction enzymes at 37 ° c . overnight . the digested plasmid was fractionated on agarose gel to remove the stuffer fragment . the plasmid band was excised and eluted by qiagen gel elution system and stored in − 20 ° c . until further use . the insert was released from the recombinant pgem - te plasmid carrying the immunodominant tandem repeats ( pgem - teasy / dd8 / lkf2564 and pgem - teasy / ke16 / lkf2564 ) by digesting it with psti and ncoi . the inserts was excised from the gel , eluted by qiagen gel elution system and ligated in psti and ncoi digested prset c vector in - frame and transformed to bl21 competent cells by the standard protocol [ sambrook et al ., 1989 ]. the clones were selected on the ampicillin plates and plasmid dna was isolated and digested with psti and ncoi to check for the insert . the clones , which released the insert , were selected for protein induction . then , a single recombinant e . coli ( bl 21 ) colony containing the insert was inoculated in 2 ml of sob containing ampicillin ( 50 μg / ml ) and incubated overnight at 37 ° c . with shaking ( 225 rpm ). the next day , 25 ml of sob was inoculated with the overnight culture and allowed to grow at 37 ° c . with shaking to an od 600 of 0 . 6 . removed 1 ml aliquot of cells , centrifuged and frozen in − 20 ° c . to the culture added iptg to a final concentration of 1 mm and incubated at 37 ° c . with shaking . a time course of expression to determine the optimal induction time for maximum expression of protein was done by taking aliquot of cells at 1 , 2 , 3 , 4 & amp ; 5 hours after induction with iptg and analyzed by sds - page and western blot . further , to determine the protein solubility , a single recombinant e . coli ( bl 21 ) colony containing the insert was inoculated in 2 ml of sob containing ampicillin ( 50 μg / ml ) and incubated overnight at 37 ° c . with shaking ( 225 rpm ). the next day , 25 ml of sob was inoculated with the overnight culture and allowed to grow at 37 ° c . with shaking to an od 600 of 0 . 6 . removed 1 ml aliquot of cells , centrifuged and frozen in − 20 ° c . to the culture added iptg to a final concentration of 1 mm and incubated at 37 ° c . with shaking for additional 4 hours . the cells were harvested by centrifugation at 4000 × g for 20 minutes and the pellet was resuspended lysis buffer for purification under native conditions ( qiagen , germany ) and lysed by sonication , 6 × 10 s with 10 s pauses at 200 - 300 w . the lysate was centrifuged in a refrigerated centrifuge for 10 minutes at 10 , 000 × g . the supernatant was transferred to a fresh tube and the pellet was resuspended in lysis buffer and preserved on ice . the pellet and the supernatant was mixed with 2 × sds - page sample buffers separately and analyzed by using 12 % sds - page and western blot . the results show that the protein is detectable in the soluble fraction and thus can be purified under native conditions . the maximum protein - producing clones were inoculated in 20 ml of sob containing 100 μg / ml ampicillin and grew overnight at 37 ° c . with vigorous shaking . the next day , 1 liter of sob was inoculated with 1 : 50 noninduced overnight culture and allowed to grow at 37 ° c . with shaking until an od 600 of 0 . 6 is reached . a 5 ml aliquot of culture was taken immediately before induction . the cells were induced by adding iptg to a final concentration of 1 mm and continued incubation at 37 ° c . for 4 hours . the cells were harvested by centrifugation at 4000 × g for 20 minutes and stored in − 20 ° c . until purification . the recombinant protein was purified using ni - nta agarose column ( qiagen , germany ) tinder native conditions following the manufacturer &# 39 ; s protocol . the cell pellets were thawed for 15 minutes on ice and resuspended in lysis buffer at 5 ml per gram wet weight . to this lysozyme was added at 1 mg / ml and incubated for 30 minutes on ice and sonicated on ice using six 10 s bursts at 200 - 300 w with a 10 s cooling period between each burst . the lysate was centrifuged at 10 , 000 × g for 30 minutes at 4 ° c . to sediment the cellular debris . the supernatant was saved . all aliquot of 50 μl supernatant was taken and stored in − 20 ° c . for analysis later . added 1 ml of the 50 % ni - nta slurry to 4 ml of cleared lysate and mixed gently by shaking ( 200 rpm on a rotary shaker ) at 4 ° c . for 60 minutes . the lysate - ni - nta mixture was loaded on to a column with the bottom outlet capped . the flow - throw was collected after removing the bottom cap and the column was washed twice with 4 ml of wash buffer and the fractions were collected for sds - page analysis . the protein was eluted 4 times with 0 . 5 mm of elution buffer and the eluate was collected in 4 tubes and analyzed by sds - page . the protein expression after purification was found to be 4 mg / liter . for analysis of the expressed protein , mini gel electrophoresis unit ( bangalore genei , india ) was used . the sds - page was carried out as per defined protocols [ laemmeli , 1970 ]. a 10 % resolving gel was prepared by mixing the following components : 2 . 66 ml water , 1 . 562 ml 30 % acrylamide , 1 . 250 ml 1 . 5m tris - hcl , 50 μl 10 % aps , 10 % temed . the gel top was with ˜ 400 μl of water saturated butanol and allowed to polymerize . after polymerization , butanol was drained off and washed with distilled water . then , 2 ml of stacking gel mix ( 1 . 25 ml water , 250 μl 30 % acrylamide , 500 μl 0 . 5m tris - hcl , 30 μl 10 % aps and 5 μl temed ) was poured carefully avoiding any air bubble on the top of the resolving gel . the comb was inserted in the stacking gel portion and left for polymerization . following the polymerization the wells were washed with excess amount of water . the samples were applied to the defined wells in the stacking gel and electrophoresis carried out at a constant voltage ( 60v for stacking and 100v for resolving gel ). standard molecular protein markers ( mbi ) and control ( vector protein ) were run alongside the samples . the completed gel was then stained in coomassie brilliant blue stain , destained and visualized against white light . the purified protein from the strain mhom / in / dd8 migrated at a size of 29 kda in correlation to the predicted molecular weight and the predicted molecular weight for the strain mhom / in / ke16 / 1998 was 26 kda but the purified protein from this strain had aberrant page mobility and was migrating at higher molecular weight ˜ 32 kda ( fig1 a ). the reported antigens are entirely different from the leishmania chagasi 230 kda antigen . the antigen varies significantly , more than 60 % in its predicted amino acid sequence to that of k39 repeat antigen from l . chagasi . also disclosed are dna encoding the 29 kd and 26 kd antigen and therapeutic and vaccine compositions comprising the antigens . purified recombinant polypeptides from the strains mhom / in / dd8 and mhom / in / ke16 / 1998 was used for immunization in rabbits . 100 micro gram of each polypeptide was emulsified with equal volume of freund &# 39 ; s complete adjuvant ( sigma , usa ). the mixture was injected intradermally at multiple sites . three booster doses of the same amount of antigen emulsified with incomplete freund &# 39 ; s adjuvant were given at 30 - day intervals . prior to the first immunization , preimmune serum was collected and tested on western blots . blood was collected aseptically from the immunized animals 12 days after the last booster , sera was separated and stored on − 70 ° c . until use . the e . coli expressed protein was resolved on to 12 % sds - page followed by electro transfer to nitrocellulose membrane [ towbin et al ., 1976 ]. the transfer was carried out using transfer buffer ( tris base 3 . 08 g , glycine 14 . 66 g , methanol 200 ml , water to the final volume of 1000 ml ) in bio - rad semi dry transfer unit . for transfer , 15v was applied and allowed to run for 45 minutes at room temperature . after transfer the membrane was put in a blocking solution containing 2 . 5 % bsa in phosphate buffered saline ( pbs , nacl 8 . 00 g , kcl 0 . 20 g , nah 2 po 4 1 . 44 g , kh 2 po 4 0 . 24 g , ph 7 . 4 ). the blocked membrane was washed three times with pbs containing 0 . 02 % tween - 20 ( pbs - t ). the membrane was incubated with kala - azar patient serum ( 1 : 200 diluted in pbs ) for 1 hour at 25 ° c . further it was washed 4 times in pbs - t and incubated in 1 : 4000 diluted anti - human igg antibodies , conjugated with alkaline phosphatase for 45 minutes at room temperature . after several washes with pbs , the membrane was developed by addition of bcip - nbt ( amresco , usa ). the purified antigens were first run on sds - page and the subjected to immunoblot with penta - anti - his hrp conjugate antibody ( fig1 a and 10 b ). to study the antigenicity of the purified proteins , sds - page followed by immunoblot was carried out with kala - azar patient &# 39 ; s sera ( fig1 a and 11 b ) and immunoblot with pkdl patient &# 39 ; s sera ( fig1 a and 12 b ) followed by western with pooled ( n = 5 ) healthy individuals sera as control was done and presented in fig1 a and 13 b . also , immunoblot with only ap - labelled anti - human secondary antibody was done as shown in fig1 a and 14 b . the results of the immunoblot and sequence analysis reveal that , the recombinant antigens from l . donovani are specific in spite of having different epitope . after protein estimation by bca method ( sigma , the purified antigen was taken for evaluation by elisa . the elisa was standardized initially at different parameters with appropriate serum and reagent controls . the elisa at 50 ng / well and 1 : 100 dilutions of sera was chosen optimal and used the same conditions for elisa with two antigens from l . donovani viz . mhom / in / dd8 and mhom / in / ke16 / 1998 . in each plate one positive control ( parasitologically and serologically positive for rk39 ) and one negative control with sera from non - endemic region another with reagent control was included . the plates were coated as below ; polystyrene micro titer plates with 96 flat - bottom wells were coated with antigen following standard protocol with minor modifications [ singh s et al ., 1995 ] by adding 50 ng of purified kinesin antigen in 200 μl of 0 . 1m bicarbonate buffer , ph 9 . 2 . the plates were covered and incubated overnight at 4 ° c . the antigen solution was then removed and plates were washed 3 times in pbst ( pbs with 0 . 05 % tween 20 ). the wells were then blocked with 200 μl of 1 % bsa for 1 hour , washed 3 times with pbst . the plates were dried at room temperature , sealed , and stored at 4 ° c . until use . the sensitized plates were incubated for 2 hours with 50 μl of patient serum diluted from 1 : 100 to the end point in pbst . the wells were washed again with pbst and incubated with 50 μl of goat anti - human igg conjugated with alkaline phosphatase at 10 − 3 dilution for another 2 hour , followed by washing 3 times with pbst . after incubation for 30 minutes at 37 ° c . with 50 μl of p - nitrophenylphosphate in diethylamine buffer , the reaction was stopped with 50 μl of 3n naoh . the optical density of each well was measured at 450 nm in a tritrius plate reader . the antibody titers to the recombinant protein from mhom / in / dd8 ( seq id no : 5 ) were considerably lower to that of mhom / in / ke16 / 1998 ( seq id no : 6 ) for the same group of samples . the preliminary studies with 72 endemic and 80 non - endemic healthy controls , 92 tuberculosis positive , 32 hiv positive , 31 hcv positive and 27 hbsag positive sera showed no cross reactivity . however , the rk39 positive previously confirmed cases of 10 vl and 10 pkdl sera show high antibody titer with both the antigens at 50 ng / well concentration . the data show that , these two well - characterized antigens from the indian strains will be highly useful for the kala - azar diagnosis in india . the fig1 depicts the mean titer value of different samples subjected to elisa using purified antigen of mhom / in / dd8 origin and the fig1 show the mean titer value of different set of samples for the purified antigen of mhom / in / ke16 / 1998 origin . purified recombinant polypeptides from the strains seq id no : 5 and seq id no : 6 were used for immunization in rabbits . 100 micro gram of each polypeptide was emulsified with equal volume of freund &# 39 ; s complete adjuvant ( sigma , usa ). the mixture was injected intradermally at multiple sites . three booster doses of the same amount of antigen emulsified with incomplete freund &# 39 ; s adjuvant were given at 30 - day intervals . prior to the first immunization , preimmune serum was collected and tested on western blots . blood was collected aseptically from the immunized animals 12 days after the last booster , sera were separated and the antibody present in the sera was affinity purified and stored at 4 ° c . until use . polystyrene micro titer plates with 96 wells were coated by diluting the antibody stored at 4 ° c . in coating buffer ( ph 9 . 2 ) [ 1 . 59 g sodium carbonate ( na 2 co 3 ), 2 . 93 g sodium bicarbonate ( nahco 3 ), 0 . 20 g sodium azide ( nan 3 ), dissolved in 900 ml h 2 o , adjusted ph to 9 . 6 with hcl and make up to 1 l ] and added 200 μl to each well of a microtitre plate . the plates were incubated at 37 ° c . for 4 h and washed with pbs - tween , three times . the plates dried by tapping upside down on tissue paper . the antibody coated to the solid support then was used for the detection of leishmania antigen this method called a double antibody or sandwich elisa , and was performed as below . to each well added 200 μl aliquots of the test sample and incubated for 4 - hours at 37 ° c . the plates are washed three times with pbst and then incubated with antibody conjugate and incubated at 37 ° c . for 2 hours washed three times with pbst and added 200 μl aliquots of substrate to each well and incubated at room temperature for 60 min . the optical density of each well was measured at 405 nm in a tritrius ® plate reader . the sandwich elisa measures the amount of antigen between two layers of antibodies . the leishmania antigen to be measured contains at least two antigenic sites , capable of binding to antibody , since at least two antibodies act in the sandwich . so sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides . sandwich elisa for quantitation of antigens are especially valuable when the concentration of antigens is low and / or they are contained in high concentrations of contaminating protein the following examples illustrate the invention , which should not be construed to limit the scope of the invention . this example illustrates the cloning of kinesin immunodominant repeat region from indian isolates of l . donovani . a pcr cloning strategy was followed using a primer set lkf 2564 and lkr 3266 ( seq id no : 10 and seq id no : 11 respectively ) to amplify the immunodominant 117 tandem repeat region . this primer set amplified a fragment of size approximately 470 bp corresponding to the four 117 bp tandem repeat from l . donovani strain mhom / in / ke16 / 1998 and approximately 590 bp size product corresponding five immunodominant tandem repeat from l . donovani strain mhom / in / dd8 . the kinesin gene following amplification by pcr was cloned in a ta cloning vector as below . the pcr amplified dna were resolved on agarose gel and the portion containing the band of interest was excised with a sterile scalpel . the dna was eluted from the gel using gel elution kit ( qiagen , germany ) following the manufacturer &# 39 ; s protocol . concentration of eluted dna was measured by absorbance at 260 nm in spectrophotometer . the gel purified pcr product of interest was ligated directly in pgemt - easy vector ( promega , usa ). in a 1 . 5 ml micro centrifuge tube the following components were added . pgem - t easy ( 100 ng / μl ) 1 . 00 μl 10 × ligation buffer 1 . 00 μl dna ( 200 ng ) 5 . 00 μl t4 dna ligase ( 2 u / μl ) 1 . 00 μl water to make the volume upto 10 . 00 μl after mixing gently , the tubes were incubated at 4 ° c . overnight and heated for 10 minutes at 70 ° c . the samples were stored at − 20 ° c . until transformation . the ligated mixture was then transformed by heat sock treatment . the competent cells were prepared by using calcium chloride method [ sambrook et al ., 1989 ]. a single colony of e . coli was inoculated in 5 ml of lb medium and incubated at 37 ° c . overnight with shaking ( 200 rpm ). next day fresh stock of 100 ml lb medium was inoculated with 1 ml of the overnight culture and incubated at 37 ° c . with continuous shaking until the o . d . reached 0 . 6 at 600 nm . the culture was chilled on ice for 30 minutes and the cells were harvested by centrifugation at 4000 g , 4 ° c . for 10 min . the cell pellet was resuspended in 1 / 10 th volume of ice cold filtered sterile 50 mm cacl 2 and kept on ice for 30 min . the cells were pelleted down and finally resuspended in 1 / 25 th volume of ice - cold 50 mm cacl 2 ( with 20 % v / v of autoclaved glycerol ) and either stored at − 70 ° c . in aliquots of 200 μl or used immediately for transformation . approximately 5 μl of ligation mixture was gently mixed with competent cell ( 200 μl ) and incubated in ice for 30 min . after incubation the cells were placed in water bath set at 42 ° c . for 90 seconds ( heat shock ) and immediately transferred to ice . 800 μl of lb medium was added to the cells and kept at 37 ° c . for 90 minutes with shaking ( 150 rpm ). the cells were plated with 16 μl of x - gal and 10 μl of 1m iptg on lb agar plates containing 50 μg / ml of ampicillin . the plates were incubated at 37 ° c . for 12 - 16 hours . the white colonies were selected and checked for the insert . the recombinant clones were further characterized by restriction mapping and sequencing . all the sequencing was done by chain termination method [ sanger et al ., 1977 ] in an automated dna sequencer , abi prism version 7 . 0 . the sequences were analyzed using various software like dnasis , lasergene : edit ; megalign etc ( dna star inc . ), clustal w ( multiple alignment of various sequence files ). all the sequences obtained were aligned to form continuous stretch by using seqman ii ( lasergene package ). along with this , the sequence was searched for homology by using blast [ altschul et al ., 1997 ] option from various websites . the full - length kinesin sequence obtained after assembling the sequence was presented in the seq id no : 1 for the strain mhom / in / dd8 , it was found to be 3016 bp in length with an open reading frame of 2670 bp . the length of the sequence for the strain mhom / in / ke16 / 1998 was found to be 2937 bp with a long open reading frame of 2577 bp ( seq id no : 2 ). the sequence analysis revealed that the size of the pcr product amplifying the immunodominant region was 563 bp for the strain mhom / in / dd8 ( fig1 ) corresponding to 4 . 8 tandem repeats of 117 bp and 466 bp for the strain mhom / in / ke16 / 1998 ( fig2 ) corresponding to 4 tandem repeats of 117 bp . further , the sequence analysis shows significant variation in both at dna level as well as in the predicted amino acid sequence from that of the reported l . chagasi sequence ( genbank , accession no . l07879 ). the strain mhom / in / dd8 shows significant variation from that of l . chagasi as well as from the strain mhom / in / ke16 / 1998 . the blast search for predicted protein sequence of the strain mhom / in / dd8 ( seq id no : 5 ) using non - redundant and patent division of genbank database reveals only up to 38 % identity with that of published l . chagasi and us patent nos . i ) u . s . pat . no . 5 , 411 , 865 and ii ) u . s . pat . no . 5 , 912 , 166 respectively fig3 . the clustal w multiple sequence alignment of the immunodominant repeat region at the nucleotide level for the l . donovani strains mhom / in / dd8 and mhom / in / ke16 / 1998 ( seq id no : 3 and seq id no : 4 ) with that of the immunodominant repeat region of l . chagasi ( genbank , accession no . l07879 ) was done and presented in the fig4 . the identities are shadowed dark . the multiple sequence alignment of the immunodominant repeat region at the amino acid level between l . donovani ( seq id no : 5 and seq id no : 6 ) and l . chagasi is presented in fig5 . multiple sequence alignment of the immunodominant repeat region at the amino acid level for the l . donovani strain mhom / in / dd8 ( seq id no : 5 ) with that of the immunodominant repeat region of l . chagasi is presented in the fig6 . sequence alignments of the immunodominant repeat region at the amino acid level for the two l . donovani strains mhom / in / dd8 and mhom / in / ke16 / 1998 ( seq id no : 5 and seq id no : 6 ) is presented in fig7 . immunodominant 39 amino acid repeats unit with intra repeat variation for the indian isolates of l . donovani , strains mhom / in / dd8 and mhom / in / ke16 / 1998 ( seq id no : 5 and seq id : 6 ) are presented in fig8 and fig9 . the sequence analysis reveals that , the amino acids vary significantly having many substitutions . these substitutions are conserved or are variable at many positions in the 39 amino acid repeated region reported for l . chagasi . at least five amino acids for l . donovani are different to l . chagasi at positions 18 , 27 , 29 , 32 and 38 indicated by “*” in the table 1 . this example illustrates the reactivity of patient sera to recognize the recombinant l . donovani antigens . the insert was released from the recombinant pgem - te plasmid carrying the immunodominant tandem repeats ( pgem - teasy / dd8 / lkf2564 and pgem - teasy / ke16 / lkf2564 ) by digesting it with psti and ncoi . the inserts was excised from the gel , eluted by qiagen gel elution system and ligated in psti and ncoi digested prset c vector in - frame and transformed to bl21 competent cells by the standard protocol [ sambrook et al ., 1989 ]. the clones were selected on the ampicillin plates and plasmid dna was isolated and digested with psti and ncoi to check for the insert . the clones , which released the insert , were selected for protein expression . the maximum protein - producing clones were inoculated in 20 ml of sob medium containing 100 μg / ml ampicillin and grown overnight at 37 ° c . with vigorous shaking . the next day , 1 liter of sob medium was inoculated with 1 : 50 noninduced overnight culture and allowed to grow at 37 ° c . with shaking until an od 600 of 0 . 6 is reached . a 5 ml aliquot of culture was taken immediately before induction . the cells were induced by adding iptg to a final concentration of 1 mm and continued incubation at 37 ° c . for 4 hours . the cells were harvested by centrifugation at 4000 × g for 20 minutes and stored in − 20 ° c . until purification . the recombinant polypeptide was purified using ni - nta agarose column ( qiagen , germany ) under native conditions following the manufacturer &# 39 ; s protocol . the purified protein from the strain mhom / in / dd8 migrated at a size of 29 kda ( fig1 a , lane 2 ) that correlates with the predicted molecular weight . the purified antigens were first run on sds - page ( fig1 a , 11 a , 12 a and 13 a ) and the subjected to immunoblot with penta - anti - his hrp conjugate antibody ( fig1 b ), kala - azar patient &# 39 ; s sera ( fig1 b ), pkdl patient &# 39 ; s sera ( fig1 b ) followed by western with pooled ( n = 5 ) healthy individuals sera ( fig1 b ), with only ap - labelled anti - human secondary antibody ( fig1 b ) were done . the results of the immunoblot and sequence analysis reveal that , the recombinant antigens from l . donovani are specific in spite of having different epitope . this example illustrates the reactivity of patient sera to recognize the l . donovani antigens . protein estimation was done by bca method ( sigma , usa ) and the purified antigens were used for elisa . the elisa was standardized initially at different parameters with appropriate serum and reagent controls . the elisa at 50 ng / well and 1 : 100 dilutions of sera was chosen optimal and used the same conditions for elisa with two recombinant polypeptides from l . donovani viz . mhom / in / dd8 ( seq id no : 5 ) and mhom / in / ke16 / 1998 ( seq id no : 6 ). in each plate one positive control ( parasitologically and serologically positive for rk39 ) and one negative control with sera from non - endemic region and another with reagent control was included . the plates were coated as below ; polystyrene micro titer plates with 96 flat - bottom wells were coated with antigen following the protocol as reported by singh s et al ., [ 2002 ] with minor modifications by adding 50 ng of purified kinesin antigen in 200 μl of 0 . 1m bicarbonate buffer , ph 9 . 2 . the plates were covered and incubated overnight at 4 ° c . the antigen solution was then removed and plates were washed 3 times in pbst ( pbs with 0 . 05 % tween - 20 ). the wells were then blocked with 200 μl of 1 % bsa for 1 hour , washed 3 times with pbst . the plates were dried at room temperature , sealed , and stored at 4 ° c . until use . the sensitized plates were incubated for 2 hours with 50 μl of patient serum diluted from 1 : 100 to the end point in pbst . the wells were washed thrice with pbst and incubated with 50 μl of goat anti - human igg conjugated with alkaline phosphatase at 10 − 3 dilution for another 2 hour , followed by washing 3 times with pbst . after incubation for 30 minutes at 37 ° c . with 50 μl of p - nitrophenylphosphate in diethylamine buffer , the reaction was stopped with 50 μl of 3n naoh . the optical density of each well was measured at 450 nm in a tritrius ® plate reader . the antibody titers to the recombinant protein from mhom / in / dd8 ( seq id no : 5 ) were considerably lower to that of mhom / in / ke16 / 1998 ( seq id no : 6 ) for the same group of samples . the preliminary study carried out with sera from 72 endemic and 80 non - endemic healthy controls , 92 tuberculosis positive , 32 hiv positive , 31 hcv positive and 27 hbsag positive individuals . these elisa results showed 100 % specificity with no cross reactivity . however , elisa carried out for confirmed vl ( n = 10 ) and pkdl ( n = 10 ) patient &# 39 ; s sera with rk39 antigen from l . chagasi ( burns et al ., 1993 ), l . donovani antigen from the strains mhom / in / dd8 ( seq id no : 5 ) and mhom / in / ke16 / 1998 ( seq id no : 6 ) show high antibody titer for all three antigens at 50 ng / well concentration . the data show that , these two well - characterized antigens from the indian strains are highly specific and sensitive for the kala - azar diagnosis in india . in a study conducted with more samples it was found that considerable number of cases that could not be diagnosed by rk39 of l . chagasi was detected by these two recombinant polypeptides from l . donovani . this reveals that , the newly isolated recombinant polypeptides from indian strains of l . donovani are more specific for the diagnosis of vl and pkdl in india thus they can be used for diagnosis of this disease extensively . however , only the data pertaining to 10 positive samples and other control samples are presented in fig1 and fig1 . the fig1 depicts the mean titer value of different samples subjected to elisa using purified antigen of mhom / in / dd8 origin and the fig1 show the mean titer value of different set of samples for the purified antigen of mhom / in / ke16 / 1998 origin . this example teaches obtaining antibody against the polypeptides seq id no : 5 or seq id no ; 6 from an animal and its use in the detection of leishmania antibody as below ; 1 . purified recombinant polypeptides from the group containing seq id no : 5 and seq id no : 6 were used for immunization in rabbits . 100 micro gram of the polypeptide was emulsified with equal volume of freund &# 39 ; s complete adjuvant ( sigma , usa ). 2 . the mixture was injected intradermally at multiple sites . 3 . three booster doses of the same amount of antigen emulsified with incomplete freund &# 39 ; s adjuvant were given at 30 - day intervals . prior to the first immunization , preimmune serum was collected and tested on western blots . 4 . blood was collected aseptically from the immunized animals 12 days after the last booster , sera were separated and the anti - antibody present in the sera was affinity purified and stored at 4 ° c . until use . 5 . the antibody was first diluted in 0 . 1m bicarbonate buffer , ph 9 . 2 and then 200 μl are added to each well of the microtiter plate . 6 . the antibody coated plate was covered with paraffin and incubated in the cold room overnight in a moist box containing a wet paper towel or at room temperature and humidity for two hours . 7 . the plate is emptied and the unoccupied sites are blocked with 100 μl of blocking buffer containing 100 mm phosphate buffer , ph 7 . 2 , 1 % bsa , 0 . 5 % tween - 20 and 0 . 02 % thimerosol for 30 min at room temperature . 8 . the plate is emptied and washed three times with wash buffer ( 100 mm phosphate buffer , 150 mm nacl , 0 . 2 % bsa and 0 . 05 % tween 20 ). 9 . the antigen solution is first diluted in antigen buffer ( 100 mm phosphate buffer , 150 mm nacl ) and then added to the plate in a volume of 50 μl per well . the plate is incubated at room temperature for 45 min to one hour . 10 . the plate is emptied again and washed three times with wash buffer . 11 . the enzyme - labeled antibody against antigen is diluted appropriately in 0 . 1m bicarbonate buffer , ph 9 . 2 and then 50 μl is added to each well and incubated at room temperature for 30 min . 12 . the plate is emptied again and washed three times with wash buffer . 13 . the color development system is added and the color intensities are measured . the characterization of the kinesin related antigen at the sequence level has revealed the similarities and differences for the kinesin antigen between two species l . donovani and l . chagasi . it is clear from this invention that the immunodominant repeat of kinesin related antigen isolated from indian strains is different from the earlier reported sequences of other leishmania species . this study revealed that the indian strains are quite different from the earlier reported strains and species from other geographical regions . the study also revealed that amino acid sequence variation between the repeat units of the indian strains is very high than for previously reported sequences . this invention has led to the isolation of polypeptide , which is different in sequence from that of earlier reported polypeptide sequence . this invention has led to the development of a method for detection of antileishmanial antibodies based on the newly found antigens from indian isolates of l . donovani . this invention has led to the leishmaniasis detection kit for the detection of vl due to species of l . donovani of india and other similar species which would have been missed by k39 polypeptide 1 . altschul , s . f ., madden , t . l ., schaffer , a . a ., zhang , z ., miller , w . and lipman , d . 1997 . gapped blast and psi - 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