Antigenicity of cystatin-binding proteins from parasitic protozoan. Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA)
(1995) In Journal of Immunological Methods 182(1). p.63-72- Abstract
- A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi... (More)
- A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates. Due to the broad inhibition profile of r-cystatin C and the marked antigenicity of parasitic cysteine proteinases, such enzymes can be readily detected by PINC-ELISA, without requirement for prior knowledge of their substrate specificities. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1108933
- author
- Scharfstein, J ; Abrahamson, Magnus LU ; de Souza, C B ; Barral, A and Silva, I V
- organization
- publishing date
- 1995
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Cysteine proteinase, Cystatin, Kininogen, Immunoassay, Trypanosoma cruzi, Leishmania
- in
- Journal of Immunological Methods
- volume
- 182
- issue
- 1
- pages
- 63 - 72
- publisher
- Elsevier
- external identifiers
-
- pmid:7769245
- scopus:0029013249
- ISSN
- 1872-7905
- DOI
- 10.1016/0022-1759(95)00023-4
- language
- English
- LU publication?
- yes
- id
- 697736bd-46d9-4e68-adfc-53cc81054b6e (old id 1108933)
- date added to LUP
- 2016-04-01 16:47:14
- date last changed
- 2021-01-03 07:53:58
@article{697736bd-46d9-4e68-adfc-53cc81054b6e,
abstract = {{A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates. Due to the broad inhibition profile of r-cystatin C and the marked antigenicity of parasitic cysteine proteinases, such enzymes can be readily detected by PINC-ELISA, without requirement for prior knowledge of their substrate specificities.}},
author = {{Scharfstein, J and Abrahamson, Magnus and de Souza, C B and Barral, A and Silva, I V}},
issn = {{1872-7905}},
keywords = {{Cysteine proteinase; Cystatin; Kininogen; Immunoassay; Trypanosoma cruzi; Leishmania}},
language = {{eng}},
number = {{1}},
pages = {{63--72}},
publisher = {{Elsevier}},
series = {{Journal of Immunological Methods}},
title = {{Antigenicity of cystatin-binding proteins from parasitic protozoan. Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA)}},
url = {{http://dx.doi.org/10.1016/0022-1759(95)00023-4}},
doi = {{10.1016/0022-1759(95)00023-4}},
volume = {{182}},
year = {{1995}},
}