ABSTRACT
An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.
Subject(s)
Drug Evaluation, Preclinical/methods , Fluoroimmunoassay/methods , Interferon-gamma/biosynthesis , Antibodies, Monoclonal , Cell Line , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/standards , Fluorescent Dyes , Fluoroimmunoassay/standards , Humans , Interferon-gamma/analysis , Interferon-gamma/standards , Recombinant Proteins , Reference Standards , RoboticsABSTRACT
There are a number of problems associated with the development of standards suitable for use in the most commonly used assays to detect cytokines in biological fluids. These problems include: (i) the failure of some MoAbs used in immunoassays to detect all different <
Subject(s)
Cytokines/analysis , Growth Substances/analysis , Immunoassay/methods , Animals , Biological Assay , Body Fluids/chemistry , Cytokines/standards , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/standards , Growth Substances/standards , Humans , Immunoassay/standards , Immunoassay/statistics & numerical data , Interferon-gamma/analysis , Interferon-gamma/standards , Interleukin-1/analysis , Interleukin-1/standards , Interleukin-4/analysis , Interleukin-4/standards , Mice , Reference StandardsABSTRACT
A large number of different human and nonhuman interferon (IFN) preparations are now available for either research purposes or commercial use. Consistency of results can be achieved only through rigorous application of biologic standards and individual species designation. International standards for the potency determinations of these preparations have been produced in accordance with World Health Organization (WHO) guidelines and are available for calibrating assays. Until recently, potency has been assessed purely as a measure of antiviral activity expressed in international units. Other biologic properties are now also being considered, including antiproliferation and immunomodulation. Indirect methods of measuring IFN, such as radioimmunoassay or enzyme immunoassay, if fully validated, may also provide useful estimates of function. Reference antisera are useful for characterizing IFN preparations and for monitoring neutralization assays for detecting anti-IFN antibodies but should not have a function in assay calibration. Factors to be considered when referring to specific designations for pure IFN species include distinctions for the species of origin, any mutant or hybrid forms, the method of production, and the presence of additional glycosylation.
Subject(s)
Interferons/standards , Animals , Antiviral Agents , Cell Line , Glycosylation , Humans , Immunologic Techniques , Interferon Type I/standards , Interferon-beta/standards , Interferon-gamma/standards , Interferons/immunology , Mice , Recombinant Proteins , Species Specificity , Terminology as Topic , World Health OrganizationABSTRACT
We describe an evaluation of four ELISA methods, including three commercial kits, for measuring recombinant and natural human interferon-g (hIFN-g). Using a panel of samples, including well-characterized reference standards, we compared relative quantification between assays, within assays and, where possible, the absolute accuracy of quantification as compared to other analytical methods. The four assays generated markedly different results; up to an almost 60-fold difference between the highest and lowest values for one sample. The differences between assays were not necessarily predictable. No single correction factor could be determined to correct results from one method to another across the panel of samples tested. We conclude that investigators should be diligent to revalidate commercial methods before depending on such methods and resultant data.