Enzyme labeling

The primary use of EIA when it was first developed was for histological labeling and localization of specific cell macromolecules. Eor example, enzymes labeled with peroxidase were used to locate specific cellular compartments and stmctures for microscopic examination. The flexibiUty of EIA was recognized quickly and it was adapted for use as a laboratory assay. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Sandwich-type sensors are applicable for measuring large antigens that are capable of binding two different antibodies. Such sensors utilize an antibody that binds the analyte-antigen, which then binds an enzyme-labeled second antibody. After removal of the nonspecifically adsorbed label, the probe is placed into the substrate-containing solution, and the extent of the enzymatic reaction is monitored... 

A comparison of the structures of penicillin and Dalanyl-Dalanine (cf. structures 41 and 42) shows that there is a great deal of similarity between the two molecules. Penicillin is essentially an acylated cyclic dipeptide of Dcysteine and Dvaline (84). As such, it contains a peptide bond, that of the /3-lactam ring, that can acylate the enzyme. Labeling studies of the peptidoglycan transpeptidase of Bacillus subtilis indicate that radioactive penicillin reacts with a sulfhydryl group of a cysteine residue of the enzyme (86). 

Our standard incorporation assays contained resuspended particulate enzyme, labelled UDP-Gal (0.1 mM) and (10 mM) in resuspension buffer (Tris, pH 7.5). After incubation, reaction mixtures were heated briefly to 100°C and soluble lupin galactan was added, to ensure the precipitation of small amounts of galactan formed in the en me reaction and dissolved during the heating step. Precipitation of macromolecular products was achieved by adding methanol to a final concentration of 70%. The pellet was freed of soluble labelled products, including residual UDP-Gal, by repeated extraction with hot 70% methanol and was then analysed for labelled (l- )-P-D-galactan. The supernatant was analysed for soluble labelled products. 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

Catalase has also been used as an enzyme label in competitive heterogeneous enzyme immunoassays. Catalase generates oxygen from hydrogen peroxide with the oxygen determined amperometrically with an oxygen electrode. This approach has been demonstrated for a-fetoprotein theophylline and human serum albumin... 

A sandwich electrochemical enzyme immunoassay has been described for IgG Alkaline phosphatase was again used as the enzyme label with the conversion of phenyl phosphate to phenol being determined electrochemically by LCEC. A detection limit of 10 pg/mL was reported. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Figure 3 Immobilized antibody ELISA. Primary antibody (Y) is passively adsorbed to the surface of a polystyrene microtiter plate. Analyte (free H) and an enzyme-labeled hapten (H-E) compete for the fixed number of primary antibody binding sites. Following a wash step (dotted line), the substrate for the enzyme is added O) and a colored product formed ( ). The amount of product is inversely proportional to the amount of analyte present...

Figure 4 Sandwich immunoassay. A capture antibody (Y) is passively adsorbed on a solid phase. The target protein contained in the sample and the enzyme-labeled reporter antibody (Y-E) are added. Both the capture antibody and enzyme-labeled reporter antibody bind to the target protein at different sites, sandwiching it between the antibodies. Following a wash step, the substrate (<>) is added and colored product ( ) formed. The amount of colored product is directly proportional to the amount of target protein captured...

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

Anderson J, Rowe LW. The use of an enzyme-labelled assay as an aid to reading micro virus-neutralisation tests. J. Immunol. Methods 1982 53 183-186. 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

The two-step nature of SPDP crosslinking provides control over the conjugation process. Complexes of defined composition can be constructed by adjusting the ratio of enzyme to secondary molecule in the reaction as well as the amount of SPDP used in the initial activation. The use of SPDP in conjugation applications is extensively cited in the literature, perhaps making it one of the more popular crosslinkers available. It is commonly used to form immunoto-xins, antibody-enzyme conjugates, and enzyme-labeled DNA probes. A standard activation and coupling procedure can be found in Chapter 5, Section 1.1. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Farr, A.C., and Nakane, P.K. (1981) Immunohistochemistry with enzyme labeled antibodies A brief review. J. Immunol. Meth. 47, 129-144. 

Ishikawa, E., Yamada, Y., and Yoshitake, S. (1981a) Enzyme labeling with N,N -o-phenylenedimaleimide. In Enzyme Immunoassay (E. Ishikawa, T. Kawai, and K. Miyazi, eds.), pp. 67-80. Tokyo. 

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