WO2009130709A2

WO2009130709A2 - A non-recombinant membrane antigen, a diagnostic kit thereof and process for detection of visceral leishmaniasis and pkdl

Info

Publication number: WO2009130709A2
Application number: PCT/IN2009/000238
Authority: WO
Inventors: Nahid Ali; Samiran Saha
Original assignee: Council of Scientific and Industrial Research CSIR
Current assignee: Council of Scientific and Industrial Research CSIR
Priority date: 2008-04-23
Filing date: 2009-04-17
Publication date: 2009-10-29
Anticipated expiration: 2010-10-23
Also published as: WO2009130709A3

Abstract

The present invention deals with a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83. The membrane antigen (LAg) having following characteristics of a complex of 25-35 polypeptides and having molecular mass in the range of 18 - 155KD. The said membrane is sensitive and specific up to the range of 95% - 100% to anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL). The present invention further deals with a process for the preparation of the said membrane antigen and the method of using it for the detection of anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL). The present invention further deals with a diagnostic kit comprising the said non- recombinant membrane antigen, useful for detecting anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala- azar Dermal Leishmaniasis (PKDL). The present invention further relates to process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit.

Description

"A NON-RECOMBINANT MEMBRANE ANTIGEN, A DIAGNOSTIC KIT

THEREOF AND PROCESS FOR DETECTION OF VISCERAL LEISHMANIASIS AND PKDL"

Field of the invention:

The present invention relates to a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83, useful for diagnosing visceral Leishmaniasis (VL) and PKDL (Post KaIa azar Dermal Leishmaniasis) in a subject suspected of being infected with the parasitic protozoa Leishmania donovani.

More particularly, the present invention relates to a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83, wherein said membrane antigen (Lag) being characterized by a complex of 25 -35 polypeptides having molecular mass in the range of 18- 155kDa and having sensitivity and specificity in the range of 95% - 100% to anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL).

The present invention further relates to a process of preparing a non recombinant membrane antigen (LAg).

The present invention further relates to a diagnostic kit useful for detecting and quantifying specific anti-leishmanial IgG antibodies in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL).

The present invention further relates to process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit.

Background and prior art of the invention:

Leishmaniasis is a serious and sometimes fatal disease estimated by the World Health Organization (WHO) to affect approximately 12 million people in 88 countries. Recent epidemics of Leishmaniasis have occurred in the Africa, the Indian subcontinent and Brazil. Present concerns are significant morbidity and mortality caused by Leishmaniasis in endemic areas and the increased incidence of the disease in HIV-infected and intravenous drug-user populations. VL is endemic in 65 countries with the annual incidence of 500,000 cases. More than 90 percent of VL occurs in poor rural and suburban areas of India, Bangladesh, Nepal, Sudan, and Brazil. See Desjeux P (2004), Comp. Immunol. Microbiol. Infect. Dis 27:305-18. Nevertheless, and despite such obstacles, the past 5 years have produced tangible progress in diagnosis, treatment, and vector control. See Sundar S et al. (2002) Clin. Infect. Dis. 35:581-6; Murray HW et al (2005) Lancet. 366:1561-77.

The diagnosis of VL is complex because its clinical features are shared by a host of other commonly occurring diseases, such as malaria, typhoid, and tuberculosis. Sequestration of the parasite in the spleen, bone marrow, or lymph nodes further complicates this issue. Unfortunately, current acceptable diagnostic practices lack the means for efficiently and accurately identifying those infected or exposed to the disease-causing parasite as explained in Singh S, (2006), Indian. J. Med. Res. 123:311-330; Sundar S et al (2002), Clin. Diagn. Lab. Immunol. 9:951-958. As a result, the prevention of leishmanial epidemics is greatly hindered and patient management is difficult. Additionally, there is an imminent threat of a developing population of people co-infected with HTV and Leishmania.

In view of this, efficient immunodiagnostic field assays are age-old need for diagnosis, patient management and epidemiological studies. Different immunodiagnostic methods including gel diffusion, complement fixation test, indirect hemagglutination test, IFA test, direct agglutination test, ELISA etc. with variable sensitivity and specificity have been developed in last three decades. See Sundar S et al, (2002), Clin. Diagn. Lab. Immunol. 9:951-958. Practical difficulties to perform these tests at the peripheral laboratories and the variable sensitivity and specificity are the main drawbacks of these tests. Following are the principal immunological tests applied for field diagnosis:

1. Direct agglutination test (DAT) is routinely performed in many laboratories of India and Sudan with 91 to 100% sensitivity and 72 to 100% specificity. See Mengistu, G. R. et al, (1990), Trans. R. Soc. Trop. Med. Hyg. 84:359-362; Singh S. A. et al, (1995), J. Parasitol. 81:1000-1003; Zijlstra, E. E. et al, (1991) Trans. R. Soc. Trop. Med. Hyg. 85:474-476 and Zijlstra, E. E. et al, (1992) Trans. R. Soc. Trop. Med. Hyg. 86:505-507. However, its reproducibility across the centers was quite weak, and in difficult field conditions, the fragility of antigen, lack of cold chain, and batch to batch variations in the antigen along with nonstandardization of test readings have severely limited its widespread applicability in regions of endemicity. Unless the antigen is made indigenous and affordable, and the technique is made user-friendly with reduced time, its field use is unlikely in countries of endemicity like India.

2. Enzyme linked immunosorbent assay (ELISA) is a valuable tool and one of the most sensitive tests for the serodiagnosis of visceral Leishmaniasis. ELISA is adaptable for use with various antigens such as whole cytoplasmic (soluble antigen, SA), purified antigens such as fucosemanose (Palatnik-de-Sousa C. B. et al. (1995), Trans. R. Soc. Trop. Med. Hyg. 89 : 390-3), defined, synthetic peptides and recombinant proteins as antigen. See Maalej I. A. et al. (2003). Am. J. Trop. Med. Hyg. 68: 312-20. The sensitivity and specificity of ELISA is greatly influenced by the antigen used. Beside the most commonly used soluble promastigotes antigen, several antigenic molecules have been reported and their negative and positive predictive values (NPV & PPV) compared. See Maalej I. A. et al, (2003). Am. J. Trop. Med. Hyg. 68: 312-20. The excretory, secretary and metabolic antigens released by L. donovani promastigotes (Ld-ESM) into a protein free medium was used for the serodiagnosis of VL by ELISA. The use of this soluble antigen preparation to capture specific IgG antibodies in the sera of kala-azar patients indicated that the soluble antigens found in conditioned medium could act as the foundation for Leishmaniasis immunoassays. See Martin S. K. et al, (1998) Ann. Trop. Med. Parasitol. 92: 571-7. These findings were utilized in Leishmaniasis immunoassays, which were highly sensitive and specific and allowed the detection of specific IgG and IgM antibodies in subjects affected with visceral, cutaneous, or canine Leishmaniasis. See Ryan J. R. et al, (2002) J. Clin. Microbiol. 40: 1037-1043; Ryan, et al. (2006) US Patent No. 7,008,774.

Modifications of the basic ELISA have been carried out e.g. DOT-ELISA (Pappas M. G., Hajkowski R. and Hockmeyer W. T. (1983) J. Immunol. Methods. 64; 205-214; Badaro R., Reed S. G., Barral A., Orge G. and Jones T. C. (1986) Am. J. Trop. Med. Hyg. 35; 72-78) and use of species specific monoclonal antibodies. See Jaffe C. L, Bennett E., Grimaldi G. Jr. and McMahon Pratt D. (1984) J. Immunol. 133; 440-447; Jaffe C. L. and McMahon Pratt D. (1987) Trans. R. Soc. Trop. Med.Hyg. 81; 587-594.

3. Recombinant kinesin protein, rK39, is one of the few antigens that have been used in the development of antibody-detection immunoassays for active Visceral Leishmaniasis (VL).

It has been shown to be specific for antibodies arising during VL caused by members of the L. donovani complex. It is highly sensitive and predictive for onset of acute disease and evokes high antibody titres in VL patients. See Singh S, et al, (2002) Clin. Diagn. Lab. Immunol. 9: 568-72. In addition, rK39 ELISA, has a high predictive value for detecting VL in immunocompromised persons, like AIDS patients. See Liarte D. B. et al, (2001) Rev. Soc. Bras. Med. Trop. 34: 577-81. This antigen is now commercially available in the form of antigen-impregnated nitrocellulose paper strips adapted for use under field conditions. However, reports from Sudan and other countries revealed that this antigen showed decreased sensitivity and specificity. In Sudan the rK39 ELISA test is reported to miss 7 per cent parasitologically proven cases. See Zijlstra E. E. et al, (2001) Trop. Med. Int. Health. 6: 108-13. In its strip test format the sensitivity is further compromised to only 67 per cent in Sudan, 71.4 percent in Southern Europe and 60 to 90 per cent in Brazil. See Zijlstra E. E. et al, (2001) Trop. Med. Int. Health. 6: 108-13; Jelinek T. et al, (1999) Eur. J. Clin. Microbiol. Infect. Dis. 18 : 669-70; Schallig H. D. et al, (2002), Mem. Inst. Oswaldo. Cruz 7: 1015-8; Carvalho S. F. et al, (2003), Am. J. Trop. Med. Hyg. 68: 321-4.

Objects of the invention:

The main object of the present invention is to provide a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83, useful for diagnosing Visceral Leishmaniasis (VL) in a subject suspected of being infected with the parasitic protozoa Leishmania donovani.

Another object of the present invention is to provide a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83., wherein said membrane antigen (LAg) being characterized by a complex of 25 -35 polypeptides having molecular mass in the range of 18- 155kDa and having sensitivity and specificity in the range of 95% - 100% to anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post KaIa- azar Dermal Leishmaniasis (PKDL).

Yet another object of the present invention is to provide a process of preparing a non recombinant membrane antigen (LAg).

Yet another object of the present invention is to provide a rapid and accurate diagnostic Dipstick assay kit useful for detecting and quantifying specific anti-leishmanial IgG antibodies in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL).

Yet another object of the present invention is to develop a process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit.

Still another objective of the present invention is to develop an ELISA based quantitative/ semi quantitative method of serodiagnosis of the post kala-azar Dermal Leishmaniasis (PKDL) patients. Summary of the invention:

The present invention provides a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83, wherein said membrane antigen (LAg) being characterized by a complex of 25 -35 polypeptides having molecular mass in the range of 18- 155kD and having sensitivity and specificity in the range of 95% - 100% to anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL).

The present invention also provides a process for the preparation of the membrane antigen and the method of using it for the detection of anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala- azar Dermal Leishmaniasis (PKDL). The present invention further provides a diagnostic kit comprising the non-recombinant membrane antigen, useful for detecting anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL).

The present invention further provides a process of detection and quantifying specific antiriesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit.

In an embodiment of the present invention is provided a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83, wherein the membrane antigen (LAg) , is having sensitivity and specificity in the range of 95% - 100% to anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL).

In an embodiment of the present invention, is provided a process of preparing a non recombinant membrane antigen (LAg) , wherein the process comprising the steps of i) culturing the L. donovani strain AG83 promastigotes in Ml 99 medium supplemented with L-glutamine, sodium bicarbonate and fetal bovine • serum, ii) harvesting and lysing the promastigote cells to obtain the ghost ' membrane, iii) centxifuging and discarding the intracellular materials from the lysed cell, iv) ultrasonicating v) and centrifuging to extract the membrane protein.

In another embodiment of the present invention is provided a diagnostic kit useful for detecting and quantifying specific anti-leishmanial IgG antibodies in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL) using a non-recombinant membrane antigen (LAg) obtained directly from promastigotes of Leishmania donovani strain AG83, wherein the diagnostic kit is selected from the group consisting of ELISA kit and Dipstick assay kit • In another embodiment of the present invention, is provided a diagnostic kit useful for detecting and quantifying specific anti-leishmanial IgG antibodies in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL), wherein the ELISA kit comprises the LAg pre-coated ELISA plates, positive control, negative control, acceptable diluents, enzyme conjugated anti-human IgG, substrate chromogen, substrate buffer and an instruction manual to use the kit .

In yet another embodiment of the present invention, is provided a diagnostic kit useful for detecting and quantifying specific anti-leishmanial IgG antibodies in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL), wherein the Dipstick assay kit comprises the LAg pre-coated Dipstick-strips, positive control, negative control, acceptable diluents, enzyme conjugated anti-human IgG, substrate chromogen, substrate buffer and an instruction manual to use the kit.

In still another embodiment of the present invention, the Dipstick kit further comprises a solid base member holding a sample contact zone and test zone containing a control line portion and test line portion.

In yet another embodiment of the present invention, the base member used is poly ethylene sheet, test zone used is nitrocellulose.

In still another embodiment of the present invention, the enzyme used for conjugation is preferably horse radish peroxidase.

In yet another embodiment of the present invention, the substrate chromogen used is 3,3'-diaminobenzidine tetrahydrochloride (DAB) for Dipstick kit.

In yet another embodiment of the present invention, the substrate chromogen used is O- phenylenediamine dihydrochloride (OPD) for ELISA kit

In another embodiment of the present invention, is provided a process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit , wherein the process is selected from a group consisting of ELISA based process and Dipstick based process.

In still another embodiment of the present invention, is provided the process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit , wherein the Dipstick based process comprises the steps of: i) preparation of dipstick having control line portion and test line-portion, ii) inserting the dipstick as obtained in step i) into a container of diluted serum sample and incubating for 30 minutes, iii) removing the incubated dipstick from the container and washing with buffer, iv) inserting the washed dipstick into second container containing peroxidase conjugated polyclonal anti-human IgG and incubating for 30 minutes, v) removing and washing the incubated dipstick as obtained in step iv with wash buffer, vi) inserting the dipstick as obtained in step v) into third container containing chromagen substrate to liberate colored insoluble antigen product, vii) quantifying the concentration of antigen product using color card

In further embodiment of the present invention, is provided the process of detection and quantifying specific antilissmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit , wherein the diagnostic kit having LAg-nitrocellulose pre coated sheets are incubated at room temperature and said sheets are analyzed by using color card, without using sophisticated instrument.

In yet another embodiment of the present invention, is provided the process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit process , wherein said process is efficient for field usage.

In still another embodiment of the present invention, the serum dilution used in the process is maintained at a ratio ranging between 1 : 1000 and 1 :2000, based upon the assay method used for detection.

In yet another embodiment of the present invention, is provided an ELISA for the serodiagnosis of Visceral^" Leishmaniasis, which is based on L. donovani promastigotes membrane antigen (LAg), prepared from a culture of the parasites in liquid medium.

In yet another embodiment of the present invention, the invention relates to a membrane based dipstick immunoassay, "which is based on membrane bound and dried LAg, for the field diagnosis of Visceral Leishmaniasis.

In still another embodiment of the present invention, the assay of the invention has a high degree of sensitivity and specificity. For example, the assay of the invention has a combined sensitivity of about 100% and specificity of about 95.7%. Preferably, the dipstick assay of the present invention developed for field diagnosis has sensitivity of about 100% and a specificity of about 98%.

Brief description of the drawings:

Figure 1 represents LAg-specific IgG levels in at 1:1000 serum dilutions Figure 2 represents LAg-specific IgG levels in at 1:1000 serum dilutions with different set of control sera.

Figure 3 represents LAg-specific IgG levels in at 1:2000 serum dilutions.

Figure 4 represents Overall TLAg-specific IgG levels in at 1:1000 serum dilutions

Figure 5 represents Reference color card for visual estimation by ELISA. A, seropsitive; B, seronegative; C, blank Figure 6 represents LAg-specific IgG levels in at 1:1000 serum dilution, all steps performed at RT;

Figure 7 represents Dot ELISA - LAg specific IgG reactivity at 1:100 serum dilutions, Strips 1, 3 denote active VL; 2, 4 denote cured VL; 5, 6 denote healthy control; 7 denotes leprosy and 8 denotes filariasis. Figure 8 represents Dot ELISA - LAg specific IgG reactivity at different serum dilutions; Strips 1, 2 denote VL; 3 denotes healthy control; 4 denotes endemic control and 5 denotes filariasis. Figure 9 represents the preparation of the dipstick device

Figure 10 represents the results of dipstick assay. A, Schematic reading of +ve and -ve results; B, Representative strips with +ve and — ve results

Table 1 shows Summary of the results of Dot ELISA; Table 2 shows Results of the Dipstick serodiagnostic assay

Detailed description of the invention:

VL infection in human is characterized by the appearance of strong anti-leishmanial antibody titers in the sera of the patients. See Bray, R. S. (1976) Immunology of parasitic infections. Blackwell Scientific Publications, 65-76; Elassad, A. M. et al. (1994), Clin. Exp. Immunol. 95:294-299. Previously we described that serodiagnosis of Visceral Leishmaniasis patients is possible by L. donovani promastigotes membrane antigen (LAg)-specific ELISA₅ which had a sensitivity of 100% and specificity of 85% for visceral Leishmaniasis patients of Indian origin. See Anam K. et al, (1999), Clin. Diagn. Lab. Immunol. 6 : 231-5. With the utilization of the diagnostic potential of LAg, the present invention provides simultaneously two indigenous cost effective serodiagnostic methods, which can be used in the field.

Thus, the present invention relates to a diagnostic kit, which would be effective to differentiate active VL not only from the healthy individuals, endemic or non endemic, but also from other tropical diseases like malaria, tuberculosis, leprosy etc. The present invention relates to an immunoassay that allows the detection of IgG antibodies in subjects affected with Visceral Leishmaniasis and PKDL, and the assay is based on LAg from promastigotes cultivated in liquid medium.

With reference to the earlier report (Anam K. et al, (1999), Clin. Diagn. Lab. Immunol. 6 : 231-5), in order to develop a quantitative/semi quantitative method of serodiagnosis of the kala-azar patients using LAg specific ELISA, the present invention relates to alter the published protocol of the assay in various ways to shorten the assay time and to achieve optimal color intensity necessary for visual differentiation of the active kala-azar sera, for the practical utilization of the method at field conditions . In order to obtain the immunodiagnostic assay for utilization in the field conditions, in one aspect, the published method of ELISA was modified with shortened assay time in a format that could be performed mostly at room temperature without necessitating any mechanized instrument and to achieve optimal color intensity necessary for visual differentiation of the active VL sera. The main changes were standardized through different sets of experiments. Different unique aspects of the present invention are the coating of the wells of polystyrene plates with LAg at room temperature for 1 hour, blocking of the excess reactive sites with 1% bovine serum albumin (BSA) for 1 hour at 37°C or at room temperature, incubation with serum samples at prescribed dilutions for 45 minutes at 37°C or at room temperature, and further incubation with peroxidase conjugated anti-human IgG at prescribed dilution for 45 minutes at 37°C or at room temperature. In the other aspect, in order to develop a strip based detection system principles of ELISA were adapted onto the nitrocellulose membrane strips. Initial standardizations of the dipstick immunodetection system were done in the format of Dot ELISA. Dipsticks are developed as described in the figure. The assay is performed at room temperature.

In the ELISA studies 40 VL patients mostly from Bihar, admitted to School of Tropical Medicine, Calcutta, India, were tested. Additionally, serum/plasma samples from 13 PKDL patients, 23 healthy individuals from non-endemic area, 9 healthy individuals from endemic regions of VL, 15 leprosy patients, 6 filariasis patients, 2 tuberculosis patients, 1 lymphoma patient and 1 malaria patient were included as controls. Development of the assay is described in following figures and examples. As shown in Figure 4, the difference between negative and positive was greater in the case of VL sera than of PKDL sera. The healthy control sera subset gave a cutoff score of about 0.306 for the overall assay. The cutoff was calculated as mean of healthy control sera + 2 SD. Figure 4 is a X-Y scattered plot to illustrate specific IgG antibody levels measured in VL and PKDL patient sera samples. With respect to specific IgG, all 40 clinically confirmed positive VL patient sera and 13 PKDL sera gave OD readings above the negative cutoff (100% sensitivity).

In the dipstick assay 102 active VL patients, 10 cured VL patients, 20 PKDL patients, 17 healthy individuals from non-endemic area, 9 healthy individuals from endemic regions of VL, 15 leprosy patients, 6 filariasis patients, 2 tuberculosis patients, 1 leukemia patient. 1 viral fever patient, 1 lymphoma patient and 2 malaria patient were included as controls. Development of the assay from its Dot ELISA format is described in following figures and examples.

Flow chart of the invention

Indian Kala-azar and PKDL

Diagnosis difficult, invasive parasitological Antileishmanjal IgG level is elevated in methods are risky the sera of the patients

LAg (Leishmαniα promastigote membrane antigen) is highly sensitive

Available serological and specific to detect antiieishmania methods are insufficient serum IgG

Alternatively the presence of artfileishmanial antibody is detected by a simple LAg-based semi quantitative ELISA

LA is coated on to a nitrocellulose membrane in the form of a line and the membrane is attached on a hard plastic

The dipstick device is used for the immunodiagnostic. Briefly, the diluted serum sample is allowed to react with the membrane bound LAg, to form Ag-Ab complex on the membrane which is therefore detected by a peroxidase conjugated secondary antibody in a colorimetric method The following examples are given by the way of illustration of the present invention and therefore should not he construed to limit the scope of the present invention.

EXAMPLE 1 Sera/plasma Preparation:

Serum/ plasma was collected from Indian kala-azar and PKDL patients mostly from Bihar and admitted to School of Tropical Medicine, Calcutta, India, between 2002-2006, and who had either splenic aspirates, bone marrow aspirates or skin biopsies from lesions that tested positive for Leishmania parasites by culture and microscopy. In total, 122 Visceral Leishmaniasis and 20 PKDL patients with controls were tested. Human negative controls were from 23 non-endemic area normal donors from Indian Institute of Chemical Biology, Kolkata, India, with no documented infection or exposure to Leishmania parasites. In addition 9 individuals from healthy individuals from endemic regions of VL, 15 leprosy patients, 6 filariasis patients, 2 tuberculosis patients, 1 lymphoma patient and 1 malaria patient were included as controls.

Blood samples were collected generally in heparinized tubes. After running on a ficoll gradient the plasma was separated, hi some cases, where blood was collected without heparin the sera was separated from the clotted blood.

EXAMPLE 2

Parasites, Culture Conditions and LAg preparation:

Leishmania donovani (MHOM/IN/83/AG83) was originally isolated from an Indian Kala-azar patient (Ghosh, A. K., et al (1985), Exp Parasitol 60:404-13) and maintained in BALB/c mice and hamsters. The amastigotes, isolated aseptically from small pieces (approximately lOOmg) of infected animal spleens, were allowed to convert to promastigotes at 22⁰C in Medium- 199 (Ml 99) pH 7.4, supplemented with 20% heat- inactivated fetal bovine sera (FBS), 2 mM glutamine, 25 mM HEPES, 100 U/ml of penicillin G -sodium and 100 microgram/ml of streptomycin sulfate. Parasites were sub cultured at an average density of 2 x 10 cells/ml for 72 h in the same medium at 22⁰C. See Dey, T., et al. (2002), J Eukaryot Microbiol 49:270-4. Leishmania donovani promastigote membrane antigens (LAg) were prepared as described earlier (AfKn F et al; 1997). In a typical preparation, 2 x 10¹⁰ cells (1.0 g cell pellet) were suspended in 5mM cold Tris-HCl buffer, pH 7.6. The suspension was vortexed 6 times for 2 minutes each, with a 10 min interval in between ice. The parasite suspension was then centrifuged at 2,310 x g for 10 min. The crude ghost membrane pellet thus obtained was resuspended in 10 ml of the same buffer and sonicated at 4⁰C, 3 times for 1 min in an ultrasonicator. The suspension was finally centrifuged at 5,190 x g for 30 min. The supernatant containing the membrane proteins was harvested and stored at — 70⁰C. The amount of protein obtained from 1.0 g cell pellet, as assayed by Lowry's method (Lowry, O. H., et al. (1951), J Biol Chem 193:265-275) was approximately 14 mg. LAg thus prepared exhibited at least 33 distinct polypeptides in 10% SDS-PAGE ranging in molecular mass from 18 to 155kDa.

EXAMPLE 3 Antibody-Capture Enzyme-Linked Immunosorbent Assay:

Generally, in the solid phase enzyme immunoassay for Leishmania donovani, soluble antigens of the Leishmania promastigotes were coated on the inner surface of a test well which serve to bind specific antibody from a sample. Peroxidase conjugated antibody to anti-human IgG was added and reacted with bound antibody. A chromogenic substrate, such as OPD, for peroxidase was added. If antibody to Leishmania was present, there was a reaction that resulted in the development of color. Other fluorescent, chemiluminescent and chromogenic agents may be used with appropriate enzymes and substrates.

The LAg-specific IgG reactivity was tested for the sensitivity of the method. The assays were carried out on polystyrene flat-bottomed microtiter plates (Tarsoήs, India). Plate sensitization was affected by coating a polystyrene, 96- well microtitre plates with lOOμl of the LAg in phosphate buffer (2.5μg protein per well), pH 7.5 at 4⁰C overnight. The plates were then washed three times with PBS supplemented with 0.05% Tween20.

Excess reactive sites were blocked with 1% bovine serum albumin (BSA, SRL, India) (150 μl/well) for 1 h at 37⁰C, washed as before and subsequently incubated for 45 min at

37°C with VL and other control sera, at 1:1000 dilution in PBS containing 1% BSA (100 μl/well). After washing, peroxidase conjugated goat polyclonal antibody directed against human IgG₅ (Bangalore Genei, India) was applied at 1 :5000 dilution with PBS containing 1% BSA (100 μl/well) for 45 min at 37⁰C and washed thrice, o-phenylenediamine dihydrochloride (OPD, Sigma) was used as the chromogenic substrate, which was dissolved in 50 mM phosphate-citrate buffer (pH 5.0). 8 mg OPD were dissolved in 10 ml of phosphate-citrate buffer and 5 μl H₂O₂ was added just before use. After substrate addition (lOOμl/well) sufficient color development were allowed for 5-10 min at room temperature. The plate was read at 450 run in an ELISA reader. Cut off is calculated as the "Mean OD+2 Standard Deviation" of the antigen specific IgG reactivity of healthy control sera.

A) In the first experiment, the modified and shortened protocol was performed to evaluate whether the optimum OD values for the seropositive samples as published in the previous report could be achieved (blocking and incubation with 1^st and 2^nd Ab as mentioned above). Figure 1 is a view of the absorbance values of active VL (AVL), PKDL, other diseases (ODs) and healthy controls. The cut off OD is shown by dotted line.

All the AVL sera have shown comparable OD as in the previous report. Three serum samples of other diseases (one each from leprosy, lymphoma and filariasis) also showed false positive result with slightly higher OD above the cut off. Hence, with the restricted number of control samples the assay showed 100% sensitivity and >60% specificity.

B) The whole assay of example 1 (A) was repeated with a different set of healthy control, AVL and PKDL sera, including 3 serum samples of other diseases, which were false positive in the above experiment to confirm if the results of example 1 (A) were constant or artifact. Figure 2 shows that two of the samples of other diseases

(lymphoma and filariasis) and the suspected sera are still false positive, with the achievement of 100% sensitivity and 70% specificity of the assay.

C) Following experiments were performed by increasing serum dilution keeping the 2^nd Ab concentration at 1:5000 to achieve higher specificity. Figure 3 shows that when serum dilutions were applied at 1:2000 only the two samples with lymphoma and filariasis remained marginally above the cut off. In this experiment with small number of controls the specificity became 90%. Slight higher reaction of the two sera of lymphoma and filariasis with LAg in all these experiments, might be due to some non-specific interactions.

D) In the above format of the experiment, although a better specificity could be achieved, the OD values for the confirmed VL sera was drastically reduced and was not acceptable for the clear visual differentiation of positive and negative samples. Additionally, the cut off calculated from the absorbance values of healthy control samples has become much low than our previous experiments. Again, it is generally observed that the LAg-specific absorbance values for seropositive VL patients are much higher than the cut-off. This pattern of absorbance is quite different from the slightly false positive OD values of few samples of other diseases. Therefore, not to compromise with the strong color reaction of the sera of VL patients the serum dilution for the assay was fixed at 1:1000. Figure 4 is a view of the cumulative data for 40 VL patients in comparison to 13 PKDL patients, 19 healthy controls, 9 endemic controls and 10 other diseases including malaria, tuberculosis, leprosy, filariasis, and lymphoma. With these results finally the cutoff is achieved as 0.309, the sensitivity of the assay for both VL and PKDL patients is 100% and the specificity is > 92%. One endemic control sample and 2 samples of lymphoma and filariasis showed marginally false positive results. Therefore the lower specificity of previous examples were due to less numbers of samples included and the specificity increased with large number of samples studied.

E) For the field use of this immunoassay, a semi quantitative visual detection of the color reaction is essential. A reference color card has been prepared for differentiation of the positive and negative samples. It is evident from the readings of the absorbance values that the negative controls gave a basal level of color development. Therefore the color card has to be used as a reference for the positive and negative samples. Figure 5 is a perspective view of the optimum color references of positive samples, negative samples and blank wells.

F) In order to make the ELISA more convenient for the field assay the whole experiments were attempted to perform at room temperature. Previously, Reed et al (1990) and Ryan et al (2006) reported efficient ELISA where all the steps were performed at room temperature. These steps were adapted in the present invention with almost equal results. Figure 6 shows the results of the LAg-specific ELISA of 4 active VL samples in which the absorbance values are comparable with the above assays.

Example 4

Dot ELISA:

In order to develop a membrane based dipstick detection system by adapting the principal of ELISA onto a nitrocellulose strip, the initial standardizations were performed in the format of Dot ELISA. Nitrocellulose membranes were cut into strips and a tiny dot was drawn by pencil on one side of each strip to mark the position of antigen coating. The strips were soaked in 25 mM Tris-HCl (pH 7.6) and semi-dried in air. Antigen used was free LAg (2.0 μg in 2 μl) or as mentioned in the text for standardization. The antigen was coated on the marked dot in a form of a circle. The strips were then blocked with 0.1% Tween20 in 100 mM Tris-Buffer-Saline (TBS) for 1 h at room temperature, washed thrice with 100 mM TBS+0.05% Tween20, and then fully dried in the air for 30 min at room temperature. Sera from kala azar patients were diluted in 100 mM TBS+0.05% Tween20 (1:100), placed in test tubes and the strips were dipped into it and kept at room temperature for 1 h to allow the antigen-antibody reaction to take place. The strips were then thoroughly washed thrice in the same wash buffer to get rid of the excess sera. The strips were then incubated for 1 h with 1:2000 diluted peroxidase conjugate goat anti- human IgG (GENEI), followed by 3 washes as described above. The last wash was done without Tween20. Enzymatic activity was revealed with 15 mg of 3,3' -diaminobenzidine tetrahydrchloride (Sigma), in 30 ml of TBS containing 15 μl of 30% H₂O₂.

A) One of the most difficult tasks in serodiagnosis of VL is the differentiation of active VL from^' a past infection as the elevated level of antileishmanial antibodies persists in the circulation even after 2 years of cure. See Zilstra EE et al (1998) Clin. Diagn. Lab. Immunol. 5:717-720. Figure 7 is a view of the LAg-specific reactivity of different serum samples from active and cured VL in comparison to different negative control sera in Dot ELISA. In this experiment serum dilutions were applied at 1 : 100. Strip 1 and 3 are active VL, 2 and 4 are cured VL, strip 5 and 6 are healthy control, strip 7 is leprosy, and strip 8 is fϊlariasis sera. B) As it is evident that differentiation of active and cured VL sera is not practicable by detection of anti-leishmanial antibody by membrane based immunoassay, but VL sera could be differentiated from other negative control sera. A faint cross reaction was observed with the negative controls at 1:100 serum dilution. Therefore further experiments in order to differentiate the kala azar sera from other control sera, the sensitivity of the assay was evaluated and exact serum-dilution was determined, at which there would be no cross reaction with the negative control sera. Therefore the dot blot analysis were carried out at different serum-dilution, i.e. at 1:1000, 1:2000, 1:5000, 1:7500 and 1:10,000. The enzymatic color reaction using DAB as the substrate was optimized for one minute.

Figure 8 shows the results of Dot ELSA at different serum dilutions. In all the panels, Strip land 2 are VL sera, strip 3 is healthy control, strip 4 is endemic control, and strip 5 is filariasis sera. It was observed that at 1:2000 serum dilution there was absolute elimination of cross reactions by the negative control samples. Therefore for further experiments 1 :2000 serum dilution was fixed for the membrane based assay.

The assay was further repeated with a large number of samples. The results are summarized in the Table 1.

Table 1 : Summary of the results of Dot ELISA

Subjects Total +ve -ve

VL 20 20 0

HC 12 0 12

EC 5 0 5

Leprosy 3 0 3

Filariasis 2 0 2

TB 1 0 1 Example 5

Dipstick immunoassay:

The present format of the invention pertains to dipstick immunoassay device. In particular, the present invention comprises an immunological dipstick device, which incorporates the use of LAg in the test line and enzyme labeled antibodies to detect LAg- specific antibodies in the serum sample of VL patients. The device obviates the need for the intermediate washing step associated with dipstick type enzyme labeled antibody assays.

Figure 9 is a perspective view of a typical dipstick device of the present invention showing test and control lines in the configuration. Referring now to FIG. 9, test zone 4 is a nitrocellulose membrane, which is attached on a solid base 1. Base 1 is constructed from a rigid support made up of moisture impervious polyethelene card which provides for a zone in order to facilitate its movement between containers. LAg is immobilized onto zone 4 in the configuration of test line 3, here shown as a dotted line. The immunological component is operable to bind a serum antibody and therefore to the peroxidase conjugated anti-human IgG. thereby forming a complex to give colored band on test zone 4. An anti-human IgG is also immobilized in the configuration of control line 5₅ again shown as a dotted line. Control line 5 acts as an internal monitor with which to gauge assay completion. A) Dipstick Preparation.

A test zone is prepared by attaching a sample (2 cm width) of precoated nitrocellulose membrane (SNS) with a pore size of 0.45μm to a 1 mm thick polyethylene sheet serving as the base. The nitrocellulose membrane is previously soaked with 25mM Tris-HCl (pH 7.6) and semi-dried in air. The LAg was coated (1 μg / strip) on the marked dotted line in a form of a horizontal line on the test line zone 3. Simultaneously, anti-human IgG (Bangalore Genei) was coated (1 μl / strip at 1 :25 dilution) on the marked dotted line in the form of horizontal line on the control line zone 5. The nitrocellulose membrane were then blocked with 2 % BSA and 0.1% NaN₃ in 100 mM Tris-Buffer-Saline (TBS) for 1 h at room temperature, washed thrice with 100 mM TBS+0.05% Tween 20 (wash buffer), and then fully dried in the air for 30 min at room temperature. The membrane was then attached to the polyethylene card at one end by a double adhesive tape 2. The Dipsticks were then cut into 6 cm by 4 mm sticks, which can be stored in a dessicator at room temperature.

The methods by which the antibodies are labeled with enzymes are well known in the art, as is the chemistry by which these labels cause insoluble colored products to precipitate out of solution when brought into contact with the appropriate substrate solution. See Kang J et al, (1997) US patent 5656448.

In use, test zone 4 is inserted into a container of diluted serum sample (1:2000) sample. This is allowed to stand for 30 minutes during which time the target antibodies, if present, binds to the LAg immobilized in test line 3. A portion of the serum IgG antibody which does not bind to the LAg, binds to the anti-human IgG immobilized in control line 5. The dipstick is removed from the first container, rapidly washed in wash buffer and inserted into a second container containing peroxidase conjugated polyclonal anti-human IgG (Bangalore Genei) at 1:5000 dilution for 30 minutes, during which a 'LAg-antileishmanial IgG-enzyme conjugated anti-human IgG complex' is formed along the test line for a positive reaction and a sandwiched complex of 'unconjugated anti-human IgG-serum IgG-enzyme conjugated anti- human IgG' is formed along the control line. The dipstick is then removed, washed again rapidly and inserted into a third container containing the substrate solution of 3,3' -diaminobenzidine tetrahydrchloride (Sigma). Upon contact with substrate, the enzyme labels presently bound to the control line and test line act to liberate insoluble colored products which deposit along test line 3 and control line 5 in higher concentrations than in other areas of test zone 4. These results (i) in both a visible colored band in test line 3 and in control line 5 if antileishmanial IgG is present Le. a positive reaction or (ii) in only a single visible colored band in control line 5 if no antileishmanial IgG is present. This color reaction is allowed to develop maximally for 2 minutes, after which the dipsticks are removed and dried on a tissue paper.

The present mode of Dipstick device is applicable for further modification to an immunochromatographic device by using Protein A-gold detection system with proper standardizations for easier performance.

Figure 10 is a perspective view of a typical positive and negative result of the dipstick immunoassay for kala-azar. Figure 1OB is a view of 6 original dipsticks after j completion of the assay. 1-4 are the results with VL sera, 5-6 are negative control sera.

C) The assay was performed with a large number of positive VL sera and different negative control sera. The assay is also useful to detect the PKDL sera. The experiments included 102 active VL sera, 10 cured VL sera, 20 PKDL sera, 170 healthy control sera from individuals of non-endemic regions, 10 endemic control sera and 28 serum samples from different other tropical diseases including leprosy, filariasis, tuberculosis, malaria, viral fever, leukemia and lymphoma. The results are summarized in Table 2.

Table 2 : Results of the Dipstick serodiagnostic assay

Subjects Υotal +ve -ve

VL

Infected 102 102 0

Cured 10 10 0

PKDL 20 20 0

HC 17 0 17

EC 10 1* 9

Leprosy 15 0 15

Filariasis 6 0 6

TB 2 0 2

Malaria 2 0 2

Viral fever 1 0 1

Leukemia 1 0 1

Lymphoma 1 0 1

* HIV +ve

Advantages of the invention:

The main advantages of the present invention are:

1. The two assays based on ELISA and Dipstick are simple, sensitive and specific assay for diagnosis of visceral Leishmaniasis, which can detect the LAg-specific IgG antibodies in the serum of the active VL and PKDL patients. . 2. ine Dipstick assay is a simple, and rapid method, easy to perform; A small amount of blood is adequate and requires minimal processing. No special equipment is required for the storage of the dipstick or buffers. Multiple samples can be assessed simultaneously and no sophisticated equipment is required. Results can visually analyzed and interpretation is simple. Results can be obtained within 2 hr and it is therefore applicable at field level.

3. The method is equally effective as rK39 strips on Indian patients. Due to its much low cost of the LAg-Dipsticks (Rs. 1.00 per test) in comparison to rK39 strips (Rs. 70.00 approx), it can be widely applied in the Indian subcontinent.

4. PKDL samples, since show wide variation of antibody titers, can be confirmed for seropositivity by a confirmatory western blot analysis previously reported. See

Saha et al, 2005, J Clin Microbiol, 43:1269-1677.

:

Claims

We claim:

1. A non-recombinant membrane antigen (LAg) obtained directly from promastigotes oϊLeiskmania donovani strain AG83, wherein the membrane

10. antigen (LAg) being characterized by a complex of 25-35 polypeptides having molecular mass in the range of 18-155 KD and having sensitivity and specificity in the range of 95% - 100% to anti-leishmanial IgG antibodies present in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL).

15

2. A process of preparing a non recombinant membrane antigen (LAg) as claimed in claim 1, wherein the process comprising the steps of i) culturing the L. donovani strain AG83 promastigotes in M199 medium supplemented with L-glutamine, sodium bicarbonate and fetal bovine serum, 0 ii) harvesting and lysing the promastigote cells to obtain the ghost membrane, iii) centrifuging and discarding the intracellular materials from the lysed cell, iv) ultrasonicating and v) centrifuging to extract the membrane protein. 5

3. A diagnostic kit useful for detecting and quantifying specific anti- leishmanial IgG antibodies in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL) using a non-recombinant membrane antigen (LAg) obtained directly from promastigotes

30 of Leishmania donovani strain AG83 as claimed in claim 1, wherein the diagnostic kit is selected from the group consisting of ELISA kit and Dipstick assay kit.

4. A diagnostic kit useful for detecting and quantifying specific anti- 35 leishmanial IgG antibodies in the serum of patient suffering from Visceral

Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL)₃ wherein the ELISA kit comprises the LAg as claimed in claim 1 pre-coated ELISA plates, positive control, negative control, acceptable diluents, enzyme conjugated anti- use the kit .

5. A diagnostic kit useful for detecting and quantifying specific anti- leishmanial IgG antibodies in the serum of patient suffering from Visceral Leishmaniasis (VL) or Post Kala-azar Dermal Leishmaniasis (PKDL), wherein the Dipstick assay kit comprises the LAg as claimed in claim 1, pre-coated Dipstick-strips, positive control, negative control, acceptable diluents, enzyme conjugated anti-human IgG, substrate chromogen, substrate buffer and an instruction manual to use the kit.

6. The diagnostic kit as claimed in claim 5 , wherein the Dipstick kit comprises a solid base member holding a sample contact zone and test zone containing a control line portion and test line portion.

7. The diagnostic kit as claimed in claim 5 or 6 , wherein the base member used is poly ethylene sheet, test zone used is nitrocellulose.

8. The diagnostic kit as claimed in any of the claims 3 to 7, wherein the enzyme used for conjugation is horse radish peroxidase.

9. The diagnostic kit as claimed in any of the claims 5 to 8, wherein the substrate chromogen used is 3,3'-diaminobenzidine tetrahydrochloride (DAB) .

10. The diagnostic kit as claimed in claim 4 or 8 , wherein the substrate chromogen used is O-phenylenediamine dihydrochloride (OPD).

11. A process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using" the diagnostic kit as claimed in any of the claims 3 to 10 , wherein the process is selected from a group consisting of ELISA based process and Dipstick based process.

12. The process of detection and quantifying specific antiliesmanial IgG kala azar dermal leishmaniasis using the diagnostic kit as claimed in claim 11, wherein the Dipstick based process comprises the steps of: i) preparation of dipstick having control line portion and test line portion, ii) inserting the dipstick as obtained in step i) into a container of diluted serum sample and incubating for 30 minutes, iii) removing the incubated dipstick from the container and washing with buffer, iv) inserting the washed dipstick into second container containing peroxidase conjugated polyclonal anti-human IgG and incubating for 30 minutes, v) removing and washing the incubated dipstick as obtained in step iv with wash buffer, vi) inserting the dipstick as obtained in step v) into third container containing chromagen substrate to liberate colored insoluble antigen product, vii) quantifying the concentration of antigen product using color card.

13. The process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit as claimed in claim 11 or 12, wherein the diagnostic kit having Lag-polystyrene pre coated sheets are incubated at room temperature and said sheets are analyzed by using color card, without using sophisticated instrument.

14. The process of detection and quantifying specific antiliesmanial IgG antibodies in the serum of patients suffering from visceral leishmaniasis or post kala azar dermal leishmaniasis using the diagnostic kit process as claimed in any of the claims 11 to 13 , wherein said process is efficient for field usage.

15. The process as claimed in any of the claims 11 to 14, wherein the serum dilution is maintained at a ratio ranging between 1:1000 and 1:2000, based upon the assay method used for detection.