“…The separated polypeptides were transferred to a nitrocellulose paper sheet and the membranes were blocked with BLOTTO overnight. Vertical strips were cut from the sheets and treated for 4h at room temperature with serum sample (10,20,30,40,50,60 and 90 days post-infection) diluted 1:50 in BLOTTO. Strips were treated for 1 h at RT with peroxidase-labelled, affinitypurified rabbit anti-mouse immunoglobulins diluted 1; 500.…”
Section: Methodsmentioning
confidence: 99%
“…13 (KSAE-PKSAEPKSAEP-COOH) and SAPA (YDSTAHGTPSTPADS-SAHSTPSTPA-COOH) and the purified recombinant proteins nos. 1 and 7 [18][19][20][21][22]. The procedure used for this test was performed essentially as described previously [31].…”
Section: Methodsmentioning
confidence: 99%
“…Additionally, peptides and clones isolated from a genomic expression library of T. cruzi [18][19][20][21][22] were employed in an FLISA test to determine the reactivity of a hyperimmune rabbit serum against T. raw^e//epimastigote antigens. Among these antigens it is important to emphasise the sequence SAPA (Shed Acute Phase Antigen).…”
The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post-infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post-infection the range was between 1:5120 and 1:10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30-50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.
“…The separated polypeptides were transferred to a nitrocellulose paper sheet and the membranes were blocked with BLOTTO overnight. Vertical strips were cut from the sheets and treated for 4h at room temperature with serum sample (10,20,30,40,50,60 and 90 days post-infection) diluted 1:50 in BLOTTO. Strips were treated for 1 h at RT with peroxidase-labelled, affinitypurified rabbit anti-mouse immunoglobulins diluted 1; 500.…”
Section: Methodsmentioning
confidence: 99%
“…13 (KSAE-PKSAEPKSAEP-COOH) and SAPA (YDSTAHGTPSTPADS-SAHSTPSTPA-COOH) and the purified recombinant proteins nos. 1 and 7 [18][19][20][21][22]. The procedure used for this test was performed essentially as described previously [31].…”
Section: Methodsmentioning
confidence: 99%
“…Additionally, peptides and clones isolated from a genomic expression library of T. cruzi [18][19][20][21][22] were employed in an FLISA test to determine the reactivity of a hyperimmune rabbit serum against T. raw^e//epimastigote antigens. Among these antigens it is important to emphasise the sequence SAPA (Shed Acute Phase Antigen).…”
The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post-infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post-infection the range was between 1:5120 and 1:10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30-50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.
In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens.
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