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Kinetics study of human retrovirus antigens expression in T lymphocytic cell lines by indirect immunofluorescence
S Gallego1, A Recalde, R Gastaldello
1Institute of Virology Dr. J.M. Vanella, School of Medical Sciences, National University of Córdoba, Argentina.
Insights
Optimizing indirect immunofluorescence assay (IFA) timing is crucial for accurate human retrovirus detection. The study found specific days post-subculture yield the highest sensitivity for detecting antibodies against HTLV-I/II and HIV-1.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- Indirect immunofluorescence assay (IFA) is a standard method for confirming human retrovirus infections.
- Viral antigen expression in infected cells can fluctuate, impacting assay sensitivity.
- Optimizing cell culture conditions and assay timing is essential for reliable results.
Purpose of the Study:
- To investigate the time kinetics of indirect immunofluorescence assay (IFA) positivity for human T-lymphotropic virus type I/II (HTLV-I/II) and human immunodeficiency virus type 1 (HIV-1).
- To determine the optimal day post-subculture for slide-based IFA to maximize sensitivity and fluorescence intensity.
- To assess the influence of serum antibody titer and cell line antigen expression on IFA results.
Main Methods:
- Kinetics of IFA positivity were studied using MT-2 (HTLV-I), MO-T (HTLV-II), CEM, and H9 (HIV-1) cell lines.
- Uninfected HT cells served as a negative control.
- Reference serum panels from the CDC were tested using conventional IFA on slides prepared on different days post-subculture.
Main Results:
- Optimal fluorescence for HTLV-I/II and HIV-1/H9 was observed on day 5 post-subculture, while HIV-1/CEM showed peak sensitivity on day 4.
- Strongly positive sera showed early reactivity, with maximal fluorescence achieved later, dependent on cell line and antibody titer.
- IFA sensitivity was significantly influenced by the day of assay and serum antibody titer, with variations in fluorescence patterns noted.
Conclusions:
- The sensitivity of IFA for detecting human retrovirus antibodies is critically dependent on the day of slide assay and serum antibody titer.
- The fifth day post-subculture is optimal for HTLV-I/II and HIV-1/H9 IFA, with day 4 preferred for HIV-1/CEM.
- An optimized IFA is highly sensitive, specific, and rapid, suitable as a supplementary or confirmatory assay for retroviral infections.
Abstract:
Indirect immunofluorescence assay (IFA) is a well-accepted assay for the confirmation of human retrovirus infection. Fluctuations in HIV-1 antigen expression in infected E-B2 cells depending on several factors have been reported. Cells kept in log phase expressed the highest levels of viral antigen. Thus, we studied the time kinetics of IFA positivity in MT-2 (HTLV-I), MO-T (HTLV-II), CEM, and H9 (HIV-1) cell lines. Uninfected T cell line, HT, was used as nonspecific control. Reference HTLV-I/II and HIV-1 serum panels from the Centers for Disease Control and Prevention (CDC) were tested by conventional IFA procedure on slides of each cell line made on different days. On the second day after subculture, HTLV-I strongly positive sera reacted on MT-2 and MO-T cells with a bright pericytoplasmic fluorescence pattern. Weakly positive sera showed a faint staining from the fifth day on, when all the sera showed the highest degree of fluorescence. With HIV-1 cell lines, sera predominantly reacted with a diffuse cytoplasmic pattern, although some sera showed a granular and pericytoplasmic capping staining. The highest degree of fluorescence was found at 3-5 days after subculture. We demonstrated that the sensitivity of the IFA for the detection of antibodies against human retroviruses depended on the day when the slides were assayed and on the serum antibody titer. The fifth day was the most appropriate for HTLV-I/II and HIV-1/H9 systems, whereas for HIV-1/CEM, the fourth day was better. Furthermore, the intensity of the immunofluorescence pattern differed with the antibody titers and the level of antigens expressed on the four cell lines studied. The IFA, improved in our laboratory, proved to be very sensitive, specific, and rapid and could be used as a supplementary/confirmatory assay for retrovirus infection.