Transplantation of CD34+ hematopoietic progenitor cells.

We have developed an avidin-biotin immunoadsorption technique in conjunction with a monoclonal anti-CD34 antibody that is capable of selecting CD34+ progenitor cells from marrow and mobilized peripheral blood. Clinical studies with these CD34+ selected cells have shown that the cells are capable of rapid and durable engraftment. In addition, there is significantly less infusional toxicity to the patient because the volume in which the CD34+ selected cells are contained is much less than that of a typical marrow or apheresis buffy coat. Selection of CD34+ progenitor cells also offers other potential advantages, including T-cell depletion of allografts and tumor cell depletion of autografts. CD34+ selection can also be used to facilitate other manipulations of marrow and peripheral blood, including gene transfection, ex vivo stem cell expansion, tumor purging, and progenitor cell banking. Future graft engineering studies are expected to clarify these relationships and enable refinement of the graft to the point at which GVHD can be minimized, graft survival maximized, and relapse-free survival prolonged.

Performance and cost-effectiveness of a dual rapid assay system for screening and confirmation of human immunodeficiency virus type 1 seropositivity.

Recent studies have shown that rapid, instrument-free assays for the detection of antibody to human immunodeficiency virus (HIV) can be as sensitive and specific as enzyme-linked immunosorbent assay (ELISA) for screening of donated blood in developing countries. Currently, however, specimens which test positive on a screening assay must still be confirmed by Western blot (immunoblot), a method which is not feasible in most developing-country laboratories. We examined whether a testing hierarchy which utilizes neither conventional ELISA nor Western blot can be reliably used for screening and confirmation of HIV infection in a high-risk population. In a retrospective analysis of 3,878 specimens which were screened for antibody to HIV in Kinshasa, Zaire, we observed that a testing hierarchy consisting of duplicate HIVCHEK screening assays followed by duplicate Serodia-HIV confirmatory assays resulted in correct confirmation of all ELISA- and Western blot-positive specimens. We conclude that such a testing hierarchy can produce highly accurate results for identification of positive specimens in routine HIV testing and provides a practical alternative to conventional methods of HIV screening and confirmation.

Phase-separation immunoassays.

Solid-phase-based immunoassays have traditionally been plagued by nonspecific binding to the solid phase and by slow reaction kinetics relative to reactants that are free to diffuse in solution. We have developed two novel immunoassays in which the solid phase is generated in situ after the specific binding reaction has occurred, thereby enhancing reaction kinetics and minimizing the opportunities for non-specific binding. In the first system, the capture antibody is conjugated to an organic monomer, polymerization of which to form insoluble polymer particles is initiated by a reaction involving free radicals. The amount of signal-labeled antibody incorporated into the resulting particles is directly proportional to the concentration of antigen. The principle is illustrated for the simultaneous assay of IgG and IgM in a single sample. In the second system, capture antibody is conjugated to a polymer, the solubility of which is a function of temperature. Specific binding is conducted below the critical solution temperature of the polymer, which is then separated from solution by increasing the temperature above the critical temperature. The incorporation of signal-labeled antibody into the precipitated polymer is directly proportional to the concentration of antigen. This principle is illustrated for the assay of hepatitis B surface antigen and Chlamydia trachomatis.

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies.

Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10(-6) or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (1:2). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes. In addition, three new strains of mice differing from existing strains in the region of the Lyt-2 and Lyt-3 loci have been constructed. They are: C.C58-Lyt-2a, Lyt-3a and C.AKR-Lyt-2a, Lyt-3a, congenic with Balb/cAn and bearing Lyt-2a and Lyt-3a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2b, Lyt-3b congenic with AKR/J and bearing the Lyt-2b and Lyt-3b alleles of Balb/cJ.