Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Development of a new automated IL-6 immunoassay.

OBJECTIVES: Quantitative detection of interleukin-6 (IL-6) in serum and plasma can help monitor immune responses and the development of acute inflammation to guide patient management. We developed an IL-6 immunoassay for use with the automated ARCHITECT system for detecting an increase in the inflammatory response. METHODS: Immunized mouse sera were tested and selected B-cells were harvested for fusion with myeloma cells. A panel of monoclonal antibodies were produced, from which capture and detection monoclonal antibodies for the prototype IL-6 immunoassay were selected and screened on the ARCHITECT instrument. The antibody pair that most effectively captured and detected IL-6 was selected to develop a prototype IL-6 immunoassay. Calibrator and panel preparations using an internal recombinant IL-6 standard were compared to serum panels prepared with the IL-6 International Standard 89/548. Assay specificity and spike recovery were determined, and assay sensitivity was compared with the Roche EUA Elecsys IL-6 assay run on the cobas analyzer. RESULTS: Twenty-one antibodies in 441 antibody pairs were screened. The prototype IL-6 assay showed high sensitivity with an estimated limit of detection of 0.317 pg/mL and limit of quantitation of <1.27. Spike recovery was 90%-110% in serum and plasma. The internal recombinant human IL-6 calibrator showed excellent stability for 63 days at 2-8 °C. The prototype IL-6 immunoassay was specific for IL-6, exhibited no cross reactivity to related cytokines and interleukins, and was 10-fold more sensitive than the Elecsys IL-6 assay. CONCLUSIONS: The prototype ARCHITECT IL-6 automated immunoassay is a reliable and robust method for the quantitative determination of IL-6 in human serum and plasma.

Enhanced Immune Response Following DNA Immunization Through In Vivo Electroporation.

DNA immunization offers the advantage of allowing for the initiation of animal immunogenicity studies while work to produce and purify the protein of interest is completed. In this study, we sought to evaluate in vivo electroporation (EP) as a means to enhance the antigen-specific immune response from DNA immunization. Mice were immunized thrice with DNA encoding the protein of interest through intramuscular (IM) or intradermal (ID) injections. Test animals were administered an electrical pulse into the muscle or dermis at the site of injection immediately following immunization. In addition, cardiotoxin was injected into the muscle of a subset of test animals 5 days before each DNA injection. Nine weeks following the final DNA immunization, mice were immunized with the encoded purified protein emulsified in Freund's adjuvant. Sera from EP mice taken 2 weeks following the final DNA immunization showed a significant enhancement in antibody response. Specifically, those mice treated with cardiotoxin, immunized IM and given EP showed a strong response, but this was only observed versus solid phase and not solution phase antigen, suggesting the resulting antibody was of low titer and affinity. Similar testing following the protein immunization revealed a significant improvement in relative affinity versus sera taken following DNA immunization. Our results suggest EP can enhance the immune response elicited by DNA immunization.

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.

Rapid determination of antigenic epitopes in human NGAL using NMR.

The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two-dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high-affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase-associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in (1)H-(15)N-TROSY HSQC and (1)H-(13)C HSQC spectra of isotope-labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual-color fluorescence cross-correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel-like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR-based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest.

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.