Poulin L, Delage R, Darveau A; International Conference on AIDS.

Int Conf AIDS. 1992 Jul 19-24; 8: A30 (abstract no. PoA 2166).

Centre Hospitalier Universite Laval, Que., Canada.

OBJECTIVES: To elaborate a long term B lymphocytes in vitro culture system suitable for the analysis of humoral immunity in HIV-1 seropositive individuals. METHODS: B lymphocytes from HIV-1 infected individuals and HIV-1 seronegative subjects immunized with tetanus toxoid were purified from peripheral blood by centrifugation on Ficoll and negative selection using anti-CD2 monoclonal antibodies coupled to magnetic beads. The resulting B lymphocytes were cultured at different concentrations in 96 wells plates in the presence of recombinant human IL-4 and anti-CD40 monoclonal antibodies on irradiated 16.2 CG7 cells (a murine Ltk- cell line expressing the Fc receptor Fc gamma RII/CDw32). Culture supernatants were screened for the presence of specific antibodies by ELISA. Antibodies were further characterized by Western blotting. RESULTS: In this in vitro culture system, purified B lymphocytes isolated from HIV-1 seropositive individuals proliferated similarly to B lymphocytes isolated from HIV-1 seronegative subjects and could be grown for more than 21 days. Moreover, specific anti-HIV-1 antibodies were readily detected in culture supernatants by ELISA. In a typical experiment, specific anti-HIV-1 antibodies were detected in more than 50% of the wells when B lymphocytes were seeded at 2.5 x 10(4) cells/well. This was in sharp contrast to anti tetanus toxoid antibodies detected in less than 5% of B cell cultures prepared from immunized HIV-1 seronegative individuals. Antibody specificity was further defined by Western blotting using culture supernatants. Anti-gp160 (Env) and anti-p24 (Gag) were the most frequently detected antibodies followed by anti-p65 and anti-p51 (Pol). Other anti-HIV-1 antibodies were also detected. CONCLUSION: The in vitro system that has been used stimulates the proliferation and differentiation of B lymphocytes isolated from seropositive individuals leading to the secretion of specific anti-HIV-1 antibodies. This system will enable us to perform detailed qualitative and quantitative analysis of the humoral response in seropositive individuals and to correlate the resulting pattern with the progression of the disease.

Publication Types:

• Meeting Abstracts

Keywords:

• Animals
• Antibody Formation
• Antibody Specificity
• B-Lymphocytes
• Blotting, Western
• Culture
• Enzyme-Linked Immunosorbent Assay
• Gene Products, env
• Genes, env
• Genes, gag
• Genes, pol
• HIV Antibodies
• HIV-1
• Humans
• In Vitro
• genetics
• immunology

Other ID:

• 92400462

UI: 102198175

From Meeting Abstracts