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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 uorescent 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 modern immunoassays.1
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TABLE 6.1. Immunoassay Labelsa
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Immunoassay Type Heterogeneous Label Type Fluorophore Label Fluorescein Europium chelate Phycobiliproteins Acridinium ester Phenanthridinium ester Alkaline phosphatase b-Galactosidase Peroxidase Urease ATP NAD Mellitin Property Measured Fluorescence intensity Time-resolved uorescence Fluorescence intensity Ester hydrolysis Ester hydrolysis Enzyme activity Enzyme activity Enzyme activity Enzyme activity Kinase activity Dehydrogenase activity Release of liposometrapped enzyme (activity) Binds avidin-enzyme and streptavidin-enzyme conjugates (activity) Fluorescence polarization Fluorescence quenching Fluorescent energy transfer Ester hydrolysis Light emission Glucose oxidase activity
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Chemiluminescent Enzyme
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Cofactor Lysing agent
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Secondary label
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Chemiluminescent Prosthetic group Enzyme Enzyme activity
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Acridinium ester Isoluminol FAD Glucose-6-phosphate dehydrogenase Malate dehydrogenase Peroxidase Hexokinase Ferrocenes Nitroxides Galactosyl-umbelliferone
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Electroactive Spin label Substrate
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Enzyme activity Aggregation and enzyme activity Substrate channeling Oxidation current Broadening of ESRb signal Enzymatic hydrolysis
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See Ref. 1. Electron spin resonance ESR.
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6.2. LABELING REACTIONS Reagents used for the labeling of protein antigens and antibodies are similar to those used for the immobilization of enzymes by covalent and cross-linking methods (cf. 4). Homobifunctional and heterobifunctional cross-linking reagents are common, using NHS ester, maleimido, aldehyde, thiocyanate, and isothiocyanate groups to provide selectivity. Glycoproteins such as antibodies may also be bound to labels containing primary amine groups through the initial oxidation of sugar residues by periodate; the resulting aldehyde groups form Schiff bases with primary amines, which may be subsequently reduced to secondary amines under mild conditions with sodium borohydride.2 Following the coupling reactions, labeled macromolecular reagents are usually puri ed by gel ltration chromatography: Porous stationary phases retain small solutes, but allow high molecular weight species to elute unretarded in aqueous buffer mobile phases. Dialysis or af nity chromatography may also be used for conjugate puri cation. The preparation of labeled haptens may occur under more extreme conditions, since protein denaturation is only a factor if the label is an enzyme. The rst critical step in hapten labeling is the introduction of a reactive group onto the hapten, which may be done by the alkylation of O or N substituents with haloesters,3 followed by hydrolysis (Eq. 6.1): 6:1 A second reaction converts hydroxyl groups into carboxylates using succinic anhydride (Eq. 6.2):4
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Alternatively, ketone groups may be used to generate reactive carboxylates (Eq. 6.3):5 6:3 Following the introduction of a carboxylic acid group onto the hapten, a variety of reagents may be used to activate these groups toward nucleophiles such as primary amine groups. The mixed-anhydride method uses isobutylchloroformate6 to generate a mixed anhydride in the presence of a base such as triethylamine (Eq. 6.4): 6:4
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The product of the mixed-anhydride reaction is reactive toward primary amine groups as well as hydroxyl groups, but is susceptible to hydrolysis. Yields of 20 30% conjugate have been obtained using a 10- to 20-fold excess of isobutylchloroformate. Carboxylic acids may also be activated using carbodiimide and carbodiimide hydroxysuccinimide reagents, as described in 4 for enzyme immobilization. For the carbodiimide reaction alone, average yields of 20% conjugate have been reported, due to a competing side reaction that converts the intermediate Oacylisourea to a stable N-acylurea. The NHS esters may be generated in situ and reacted directly with the label; for the progesterone b-galactosidase reaction, a 10% loss in activity was reported, and 26% of the active enzyme was found to be immunoreactive. However, the stability of the NHS ester allows its isolation and puri cation; the puri ed NHS ester of progesterone, following reaction with enzyme, yielded a 100% immunoreactive enzyme hapten conjugate, and the steroid/enzyme molar ratio was found to be 2:1.7 Excess carbodiimide coupling reagent has been found to deactivate many enzymes when the in situ method is used, and the isolation of the NHS ester intermediate allows direct control over stoichiometry in the coupling step. Conjugates are generally characterized by determining the hapten/label or antibody/label molar ratio, and by examining the characteristics of the label to determine whether conjugation has resulted in property changes. For example, the speci c activity of enzyme labels is determined, and the molar absorptivity and quantum yields of uorescent labels are compared before and after coupling. Enzymatic labels may be protected from deactivation by including a competitive inhibitor in the reaction mixture during coupling; the presence of this species can protect reactive groups at the active site from modi cation.8 Immunoreactivity, the fraction of the hapten label or antibody label conjugate that can be bound by excess antibody or antigen, respectively, is often used to characterize a labeled reagent, and binding af nities may also be determined. The above reactions are given as examples of the methods used to link haptens and antibodies to detectable labels; a comprehensive listing of conjugation procedures is beyond the scope of this book.
6.3. HETEROGENEOUS IMMUNOASSAYS Many immunoassays require a separation step prior to quantitation, in order to separate the bound and free fractions of the labeled species. Consider an immunoassay in which a labeled ligand, Ag*, competes with unlabeled analyte, Ag, for a limited quantity of antibody binding sites. This competitive equilibrium is represented by Eqs. 6.5 and 6.6, ! Ab Ag Ab:Ag ! Ab Ag Ab:Ag 6:5 6:6