Method for immunodiagnostic testing of visceral leishmaniasis, kit and use

Abstract

METHOD AND KIT FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS USING ANTIGENIC PROTEINS OF LEISHMANIA INFANTUM. This technology relates to a method and a kit for the sensitive and specific serological immunological diagnosis of canine and human visceral leishmaniasis, comprising the use of 17 (seventeen) antigenic proteins of Leishmania infantum, individually or in combination, as well as in recombinant form or as specific B-lymphocyte epitopes, individually or in combination.

Description

1 / 22 “METHOD FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, KIT AND USE”

[001] The present technology relates to a method and a kit for the sensitive and specific serological immunological diagnosis of canine and human visceral leishmaniasis, comprising the use of an antigenic protein of Leishmania infantum.

[002] Visceral leishmaniasis (VL) is a neglected but potentially fatal disease that occurs in several countries around the world. It is caused by protozoan parasites of the genus Leishmania, belonging to the Leishmania donovani complex in Africa and the Indian subcontinent, and by the species Leishmania infantum in Europe and Latin America (Alvar J, et al. WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One, 7 e35671, 2012). An incidence of 500,000 new cases of VL is estimated, and more than 50,000 deaths occur each year. With the increasing number of cases of parasite resistance to currently available drugs for the treatment of the disease, early detection through accurate diagnosis is important to ensure immediate treatment for the patient, and is also an important control measure for VL, aiming to interrupt the parasite's transmission cycle (Srivastava P, et al.Diagnosis of Indian visceral leishmaniasis by nucleic acid detection using PCR. PLoS One 6:e19304, 2011).

[003] In endemic areas, infected individuals who are unable to control the infection develop acute and symptomatic VL. However, a significant portion of individuals residing in such areas remain infected but without developing active disease, behaving as asymptomatic patients (Caldas AJ, et al. Risk factors associated with asymptomatic infection by Leishmania chagasi in north-east Brazil. Trans R Soc Trop Med Hyg.96:21-8, 2002). This fact may suggest that infections Petition 870260018109, dated 26 / 02 / 2026, page 9 / 32 2 / 22 repeated infections are common in such areas, and a percentage of infected individuals may develop immunity and resistance to the disease (Das VN, et al. Asymptomatic infection of visceral leishmaniasis in hyperendemic areas of Vaishali district, Bihar, India: a challenge to kalaazar elimination programmes. Trans R Soc Trop Med Hyg. 105(11):6616, 2011). In a study conducted in Brazil, it was observed that only 20% of patients infected with L. infantum develop acute and symptomatic VL, while the vast majority of patients remain asymptomatic (Barão SC, et al. Human asymptomatic infection in visceral leishmaniasis: a seroprevalence study in an urban area of ​​low endemicity. Preliminary results. Am J Trop Med Hyg 77:1051-3, 2007).

[004] Patients with symptomatic VL typically present with clinical signs and symptoms of systemic infection, including fever, fatigue, loss of appetite, and weight loss. However, clinical findings are often inconclusive and confused with other co-endemic diseases of VL that have a more common incidence, such as Chagas disease, malaria, tuberculosis, AIDS, among others (Srividya G, et al. Diagnosis of visceral leishmaniasis: developments over the last decade. Parasitol Res. 110:1065-1078, 2012).

[005] Accurate diagnosis of VL is the first step towards achieving the goal of eliminating the disease, as well as ensuring the patient receives rapid and possibly more effective treatment. However, parasitological diagnosis is considered complex and invasive, being based on direct microscopic examination of aspirates from the spleen, lymph nodes, or bone marrow of patients. This diagnosis occurs by demonstrating the amastigote forms of the parasites through such microscopic examinations in the collected samples (Reus M, et al. Visceral leishmaniasis: diagnosis by ultrasound-guided fine needle aspiration of an axillary node. Br J Radiol 78:158-160, 2005; Artan R, et al. Liver biopsy in the diagnosis of visceral Petition 870260018109, dated 26 / 02 / 2026, page 10 / 32 3 / 22 leishmaniasis. J Gastroenterol Hepatol 21:299-302, 2006). The technique is capable of detecting the presence of the parasite and is low-cost. Its specificity is high, but its sensitivity is variable and depends on the presence of the parasite in that small sample collected. This occurs because the parasites may not be present in the sample collected from patient aspirates, leading to false-negative results (Del Olmo ML, et al. Visceral leishmaniasis diagnosed by duodenal biopsy. Rev Esp Enferm Dig 101:439-440, 2009). Also, the collection procedure carries risks of internal bleeding for patients, which can be fatal. The results also depend on the professional's technical skills and the quality of the prepared slides (Del Olmo ML, et al. Visceral leishmaniasis diagnosed by duodenal biopsy. Rev Esp Enferm Dig 101:439-440, 2009).

[006] Thus, no method currently available for the diagnosis of VL presents all the desirable characteristics of high sensitivity and specificity, in addition to being easy to perform and low cost. This fact emphasizes the importance of combining immunological, parasitological, and molecular tests in order to achieve high positivity rates in the diagnosis of the disease (Cota GF, et al. Comparison of parasitological, serological, and molecular tests for visceral leishmaniasis in HIV-infected patients: a cross-sectional delayed-type study. Am J Trop Med Hyg.89(3):570-7, 2013).

[007] Polymerase chain reaction (PCR) is a molecular parasitological test that has been used with a relatively high degree of success in the diagnosis of VL. The technique is considered more sensitive and specific than conventional parasitological diagnostic methods. However, factors such as differences in methodology, such as the use of blood or bone marrow aspirates, as well as DNA extraction procedures, the choice of target gene, and the use of controls, remain. Petition 870260018109, dated 26 / 02 / 2026, page 11 / 32 4 / 22 appropriate resources and the presence of well-trained technicians have been limiting factors for the dissemination of PCR as a standard diagnostic method (Antinori S, et al. Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single-center, 8-year experience in Italy and review of the literature. Clin Infect Dis 44: 1602-1610, 2007).

[008] The need to develop diagnostic tools that can identify patients with asymptomatic and symptomatic VL, as well as distinguish them from individuals residing in endemic areas of the disease or from patients developing diseases that cause cross-reactivity in the diagnosis of VL, justifies the search for new methods (Boelaert M, et al. Diagnostic tests for kala-azar: a multi-centre study of the freeze-dried DAT, rK39 strip test and KAtex in East Africa and the Indian subcontinent.

[009] Serological methods based on the identification of anti-Leishmania antibodies in patient sera have been developed and / or improved. Recombinant antigens have considerably improved the sensitivity and specificity of immunological diagnosis of VL, compared to the use of crude extracts or semi-purified fractions. Such antigens have high diagnostic value for immunocompetent patients, but have compromised efficacy in immunosuppressed patients (Nigro L, et al. Comparison of the indirect immunofluorescent antibody test and the direct agglutination test for serodiagnosis of visceral leishmaniasis in HIV-infected subjects. Eur J Clin Microbiol Infect Dis.15(10):832-5, 1996).

[0010] Tests such as the Enzyme-Linked Immunosorbent Assay (ELISA), Indirect Fluorescent Antibody Test (IFAT), Western blot, and the direct agglutination test (DAT) have been used for such purposes. There is the advantage Petition 870260018109, dated 26 / 02 / 2026, p. 12 / 32 5 / 22 in improving such a diagnostic condition, given that sample collection is based on venipuncture to withdraw a blood aliquot, and is considered less invasive and with fewer risks to patients compared to the aspirates required for sample collection and execution of parasitological examinations (Peixoto HM, et al. Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis. Trop Med Int Health.20(3):334-52, 2015).

[0011] ELISA and Western blot techniques have played an important role in the serodiagnosis of VL (Salotra P, et al. Western blot analysis of humoral immune response to Leishmania donovani antigens in patients with post-kala-azar dermal leishmaniasis. Trans R Soc Trop Med Hyg. 93:98-101, 1999). Among the recombinant antigens used, a kinesin-related protein K39 has been used to detect patients with active VL. This protein is encoded by the kinesin-related gene and contains repetitive sequences of 39 amino acid residues. The recombinant protein rK39 derived from L. chagasi has been shown to be an effective marker for the diagnosis of VL using the ELISA technique (Badaró R, et al. rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis. 173(3):75861, 1996).The protein has been used in the serodiagnosis of the disease in several countries around the world, showing varying values ​​of sensitivity and specificity (Singh DP, et al. In search of an ideal test for diagnosis and prognosis of kala azar. J Health Popul Nutr 28:281-285, 2010).

[0012] The commercial kit (Dipstick rK39®) is a test for the detection of human and canine leishmaniasis, and is recommended by the World Health Organization (WHO). However, its validation for detecting cases of L. infantum infection has varied according to the strain and culture of the parasites, the recombinant protein production process, and the Petition 870260018109, dated 26 / 02 / 2026, page 13 / 32 6 / 22 clinical and serological variability of patients in different countries (Zijlstra EE and El-Hassan AM. Leishmaniasis in Sudan: post kala-azar dermal leishmaniasis. Trans R Soc Trop Med Hyg. 95:S59-S76, 2001).

[0013] Other Leishmania proteins have also been tested for serodiagnosis of VL. Parasite surface antigens, cytoplasmic or nuclear proteins have been shown to exhibit a specific humoral response in patients with VL, thus indicating their potential for serodiagnosis of the disease. Other kinesin-related proteins, such as K26, K9, KRP42, and KE16, have been tested in the serological diagnosis of VL, and have shown relative degrees of success (Takagi H, et al. Short report: production of recombinant kinesin-related protein of Leishmania donovani and its application in the serodiagnosis of visceral leishmaniasis. AmJTrop Med Hyg.76:902-905, 2007).

[0014] The humoral response against the A2 protein, a specific protein for amastigote forms of some Leishmania species, has been investigated in India, Sudan, and the Americas, and the results have shown varying degrees of sensitivity and specificity (Carvalho FA, et al. Diagnosis of American visceral leishmaniasis in humans and dogs using the recombinant Leishmania donovani A2 antigen. Diagn Microbiol Infect Dis.43:289-295, 2002; Ghedin E, et al. Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis. Clin Diagn Lab Immunol.4:530-535, 1997). The amastin protein has also been evaluated with a relatively high degree of success in the serodiagnosis of VL (Rafati S, et al. Amastin peptide-binding antibodies as biomarkers of active human visceral leishmaniasis. Clin Vacc Immunol.13:1104-1110, 2006).

[0015] An ideal serodiagnostic test for VL should focus on the use of antigens that can be reactive with the sera of infected patients, regardless of clinical symptoms, without being Petition 870260018109, dated 26 / 02 / 2026, page 14 / 32 7 / 22 recognized by individuals without the disease or by sera from patients developing diseases related to VL, such as Chagas disease, malaria, tuberculosis, among others. However, current antigens present problems related to cross-reactivity with sera from such patients (Abass E, et al. rKLO8, a Novel Leishmania donovani Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan. PloS NTDs. 7, e2322, 2013).

[0016] The heterogeneity of the humoral response observed in patients with VL indicates that, for effective serodiagnosis of VL, a combination of antigens in the same ELISA could be desirable, compared to the use of antigens isolated from the parasites (Singh DP, et al. In search of an ideal test for diagnosis and prognosis of kalaazar. J Health Popul Nutr. 28:281-285, 2010). A combination of different antigens with diagnostic value could also detect asymptomatic infections, thus being highly desirable for serodiagnosis of the disease. Monitoring the effectiveness of treatment and preventing disease relapses are considered fundamental for the control of VL. Currently available serological tests can remain positive for long periods of time after cure (Gidwani K, et al. Persistence of Leishmania donovani antibodies in past visceral leishmaniasis cases in India. Clin Vaccine Immunol. 18:346-348, 2010).Therefore, research into new antigenic markers that have the ability not only to diagnose acute cases of the disease, but also to allow for monitoring of treatment and cure of patients, should also be considered (Sakru N, et al. Investigation of asymptomatic visceral leishmaniasis cases using Western blot in an endemic area in Turkey. New Microbiol. 30:13-18, 2007). Research into antigens expressed in the promastigote forms of Leishmania could be considered relevant for improving serodiagnostic conditions. Petition 870260018109, dated 26 / 02 / 2026, page 15 / 32 8 / 22 of LV, since this morphological form is the one that comes into initial contact with the mammalian host and triggers the infection process, as well as the beginning of the antileishmanial immune response in the infected host. Thus, the first antibodies are produced against proteins expressed in this morphological form of the parasites (Tripathi P, Gupta SK, Sinha S, Sundar S, Dube A, Naik S. Prophylactic efficacy of high-molecular-weight antigenic fractions of a recent clinical isolate of Leishmania donovani against visceral leishmaniasis. Scand J Immunol. 2008 Nov;68(5):492-501. Santos-Gomes GM, Rodrigues A, Teixeira F, Carreira J, Alexandre Pires G, Carvalho S, Santos-Mateus D, Martins C, Vale-Gato I, Marques C, Tomás AM. Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells. Vaccine. March 5, 2014;32(11):1247-53).

[0017] Thus, using such antigenic markers from this stage, it would be possible to develop a serodiagnostic kit for VL that would be able to detect the disease in its initial phase, thus allowing for faster and more effective treatment, as well as enabling a lower incidence of side effects from currently used drugs, since treatment, if carried out at the beginning of the infection and before the establishment of chronic disease, should probably be more effective against the parasites.

[0018] Research using proteomics to evaluate protein expression models in Leishmania offers the possibility of analyzing the functions of various proteins, including those previously identified only by genomics and considered hypothetical, as well as new diagnostic markers, vaccine candidates and / or therapeutic targets against the disease (Coelho, VT, et al. Identification of proteins in promastigote and amastigote-like Leishmania using an Petition 870260018109, dated 26 / 02 / 2026, page 16 / 32 9 / 22 immunoproteomic approach. PLoS neglected tropical diseases 6 e1430., 2012; Duarte MC, et al. Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis. Clin Vaccine Immunol. 22(11):1187-96, 2015).

[0019] Studies have been developed to analyze the expression and differentiation of stage-specific proteins in Leishmania (El Fakhry Y, et al. Proteomic approach to identify developmentally regulated proteins in Leishmania infantum. Proteomics. 2:1007-1017, 2002; Brotherton MC, et al. Analysis of stage-specific expression of basic proteins in Leishmania infantum. J Proteome Res. 9:3842-3853, 2010). The specific binding of antibodies to parasite antigens, produced during the different stages of disease progression in patients, certainly contributes to refining this analysis, which aims to identify not only differentially expressed proteins, but also new antigens recognized by the host's immune system during active infection (Costa, MM et al., 2012. Analysis of Leishmania chagasi by 2-D difference gel electrophoresis (2-D DIGE) and immunoproteomics: identification of novel candidate antigens for diagnostic tests and vaccine. J. Proteome Res.10, 2172-2184; Martins, VT et al., 2013. Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the superoxygenase family, against visceral leishmaniasis. PLoS Negl. Trop. Dis. 7, e2148. Martins, VT et al, 2015. A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis. Parasite. Vectors 8, 363).

[0020] Patent document BR102015010593, entitled “Kit for serological diagnosis of leishmaniasis, based on immunoproteomics and use” deals with the identification of 19 (nineteen) proteins isolated from Petition 870260018109, dated 26 / 02 / 2026, page 17 / 32 10 / 22 Leishmania braziliensis parasite for application in the serological diagnosis of cases of VL and Human Cutaneous Leishmaniasis (CL). In this technology, the species Leishmania braziliensis and sera from patients with cutaneous leishmaniasis were used. This differs from the present application because no immunoproteomic study was performed with the species Leishmania infantum and sera from patients with visceral leishmaniasis were used. In other words, different models were used between the technologies and, in addition, the proteins in question are not similar.

[0021] Patent document US6365165, entitled “Leishmania antigens for use in the therapy and diagnosis of leishmaniasis,” describes a method for stimulating the immune system and a vaccine composition for the treatment of leishmaniasis. This technology used the species *Leishmania donovani* and immunostimulation and T-cell proliferation experiments in BALB / c mice to identify proteins exhibiting a more immunogenic character. This technology differs from the present application because the experimental approach is different; that is, the present technology conducts an immunoproteomic study with the species *Leishmania infantum* and uses sera from patients with visceral leishmaniasis to identify antigenic proteins.Furthermore, the present technology is based on a humoral response model using antibodies from sick individuals, unlike the aforementioned technology which used a cellular response model to identify the proteins of interest.

[0022] Patent document WO2014091463, entitled “Method for producing leishmania recombinant proteins and use in a diagnostic kit and vaccine against leishmaniases,” refers to the use of recombinant proteins HSP 83-1, MAPK, and MAPK3, selected by bioinformatics methods involving immunoproteomics and phylogenetic algorithmic analyses, for the production of a vaccine and diagnostic kit for Petition 870260018109, dated 26 / 02 / 2026, page 18 / 32 11 / 22 leishmaniasis. The aforementioned technology differs from the present application due to the different experimental approach and the proteins in question being distinct from those contained in this document.

[0023] The present technology deals with the selection of L. infantum antigenic proteins by immunoproteomics that can be used, individually or in combination, as well as in recombinant form or of their specific B lymphocyte epitopes, individually or in combination; for the sensitive and specific serological immunological diagnosis of canine and human VL.

[0024] In the state of the art, no similar technology has been found using the proteins contained in the present application selected from L. infantum by immunoproteomic analysis, which characterizes its inventive activity. BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure 1 represents the immunoblotting of the total protein extract of stationary-phase promastigote forms of L. infantum using different pools containing serum samples from patients. Two-dimensional gels were prepared with the total protein extract (200 μg) of L. infantum, with the first dimension in IEF pH 4.0 to 7.0 and the second dimension in SDS-PAGE 12%. These gels were then subjected to immunoblotting experiments and reacted with serum pools from healthy individuals residing in an endemic area of ​​VL (A), with serum from patients with VL (B), or from patients with Chagas disease (C). Spots recognized by the VL patient serum pool are marked in red, and by the Chagas disease patient serum pool are marked in yellow.

[0026] Figure 2 shows the spots selected for identification of reactive proteins in immunoblotting experiments with sera from patients with VL. After performing the experiments of Petition 870260018109, dated 26 / 02 / 2026, page 19 / 32 In 12 / 22 immunoblotting, spots that were not reactive with sera from healthy individuals or with sera from patients with Chagas disease were marked (red circle) and excised from four different gels for subsequent analysis by mass spectrometry and identification of native proteins. DETAILED DESCRIPTION OF THE TECHNOLOGY

[0027] The present technology relates to a method and a kit for the sensitive and specific serological immunological diagnosis of human and canine visceral leishmaniasis, comprising the use of 17 (seventeen) antigenic proteins of Leishmania infantum, individually or in combination, as well as in recombinant form or of their specific B lymphocyte epitopes, individually or in combination.

[0028] These proteins were selected using an immunoproteomic tool composed of two-dimensional electrophoresis, immunoblotting and mass spectrometry techniques, developed with total protein extracts from stationary-phase promastigote forms of L. infantum and sera from patients with symptomatic and asymptomatic visceral leishmaniasis (VL), as well as from patients with Chagas disease and healthy individuals residing in an endemic area of ​​the disease.

[0029] More specifically, the 17 (seventeen) Leishmania infantum proteins used in the proposed method and kit are defined by SEQ ID No. 1 to 17.

[0030] The proposed method for serodiagnosis of visceral leishmaniasis is characterized by comprising the following steps: a) Exposure of a sample to at least one of the proteins defined by the SEQ ID sequences No. 1 to 17, or their corresponding epitopes, with such antigens bound to a solid support or carrier; Petition 870260018109, dated 26 / 02 / 2026, p. 20 / 32 13 / 22 b) Addition of an antibody or a protein, which are conjugated to an enzyme or a marker and which bind to the antibodies in the sample from step (a); c) Detection of specific antibodies for visceral leishmaniasis in the sample mentioned in step (a), using reagents capable of detecting the enzyme or marker mentioned in step (b).

[0031] In step “a”, the sample may be selected from the group comprising blood, serum, plasma or other body fluid, without the need for large quantities thereof. The solid support may comprise materials consisting of nitrocellulose, nylon, latex, polypropylene, polystyrene or other used for the same purpose as those described above.

[0032] In step “b”, the secondary antibody may comprise IgG, IgM, IgA, IgE and their respective subclasses. The protein related to this step may be Protein A and / or Protein G. The enzyme described in this step may be selected from the group consisting of peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. Furthermore, the marker may be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents.

[0033] In step “c”, the detection antibody can be selected from the group comprising fluorescence, immunofluorescence, immunoluminescence, absorbance or radioisotope detection.

[0034] The kit for the diagnosis of visceral leishmaniasis proposed in the present invention is characterized by comprising the proteins defined in SEQ ID No. 1-17, which can be expressed in recombinant form or their specific B lymphocyte epitopes produced in synthetic form, and in all forms can be used alone or in combination. Petition 870260018109, dated 26 / 02 / 2026, page 21 / 32 14 / 22

[0035] The proteins contained in the present invention, in recombinant form or as their synthetic epitopes, may be used for serodiagnosis not only of human VL, but also of canine VL, due to the similarity of recognition of these proteins by the immune system of these mammalian hosts.

[0036] These proteins were specifically recognized by serum samples from patients with symptomatic and asymptomatic VL, but not by antibodies in the sera of healthy individuals residing in an endemic area of ​​the disease or of patients with Chagas disease.

[0037] The present invention can be better understood through the following examples, which are not limiting in the technology. EXAMPLE 1. PROTEIN SELECTION BY IMMUNOPROTEOMICS

[0038] Preparation and extraction of total proteins from L. infantum. Promastigote forms (1 x 1010) were washed 3 times in 40 mM Tris-HCl, pH 7.5, centrifuged at 5,000 x g for 10 minutes at 4°C. The precipitate was resuspended in lysis buffer [7 M urea, 2 M thiourea, 4% chol-amidopropyl dimethylammonio-1-propanesulfonate (CHAPS), 40 mM dithiothreitol (DTT), 2% IPG (pH 4-7) and 40 mM Tris base], adding a protease inhibitor cocktail (GE Healthcare, Uppsala, Sweden). Samples were incubated for 1 hour at room temperature with constant stirring. Protein purification was performed by precipitation using the 2D Clean Up Kit (GE Healthcare), according to the manufacturer's instructions. The extract was quantified using the 2-D Quant-Kit (GE Healthcare), and aliquots were kept at -80°C until use.

[0039] Isoelectric focusing. Gels with pH gradients immobilized on strips (Immobiline DryStrips Gels, GE Healthcare) of constant size (7 cm) were used, with a linearly determined pH range from 4.0 to 7.0, using the Ettan IPGphor III system (GE Healthcare). As Petition 870260018109, dated 26 / 02 / 2026, p. 22 / 32 15 / 22 samples were thawed and diluted in DeStreak and 1% IPG buffer (GE Healthcare) to a final volume of 125 μL, containing 150 μg of protein. Rehydration was performed by adding the strip, gel side down, to the diluted sample volume within a suitable holder containing separate wells for each strip. To seal the strips, 3 mL of mineral oil (Cover Fluid, GE Healthcare) was used per well, completely covering them and keeping the sample and strips isolated from the external environment. Rehydration was performed for a minimum of 12 and a maximum of 20 hours, as per the manufacturer's technical recommendations. After rehydration, the strips were transferred from the plastic holder to the appropriate sarcophagus for use in the focusing device. The strips were placed with the gel side up and covered with mineral oil.The apparatus was assembled by adding current-conducting paper moistened with water (Paper Wick, GE Healthcare) to each end of the tapes, along with supports that allow the passage of current.

[0040] Reduction, alkylation and second dimension. The strips were equilibrated using equilibrium buffer (75 mM Tris-HCl, pH 8.8, 6 M urea, 29% glycerol PA, 2% SDS and 0.001% bromophenol blue) supplemented with 1% DTT, incubated for 15 minutes to reduce the thiol group of the cysteine ​​residues, causing the cleavage of disulfide bonds. Subsequently, the strips were transferred to equilibrium buffer containing 2.5% iodoacetamide and incubated for 15 minutes to alkylate the free thiol group of the cysteine ​​residues. For the second dimension, 12% SDS-PAGE gels were prepared using the mini VE Vertical Electrophoresis system (GE Healthcare). The running buffer consisted of 25 mM Tris base, 0.2 M glycine, and 0.5% SDS, pH 8.8, and was maintained at 10°C throughout the run and under cooling. After polymerization of the gels, the reduced and alkylated strips were obtained in step [previous step]. Petition 870260018109, dated 26 / 02 / 2026, page 23 / 32 16 / 22 previous strips were washed and positioned in the channels, with the acidic region of the strip positioned to the right of the standard molecular weight marker (BioRad, Broad Range). The strips were then sealed with 0.5% agarose solution. Migration occurred at 15 mA / gel for 30 minutes and subsequently at 20 mA / gel until the end of the run (Figure 1).

[0041] Staining, digitization, and image analysis. Gel staining was performed using a succession of timed baths under gentle agitation to ensure reproducibility. Solution volumes were standardized to: 100 mL solution / gel for 7 cm strips and 250 mL solution / gel for 13 cm strips. Gel staining was performed with Coomassie colloidal Brilliant Blue G-250. Storage was carried out at 4°C in a solution containing 1% acetic acid. Images were documented using the Typhoon scanner (GE Healthcare), and analyses were performed using PDQuest™ software (BioRad). The theoretical isoelectric point (pI) of the proteins identified by de novo sequencing was calculated on the website (http: / / scansite.mit.edu / calc_mw_pI.html).

[0042] Immunoblotting assays. To identify protein spots reactive with antibodies present in serum pools from patients with symptomatic and asymptomatic VL, healthy individuals residing in VL-endemic areas, and patients with Chagas disease, immunoblotting experiments were used. For this purpose, the total extract of stationary-phase promastigote forms of L. infantum was separated on 2-DE gels and transferred to cellulose membranes (Schleicher & Schull, Dassel, Germany) using a semi-dry transfer apparatus for 2 hours at 400 mA in transfer buffer. Afterwards, the membranes containing the transferred proteins were incubated in 1x PBS-T blocking solution pH 7.4 (5% skimmed milk powder in PBS, plus 0.05% Tween 20) for 2 hours. Petition 870260018109, dated 26 / 02 / 2026, page 24 / 32 17 / 22 room temperature. After blocking, the membranes were incubated in 0.05% PBS-Tween 20 solution, and the serum pools, diluted 1:400, were added. The membranes were incubated for 1 hour, then washed with 0.05% PBS-Tween 20 three times, for 10 minutes each time; they were then incubated in a new 0.05% PBS-Tween 20 solution containing the anti-human IgG conjugate linked to the peroxidase enzyme, at a dilution of 1:10,000, for 1 hour. Subsequently, the membranes were washed in 0.05% PBS / Tween 20 solution three times, for 10 minutes each time, and two further washes with PBS were performed twice, for 10 minutes each time.

[0043] The reaction was developed using a solution composed of 25 mg of chloronaphytol, 8.5% methanol, 50 mg of diaminobenzidine, and 20 DL of 30 vol. hydrogen peroxide (H2O2) for each 100 mL of 1x PBS. The reaction was stopped by adding distilled water. The 2-DE gels and membranes were digitized using ImageScanner III (GE Healthcare) and analyzed using Melanie Viewer 7.0 2D Gel Analysis software, available at http: / / world2dpage.expasy.org / melanie / 2DImageAnalysisViewer.html (Expasy). (Bioinformatics Resource Portal). Protein spots recognized only by antibodies in serum samples from patients with VL, but not recognized by antibodies from healthy individuals or patients with Chagas disease, were collected from the gels for subsequent protein identification (Figure 2).

[0044] Tryptic digestion in polyacrylamide gel. Selected protein spots were excised from promastigote gels and decolorized in a 50% acetonitrile and 25 mM ammonium bicarbonate solution, pH 8.0. Three 15-minute washes were performed under vortex agitation. Subsequently, the gels were dehydrated in acetonitrile for 5 minutes and dried. The dehydrated gel fragments were subjected Petition 870260018109, dated 26 / 02 / 2026, p. 25 / 32 18 / 22 samples were digested with trypsin (Promega cat.#V511) at 20 ng / L in 25 mM ammonium bicarbonate for 18 hours at 37°C. The generated peptides were extracted with 5% formic acid and 50% acetonitrile for 30 minutes under constant stirring. Finally, the peptides were concentrated to a volume of 10 µL. The samples were passed through Zip-Tip columns (C18 resin; P10, Millipore Corporation, Bedford, MA) to remove salts. Elution of the peptides from the column was performed in 50% acetonitrile and 0.1% trifluoroacetic acid.

[0045] MALDI-TOF / TOF mass spectrometry. Trypsin-digested peptides were confirmed for purity and identity using an Ultraflex III MALDI-TOF / TOF mass spectrometer (Bruker, Daltonics, Germany), equipped with a SmartBeam laser and data analysis in a computer program controlled by FlexControl 3.0 software. Samples were resuspended in ultrapure water and mixed with a matrix of α-cyano-4-hydroxycinnamic acid (5 mg) dissolved in distilled water (200 pL), acetonitrile (250 pL) and 3% aqueous TFA solution (50 pL), in a 1:3 (v:v) ratio; then, they were applied in duplicate to an MTP Massive plate (384 wells) and allowed to dry at room temperature.Molecular weights were determined by MS in reflected / positive mode, and peptide sequences were confirmed by MS / MS in LIFT™ mode, both with external peptide mixture calibration, according to the manufacturer's instructions (Peptide Calibration Standard I, Bruker Daltonics, Germany). Mass spectra were acquired after 200 laser shots at a frequency of 100 Hz, and mass detection was performed in the range of 1,000 to 4,000 m / z. EXAMPLE 2. IDENTIFICATION OF PROTEINS OF INTEREST

[0046] The primary sequence of the peptides was interpreted by de novo sequencing of the spectra obtained by MS / MS analyses. Petition 870260018109, dated 26 / 02 / 2026, page 26 / 32 19 / 22 Spectra were processed and visualized using Flex Analysis 3.0 software. Amino acid sequences of proteins and peptides were compared with other sequences available in the NCBI database (http: / / blast.ncbi.nlm.nih.gov) using the Leishmania organism (taxid: 5658). Low-quality spectra were not considered for the database search; only reliable sequences of sequenced peptides were used for correct identification by alignment. Peptide sequences were extended to the nearest tryptic cleavage site, and the coincidence between theoretical and experimental masses, as well as the quality of the alignment, were considered as criteria for identification. Alignments were performed using the Clustal W multiple alignment tool, available in Bioedit v.7.0.4.1 software. Protein identification is presented in Table 1. Table 1. Proteins of interest identified in immunoblotting experiments. Legends: 1) Name of the identified protein; 2) Accession code on the NCBI website; 3) Species in which the protein was first identified. Protein name1 Accession code2 Species3 CAS / CSE / importin domain XP_001566967.1 L. braziliensis protein Endonuclease III XP_001562623.1 L. braziliensis Paraflagellar rod protein 1C Q86PL5 L. Mexican Elongation factor 2 Q4Q259 L. major Paraflagellar rod protein 1D XP_003392693.1 L. infantum Pyridoxal kinase XP_001566767.1 L. braziliensis GTP-binding protein XP_001466177.1 L. infantum Polynucleotide Q4QFL6 L. major adenylyltransferase Elongation factor 1-beta CBZ37569.1 L. donovani Hypothetical protein Q4Q534 L. major Hypothetical protein Q4Q8Z6 L. major Protein hypothetical XP_003878089.1 L. Mexicana hypothetical protein XP_003886492.1 L. infantum Petition 870260018109, dated 26 / 02 / 2026, page 27 / 32 20 / 22 Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein CBZ32527.1 Q4QGG5 CBZ35811.1 XP 001566388.1 L. donovani L. major L. donovani L. braziliensis EXAMPLE 3. CLONING, EXPRESSION AND PURIFICATION OF PARAFLAGELLAR ROD PROTEIN 1D AND HYPOTHETICAL PROTEIN

[0047] The nucleotide sequences of paraflagellar rod protein 1D (XP_003392693.1) and hypothetical (XP_003886492.1) proteins were obtained from the NCBI (National Center for Biotechnology Information) website (http: / / www.ncbi.nlm.nih.gov). The recombinant proteins were obtained after cloning their coding genes from L. infantum DNA. They were expressed in Escherichia coli strain M15 and purified by affinity chromatography on a nickel column (Qiagen GmbH, Germany), after induction for 4 hours using 1.0 mM IPTG at 37°C. EXAMPLE 4. VALIDATION OF PROTEINS FOR SERODIAGNOSIS OF LV

[0048] Although in immunoblotting experiments the 17 proteins can already be considered validated for the serodiagnosis of VL, since they were recognized by antibodies in sera from patients with VL, but not by antibodies from patients with Chagas disease or healthy individuals, an additional validation was performed with 2 of the 17 randomly selected proteins. For this, they were cloned and their recombinant forms were evaluated in ELISA experiments. To this end, titration curves were performed to determine the most appropriate concentration of antigens and dilutions of serum samples for use in the experiments. Microtiter plates (Falcon) were sensitized with the paraflagellar rod protein 1D and a hypothetical protein or with SLA from L. infantum (in Petition 870260018109, dated 26 / 02 / 2026, page 28 / 32 21 / 22 concentrations of 0.5, 0.5, and 2.0 pg per well, respectively), dissolved in 100 pL of coating buffer (50 mM carbonate buffer, pH 9.6), for 16 hours at 4°C. Then, the free sites were blocked using 200 pL of a PBS-T solution (saline plus 0.05% Tween 20), containing 5% albumin, for 1 hour at 37°C. After washing the plates three times with PBS-T, serum samples from patients with asymptomatic (n=10) and symptomatic (n=20) visceral leishmaniasis, patients with Chagas disease (n=20), and healthy individuals from endemic (n=30) or non-endemic (n=30) areas were added to the wells and diluted 1:200 in PBS-T. The incubation was carried out for 1 hour at 37°C. Afterwards, the plates were washed five times in PBS-T and incubated with anti-human IgG conjugate linked to the peroxidase enzyme, at a dilution of 1:10,000 in PBS-T, for 1 hour at 37°C.Afterwards, the plates were washed seven times with PBS-T and the reactions were carried out by incubation with a solution composed of 2 mL of H2O2, 2 mg of orthophenylenediamine and 10 mL of citrate-phosphate buffer, pH 5.0, for 30 minutes in the dark. The reaction was stopped by the addition of 25 µL of 2N H2SO4. The absorbances of the samples were measured using a spectrophotometer (Molecular Devices Spectra Max Plus, Canada) at a wavelength of 492 nm. The results of the sensitivity and specificity values ​​are shown in Table 2. Table 2. Sensitivity and specificity results using recombinant paraflagellar rod protein 1D and hypothetical proteins for serodiagnosis of visceral leishmaniasis. Abbreviations: Se: sensitivity. Sp: specificity. CI: confidence interval. SLA: soluble protein extract of the parasites. Antigen Se(%) CI 95% Sp(%) CI 95% Paraflagellar rod protein 1D 100.0 79.4-100.0 100.00 94.13-100.00 Hypothetical 100.0 79.4-100.0 100.00 94.22-100.00 SLA L. infantum 62.5 42.5-85.5 45.5 25.5-65.0 Petition 870260018109, dated 26 / 02 / 2026, page 29 / 32 22 / 22

[0049] It can be observed that the two proteins that were identified in the immunoproteome and randomly selected from among the 17 proteins for validation in the serodiagnosis of VL presented sensitivity and specificity values ​​of 100% for the serological detection of patients with VL. Petition 870260018109, dated 26 / 02 / 2026, pp. 30 / 32

Claims

1 / 2 CLAIMS 1- METHOD FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS characterized by comprising: a) exposure of a sample of blood, serum, plasma or other body fluid to the polypeptide defined by the sequence SEQ ID No1, linked to a solid support or carrier; b) addition of a secondary antibody selected from the group comprising IgG, IgM, IgA, IgE and their respective subclasses or a protein selected from the group comprising Protein A and / or Protein G, conjugated to an enzyme or marker and which bind to the antibodies possibly present in the sample from step (a); c) detection of Leishmania-specific antibodies in the aforementioned sample using reagents capable of detecting the enzyme or marker mentioned in step (b). 2- METHOD FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, according to claim 1, step “a”, characterized by the solid support being selected from a group of materials consisting of nitrocellulose, nylon, latex, polypropylene, polystyrene or other used for the same purpose as those described above. 3- METHOD FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, according to claim 1, step “b”, characterized by the enzyme being selected from the group consisting of peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. 4- METHOD FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, according to claim 1, step “b”, characterized by the marker being selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. 5- KIT FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, characterized by comprising: a. the protein defined by SEQ ID No. 1; b. a secondary antibody selected from the group comprising IgG, IgM, IgA, IgE and / or their respective subclasses or a protein selected from the group comprising protein A and / or protein G, where the secondary antibody or protein is conjugated to an enzyme or a marker; c. a reagent for detecting the enzyme or marker defined in item “b”. 6- KIT FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, according to claim 5, characterized in that the protein is bound to a solid support, selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene, or to a carrier. 7- KIT FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, according to claim 5, characterized by the marker being selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. 8- KIT FOR IMMUNODIAGNOSTIC TESTING OF VISCERAL LEISHMANIASIS, according to claim 5, characterized by the enzyme being selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. 9- USE of the protein defined by SEQ ID No. 1, characterized as being in the diagnosis of visceral leishmaniasis in humans and dogs. Petition 870260018109, dated 02 / 26 / 2026, page 8 / 32

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