Enzyme-antibody labels

The first application of immunologically based technology to pesticides was not reported until 1970, when Centeno and Johnson developed antibodies that selectively bound malathion. A few years later, radioimmunoassays were developed for aldrin and dieldrin and for parathion. In 1972, Engvall and Perlman introduced the use of enzymes as labels for immunoassay and launched the term enzyme-linked... 

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. 

The devices usually contain antibody immobilized on a membrane surface above a filter and absorbent pad (Figure 7.16). The sample is placed in the device and passes through the membrane into the absorbent pad. Where analyte is present in the sample it is sequestered by the antibody on the membrane and is then visualized by addition of a labelled second antibody. Labelled second antibody not bound to analyte also passes into the absorbent pad, sometimes with the aid of a wash solution. The labelled molecule may be an enzyme, which would then require addition of a suitable substrate, or a label such as colloidal gold, which has the virtue of being visible without the aid of a second reagent. 

AVIDIN OR STREPTAVIDIN LABELED ENZYME ANTIBODY TO BIOTIN AND A... 

Figure 3.25 Scheme of a SWNT immunosensor using secondary antibody labeled with HRP enzyme. HRP catalyses H2O2 and generates electrons that can be amperometrically detected, (a) Treatment with a conventional HRP-labeled secondary antibody providing one label per binding event and (b) treatment with an HRP-... 

With this technique, the often numerous primary antibodies used do not all have to be labeled with enzyme, provided they are all of the same species that a secondary antibody raised against immunoglobulin will recognize. The secondary antibody, labeled with enzyme, provides for the detection of all of the primary antibodies without the need for them to be individually conjugated. The problems of background and denaturation associated with conjugation are still present and can be troublesome. However, an increment of sensitivity can be achieved greater than that obtained by the previous method. 

Figure 2. Labeling with anti-crude enzyme mixture. A and B show the specificity of the antibodies compared to the preimmune-treated control C, plasmatic and cytoplasmic localization of the enzymes. No labeling in the fungal wall. (Pm, plasmalemma W, hyphal wall.)...

The actual response of monoclonal antibodies with individual cells is usually visualized either directly (typically using fluorescent stains) or indirectly [using the reaction of antibody labeled with horseradish peroxidase (HRP) or other enzymes] with diaminobenzidine (DAB) (or other substrate while using other enzymes) under the microscope or in the flow cytometer. The latter, however, is not employed routinely in CSF immunocytology, although it has an advantage in clinical hematology. 

As shown in Fig. 21, in a direct ELISA the unlabeled antigen (a range of standard antigen concentrations or unknown samples) is attached to the solid phase. Enzyme-conjugated (labeled) primary antibody is then added. After incubation and washing of the plate... 

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

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

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

An example of the immobilization of antibodies on channel surfaces was presented by Eteshola and Leckband [395]. A microfluidic sensor chip was developed to quantify a model analyte (sheep IgM) with sensitivities down to 17 nM. This was achieved by first immobilizing a layer of bovine serum albumine (BSA) onto the channel wall, followed by specific adsorption of protein A to which the primary antibody for IgM was coupled covalently. This antibody could capture IgM, which was detected with the secondary antibody, labeled with horseradish peroxidase (Scheme 4.91). This enzyme catalyzes the conversion of the fluorogenic substrate 3-(p-hydroxyphenyl)propioni c acid into a fluorophore, which was quantified off-chip with a spectrofluorometer. The measured fluorescence signal was proportional to the analyte concentration in the test sample. 

Enzyme immunoassay (ElA) These assays exploit the catalytic properties of enzymes. Typically, antibodies labelled with an enzyme are used, for example horseradish peroxidase. The enzyme, which is bound and remains after washing, is able to convert added substrate to generate a coloured product that can be measured. The major enzyme immunoassay (ElA) is ELISA, which is covered in more detail later. 

The advantages and limitations of enzymes as labels depend on the properties of both the enzyme and the molecule to be labeled. The widespread use of enzymes as antibody labels requires consideration of some aspects of the antibody molecule itself. The availability and characteristics of antibody reagents (native or recombinant) will always be the most important factor controlling assay per-... 

Labeling reactions can be broadly divided into those employing specific or nonspecific linkages (39, 40). Therefore, we will not review labeling reactions, but will instead outline the principal considerations salient to the use of enzyme-antibody conjugates in bioanalytical systems. 

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