Generation of human neutrophil gelatinase-associated lipocalin monoclonal antibodies for use in ARCHITECT® assay.

Development of a robust immunoassay requires the selection of monoclonal antibodies with desired properties. These properties generally include kinetics parameters such as on-rate and off-rate (i.e., binding affinity), and, often times, the ability to form a sandwich with the analyte of interest. We sought to obtain antibodies suitable for development of an immunoassay capable of detecting human neutrophil gelatinase-associated lipocalin (NGAL), a glycosylated lipocalin of 25 kDa expressed in kidney tubules in response to injury that has been shown to be a urinary biomarker capable of diagnosing acute kidney injury. We immunized CAF1/J and RBF/DnJ mouse strains with recombinant NGAL, and a robust immune response, as measured by serum antibody titer, was observed among all CAF1/J mice. Antibodies secreted from mouse B cell-myeloma hybridomas were screened by enzyme immunoassay (EIA) and by surface plasmon resonance using a method we termed hybrid supernatant kinetic screening. Approximately 300 hybrid clones were evaluated by this technique to identify antibodies with the kinetic binding parameters meeting criteria required for further assay development (i.e., rapid association and slow dissociation). This data, along with epitope grouping, cell growth, cell viability, and antibody secretion, were used to identify antibodies for testing in the ARCHITECT assay.

Generation and characterization of chimeric antibodies against NS3, NS4, NS5, and core antigens of hepatitis C virus.

Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (V(H)) and light-chain (V(L)) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (C(H)) and light-chain (C(L)) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthine- and thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.

Affinity maturation of tacrolimus antibody for improved immunoassay performance.

BACKGROUND: Organic solvents used for extraction of tacrolimus from whole blood samples lower the apparent affinity of the antibody used in a diagnostic immunoassay, thereby affecting the detection limit. METHODS: We used in vitro recombinant antibody engineering to screen and isolate clones from diverse libraries with mutagenic complementarity regions (CDRs) from tacrolimus 1-60-46 hybridoma cell line, with improved binding to tacrolimus in the presence of 10% methanol organic solvent solution. RESULTS: We isolated a number of clones with mutations in variable heavy (VH) CDR 2, variable light (VL) CDR 1, and VL CDR 3 with improved binding. Various combinatorial pairings constructed from these individual mutations contained >10-fold improvements in both the dissociation rate and overall equilibrium affinity constants. Selected clones produced as IgG have increased functional sensitivity, with a 3- to 6-fold reduction in the limit of detection relative to the parental tacrolimus 1-60-46 monoclonal antibody in the Architect Tacrolimus immunodiagnostic assay. CONCLUSIONS: The recent advent of recombinant in vitro antibody display technologies in general, and yeast surface display in particular, allows the flexibility to engineer new or augment specific analytical characteristics, such as affinity, specificity, or stability, into previously isolated and otherwise desirable antibodies to enhance assay performance. These in vitro selections can also be performed under conditions meant to mimic the assay in which the reagent will ultimately be used, to increase the likelihood of successful assay development.