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).
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.
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.
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