WO2009130709A2 - Antigène membranaire non recombinant, kit de diagnostic l'employant et procédé de détection de la leishmaniose viscérale et de la pkdl - Google Patents

Antigène membranaire non recombinant, kit de diagnostic l'employant et procédé de détection de la leishmaniose viscérale et de la pkdl Download PDF

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WO2009130709A2
WO2009130709A2 PCT/IN2009/000238 IN2009000238W WO2009130709A2 WO 2009130709 A2 WO2009130709 A2 WO 2009130709A2 IN 2009000238 W IN2009000238 W IN 2009000238W WO 2009130709 A2 WO2009130709 A2 WO 2009130709A2
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serum
leishmaniasis
dipstick
diagnostic kit
lag
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PCT/IN2009/000238
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WO2009130709A3 (fr
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Nahid Ali
Samiran Saha
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Council Of Scientific & Industrial Research
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa

Definitions

  • 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.
  • LAg membrane antigen 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.
  • 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).
  • LAg membrane antigen
  • 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).
  • VL Visceral Leishmaniasis
  • PKDL Post Kala-azar Dermal Leishmaniasis
  • 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.
  • 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.
  • VL 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.
  • 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.
  • 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.
  • DAT Direct agglutination test
  • Enzyme linked immunosorbent assay 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.
  • ELISA 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.
  • Ld-ESM L. donovani promastigotes
  • 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).
  • 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.
  • LAg membrane antigen
  • 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).
  • VL Visceral Leishmaniasis
  • PKDL Post KaIa- azar Dermal Leishmaniasis
  • 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).
  • VL Visceral Leishmaniasis
  • PKDL Post Kala-azar Dermal Leishmaniasis
  • 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).
  • LAg membrane antigen 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
  • 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.
  • 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).
  • VL Visceral Leishmaniasis
  • PKDL Post Kala-azar Dermal Leishmaniasis
  • a process of preparing a non recombinant membrane antigen (LAg) 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.
  • 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 •
  • the ELISA 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
  • 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.
  • VL Visceral Leishmaniasis
  • PKDL Post Kala-azar Dermal Leishmaniasis
  • 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.
  • the base member used is poly ethylene sheet
  • test zone used is nitrocellulose
  • the enzyme used for conjugation is preferably horse radish peroxidase.
  • the substrate chromogen used is 3,3'-diaminobenzidine tetrahydrochloride (DAB) for Dipstick kit.
  • DAB 3,3'-diaminobenzidine tetrahydrochloride
  • the substrate chromogen used is O- phenylenediamine dihydrochloride (OPD) for ELISA kit
  • 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.
  • 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
  • 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.
  • 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.
  • LAg membrane antigen
  • 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.
  • the assay of the invention has a high degree of sensitivity and specificity.
  • the assay of the invention has a combined sensitivity of about 100% and specificity of about 95.7%.
  • the dipstick assay of the present invention developed for field diagnosis has sensitivity of about 100% and a specificity of about 98%.
  • 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
  • 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.
  • L. donovani promastigotes membrane antigen (LAg)-specific ELISA 5 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.
  • LAg membrane antigen
  • 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.
  • 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 .
  • 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.
  • BSA bovine serum albumin
  • 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).
  • Diagnosis difficult, invasive parasitological Antileishmanjal IgG level is elevated in methods are risky the sera of the patients
  • LAg (Leishm ⁇ ni ⁇ promastigote membrane antigen) is highly sensitive
  • 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.
  • 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.
  • 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, Marie, India, with no documented infection or exposure to Leishmania parasites.
  • 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.
  • 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.
  • amastigotes isolated aseptically from small pieces (approximately lOOmg) of infected animal spleens, were allowed to convert to promastigotes at 22 0 C 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 22 0 C. See Dey, T., et al.
  • LAg Leishmania donovani promastigote membrane antigens
  • the suspension was finally centrifuged at 5,190 x g for 30 min.
  • the supernatant containing the membrane proteins was harvested and stored at — 70 0 C.
  • 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.
  • 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.
  • 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 (Tarso ⁇ s, India). Plate sensitization was affected by coating a polystyrene, 96- well microtitre plates with lOO ⁇ l of the LAg in phosphate buffer (2.5 ⁇ g protein per well), pH 7.5 at 4 0 C overnight. The plates were then washed three times with PBS supplemented with 0.05% Tween20.
  • BSA bovine serum albumin
  • 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.
  • example 1 (A) 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
  • 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.
  • 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.
  • FIG. 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 f ⁇ lariasis sera.
  • 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.
  • Strip land 2 are VL sera
  • strip 3 is healthy control
  • strip 4 is endemic control
  • 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 present format of the invention pertains to dipstick immunoassay device.
  • 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.
  • FIG. 9 is a perspective view of a typical dipstick device of the present invention showing test and control lines in the configuration.
  • 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 5 5 again shown as a dotted line. Control line 5 acts as an internal monitor with which to gauge assay completion.
  • a test zone is prepared by attaching a sample (2 cm width) of precoated nitrocellulose membrane (SNS) with a pore size of 0.45 ⁇ m 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 ⁇ g / strip) on the marked dotted line in a form of a horizontal line on the test line zone 3.
  • anti-human IgG (Bangalore Genei) was coated (1 ⁇ l / strip at 1 :25 dilution) on the marked dotted line in the form of horizontal line on the control line zone 5.
  • nitrocellulose membrane were then blocked with 2 % BSA and 0.1% NaN 3 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.
  • TBS Tris-Buffer-Saline
  • 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.
  • 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.
  • peroxidase conjugated polyclonal anti-human IgG Beangalore Genei
  • the dipstick is then removed, washed again rapidly and inserted into a third container containing the substrate solution of 3,3' -diaminobenzidine tetrahydrchloride (Sigma).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PKDL samples since show wide variation of antibody titers, can be confirmed for seropositivity by a confirmatory western blot analysis previously reported. See

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Abstract

La présente invention concerne un antigène membranaire non recombinant (LAg) directement obtenu à partir de promastigotes de Leishmania donovani souche AG83. L'antigène membranaire (LAg) a les caractéristiques suivantes d'un complexe de 25-35 polypeptides et une masse moléculaire dans la plage de 18 - 155 KD. Ladite membrane est sensible et spécifique jusqu'à la plage de 95 % à 100 % aux anticorps IgG anti-Leishmania dans le sérum d'un patient soufrant de leishmaniose viscérale (VL) ou de leishmaniose cutanée post kala-azar (PKDL). La présente invention concerne aussi un procédé de préparation dudit antigène membranaire et son procédé d'utilisation pour la détection d'anticorps IgG anti-Leishmania présents dans le sérum d'un patient soufrant de leishmaniose viscérale (VL) ou de leishmaniose cutanée post kala-azar (PKDL). La présente invention concerne en outre un kit de diagnostic comprenant ledit antigène membranaire non recombinant, utile pour détecter des anticorps IgG anti-Leishmania présents dans le sérum d'un patient soufrant de leishmaniose viscérale (VL) ou de leishmaniose cutanée post kala-azar (PKDL). La présente invention concerne également un procédé de détection et de quantification d'anticorps IgG anti-Leishmania présents dans le sérum de patients soufrant de leishmaniose viscérale (VL) ou de leishmaniose cutanée post kala-azar (PKDL) au moyen du kit de diagnostic.
PCT/IN2009/000238 2008-04-23 2009-04-17 Antigène membranaire non recombinant, kit de diagnostic l'employant et procédé de détection de la leishmaniose viscérale et de la pkdl WO2009130709A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016113749A1 (fr) * 2015-01-13 2016-07-21 Council Of Scientific & Industrial Research Kit utile pour mesurer des anticorps dirigés contre un antigène membranaire non recombiné (lag) dans un échantillon d'urine
US10422806B1 (en) * 2013-07-25 2019-09-24 Theranos Ip Company, Llc Methods for improving assays of biological samples

Citations (1)

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EP0293827A2 (fr) * 1987-06-01 1988-12-07 Yeda Research And Development Company, Ltd. Essai pour leishmaniose

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EP0293827A2 (fr) * 1987-06-01 1988-12-07 Yeda Research And Development Company, Ltd. Essai pour leishmaniose

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Title
AFRIN ET AL: "Adjuvancity and protective immunity elicted by Leishmania donovani antigens encapsulated in positively charged liposomes" INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, US, vol. 65, no. 6, 1 June 1997 (1997-06-01), pages 2371-2377, XP002991927 ISSN: 0019-9567 *
ANAM K ET AL: "Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients." CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY MAR 1999, vol. 6, no. 2, March 1999 (1999-03), pages 231-235, XP002541685 ISSN: 1071-412X *
BANDYOPADHYAY S ET AL: "Purification, characterization of O-acetylated sialoglycoconjugates-specific IgM, and development of an enzyme-linked immunosorbent assay for diagnosis and follow-up of indian visceral leishmaniasis patients" DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASES, ELSEVIER SCIENCE PUBLISHING CO., AMSTERDAM, NL, vol. 50, no. 1, 1 September 2004 (2004-09-01), pages 15-24, XP004565933 ISSN: 0732-8893 *
MARTIN S K ET AL: "A DIAGNOSTIC ELISA FOR VISCERAL LEISHMANIASIS, BASED ON ANTIGEN FROM MEDIA CONDITIONED BY LEISHMANIA DONOVANI PROMASTIGOTES" ANNALS OF TROPICAL MEDICINE AND PARASITOLOGY, ACADEMIC PRESS, LONDON, GB, vol. 92, no. 5, 1 January 1998 (1998-01-01), pages 571-577, XP002942877 ISSN: 0003-4983 *
MUKERJI K ET AL: "Direct enzyme-linked immunosorbent assay: a simple immunoassay using Leishmania donovani promastigote for diagnosis of kala-azar." JOURNAL OF CLINICAL LABORATORY ANALYSIS 1991, vol. 5, no. 4, 1991, pages 299-301, XP002942878 ISSN: 0887-8013 *
SAHA SAMIRAN ET AL: "Immune responses in kala-azar." THE INDIAN JOURNAL OF MEDICAL RESEARCH MAR 2006, vol. 123, no. 3, March 2006 (2006-03), pages 245-266, XP002541684 ISSN: 0971-5916 *
SAHA SAMIRAN ET AL: "Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis." JOURNAL OF CLINICAL MICROBIOLOGY MAR 2005, vol. 43, no. 3, March 2005 (2005-03), pages 1269-1277, XP002541682 ISSN: 0095-1137 *
TRULLOLS E ET AL: "Validation of qualitative analytical methods" TRAC, TRENDS IN ANALYTICAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 23, no. 2, 1 February 2004 (2004-02-01), pages 137-145, XP004566146 ISSN: 0165-9936 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10422806B1 (en) * 2013-07-25 2019-09-24 Theranos Ip Company, Llc Methods for improving assays of biological samples
WO2016113749A1 (fr) * 2015-01-13 2016-07-21 Council Of Scientific & Industrial Research Kit utile pour mesurer des anticorps dirigés contre un antigène membranaire non recombiné (lag) dans un échantillon d'urine

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