Simultaneous determination of total IgE and allergen-specific IgE in serum by the MAST chemiluminescent assay system.

We have developed a chemiluminescent immunoenzymometric system. The first commercial application of this chemiluminescent assay (CLA) is the measurement of total IgE and allergen-specific IgE in human serum. The CLA system is a second-generation adaptation of the MAST RIA allergy profiling system. The MAST CLA system assay protocol consists of three steps: overnight incubation of serum, a 4-h incubation with enzyme-labeled antibody, and a 30-min chemiluminescent reaction, which produces a visible image (immunograph) on high-speed Polaroid instant film. The densities of the bands produced on the film are quantified with an inexpensive microprocessor-controlled infrared transmittance densitometer. The novel luminogenic substrates used yield a constant light output for over 2 h with an intensity at least 10-fold greater than that of commercial chemiluminescent reagents. The MAST CLA system exhibits sensitivity, specificity, and precision equal to that of the MAST RIA system (r = 0.96 for 40 serum samples analyzed with 25 allergens). As many as 35 different allergens per sample can be quantified in a single assay. The MAST CLA system requires no standard curve or volume-dependent pipetting steps, incorporates both positive and negative controls for each sample, and quantifies allergen-specific IgE at picomolar concentrations.

Application of the MAST Immunodiagnostic System to the determination of allergen-specific IgE.

The MAST Immunodiagnostic Test System was developed to provide a comprehensive, simple means for the in vitro measurement of multiple antigens or antibodies. The first commercial application of the MAST system incorporates several novel features for cost-effective diagnosis of IgE-mediated allergy in a clinical laboratory or a physician's office. The basis of the MAST system is a unique analytical test chamber, which contains cellulose thread as the solid-phase matrix and allows multiple test results from a single assay. This test chamber incorporates both positive and negative controls and requires no volume-dependent pipetting steps. Immunographic exposure onto high-speed Polaroid instant film allows for quantifying results with an automatic recording infrared-transmittance densitometer. Test results are easily interpreted by using a patient test record provided with the system. The MAST system greatly simplifies testing for allergen-specific IgE, while retaining specificity and sensitivity. Currently, with the MAST system one can simultaneously measure picomoles of allergen-specific IgE in up to 35 different allergen classes. In addition to allergy testing, the MAST technology is applicable to other immunodiagnostic profiles.

Surface IgG-mediated mixed lymphocyte culture and cell-mediated lympholysis responses by immunocompetent thymus cells.

Radiolabeled preparations of murine surface immunoglobulin have been shown to bind selectively to immunocompetent responder thymus cells. Such bound immunoglobulin (especially IgG) facilitate the mixed lymphocyte culture and cell-mediated lympholysis responses between immunocompetent thymus-cell subsets. Papain digest of IgG also promote alloantigen recognition between immunocompetent thymocytes; most of the activity residues in the Fc piece. The binding of IgG per se does not activate responder thymocytes. Alloantigen-specific MLC and CML responses in vitro appear to require the participation of alloantigen receptors on the responder cell, the expression of alloantigens by the stimulator thymus cells and the binding of B-cell immunoglobulin by the immunocompetent responder thymocytes. When any of these processes are interrupted an alloantigen-specific response is not generated in vitro.

Binding of surface IgG by immunocompetent thymus cells.

We have previously shown that myeloma-derived or serum-derived immunoglobulin could facilitate in vitro alloantigen recognition by immunocompetent thymus cells ('subset-I thymocytes'). The present report demonstrates the isolation of surface immunoglobulins from radiolabeled splenic B lymphocytes. Solubilized radiolabeled preparations of surface immunoglobulin are shown to bind in vitro to lymphoid cells, especially to immunocompetent thymus cells. Immunocompetent thymus cells (subset-I thymocytes preferentially bind more IgG than IgM. Papain digests of IgG are also bound by subset-I thymocytes; preferential binding of the Fc piece is observed. Although the binding of IgG or its pieces per se does not activate lymphoid cells in vitro a role for the binding of IgG during alloantigen recognition in vitro between immunocompetent thymus cells is demonstrated.

Generation of cytotoxic effector cells by immunocompetent thymus cell subpopulations.

Immunocompetent mouse thymus cell subsets (TH-2) cultured with allogeneic TH-2 cells, required a peripheral lymphoid cell in order to generate specifically cytotoxic T cells (CTL) in vitro. The helper cell is identified as a B cell, and can be supplied either in the mixture of target or responding peripheral cells, or in optimal ratios of 10:1 to 20:1 as nonproliferating cells syngeneic to the responder, when both responder and target are TH-2. Allogeneic helper cells are much less effective. Kinetic studies indicate that helper cell activity involves initial interaction with stimulator cell and is then required throughout the period of generation of cytotoxic lymphocytes. Addition of critical levels of certain IgG preparations, particularly IgG2A appears to support generation of CTL in TH-2/TH-2 cultures but is considerably less effective than intact B cells. Cytotoxicity generated under these conditions has the same genetic requirements for I-region histoincompatibility between stimulator and responder, and for K or D identity of stimulator and target cell, as has been reported for peripheral cell CTL.