Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1–3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections.

Peripheral blood leukocytes incubated with a semisynthetic phage antibody library and fluorochromelabeled CD3 and CD20 antibodies were used to isolate human single-chain Fv antibodies specific for subsets of blood leukocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hitherto-unknown staining patterns of B-lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. This approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries. The effectiveness of such 4Abs in biochemical and functional assays varies; typically, the procedure used to select 4Abs determines their utility (2-6).Human monoclonal antibodies that bind to native cell surface structures are expected to have broad application in therapeutic and diagnostic procedures (7). An important extension of #Ab display technology would be a strategy for the direct selection of specific antibodies against antigens expressed on the surface of subpopulations of cells present in a heterogeneous mixture. Ideally, such antibodies would be derived from a single highly diverse library containing "every" conceivable antibody specificity. We have recently constructed a library from 49 human germline immunoglobulin heavychain variable (VH) genes fused to a heavy-chain joining segment 4 (JH4) gene and partly randomized complementarity-determining region 3 (CDR3) sequences varying in length between 6 and 15 amino acids. The CDR3s were designed to contain short stretches of fully randomized amino acid residues flanked by regions of limited variability. Residues in the latter portion of CDR3 were selected on the basis of their frequent occurrence in CDRs of natural antibody molecules (8-10). We reasoned that a designed, semirandom CDR3 would result in an increased frequency of clones producing functional antigen binding sites and, in addition, in a more efficient use of the restricted sequence space within phage display libraries. The synthetic VH segments were combined with seven different light-chain variable (VL) genes and expressed as gene III-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3938 single-chain Fv (scFv) fragments on the surface of phage, resulting in a library of 3.6 X 108 clones. From this library we isolated monoclonal 4Abs (m4Abs) to a variety of different structures (haptens, proteins, and polysaccharides) by selection on solid-phase bound antigen. These phage antibodies proved to be useful reagents in a variety of biochemical assays (6).In the present experim...

No abstract

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.

An interlaboratory study using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts (n ‫؍‬ 35) was performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases.