ELISA and western blotting for the detection of Hsp70 and Hsp83 antigens of Leishmania donovani

Kaur, Jaspreet; Kaur, Sukhbir

doi:10.1007/s12639-012-0133-0

Cited by 11 publications

(11 citation statements)

References 40 publications

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“…The target regions for the recombinant antigen development in this study were selected based on their high potency for the diagnosis of leishmaniasis that had been employed in various immunological approaches (Celeste et al. ; Kaur and Kaur ; Maia et al. ; Reed et al.…”

Section: Discussionmentioning

confidence: 99%

“…This study characterized and cloned four recombinant antigens from L. martiniquensis, a newly emerging Leishmania species in Thailand and neighboring countries (Leelayoova et al 2017). The target regions for the recombinant antigen development in this study were selected based on their high potency for the diagnosis of leishmaniasis that had been employed in various immunological approaches (Celeste et al 2014;Kaur and Kaur 2013;Maia et al 2012;Reed et al 1987Reed et al , 1990. This study also evaluated the diagnostic efficiency of these recombinant antigens to diagnose VL using the western blot method.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection

Siripattanapipong

Kato

Tan‐ariya

et al. 2017

J Eukaryotic Microbiology

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Leishmania martiniquensis, a zoonotic hemoflagellate, is a causative agent of cutaneous (CL) and visceral leishmaniasis (VL) among humans and animals. This organism, first reported in Martinique Island, now has become an emerging infectious agent in Thailand. Symptomatic cases of L. martiniquensis infection among humans have continuously increased. In the meantime, asymptomatic infection of this novel species has seriously created national public health awareness and concern to prevent and control disease transmission. The unsuccessful serological test using the commercial rK39 dipstick based on antigen from Leishmania donovani to detect the antibodies against VL among infected Thai patients has encouraged us to further explore a new sensitive and specific antigenic epitope. In this study, we determined the sequences and expressed recombinant proteins of kinesin 39 (k39), heat shock protein 70 (hsp70), heat shock protein 83 (hsp83), and glycoprotein 63 (gp63) of L. martiniquensis to evaluate the diagnostic efficiency to detect antibodies against L. martiniquensis in patient sera. The preliminary results from western blot analysis have suggested that K39 is the most sensitive recombinant protein to detect L. martiniquensis. Moreover, this recombinant protein reacts with antibodies against L. donovani and Leishmania infantum, making it a promising antigen for further development of a universal rapid diagnostic tool for VL.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection

Siripattanapipong

Kato

Tan‐ariya

et al. 2017

J Eukaryotic Microbiology

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“…Previous studies have described that heat shock proteins, such as HSP83 and other chaperones, are among the most abundant proteins detected in Leishmania antigenic extracts and have obtained promising results that can be applied to the development of diagnostic kits (38)(39)(40)(41)(42)(43)(44). Here, the antigenicity of recombinant HSP83.1 (rHSP83.1) and three epitopes predicted in this protein were tested with sera from the TL, VL, and CVL groups.…”

Section: Discussionmentioning

confidence: 99%

Epitope Mapping of the HSP83.1 Protein of Leishmania braziliensis Discloses Novel Targets for Immunodiagnosis of Tegumentary and Visceral Clinical Forms of Leishmaniasis

Menezes‐Souza

Mendes

Gomes

et al. 2014

Clin. Vaccine Immunol.

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Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.

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“…Este estudo caracterizou esses antígenos como carboidratos de baixo peso molecular através de técnicas de precipitação e marcação específica com biotina hidrazida (Sarkari et al, 2002). (Kaur, Kaur, 2013). A presença de antígenos na LV experimental também foi demonstrada em ratos através da detecção de antígenos urinários, em que o sistema detectou amostras positivas 1 semana após infecção e ao longo de 26 semanas e foi negativo após início de tratamento (Attar et al, 2001 (Szittner et al, 2016), tuberculose (Imaz et al, 2008), doença de Alzheimer (Gustaw et al, 2008), doenças alérgicas (Paganelli et al, 1981) (Johansson et al, 2002).…”

Section: Antigenicidade De Proteínas E Carboidratos Biotiniladosunclassified

“…A detecção de antígenos pode fornecer melhores resultados laboratoriais, pois os níveis de antígenos podem ser relacionados a carga parasitária de um indivíduo com LV (Kaur, Kaur, 2013). Entretanto a detecção de antígenos por ELISA é pouco usada devido a sua baixa sensibilidade e a interferência causada por imunocomplexos circulantes, podendo ocasionar resultados falsos negativos (Ferrua et al, 2009).…”

Section: Introductionunclassified

Detecção de antígenos circulantes como abordagem diagnóstica em leishmaniose visceral.

Carvalho¹

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Carvalho CA. Detection of circulating antigens as a diagnostic approach in visceral leishmaniasis [PhD thesis (Parasitology)]. São Paulo: Instituto de Ciências Biomédicas da Universidade de São Paulo; 2017. Visceral leishmaniasis is a parasitic disease characterized by high levels of IgG antibodies, important for the diagnosis of the infection, but without discriminating active disease. Without effective role during the immune response, these antibodies participate in the formation of circulating immune complexes, indicating the presence of circulating antigens. Despite the high diagnostic specificity, the detection of antigens in serum is poorly explored in visceral leishmaniasis. The detection of antigens and immunocomplexes depend on both the specificity of antibodies and the characteristics of the antigens, which may be from large proteins or small polysaccharides. In this study, we evaluated the detection of free circulating antigens or immunocomplexes, the characteristics and antigenic specificity of IgG antibodies to proteins or carbohydrates of L. (l) infantum in experimental visceral leishmaniasis. Hamster samples with experimental infection by L. (L.) infantum chagasi was evaluated at 15, 30, 45, 60 and 90 days of infection. High concentrations of specific IgG antibodies were detected in samples with experimental and natural visceral leishmaniasis, always with high avidity, even in initial periods of infection. Immunoaffinity purification of low avidity IgG also confirmed this high concentration. Proteins and carbohydrates were isolated from the total antigenic extract of promastigotes by molecular exclusion chromatography or ethanol precipitation and labeled with biotin for amines or sugars. For use in ELISA, carbohydrates were conjugated to BSA, which allowed the detection of high levels of carbohydrate-specific IgG antibodies. The presence of antigenic carbohydrates was also demonstrated by the detection of IgG increase in samples with experimental infection, which confirmed the presence of circulating immunocomplexes specific to carbohydrates. Our data suggest that the involvement of the humoral immune response in visceral leishmaniasis should have the involvement of carbohydrates of the parasite. The development of new diagnostic tools for the detection of antigenic carbohydrates may allow the identification of immunocomplexes and circulating antigens in visceral leishmaniasis.

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ELISA and western blotting for the detection of Hsp70 and Hsp83 antigens of Leishmania donovani

Cited by 11 publications

References 40 publications

Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection

Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection

Epitope Mapping of the HSP83.1 Protein of Leishmania braziliensis Discloses Novel Targets for Immunodiagnosis of Tegumentary and Visceral Clinical Forms of Leishmaniasis

Detecção de antígenos circulantes como abordagem diagnóstica em leishmaniose visceral.

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