Quantitative Immunoassays with Labels

The classification of immunoassay methods is based on (a) whether they are homogeneous, with no separation step needed prior to measurement, or heterogeneous, where a separation step is required (b) which species, antibody or antigen, is labeled and (c) the type of label employed. 

Bianalytical Chemistry, by Susan R. Mikkelsen and Eduardo Coiton ISBN 0-471-54447-7 Copyright 2004 John Wiley Sons, Inc. 

While no real labels meet all of these needs, the properties of some of the more recently introduced labelling systems are approaching the ideal. Radioisotopes, once the only type of label used for immunoassays, have clearly been overwhelmed by current applications of fluorescent labeling methods, enzyme labels, and even coenzyme and prosthetic group labels. A variety of alternative labels has also been investigated, including red blood cells, latex particles, viruses, metals, and free radicals. Table 6.1 shows a representative listing of labels used in modem immunoassays.1 

Immunoassay Type Label type Label Property Measured 

Heterogeneous Fluorophore Fluorescein Europium chelate Phycobiliproteins Fluorescence intensity Time-resolved fluorescence Fluorescence intensity 

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Christopoulos TK, Chiu NH. Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments. Anal Chem 1995 67(23) 4290 1294. 

ABA is detected and quantified by enzyme immunoassay with a monoclonal antibody.578 The detection limit of immunoassay with a monoclonal antibody is about 0.02 pmol however, enzyme immunoassay cannot exclude cross-reactivity with unknown substances, so GC- or LC-SIM analysis using the stable isotope dilution method is recommended for correct quantitation. As internal standards labeled with the stable isotope, deuterated derivatives of ABA such as [3, 5, 5, 7, 7, 7 -2H6]ABA (ABA-//6)579 and [7, 7, 7 -2H3] derivatives of phaseic and dihydrophaseic acids580 are used. 

One of the earliest efforts of qualitative measurement of a protein (human serum albumin) in a microchip-based device was based on bead agglutination in a microchamber (approximately lOjaL). Subsequently, several quantitative immunoassays have been performed using microchip electrophoretic systems that permit separation and quantitation of free- and bound-labeled antigens in competitive assays (see Chapter 5). Most are carried out in channels micro-machined into fused silica substrates. Early work on quantitative assays achieved measurement of cortisol in serum.The assay used cortisol labeled with fluorescein and an argon laser detector at 488 nm and required only 80 pL of a 40x dilution of serum as the sample. Other capillary electrophoresis-based assays for a variety of antibodies have also been developed that include immunoglobulins (IgG, IgA, and IgM), antibovine serum albumin, and antiestradiol. ... 

In quantitative immunoassays, e.g., enzyme-linked immunosorbent assay and radioimmunoassay, a known amount of labeled vitamin is mixed with sample extract in which the vitamin content should be determined. Methods of labeling include radioisotopes (e.g., cobalamine), fluorescence, or luminescence markers (e.g., folate). The mixture is subjected to binding agent, equally forming complexes with both labeled and unlabeled vitamin. This complex is then isolated, and the amount of labeled vitamin present is measured. Sample vitamin concentration can be deduced from the ratio of labeled vitamin added to labeled vitamin measured after isolation. Advantages of immunoassays are short analysis time, and the possibility of automating them on clinical analyzers. 

The first quantitative immunoassays were based on the use of radioactive reporter molecules, mainly and " C. These radioimmunoassays (RIAs) have gradually been replaced by nomadioactive reporter molecules or labels, e.g., enzymes, fluorescent, and luminescent molecules and combinations of these. The nonradio-active labels facilitated development of automated immunoassays, and they also contributed to the design of assays with substantially reduced detection limits. However, nomadioactive assay have an advantage only in immunometric sandwich assays in inhibition assays, the use on nonradioactive labels has not provided any improvement in assay sensitivity as compared to RIA [1]. The development in immunoassay technology has been reviewed by Ekins [2]. 

Phase-separation immunoassays have been reported, in which the solid phase particles are formed after the immunoreaction is completed.(42) Phase-separation immunoassays are advantageous since the unstirred layer of solution near a solid surface alters diffusion and binding kinetics at the surface in comparison with the properties of the bulk solution. In phase-separation assays for IgG and IgM, capture antibodies are bound with monomers suitable for styrene or acrylamide polymerization.(42) Monomer-labeled capture antibodies are reacted with analyte and with fluorescein- and/or phycoerythrin-labeled antibodies in a sandwich assay, followed by polymerization of the monomers. Fluorescence of the resulting particles is quantitated in a FACS IV flow microfluorometer, and is directly proportional to analyte concentration. 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

Immunosensors have been designed which use both direct and indirect immunoassay technology to detect specific analytes within a minute or less in a variety of matrices (see Fig. 9). Indirect immunosensors may employ ELA, FLA, or CLIA principles whereby enzyme-, fluorophore- or chemiluminescent-labeled analyte competes with the target (nonlabeled) analyte for binding sites on the immobilized antibody. Unbound (free) labeled analyte is then quantitated using an electrochemical, optical, or electromechanical transducer and compared to the amount of target analyte in the sample. 

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