Transgenic mice and transhybridomas producing chimeric mouse/human anti-human interleukin-2 receptor recombinant antibodies.

Transgenic mice were developed that secreted chimeric mouse/human anti-human interleukin-2 receptor (IL-2R) antibodies (Ab) into their serum. In addition, hybridomas producing the chimeric Ab in tissue culture were generated from the transgenic mice. The presence of the mouse/human immunoglobulin (Ig) transgene did not appear to affect rearrangement of endogenous murine Ig in the hybridomas. Serum levels of the chimeric Ab correlated with transgene copy number. Although many of the transgenic lineages had serum titers of the chimeric Ab comparable to endogenous mouse IgG, there was no apparent correlation with endogenous mouse IgG levels.

An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase.

A sandwich capture ELISA technique is presented for detection of recombinant proteins sharing a common affinity domain or reagents such as antibodies that bind to these proteins. An activated carrier protein (BSA) is modified with reduced glutathione (GT), forming an affinity capture reagent for glutathione-S-transferase (GST) and recombinant fusion proteins bearing the GST moiety. GT-BSA is immobilized on microtiter plates, and a sandwich is formed consisting of the recombinant fusion protein, reactive antibodies, and detection antibodies. An example is given that demonstrates that this format yields equivalent results to a conventional ELISA test with a panel of newly diagnosed diabetic sera reacting with an islet cell autoantigen.

Use of recombinant fusion proteins for generation and rapid characterization of monoclonal antibodies. Application to the Kunitz domain of human beta amyloid precursor protein.

Production of peptides by recombinant DNA techniques is an efficient alternative to chemical synthesis of peptides. Proteins and peptides produced by recombinant DNA methods in E. coli are routinely used as antigens for the production of antibodies. However, most small peptides are rapidly degraded within the E. coli cell, and therefore, must initially be expressed as components of larger, more stable fusion proteins. The peptide of interest must be cleaved from the fusion protein, and purified prior to immunization to eliminate epitopes contributed by the fusion partner. We have now established methods for the production and characterization of monoclonal antibodies using partially purified, uncleaved fusion proteins. We have also described a method for efficient production and detection of the fusion protein, an EIA for rapid differential screening of hybridoma supernatants, and a strategy for epitope mapping of the antibodies. These methods have been applied to the production and characterization of monoclonal antibodies specific for a 75-amino-acid internal segment of the Alzheimer amyloid precursor protein, and should be applicable to a wide variety of other peptides and proteins.

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.