Detection of immunoglobulin isotypes from dried blood spots.

The study was designed to determine the sensitivity and reproducibility of recovering immunoglobulin (Ig) isotypes (IgG subclasses, IgA, IgE and IgM classes) from dried blood spots (DBS), a methodologic subcomponent of the Upstate KIDS Study. A multiplexed Luminex assay was used for IgG1/2/3/4, IgA and IgM analysis; an ELISA was used for IgE. Plasma samples from de-identified patients were used to compare the Luminex assay with nephelometry, which is routinely used to quantify IgA, IgG and IgM in clinical samples. The IgE ELISA was compared to an immunofluorescence assay. Prior to evaluation of punches from newborn dried blood spots (NDBSs), recoveries of Ig from punches of cord blood DBSs (CBDBSs) vs. plasma from the same cord bloods were compared. Although the recoveries of Ig from plasma and DBSs were not comparable, which could be due to cell lysates in the DBS samples, the analyses were reproducible. Additionally, the levels of IgA, IgG2, IgG4, and IgM recovered from CBDBSs positively correlated with those in plasma. The DBS data is a relative value since it is not equivalent to the plasma concentration. The majority of Ig concentrations recovered from 108 newborns of the Upstate KIDs Study were within the range of newborn plasma Ig levels with the exception of IgG3. The IgG4 values displayed the greatest variance with a wide range (0.01-319 mg/dl), whereas, IgG1 values had the narrowest range (85.2-960.4 mg/dl).

Enhanced antigen-specific antibody and cytokine responses when targeting antigen to human FcGAMMA receptor type I using an anti-human FcGAMMA receptor type I-streptavidin fusion protein in an adjuvant-free system.

There is a continuing need for alternatives to current human adjuvants. Recombinant protein vaccines, which target antigen to human Fc gamma receptor type I (hFcgammaRI) on hFcgammaRI-expressing antigen presenting cells, provide one potential alternative. Using a recombinant anti-hFcgammaRI-antigen fusion protein and adjuvant independent mouse model, we demonstrate enhanced antigen-specific antibody responses to low doses of antigen, when targeting antigen to hFcgammaRI in vivo. Enhanced antibody production to hFcyRI-targeted antigen is evident in both primary and secondary immune responses, as compared to that of non-targeted antigen. Furthermore, antibody isotype and cytokine responses following immunization with hFcgammaRI-targeted antigen, suggest enhancement of both Th1 and Th2 responses.

Low-level signaling generated by FcgammaRIIB-B cell receptor co-ligation establishes a state of global B cell receptor nonresponsiveness.

In addition to the stimulatory, antigen-specific B cell receptor (BCR), B lymphocytes also express multiple inhibitory receptors, including Fc gamma receptor type IIB (FcgammaRIIB). Moreover, many laboratories have demonstrated that co-ligation of BCR molecules to inhibitory FcgammaRIIB molecules with high concentrations (10-15 microg/ml) of ligand results in altered BCR signaling. However, there are no reports on the effect of low concentrations of ligand on BCR-FcgammaRIIB co-ligation and subsequent signaling. This knowledge will be critical for optimizing the in vivo use of such reagents. Accordingly, the effect of low ligand concentration on the level of BCR-FcgammaRIIB co-ligation and subsequent BCR signaling was analyzed. The results demonstrate that co-ligation of BCR and FcgammaRIIB molecules at low concentrations (0.5-1.5 microg/ml) of cross-linking reagent, establishes a condition that prevents the B cell from responding to subsequent stimulation, even when the initial exposure to cross-linking reagent fails to generate a calcium flux. Moreover, analysis of the effect of BCR-FcgammaRIIB co-ligation in cells expressing a nonsignaling competent BCR suggest that FcgammaRIIB-mediated inhibition of BCR signaling requires co-ligation of FcgammaRIIB with signaling competent BCR molecules. These results suggest that in vivo treatments with low levels of BCR-FcgammaRIIB cross-linking reagent can induce BCR-FcgammaRIIB co-ligation and establish a condition of B cell nonresponsiveness.

Generation of a human IgG3-streptavidin fusion protein. Implications for the inhibition and elimination of auto-reactive B cells.

Antibodies against self-molecules play a significant role in the development and progression of Systemic Lupus Erythematosus, as well as a number of other autoimmune disorders. Immunosuppressive drugs have been used to control this process. However, they are normally not specific to the offending cell, and can actually suppress beneficial immune responses to pathogens. In this paper a genetically engineered targeting molecule is described, which has the capacity to target antigen-specific B cells for inhibition or elimination. The targeting molecule is a fusion of streptavidin subunit to the constant region of human IgG3 (IgG3-Av). It is demonstrated by ELISA and flow cytometry that IgG3-Av binds biotinylated antigen as well as human Fc gamma receptors present on myeloid cells. It is also shown by confocal microscopy and flow cytometry, that IgG3-Av can mediate Fc receptor-dependent phagocytosis of latex microspheres adsorbed with biotinylated antigen. Furthermore, the IgG3-Av construct can modulate Ca++ flux, characteristic of B cell inhibition as well as ADCC of B cells in an antigen-specific manner. In summary, these studies describe an approach, which has the potential to be used as a treatment to inhibit or remove antigen-specific (auto-reactive) B cells.

Production of genetically engineered biotinylated interleukin-2 and its application in a rapid nonradioactive assay for T-cell activation.

The development of reliable assay systems that can measure lymphocyte activation in vitro has been a major goal of immunodiagnostics. Traditionally, tritiated thymidine incorporation has been used to monitor T-cell activation. Other methods include enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay, and colorimetric assays. We have established a lymphocyte activation assay that utilizes fluorescein isothiocyanate (FITC)-streptavidin bound to recombinant biotinylated human interleukin-2 (IL-2). Utilizing recombinant DNA technology, a unique monobiotinylated human IL-2 has been created and isolated using the Promega PinPoint vector system. ELISA has been used to demonstrate streptavidin binding and recognition by a human IL-2-specific antibody. IL-2 function has been demonstrated using a murine IL-2-dependent T-cell line, CTLL-2, responsive to human IL-2. Recombinant biotinylated human IL-2 conjugated to streptavidin-FITC or streptavidin-horseradish peroxidase has been used to monitor T-cell activation in the presence of antigen as well as mitogen. The sensitivity and convenience of this method make this lymphocyte activation assay an attractive alternative to tritiated thymidine incorporation as a method for monitoring T-cell activation. In addition, the availability of a recombinant biotinylated human IL-2 will permit the production of a uniform product suitable for diagnostic and clinical application.

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcgammaRI-streptavidin construct and microspheres coated with biotinylated-antigen.

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.