Optimal conditions for in vitro anti-human immunodeficiency virus type 1 (HIV-1) antibody (Ab) synthesis and detection were re-appraised. Western blot (WB) and radioimmunoassay (RIA) could detect about 1 and 10 ng, respectively, of HIV-1-specific Ab (HIV-Ab), while the sensitivity of an enzyme-linked immunosorbent assay (ELISA) was much lower. Optimal HIV-Ab recovery was obtained by culturing 2.5 x 10(6) peripheral blood mononuclear cells (PBMC)/ml from seropositive subjects for 16 days in the absence of mitogens; at higher cell concentrations, background levels were unacceptably high. The background of non-de novo synthesized HIV-Ab was due to insufficient PBMC washing and/or cytophilic immunoglobulin (Ig); a particular washing procedure, as well as 24 h peripheral blood mononuclear cells (PBMC) pre-culture, might help in limiting this phenomenon. However, results should be compared with those obtained in cultures containing puromycin especially in infants, where a higher CD16 antigen expression in lymphocytes is likely responsible for increased amounts of cytophilic Ig released in culture supernatants, compared to adults.
Standardization of in vitro synthesis and detection of HIV-1-specific antibodies
COLOMBATTI, Marco;
1993-01-01
Abstract
Optimal conditions for in vitro anti-human immunodeficiency virus type 1 (HIV-1) antibody (Ab) synthesis and detection were re-appraised. Western blot (WB) and radioimmunoassay (RIA) could detect about 1 and 10 ng, respectively, of HIV-1-specific Ab (HIV-Ab), while the sensitivity of an enzyme-linked immunosorbent assay (ELISA) was much lower. Optimal HIV-Ab recovery was obtained by culturing 2.5 x 10(6) peripheral blood mononuclear cells (PBMC)/ml from seropositive subjects for 16 days in the absence of mitogens; at higher cell concentrations, background levels were unacceptably high. The background of non-de novo synthesized HIV-Ab was due to insufficient PBMC washing and/or cytophilic immunoglobulin (Ig); a particular washing procedure, as well as 24 h peripheral blood mononuclear cells (PBMC) pre-culture, might help in limiting this phenomenon. However, results should be compared with those obtained in cultures containing puromycin especially in infants, where a higher CD16 antigen expression in lymphocytes is likely responsible for increased amounts of cytophilic Ig released in culture supernatants, compared to adults.
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