studies have demonstrated that certain cytokines may either stimulate the growth of B cells precommitted to a given isotype [4] or induce a real isotype switching of IgM-bearing B lymphocytes [5C8]. 2 T-independent response, mainly of IgG1 and IgG2 isotypes [10, 11]. The respective roles of the cytokine microenvironment and the antigenic nature in determining the isotype profile of humoral responses are still unclear. In this respect, studies in infections may be particularly revealing because of the clear Th1CTh2 dichotomy [12, 13]. Leprosy is usually a spectral disease, where the clinical presentations correlate with the level of cell-mediated immunity. At one end of the spectrum, patients present with a resistant and localized form (tuberculoid leprosy), associated with a strong and efficient cell-mediated immune response driven by IFN-. At the opposite end of this spectrum, patients present with a susceptible and disseminated form (lepromatous leprosy) associated with the absence of cell-mediated response and the predominance of a non-protective humoral response induced by IL-4. In between these two extremes are various intermediate clinicalCimmunological forms known as borderline leprosy. This dichotomy has not been demonstrated in contamination by [14]. Although Melittin the localized forms of the disease are associated with a strong IFN- response [15], the disseminated forms are found to have a IFN- decrease, but no elevated IL-4 production [16]. The other attractive feature that mycobacteria offer for our purpose is usually that they elicit an antibody response against a variety of antigenic determinants. Mycobacteria have a cell wall made of lipids and polysaccharides, and during multiplication they secrete proteins. In this study, we analyse the isotypic distributions of antibodies directed to mycobacteria antigens of different biochemical nature, namely proteins (38-kD antigen, and a cell-free extract Melittin (CFX) from (batch no. 228 [21], kindly provided by J. Colston, London, through the IMMLEP programme of WHO) and LAM (gift from P. Brennan, Colorado). Sera from Melittin TB patients were tested against 38-kD recombinant protein [22] and LAM antigens. Immulon 4 plates (Dynatech, Chantilly, VA) were coated overnight at 4C with antigens diluted in 01 m carbonate buffer pH 96 at concentrations of 1 1 g/ml, 10 g/ml and 5 g/ml for LAM, 38-kD and CFX, respectively. Optimal working dilutions of the test sera were determined in preliminary experiments. Serial five-fold dilutions of each serum sample in 015 m NaCl 10 mm PBS pH 74 with 2% bovine serum albumin (PBSCBSA) were incubated for 3 h at room temperature in a plate coated with the corresponding antigen. After washing, bound IgG was revealed by sequential probing for 1 h at room heat with an anti- MoAb (clone GG7; Sigma, St Louis, MO), and peroxidase-conjugated rabbit anti-mouse IgG antibody preabsorbed with cross-reacting human serum proteins (Jackson Immunochemicals). Dilutions yielding OD at 70% of the plateau were chosen for further isotype-specific antibody level determinations, in order to ensure that solid-phase antigen was in excess. When no plateau was evident at a dilution of 1 1:5, samples were further tested at 1:10 dilution. In the antibody quantification experiments, each assay included a calibration curve obtained with either purified polyclonal IgG (for IgG1 determination), or IgG2, IgG3, or IgG4 myeloma proteins previously calibrated by spectrophotometry at 280 nm. For the standard curve, 12 wells were coated with 50 l/well of goat antibodies specific for the human IgG F(ab)2 fragment (Sigma) at 17 g/ml in 01 m sodium carbonate buffer pH 96, at 4C overnight, and the remaining wells in the plate were coated with the bacterial antigens for the test samples. All samples were diluted in PBSCBSA, and 50 l were Melittin incubated for 3 h at room heat in triplicates in antigen-coated wells for the samples. Standard IgG proteins were incubated in duplicates in anti-F(ab)2 antibody-coated wells. Concentrations of standard immunoglobulin were 10, 50, 250, 1250 and 6250 ng/ml polyclonal IgG (corresponding to 8C5000 ng/ml IgG1) for IgG1, 16, Melittin 8, 40, 200 and 1000 ng/ml for IgG2, 08, 4, 20, 100 and 500 ng/ml for IgG3, and 024, 12, 6, 30 and 150 ng/ml for IgG4. After washing five occasions with PBSC05% Tween 20, 50 l/well of IgG subclass-specific MoAbs anti-IgG1 (clone NL16; Unipath), anti-IgG2 (clone Rabbit Polyclonal to PHACTR4 HP6014; Sigma), anti-IgG3 (clone ZG4; Unipath), or anti-IgG4 (clone RJ4; Unipath) were added at 1:5000, 1:3000, 1:10 000 and 1:3000 dilutions, respectively, in PBSCBSA. Following overnight incubation at 4C, plates were washed as before and revealed for 1 h at room temperature by a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Caltag, Burlingame, CA) diluted 1:2000 in PBSCBSA. Substrate answer.
