Differential responses of human B-lymphocyte subpopulations to graded levels of CD40-CD154 interaction.

Naïve and memory B-lymphocyte populations are activated by CD154 interaction through cell-surface CD40. This interaction plays an important role in the regulation of the humoral immune response, and increasing evidence indicates that fine variation in CD40 binding influences B lymphocytes, macrophages and dendritic cells in murine models. Here we have investigated whether and how variations in the intensity of the CD40-CD154 interaction could contribute to differential regulation of human B-lymphocyte populations. Proliferation and differentiation of B lymphocytes were monitored in response to graded levels of CD40 stimulation in the presence of interleukin (IL)-2, IL-4 and IL-10. Our results show that the level of CD154 binding to CD40 on B lymphocytes can directly influence the evolution of CD19(+) CD27(-) and CD19(+) CD27(+) cell populations. Furthermore, proliferation, global expansion of CD19(+) cells and emergence of CD38(++) CD138(+) cells, as well as immunoglobulin G (IgG) and IgM secretion, were affected by the level of exposure of B lymphocytes to CD154. These results suggest that the CD40-CD154 interaction is more like a rheostat than an on/off switch, and its variation of intensity may play a role in the regulation of B-lymphocyte activation following the primary and/or secondary humoral immune response.

Sensitive detection of human IgG in ELISA using a monoclonal anti-IgG-peroxidase conjugate.

Enzyme-antibody (Ab) conjugates specific for IgG are widely used in indirect immunological assays and have been until recently routinely prepared with polyclonal IgG-specific animal Abs. The use of monoclonal Abs (MAbs) could permit a better standardization of the IgG-specific conjugate reagents but is expected to result in lower reactivity due to the recognition of a single epitope by the MAbs. In this work, we have characterized a monoclonal anti-human IgG-peroxidase (HRP) reagent and compared its reactivity with commercial reagents. The murine C5-1 anti-human IgG MAb was selected for conjugation because of its high affinity (K(a) = 1.9 x 10(10)M), pan-IgG reactivity and absence of cross-reactivity with various structures including animal IgGs. The specific activity and binding kinetics of the C5-1:HRP conjugate were similar to the ones of two polyclonal anti-IgG:HRP conjugates when tested with immobilized human IgG. The C5-1:HRP conjugate could detect low amounts of human IgG much more effectively than two commercial monoclonal conjugates although it was slightly less effective than a polyclonal conjugate. However, the C5-1 conjugate yielded reduced background reactivity compared to the polyclonal conjugate, resulting in similar signal-to-noise ratios. These results indicate that the C5-1:HRP conjugate could be a suitable substitute for anti-human IgG conjugates prepared from animal antisera.