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Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in
M R Luz1, M de N Soeiro, T C Araujo-Jorge
1Departamento de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil.
Insights
This study introduces an improved ELISA assay for detecting Trypanosoma cruzi. Preincubating chagasic human sera with cell suspensions significantly reduces background noise and enhances detection sensitivity for Chagas disease diagnosis.
Area of Science:
- Parasitology
- Immunology
- Biotechnology
Background:
- Trypanosoma cruzi causes Chagas disease.
- Existing diagnostic assays often suffer from high background noise, particularly in certain cell cultures.
- This limits their sensitivity and reliability in detecting T. cruzi infections.
Purpose of the Study:
- To develop an improved assay for detecting Trypanosoma cruzi in infected cell cultures.
- To reduce high background levels observed in previous detection methods.
- To enhance the sensitivity of T. cruzi detection using chagasic human sera (CHS).
Main Methods:
- A modified ELISA method was developed.
- Chagasic human sera (CHS) were preincubated with uninfected peritoneal macrophages or heart muscle cells (MHC).
- Preincubation was performed using both cell monolayers and cell suspensions, with the L929 cell line also tested for preadsorption.
Main Results:
- Preincubation of CHS with cell suspensions significantly reduced background levels by up to three times.
- Sensitivity for T. cruzi detection increased by up to twenty times when using cell suspensions for preincubation.
- The L929 cell line proved suitable for CHS preadsorption, further improving assay performance.
Conclusions:
- High background in previous assays may stem from human autoantibodies reacting with cell components.
- The modified procedure, involving preincubation with cell suspensions, substantially enhances ELISA performance.
- This improved assay is valuable for detecting T. cruzi, especially in low-infection or high-background scenarios.
Abstract:
We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels--hitherto observed in all experiments especially in those using HMC--CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continuous fibroblastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background.
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