WO2016113749A1 - Kit useful for measuring antibodies directed to a non-recombinant membrane antigen (lag) in a urine sample - Google Patents

Abstract

The present invention relates to a dipstick for the non-invasive detection of visceral leishmaniasis (VL) or kala-azar in urine sample. The present invention provides two assays, ELISA and dipstick for non-invasive diagnosis of VL using urine samples. As the assays are urine-based the samples are easier and risk-free in collection and storage. The urine-based ELISA shows 93.47% sensitivity and 100% specificity and can be used for diagnosis of a number of patients simultaneously. The dipstick assay is a urine-based rapid test, 100% sensitive and 100% specific for diagnosis of VL. The dipstick assay is a simple method in which no any sophisticated equipment is required and all the steps of the assay are carried out at room temperature where results can be obtained within 2 hr and can be seen by the naked eye, therefore ideal for field settings.

Description

USEFUL FOR MEASURING ANTIBODIES DIRECTED TO ANON-RECOMBINANT MEMBRANE ANTIGEN (LAG) IN A URINE SAMPLE

FIELD OF THE INVENTION

[001] The present invention relates to a method for detecting non-recombinant membrane antigen (LAg) specific antibodies in a urine sample. Further it relates to a non-invasive detection of visceral l ishmaniasis (VL) or kala-azar in a urine sample. The current invention further relates to a method of developing a nitrocellulose membrane based dipstick kit for diagnosis of VL through urine samples. The said dipstick is 100% sensitive and 100% specific where result can be seen as a colour band to the naked eye. The present application more particularly relates to a method for the detection of antibodies in urine of VL infected subjects using non- recombinani promastigote membrane antigen (LAg) from Leishmania donovani strain AG83. The present invention also relates to a process of an indirect Enzyme Linked Immuno Sorbent Assay (ELISA) for the diagnosis of VL through urine samples.

BACKGROUND OF THE INVENTION

[002] Visceral leishmaniasis (VL) or Kala-azar is one of the severe infectious diseases, which is potentially lethal if not treated in time. Tropical and subtropical regions in about 88 countries world-wide are endemic for VL with 0.2-0.4 million estimated cases annually [1]. India, Bangladesh, Sudan, Brazil, Ethiopia and south Sudan face 90% of all VL burden globally [1]. In the Indian subcontinent and in East Africa, the disease is caused by L. d * onovani and transmitted by the infected sand fly Phleboiomus argentipes. Another species i.e. Leishmania infantum (syn. L chagasi) is the causative agent for VL in Europe, North Africa and Latin America and the vector is Liiizomia longipalpis [2-3]. In spite of complete treatment, VL may also emerge as skin lesions called post kala-azar dermal leishmaniasis (PKDL) [4]. PKDL subjects are a potent reservoir for disease transmission as the skin lesions also contain Leishmania parasites [5-6].

[003] The common symptoms during VL infection are prolonged fever, enlargement of lymph nodes, spleen, and liver, pancytopenia and anaemia [7]. Symptom based diagnosis of VL is challenging because the disease shares many clinical features that are common in diseases like typhoid, malaria, and tuberculosis. Hence, it is desirable to have a confirmatory test for diagnosis of the disease. As the parasite invades the reticulo-endothelial system, detection of the parasite from 52% to 99% among different tissue smears [8]. Moreover, detection of parasite through tissue aspirate is invasive, risky, time taking and labour intensive technique which demands highly skilled practitioner. Apart from parasitology, immunological and molecular diagnoses are also used for VL.

[004] Immunological diagnosis of VL is based on either detection of leishmanial antigens or anti- leishm nial antibodies present in the serum of infected subjects. Direct agglutination test (DAT), enzyme linked immunosorbent assay (ELISA), and strip test are some of the principal antibody detection tests presently in use for diagnosis of VL in different endemic regions [9]. DAT has been routinely used in many regions for VL diagnosis, based on agglutination reaction between parasite antigen and host serum antibody. DAT offers sensitivities and specificities in a range of 1 -100% and 85-100% respectively [10-1 1]. However, differences in antigen quality, long incubation time, need of serial serum dilution, and variations in the cut off titre across the centres limited its applicability.

[005] Detection of serum antibody through ELISA is a frequently used technique for VL diagnosis in many endemic areas. The sensitivity and specificity of ELISA significantly depends on the coated antigen. Crude antigens showed cross reactivity so recombinant antigens have been cloned for VL diagnosis. Recombinant antigens such as heat shock proteins (rHSP83, rGRP78), enzymes (l LACK. rCP), surface proteins (rGP63), and kinesin-related proteins (rK9, rK26, rK28, rKE16 and rK39), have been evaluated for diagnostic potential [12]. Among all, rK39 which is a 39 amino acid peptide was found very appealing for VL diagnosis. The antigen showed good sensitivity and specificity i.e. 67-100% and 93-100% respectively, especially in the Indian subcontinent [13]. However, the procedure of ELISA is lengthy, cumbersome and not applicable for the field settings. To overcome these shortcomings, antibody-capture ELISA has been further evolved into a rapid immunochromatographic strip test in which recombinant antigen is impregnated onto nitrocellulose membrane, such as rK39 strips which is commercially available and extensively used in the Indian subcontinent with sensitivities and specificities ranging from 88 to 100% and 71 to 100%, respectively. The performance is however not up to the mark in East Africa and Brazil where it shows lower sensitivities between 70 and 94% [8].

[006] Several new rapid diagnostic strip tests like r E-16 and r 28 have been developed in the recent years and their performance in different endemic regions are being conducted [14-15]. Previous studies have reported an ELISA and a dipstick for detection of serum antibodies of Indian and Brazilian VL, and PKDL subjects [16]. PCR is another method to diagnose VL where specific sequence of parasite genome is detected [17]. PCR gives very good specificity and sensitivity but the test is subjective and requires a laboratory facility which is not available in field conditions [18].

[007] Atex is the only urine based diagnostic tool commercially available for VL. KAtex is a rapid and field adaptable test which detects 5-20 kDa molecular weight leishmanial antigen in the urine of VL infected subjects [19]. Although KAtex is a non-invasive technique its sensitivity is poor and ranges from 36 to 94% [20-21]. Moreover, to remove false-positives, boiling of the urine sample prior to the assay is required which is an awkward step to do [19], Apart from KAtex, PCR, DAT, ELISA and rK39 strip tests have also been evaluated with fluids other than serum like urine, saliva or buccal swab. DAT has been tested with urine samples and found to be 90.7% sensitive and 96.4% specific [22]. rK39-ELISA was performed in India with saliva of the patients and showed 100% specificity with non-endemic healthy controls but sensitivity was 83.3% only [23]. 18S rRNA-based PCR was evaluated using buccal swab samples of VL infected patients and found 90.56% specificity with only 83% sensitivity [24]. PCR was also evaluated with urine samples and found 100% specificity and 88% sensitivity [25]. Recently, performance of rK39 strips have been evaluated with urine samples in different endemic regions of the Indian subcontinent. In Bangladesh, rK39 strip test with urine showed sensitivity and specificit of 95% and 93.3%, respectively [26]. In India, a study showed that the sensitivity of rK39 strip test with urine samples was 96.4% and specificity ranged from 62.2 to 77.08% [27]. Another study in India with rK39 strips using urine samples showed 96.1% sensitivity and 100% specificity [28]. 100% sensitivity and 86.33% specificity were reported with still another study for the diagnosis of VL in India with rK39 strips using urine [29].

[008] At present, available diagnosis of VL includes risk of invasive tissue aspiration or blood collection. There is only one kit available for VL diagnosis which is urine based (KAtex), but its performance is not encouraging in all endemic areas. The current invention fulfils the need for a non-invasive diagnosis of VL. OBJECTIVES OF THE INVENTION [009] The main object of the present invention is to provide a dipstick for the non-invasive detection of visceral leishmaniasis (VL) in urine sample.

[0010] Another object of the present invention is to provide a method for the detection of antibodies in the urine of VL infected subjects using non-recombinant promastigote membrane antigen (LAg) isolated from Leishmania donovani strain AG83 (ATCC PAR-413).

[0011] Yet another object of the present invention is to provide a process of ELISA to diagnose VL through urine samples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0013] Figure 1 depicts ELISA showing LAg specific antibodies in urine of VL patients with different set of control samples.

[0014] Figure 2 depicts ELISA showing LAg specific antibodies in urine of VL patients and controls at different concentrations of LAg.

[0015] Figure 3 depicts ELISA showing LAg specific antibodies in urine of VL patients and controls at different urine dilutions.

[0016] Figure 4 depicts ELISA showing LAg specific antibody subtypes present in urine of VL patients with different set of controls.

[0017] Figure 5 depicts ELISA showing overall LAg specific antibodies in urine of VL patients with different set of controls.

[0018] Figure 6 depicts dot-blot showing LAg specific IgG reactivity at different concentrations of LAg.

[0019] Figure 7 depicts dot-blot showing LAg specific IgG reactivity at different urine dilutions.

[0020] Figure 8 depicts dot-blot showing LAg specific IgG reactivity at different BSA percentage for blocking. [00211 Figure 9 depicts dot-blot showing LAg specific IgG reactivity from urine of VL patients and different set of control samples.

[0022] Figure 10 depicts dot-blot showing IgG reactivity at different dilutions of anti-human IgG.

[0023] Figure 1 1 depicts dot-blot showing LAg specific IgG reactivity and anti-human IgG reactivity from urine of VL patients and different set of control samples.

[0024] Figure 12 depicts the preparation of the dipstick device.

[0025] Figure 13 depicts the dipstick tests with the urine of VL patients with different set of controls.

[0026] Figure 14 depicts the comparative evaluation of ELISA reactivity of Leishmania antibodies in urine against L Ag at cut-off 0.1815. The study groups were composed of a panel of VL patients (VL; n=76). healthy endemic controls (HEC; n=8), non-endemic healthy controls (NEHC; n=15) and other diseases (OD; n=12) including malaria (n=2), viral fever (n=2), tuberculosis (n=4) and typhoid (n=4). At cut-off 0.1815, 97.37% sensitivity and 100% specificity was determined for 76 VL urine samples and 35 non VL urine samples which included 15 healthy non-endemics, 8 healthy endemics and 12 other symptomatically similar diseases by using a receiver-operator characteristic (ROC) curve in GraphPad Prism software (version 5. (J). The total number of samples used and the overall result of ELISA are summarized in Table 3.

SUMMARY OF INVENTION

[0027] In an aspect of the present invention, there is provided a non-invasive method for the detection of visceral leishmaniasis using a dipstick comprising of: a) about 1.5 μg of leishmanial antigen; and b) about 1 :20 dilution of anti-human IgG antibody, said non-invasive method comprising the steps of: i) providing a subject urine sample having 1 :5 dilution; ii) dipping the dipstick into urine sample obtained in step (i) at room temperature (at least 22°C) for a minimum 30 minutes; iii) washing the dipstick obtained in step (ii) with wash buffers; iv) dipstick obtained in step (iii) is further dipped into a solution having diluted (1 :2000) peroxide conjugated anti-human IgG at room temperature (at least 22°C) for 30 minutes after that remove dipstick from the urine sample and perform washing with known wash buffers; and v) incubation of dipstick obtained in step (iv) with substrate solution to detect visceral leishmaniasis.

|0028] In an aspect of the present invention, there is provided a kit for measuring non- recombinant membrane antigen (LAg) specific antibodies in a urine sample, said kit comprising of nitrocellulose strips pre-coated with said LAg as dried antigen, positive control (optimised urine dilution), enzyme conjugated anti-human IgG, substrate chromogen, and buffers for washing and dilutions, and an instruction manual.

[0029] In an aspect of the present disclosure, there is provided an ELISA to detect antibodies against L. onovani membrane antigen (LAg) in urine.

[0030] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0031]

DETAILED DESCRIPTION OF THE INVENTION

[0032] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions:

[0033] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below. [0034] The term "room temperature" refers to a temperature that is at least 22°C, in the range of 22-28°C.

[0035] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0036] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0037] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0038] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning LIS commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0040] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally- equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0041 ] It has already been documented that in VL infection, large number of polyclonal antibodies are produced by the host immune system against the infecting parasites, specially IgG antibodies [30]. Previously, our lab has described the sero-diagnostic abilities of non- recombinant L. donovani promastigote membrane antigen (LAg) through ELISA, which had a sensitivity of 100% and specificity of 85% [31 ]. Later on this LAg-based ELISA was improved up to 100% sensitive and 96.3% specific for the diagnosis of VL through serum samples [32], Transformation of LAg-based ELISA to dipstick assay has been proved 100% sensitive and equally speci fic for sero-diagnosis of VL and PKDL [16]. [0042] Therefore, the present invention relates to a process of ELISA and dipstick assay to differentiate active VL from healthy individuals and other diseases through urine samples. The present invention is based on detection of LAg-specific antibodies in the urine of subjects affected from VL. Advantage of the urine based assay is that it is a non-invasive technique which is easier and risk free in collection and storage.

[0043] Accordingly, the present invention relates to a method for the non-invasive detection of visceral leishmaniasis (VL) or kala-azar in urine sample. The diagnosis of VL relies largely on risky and painful techniques of detecting parasites in splenic and bone marrow aspirates or invasive sero-diagnostics.

[0044] Moreover the present invention provides two assays ELISA and dipstick for non-invasive diagnosis of VL using urine samples. As the assays are urine-based the samples are easier and risk-free in collection and storage.

[0045] The urine-based ELISA shows 93.47% sensitivity and 100% specificity and can be used for diagnosis of a number of patients simultaneously.

[0046] The dipstick assay is a urine-based rapid test, 100% sensitive and 100% specific for diagnosis of VL.

[0047] The dipstick assay is a simple method in which no any sophisticated equipment is required and all the steps of the assay are carried out at room temperature where results can be obtained within 2 hr and can be seen by the naked eye, therefore ideal for field settings.

[0048] In order to obtain a urine-based ELISA for immunodiagnosis of VL, the earlier published method of ELISA with serum samples was modified and different parameters of the experiment were standardized accordingly.

[0049] The unique aspects of the present invention are coating of adequate LAg onto ELISA plates overnight at 4 C, blocking of excessive reactive sites with bovine serum albumin (BSA) for 2 hr at 37 C, incubation with urine samples at prescribed dilution for 1 hr at 37 C, and subsequently addition of peroxidase-conjugated anti-human IgG at prescribed dilution and then substrate to get the absorbance of anti-leishmanial antibodies present in the urine.

[0050] In the other aspect, in order to develop a urine-based field adaptable immunodiagnostic assay, principles of ELISA was utilised in the dot blot format where polystyrene ELISA plates were replaced with nitrocellulose membrane strips. After standardization of different parameters of dot blot assay dipsticks were developed which is cost effective, easy to use and ideal for field adaptable diagnosis of VL.

[0051] The unique aspects of the dipstick assay are incubation of dipstick with urine samples at prescribed dilution for 30 min at room temperature, addition of peroxidase-conjugated anti- human lgG at prescribed dilution for 30 min at room temperature and subsequently substrate to achieve a colour band as an evidence of anti-laishmanial antibodies present in the urine.

[0052] In EL1SA, 46 confirmed VL patients from Shri Krishna Medical College and Hospital, Muzaffarpur (Bihar, India) and School of Tropical Medicine (Kolkata, India) were tested in these studies. Additionally, 8 non-endemic healthy controls (NEHC), 2 endemic healthy controls (EHC), and 6 other diseases including 2 malaria. 2 viral fever and 2 tuberculosis were also tested to know the cross reactivity of the assay. The mean and standard deviation of NEHC urine gave the cut off value for the assay which was 0.204. Figure 5 shows the X-Y scattered plot of absorbance value to illustrate specific IgG antibody levels in the urine. With respect to LAg- specific IgG antibodies, 3 VL samples gave titre below the cut off line so they are false negative and the sensitivity calculated was 93.47%. All the healthy and negative controls were below the cut off so specificity of the assay was 100% without any false positives. The overall ELISA results are .summarized in Table 01 .

[0053] In the dipstick assay, 50 confirmed VL patients were tested with 8 non-endemic healthy controls. 2 endemic healthy controls (EHC), and 10 other diseases including 2 malaria, 2 viral fever. 2 tuberculosis and 4 typhoid cases. All the VL patients showed clear positive result with two coloured bands where as all the 20 healthy and negative controls gave clear negative result with only one band at the control line. Therefore the dipstick assay is 100% sensitive and 100% specific in our laboratory conditions. The overall dipstick results are summarized in Table 02.

[0054] In an aspect of the present invention, there is provided a non-invasive method for the detection of visceral leishmaniasis using a dipstick as described herein, wherein the substrate solution comprises of 0.05% of 3,3'-diaminobenzedene tetrahydrochloride (DAB) + 0.05% of 30% H₂0₂ in l OOmM TBS.

[0055] In an aspect of the present invention, there is provided a non-invasive method for the detection of visceral leishmaniasis using a dipstick as described herein, wherein detection of visceral leishmania is positive upon appearance two visible colour bands. [0056] In an aspect of the present invention, there is provide a kit as described herein, wherein the nitrocellulose strip comprises of two zones, namely, a sample contact zone of nitrocellulose membrane and a support zone of polyethylene sheet, the sample contact zone further comprising of a test line portion coated with said antigen (LAg) and a control line portion coated with anti- human IgG antibody.

[0057] In an aspect of the present invention, there is provide a kit as described herein, wherein said urine sample is used at a dilution of 1 :5 for the dipstick assay.

[0058] In an aspect of the present invention, there is provide a kit as described herein, wherein said enzyme is horse radish peroxidase conjugated with anti-human IgG at a dilution of 1:2000.

[0059] In an aspect of the present invention, there is provide a kit as described herein, wherein said substrate chromogen is 3,3'-diaminobenzidine tetrahydrochloride (DAB).

[0060] In an aspect of the present invention, there is provide a kit as described herein, wherein said kit is 100% sensitive and 100% specific for diagnosis of VL in field conditions.

[0061] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES

[0062] The disclosure will now be illustrated with working examples, which is intended to illustrate the w orking of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1

Study subjects and collection of urine samples

[0063] Patients with active VL admitted to Shri Krishna Medical College and Hospital, Muzaffarpur (Bihar, India) and School of Tropical Medicine (Kolkata, India) were diagnosed by the usual clinical presentations such as prolonged fever, hepatosplenomegaly, pancytopenia etc. and confirmed by microscopy of Leishmcmia parasites from splenic or bone marrow aspirate and/or rK39 strip test was included in this study. Urine samples of confirmed VL cases, healthy volunteers and patients having disease other than kala-azar (symptomatically similar diseases like viral fever, malaria, tuberculosis and typhoid) were randomly collected. Immediately after collection of urine samples, Sodium azide (NaN₃) was added to each sample at a final concentration of 0.1% as a preservative to prevent fungal growth. The samples were stored at 4°C until use.

Example 2

Maintenance of parasites

[0064] L. donovani strain (MHOM/IN/83/AGS3) (ATCC-PAR-413), initially isolated from an Indian kala-azar patient, was maintained in hamsters. Infected hamsters were bred in the animal facility of Indian Institute of Chemical Biology, Kolkata and amastigotes were allowed to transform into promastigotes at 22°C in Medium 199, pH 7.4 supplemented with 10% heat inactivated fetal bovine sera (FBS) (Sigma-Aldi ich), 25 mM HEPES, 2 mM glutamine, lOOU/ml of penicillin G-sodium and 100μ /ml of streptomycin sulphate. Further promastigotes were sub- cultured upto 3^rd to 5^th passage in the same medium at 22°C at an average density of 2xl0⁶ cells/ml [33]. Subsequently, the cells were centrifuged (1200g, at room temperature for 10 min) and pellet were washed two times for 10 min with 0.02M PBS and finally stored at -20°C until antigen preparation.

Preparation ot^' leishmanial proniastigote membrane antigen (LAg)

[0065] To isolate L. donovani membrane antigen, washed cell pellet was suspended in 5 mM cold Tris-HCl buffer, pH 7.6 (l of cell pellet in 50 ml of cold 5 mM Tris-HCl buffer ) and vortexed 6 limes for 2 minutes with 10 min interval in cold. After vortexing, the suspension was centrifuged at 2310g for 10 min to obtain the ghost membrane pellet. Pellet was then re- suspended in 10 ml of the same buffer and sonicated 6 times with one min pulse and one min interval at 4°C. Subsequently, the suspension was centrifuged at 5190 g for 30 min and L. donovani promastigote membrane proteins were collected from the supernatant and stored at - 80°C until use [34]. Protein concentration was determined by Lowry method [35].

Example ELISA to detect antibodies in urine

[0066] In an antibody-capture enzyme immunoassay, antigens were coated on the wells of ELISA plates. Antigens bind with specific anti bodies present in the sample. Enzyme conjugated anti-antibody is added which binds with antigen -antibody complex and catalyzes the substrate into a coloured product. Intensity of coloured product is correlated with the antibody present in the sample.

[0067] To develop a urine based diagnosis of VL, antibodies in the urine samples were detected through ELISA which was performed as fol lows. Flat bottom 96-well microtitter plates (Maxisorp; Nunc) were coated with 2.5

of leishmanial antigen in 100 μΐ phosphate buffer and incubated overnight at 4°C. Next day plates were blocked with 200 μΐ of 0.02M phosphate buffer saline (PBS) (pi I 7.2) containing 1% of bovine serum albumin (BSA) (SRL) for two hours at 37°C. After three times w ashing with PBS containing 0.1% Tween-20 (PBS-T), wells were loaded with urine sample ( 100 μΐ/wel l) and incubated at 37°C for an hour. After three washes with same PBS-T, peroxidase conjugated goat anti-human IgG (Banglore GeNei), diluted 1 :4000 with blocking buffer (100 μΐ. W ell ), was added and incubated at 37°C for an hour. Plates were then washed three times in PBS-T and incubated with 0.08% of substrate OPD (o- Phenylenediamine dihydrochloride (Signia-Aldrich) and 0.04% of 30%H₂02 (Merck) in 50mM phosphate-citrate buffer (100 μΐ/well) (pH 5.0). Substrate was left for colouration for 15 minutes at room temperature. The reaction was stopped by the addition of 2N sulphuric acid (50 μΐ/well), and the optical density was determined at 492 nm in an ELISA reader (Thermo RS232C). Each sample was assayed in duplicate in which, if the absorbance values of the duplicate differ>40% from their average, the sample w as retested. For comparison of quantitative result between the group, the absorbance of ELISA were evaluated by unpaired t test and considering significant at P <0.05 with two-tailed. The cut-off point was determined as the mean of the healthy controls plus 3 standard deviations.

A. Pilot study

[0068] In the first experiment, ELI SA was performed according to the procedure mentioned above with four leishmanial antigens to evaluate Leishmania-specific antibodies in urine. Out of four antigens tested, two were crude antigens, Leishmania promastigote membrane antigen (LAg) and soluble leishmanial antigen (SLA), and two were recombinant antigens, glycoprotein 63 (rGP63) and cystein proteaseA (rCPA). Urine sample of six VL patients and four healthy controls were tested for this study. Antibodies present in the urine were higher for LAg than other antigens tested (data not shown for antigens other than LAg). Urine from all six VL patients showed significantly higher antibody titre at 492nm as compared to the healthy controls [Fig 1]. P value for the two tailed unpaired t test was O.0001, considered statistically significant with cut off value of 0.315 calculated as mentioned above. So in the very first experiment we found that urine antibodies specific to LAg were all above the cut off line and healthy controls below the cut off line. Therefore after this experiment LAg was established as our target antigen for diagnosis purpose of VL.

B. Determination of optimum LAg concentration

[0069] Further, experiment with different sets of urine samples, some cross reactivity with other diseases (OD) were observed for LAg al 2^g/well (data not shown). So LAg was taken in different concentrations to optimize at which concentration infected VL patients were significant ly different from the healthy and negative controls. ELISA was done with five VL urine, three healthy controls, two malaria and two viral fever negative control samples according to the protocol discussed earlier with varying LAg concentrations. Five different LAg concentrations, 0Λ μg, 0.25μg, O.S tg. 1 μ«, and 2.5μ¾¾Λνβ11 were used for coating ELISA plates. It was found that ^g/well of LAg was adequate for diagnosis of VL in which mean ± standard deviation of optical density for VL patients was observed as 1.93 ± 0.360 and for healthy and negative controls as 0.214 ± 0.075 [fig 2]. LAg at 2^g/well showed cross reactivity with one malaria and one viral fever urine sample whereas LAg lower than ^g/well showed lower antibody titre with VL urine. Therefore ^g/well of LAg was taken as a standard for further ELISA experiments.

C. Optimization of appropriate urine dilution

[0070J Folio ving experiment was performed to determine the appropriate urine dilution at which ^g/well of LAg would not show any cross reactivity. Three different urine dilutions i.e.

1 :10, 1 : 100, and 1 :500 and one set of urine without dilution (N) were tested in this experiment.

Five VL urine samples, three healthy controls, two malaria and two viral fever samples were used for the assay. It was found that urine without dilution and at 1 : 10 urine dilution, antibody titre was significantly different from the healthy and negative controls (P < 0.0001) but urine at 1 : 10 dilution showed lower standard error of mean (0.017) for controls than urine without dilution (0.105). Therefore 1 : 10 urine dilution was decided for further ELISA experiments to get more specific result without compromising with the sensitivity [fig 3].

D. Isotypes IgG, IgA, IgM and IgE in urine samples.

[0071] All the experiments done earlier were to detect LAg-specific IgG antibodies in urine of VL patients. In the following experiment, other LAg-specific antibody isotypes in addition to IgG i.e. IgA, IgM and IgE were evaluated in the urine sample. ELISA was performed with of LAg and 1 : 10 urine dilution as standardized earlier. HRP-conjugated goat anti- human IgG, IgA, IgM and IgE were used for detection of IgG, IgA, IgM and IgE antibody in urine, respectively. Assay was done with five VL urine samples, three healthy controls, two malaria and two viral fever samples. Among the four antibody isotypes evaluated, the only LAg- specific antibodies present in a very significant amount in all the urine samples of VL patients was IgG [fig 4]. Therefore ELISA was restricted with the detection of LAg-specific IgG antibody in the urine of VL patients.

E. Final ELISA after standardization

[0072] For immunological diagnosis of VL different parameters of urine based ELISA was standardized and the outcome emerged as a non-invasive immunological method for diagnosis of the disease. Figure 5 is a view of the cumulative data for 46 VL cases, 8 non-endemic healthy controls. 2 endemic healthy controls and 6 other diseases including 2 malaria, 2 viral fever and 2 tuberculosis. ELISA was performed with these cases and cut off value (0.204) was obtained from absorbance value of healthy control samples according to the formula mentioned earlier. P value of VL with healthy controls was observed O.0001 and with other diseases it was found 0.0002. Three VL samples were found false negative where as healthy controls and other diseases did not show any false positive result. Therefore sensitivity of the assay was obtained 93.47% and the specificity was 100%. The total number of samples used and the overall results of ELISA are summarized in Table 01.

[0073] Table 01 : overall result of urine-based ELISA

NEHC 8 0 8

EHC 2 0 2

Malaria 2 0 2

Viral fever 2 0 2

Tuberculosis 2 0 2

[0074] ELISA was standardized for diagnosis of VL with the urine of infected patients using LAg as antigen but ELISA is not convenient for the field setting. It is a time consuming process which requires ELISA reader for final reading so, can only be performed in the labs or hospitals. To address all these limitations of ELISA in VL diagnosis, antibody-capture ELISA was adopted to dot-blot assay.

Example 4

Dot blot assay

[0075] To develop a nitrocellulose membrane based diagnostic assay that can easily used in the field conditions, principles of the ELISA method was adapted in the form of dot blot.

[0076] Nitrocellulose membrane (Amersham) was cut into small rectangular strips which were then soaked in 25mM Tris-HCl (pH 7.6).

[0077] Strips were then taken out and in semidried condition; LAg (l ^g in 2μ1 25mM Tris- HCl/strip) was coated on to the centre of the nitrocellulose membrane strip in the form of dot.

[0078] After drying, strips were blocked with 5% BSA+ 0.01% NaN₃ and 0.1% Tween-20 in lOOmM Tris Duffer Saline (TBS) and incubated at 4°C overnight. Next day, strips were washed thrice with lOOmM TBS i-0.05% Tween-20 (wash buffer) and allowed to dry completely at room temperature.

[0079] Urine samples from kala-azar patients and controls were diluted (1 :5) in lOOmM TBS+0.05% Tween-20 and kept in 2ml tubes. Strips were then dipped in diluted urine samples and incubated for 30 minutes at room temperature, then washed two times with the wash buffer described above.

[0080] The strips were then dipped in diluted (1 :2000) peroxidase conjugate goat anti-human IgG (GeNei) for 30 minutes at room temperature, then washed two times with the same wash buffer and the third wash was with lOOmM TBS only (without Tween-20).

[0081] After washing strips were dipped for 5 minutes into freshly prepared substrate solution having 0.05% of 3,3'-diaminobeiizedene tetrahydrochloride (DAB) (Sigma)+0.05% of 30%H₂0₂ (Merck) in l OOmM TBS.

[0082] Finally strips were washed with distilled water properly and dried. Brown colour spot was seen at the dotted position of the strips if the urine sample is from kala-azar patients while healthy individuals and other diseases show no colour on the membrane.

A. Optimization of LAg concentration

[0083] The clot blot assay were carried out to optimize antigen concentration so that there would be no cross reactivity with the healthy controls and other diseases. Therefore LAg at four different concentrations i.e. 2.0, 1.5, 1.0, and 0^g/strip were determined [Fig 6].

[0084] It was observed that LAg lesser than l ^g gives slightly faint colour for VL cases where as LAg more than that shows cross reactivity for negative controls.

[0085] Therefore 1.5^ig LAg is adequate for dot blot assay in which clear dark brown colour appears in VL. cases and elimination οΓ cross reactivity by the healthy controls and other diseases. Therefore, 1 ^g LAg was optimized for dot blot assay for the further experiments.

B. Optimization of urine dilution

[0086] For EL1SA 1 : 10 dilution of urine was optimized but for dot blot it was seen that colour of the dots was not much dark at this dilution and urine without dilution showed cross reactivity.

[0087] Therefore appropriate urine dilution for dot blot assay was established with 1.5 μg LAg/strip as standardized earlier. It was observed that among the different urine dilutions tested i.e. without dilution (1 :0), 1 :2, 1 :5. 1 : 10, and 1 :50, at a dilution of 1 :5 there was no cross reaction with healthy controls and other diseases and colour of the dots for VL cases were satisfactorily dark [Fig 7]. Therefore urine at 1 :5 dilution was optimized for dot blot assay.

C. Blocking standardization [0088] To avoid background and non-specific binding of antibodies to the membrane it was important to find out proper blocking. Earlier 1% BSA was used for ELISA method but in dot blot assay non-specific colour was seen in the healthy controls and other diseases mainly with malaria (data not shown).

[0089] Therefore in the present standardization 1.5%, 3% and 5% BSA were tested and found that at 5% BSA blocking there were no cross reaction in healthy controls and other diseases while in VL infected patients sufficient colour was observed [Fig 8]. 0.1% Tween-20 and 5% skimmed milk were also tested but the results were unsatisfactory. So 5% BSA blocking was selected for dot blot assay.

D. Dot-blot as a test for VL diagnosis

[0090] After standardization of different experimental parameters to get distinct visual result, the dot blot was performed in optimized condition with 10 VL cases, two malaria samples, two viral fever samples, two tuberculosis samples, one typhoid sample and four healthy control samples [Fig 9],

[0091] It was observed that all VL cases show dark brown colour at the point on which LAg were coated, whereas no non-specifie colour was found in healthy controls as well as diseases other than VL. So this whole assay w s used further as a test line for VL diagnosis.

E. Optimizat ion of anti-human IgG antibody for control

[0092] For designing a dipstick it w as necessary to introduce a control line to assess the performance of the assay. Therefore rabbit anti-human IgG antibody was used and optimized for this purpose. IgG antibody was present non specifically in all urine samples healthy as well as infected, ther fore, anti- human IgG was bound to any IgG present in the urine irrespective of the condition healthy or diseased. Two VL cases, one malaria, one viral fever, and two healthy control samples were used to standardize anti- human IgG antibody. Therefore four different dilutions of anti-human IgG antibod was taken i.e., 1 :20, 1 : 100, 1 :500, and 1 : 1000 and it was found that at 1 :20 dilution clear dark brown colour was observed in all the samples tested [Fig 10].

F. Dot blot with test and control points

[0093] After standardization of LAg as a test and rabbit anti-human IgG as a control for VL diagnosis, both were coated in the same strip at two marked positions. The upper dot of the strip comprises lest point coated with LAg and below was the control point coated with rabbit anti- human IgG antibody.

[0094] Eight VL cases were tested and found distinct colour in both the points whereas in all the negative controls used i.e. one of each malaria, viral fever, tuberculosis, typhoid and four healthy cases show single colour only at the control point [Fig 1 1]. Test points in these samples were found completely clear without any colour.

Example 5

Development and evaluation of Dipstick diagnostic assay

[0095] The present invention relates to a urine-based immunological dipstick device comprises nitrocellulose membrane impregnated with LAg and anti-human IgG for field diagnosis of VL. The dipstick assay comprises incubation with urine sample followed by enzyme-conjugated anti- human IgG and then substrate chromogen including washing in each step to give dark brown colour band which could be seen directly without any sophisticated instrument.

[0096] The typical dipstick device invention is represented in figure 12 showing components of the device used in the process. Referring to the figure 12, the whole device is defined into two zones. A sample contact zone 2 is a nitrocellulose membrane fixed with the support zone 1 by a double headed adhesive tape 5. The sample contact zone 2 comprises a test line 3 impregnated with leishmanial antigen; LAg, here shown as a dotted line which binds to LAg-specific urine antibodies and further the antigen-antibody complex detecting by peroxide conjugated anti- human IgG. followed by substrate to give coloured band on test line 3. The sample contact zone 2 further comprises a control line 4 impregnated with rabbit anti-human IgG, again shown as a dotted line which binds to urine antibodies of any specificity and serves as an internal control to assess the experiment completion. The support zone 1 is a rigid base provided by moisture unreceptive polythene sheet which makes the dipstick convenient to use.

[0097] The dipstick device was prepared by referring initial optimizations of dot blot assay. Nitrocellulose membrane (Amersham) with a pore size of 0.45μπι was cut into 8cm x 2.4cm rectangular piece to make 20 strips at a time. Two faint horizontal lines were drawn with the pencil to mark the positions. Membrane was then soaked in 25mM Tris-HCl (pH 7.6) and in semidried condition 1.5pg LAg in 2μ1 of 25mM Tris-HCl/strip (30μg LAg in 40μ1 of 25mM Tris-HCl/20 strips) were coated on the nitrocellulose membrane at the upper horizontal line marked with pencil. Simultaneously, for control line marked as lower horizontal line was coated with 1 :20 of rabbit anti-human IgG in 2μ1 of 25mM Tris-HCl (40μ1 of 25mM Tris-HCl/20 strips). Coated membrane was left to dry then blocked with 5% BSA+ 0.01% NaN₃ and 0.1% Tween-20 in l OOmM Tris Buffer Saline (TBS) and incubated at 4°C overnight. Next day, strips were washed thrice with lOOmM TBS+0.05% Tween-20 (wash buffer) and allowed to dry completely at room temperature. Now coated 8cm x 2.4cm nitrocellulose membrane piece was adhered with 8cm x 7cm size plastic sheet with a double headed adhesive tape and cut into 4mm width strips. Prepared strips were stored at room temperature before use.

[0098] For dipstick assay, the strips were dipped into 2ml tube having urine sample at 1 :5 dilution and allowed to incubate for 30 minutes at room temperature. LAg-specific antibodies in the urine sample, if present binds with the immobilized LAg coated in test line 3. A portion of urine IgG antibody also binds to the rabbit anti-human IgG coated in control line 4. Strips were then taken out from 1^st tube and washed two times with the wash buffer (described above). The strips were then dipped into 2^nd 2ml tube having diluted (1 :2000) peroxidase conjugated goat anti-human IgG (GeNei) for 30 minutes at room temperature. Peroxidase conjugated goat anti- human IgG binds with the urine IgG and forms complex of LAg-specific IgG-Peroxidase conjugated goat anti-human IgG at the test line and rabbit anti-human IgG-Peroxidase conjugated goat anti-human IgG at the control line. Strips were removed from 2^nd tube and washed two times with the same wash buffer and the third wash with lOOmM TBS only (without Tween-20). After washing strips were dipped into the 3^rd 2ml tube for 5 minutes having freshly prepared substrate solution comprises 0.05% of 3,3'-diaminobenzedene tetrahydrochloride (DAB) (Sigma) + 0.05% of 30%H₂0₂ (Merck) in l OOmM TBS. The substrate is catalyzed by the peroxidase and forms an insoluble coloured product along the test and control lines depending of the complex made at the test line 3 and control line 4. Dark brown colour band was seen at the test line 3 of the strips if anti-leishmanial IgG is present in the urine sample as in VL cases while healthy individuals and other diseases show no colour at the test line 3. Control line 4 shows brown coloured band with all the urine samples infected and healthy where non-specific IgG is present. The slrips were then removed from substrate and washed with distilled water properly and allowed to dry. Two visible coloured bands at the test line 3 and control line 4 shows VL positive result and a single coloured band at the control line 4 is VL negative. [0099] The di stick assay was performed with a large number of confirmed VL urine along with various controls. The experiment included urine samples of 50 active VL, 8 non-endemic healthy controls, 2 endemic healthy controls and 10 from diseases other than VL including 2 malaria, 2 viral fever. 2 tuberculosis and 4 typhoid cases (Table 02). A 100% agreement in the reading of the dipstick test was found with the VL patients diagnosed by splenic or bone marrow aspiration. The analyses performed with the dipstick test were all valid, as the internal control of the dipstick test w a always positive. Representative positive and negative results are shown in figure 13. All the VL samples showed clear positive result with two coloured bands at the test line and control line whereas all the 20 healthy and negative controls gave clear negative result with only one band at the control line. Therefore, calculations of the sensitivity and specificity revealed that the dipstick test was 100% sensitive and 100% specific for the sample tested in the laboratory conditions.

[00100] Table 02: overall result of urine-based dipstick assay

[00101] Table 03. Overall result of urine-based

ELISA

Tuberculosis 4 0 4

Typhoid 4 0 4

[00102] Table 04. Overall result of urine-based dipstick assay

ADVANTAGES OF THE INVENTION

The main advantages of the present invention are:

[00103] The two assays ELISA and dipstick are non- invasive methods for diagnosis of visceral leishmaniasis (VL) using urine as sample. As the assays are urine-based the samples are easier and risk- free in collection and storage.

[00104] ELISA shows good sensiiivity and excellent specificity and can be used for diagnosis of a number of patients simultaneously using urine samples.

[00105] The dipstick assay is a urine-based rapid test, highly sensitive and specific for diagnosis of VL in comparison to KAtex, the only urine-based commercially available diagnostic kit for VL which shows poor sensitivity (36-85%). [00106| Anotlier advantage of the said dipstick assay is that it does not require urine to be boiled before the test as the case for KAtex.

[00107] The dipstick assay is a simple method in which no any sophisticated equipment is required and all the steps of the assay are carried out at normal temperature where results can be obtained within 2 hr and can be seen by the naked eye, therefore ideal for field settings.

Reference cited:

1. Alvar. .1.. t al., Leishmaniasis worldwide and global estimates of its incidence. PLoS One, 2012. 7(5): p. e35671.

2. Mondal, S., P. Bhattachai ya, and N. Ali, Current diagnosis and treatment of visceral leishmaniasis. Expert Rev Anti Infect Ther, 2010. 8(8): p. 919-44.

3. Chappuis. F., et al., Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol, 2007. 5( 1 1 ): p. 873-82.

4. Mondal. D., et al., Enhanced case detection and improved diagnosis of PKDL in a Kala- azar-endcinic area of Bangladesh. PLoS Negl Trop Dis, 2010. 4(10).

5. Matlashewski, G., et al., Visceral leishmaniasis: elimination with existing interventions. Lancet Infect Dis. 201 1. 1 1 (4): p. 322-5.

6. Islam, S.. M. Ashraful Alam Bhuiyan. and C. Bern, Post-Kala-Azar dermal leishmaniasis in Mymensingh. Bangladesh. Am .1 Trop Med Hyg, 201 1. 85(2): p. 193-4.

7. Antinori, S., L. Schifanella. and M. Corbellino, Leishmaniasis: new insights from an old and neglected disease. Eur J Clin Microbiol Infect Dis, 2012. 31(2): p. 109-18.

8. Ejazi. S.A. and N. Ali. Developments in diagnosis and treatment of visceral leishmaniasis during the last decade and future prospects. Expert Rev Anti Infect Ther, 2013. 11 (1): p. 79-98.

9. Srivastava, P., et al., Diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg, 2011. 105(1): p. 1 -6.

10. Mandal, J., et al., Evaluation of direct agglutination test, rk39 Test, and ELISA for the diagnosis of visceral leishmaniasis. Am J Trop Med Hyg, 2008. 79(1): p. 76-8. 1 1. Teran-Angel, G., et al., The direct agglutination test as an alternative method for the diagnosis of canine and human visceral leishmaniasis. Biomedica, 2007. 27(3): p. 447-53.

12. Srividya, G., et al., Diagnosis of visceral leishmaniasis: developments over the last decade. Parasilol Res, 2012. 1 10(3): p. 1065-78.

13. Mai a, /... et al., Comparative study of rK39 Leishmania antigen for serodiagnosis of visceral leishmaniasis: systematic review with meta-analysis. PLoS Negl Trop Dis, 2012. 6(1): p. el484.

14. Vaish, M ., et al., Evaluation of two novel rapid rKE16 antigen-based tests for diagnosis of visceral leishmaniasis in India. J Clin Microbiol, 2012. 50(9): p. 3091-2.

1 5. Paltabhi , S., et al., Design, development and evaluation of rK28-based point-of-care tests or improving rapid diagnosis of visceral leishmaniasis. PLoS Negl Trop Dis, 2010. 4(9).

16. Saha, S ., et al., Easy test for visceral Leishmaniasis and post-Kala-azar Dermal Leishmaniasis. Emerg Infect Dis, 201 1. 17(7): p. 1304-6.

17. Roolfsema, J.H., et al.. Evaluation and improvement of two PCR targets in molecular typing of clinical samples of Leishmania patients. Exp Parasitol, 201 1. 127(1): p. 36-41.

18. Srivaslava, P., et al., Diagnosis of Indian visceral leishmaniasis by nucleic acid detection using PCR. PLoS One, 201 1. 6(4): p. el 9304.

19. Halam, G . R., et al., Improvement of the newly developed latex agglutination test (Katex) or diagnosis of visceral lieshmaniasis. J Clin Lab Anal, 2009. 23(4): p. 202-5.

20. Boelaeri. M., et al., Diagnostic tests for kala-azar: a multi-centre study of the freeze- dried DA T. rK3 V strip test and KAtex in East Africa and the Indian subcontinent. Trans R Soc Trop Med Hyg. 2008. 102(1): p. 32-40.

21. Ahsan. M.M., et al., Evaluation of latex agglutination test (KAtex) for early diagnosis of kala-azar. Mymensingh Med J, 2010. 19(3): p. 335-9.

22. Islam. M.Z., et al., Direct agglutination test with urine samples for the diagnosis of visceral leishmaniasis. Am J Trop Med Hyg, 2004. 70(1): p. 78-82.

23. Vaish, M., et al., rK39 antigen for the diagnosis of visceral leishmaniasis by using human saliva. Am J Trop Med Hyg, 2012. 86(4): p. 598-600.

24. Vaish, M., et al., Noninvasive molecular diagnosis of human visceral leishmaniasis. J Clin Microbiol, 201 1 . 49(5): p. 2003-5. 25. Fisa, R.. et al., Leishmania infantum DNA detection in urine from patients with visceral leishmaniasis and after treatment control. Am J Trop Med Hyg, 2008. 78(5): p. 741-4.

26. Khan, .G., et al., Evaluation of rK-39 strip test using urine for diagnosis of visceral leishmaniasis in an endemic area in Bangladesh. Parasit Vectors, 2010. 3: p. 1 14.

27. Chakravariy. J., et al., Evaluation of rk39 immunochromatographic test with urine for diagnosis of visceral leishmaniasis. Trans Soc Trop Med Hyg, 201 1. 105(9): p. 537-9.

28. Singh, L⁾.. et al., Evaluation of rK-39 strip test using urine for diagnosis of visceral leishmaniasis in an endemic region of India. Am J Trop Med Hyg, 2013. 88(2): p. 222-6.

29. Goswami. R.P., et al., Testing urine samples with rK39 strip as the simplest non-invasive field diagnosis for vi sceral leishmaniasis: an early report from eastern India. J Postgrad Med,

2012. 58(3): p. 1 80-4.

30. Elassad. A.M.. et al., The significance of blood levels of IgM, IgA, IgG and IgG subclasses in Sudanese visceral leishmaniasis patients. Clin Exp Immunol, 1994. 95(2): p. 294- 9.

31. Anam, K., et al., Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients. Clin Diagn Lab Immunol, 1999. 6(2): p. 231 -5.

32. Saha, S., et al., Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotling for differential diagnosis of Indian post-kala-azar dermal leishmaniasis. J Clin Microbiol, 2005. 43(3): p. 1269-77.

33. Mazimider, S., et al., Potency, efficacy and durability of DNA/DNA, DNA/protein and protein/protein based vaccination using gp63 against Leishmania donovani in BALB/c mice. PLoS One, 201 1 . 6(2): p. el4644.

34. Bhowmick, S., R. Ravindran, and N. Ali, Leishmanial antigens in liposomes promote protective immunity and provide immunotherapy against visceral leishmaniasis via polarized

Thl response. Vacci ne, 2007. 25(35): p. 6544-56.

35. Lowry, O.H., et al., Protein measurement with the Folin phenol reagent. J Biol Chem, 1951. 193(1 ): p. 265-75.

Claims

I/We Claim:

1. A non-invasive method for the detection of visceral leishmaniasis using a dipstick comprising of:

a. about 1 .5 μg of leishmanial antigen; and

b. about 1 :20 dilution of anti-human IgG antibody,

said non-invasive method comprising the steps of:

i. providing a subject urine sample having 1 :5 dilution,

ii. dipping the dipstick into urine sample obtained in step (i) at room temperature (at least 22°C) for a minimum 30 minutes,

Hi. washing the dipstick obtained in step (ii) with wash buffers,

iv. dipstick obtained in step (iii) is further dipped into a solution having diluted (1 :2000) peroxide conjugated anti-human IgG at room temperature (at least 22°C) for 30 minutes after that remove dipstick from the urine sample and perform washing with known wash buffers; and

v. incubating of dipstick obtained in step (iv) with substrate solution to detect visceral leishmaniasis.

2. The method as elaimed in claim 1 , wherein the substrate solution comprises of 0.05% of 3.3'-diaminobenzedene tetrahydrochloride (DAB) + 0.05% of 30% H₂0₂ in lOOmM TBS.

3. The method as claimed in claim 1 , wherein the detection of visceral leishmania is positive upon appearance two visible colour bands .

4. A kit for measuring non-recombinant membrane antigen (LAg) specific antibodies in a urine sample, said kit comprising of nitrocellulose strips pre-coated with said LAg as dried antigen, positive control (optimised urine dilution), enzyme conjugated anti-human IgG, substrate chromogen, and buffers for washing and dilutions, and an instruction manual.

5. The kit as elaimed in claim 4, wherein the nitrocellulose strip comprises of two zones, namely, a sample contact zone of nitrocellulose membrane and a support zone of polyethylene sheet, the sample contact zone further comprising of a test line portion coated with said antigen (LAg) and a control line portion coated with anti-human IgG antibody.

6. fhe kit as claimed in claim 4, wherein said urine sample is used at a dilution of 1 :5 for the dipstick assay.

7. The kit as claimed in claim 4, wherein said enzyme is horse radish peroxidase conjugated with anti-human IgG at a dilution of 1 :2000.

8. The kit as claimed in claim 4, wherein said substrate chromogen is 3,3'-diaminobenzidine tetrahydrochloride ( DAB).

9. The kit as claimed in claim 4, wherein said kit is 100% sensitive and 100% specific for diagnosis of V L in field conditions.

10. An ELISA to delect antibodies against L. donovani membrane antigen (LAg) in urine.