Antibodies

As in all immunologic techniques using labeled compounds, the antibodies employed in immunoenzymatic procedures can be distinguished as unlabeled ones, i.e. those that take part in the first reaction with the antigen, and as enzyme-labeled antibodies that are added secondarily and, usually, in high excess. The sensitivity of these assays depends essentially on the avidity of the first, while their performance depends mainly on the qualities and characteristics of the enzyme-labeled antibody. Since high avidity antibodies are usually present in hyperimmunized animals, when possible it is preferable to use polyclonal and/or monoclonal antibodies derived from such animals. In the great majority of cases, an enzyme-labeled polyclonal antibody preparation recognizing immunoglobulins (usually a mixture of anti-|x, and anti-"Y and anti-a), or less frequently a given antigen, is used. The immunoglobulin G (IgG) fraction or the antibodies specifically isolated from the antiserum are bound to the enzyme. Although good results are obtained with labeled IgG, it is preferable, when possible, to use isolated antibodies; the antigen-specific staining is more intense and the nonspecific background is weaker.

Labeling of antibodies with enzymes

Antibodies (or antigens) are labeled with enzymes either by covalent or by biospecific noncovalent linkages. To obtain covalent antibody-enzyme conjugates a number of cross-linking agents have been tried, but only glutaraldehyde, ra-periodate and maleimide derivatives are currently used. Glutaraldehyde, reacting under mild conditions with the free amino groups of proteins, has been used for the preparation of almost all the enzyme-antibodv conjugates applied in immunoenzymatic techniques. Treatment of peroxidase (or other polysaccharide enzymes) with w-periodate results in the generation of active aldehyde groups in the peroxidase molecule which react with the free amino groups of antibodies (or antigens) added subsequently. Treatment of enzymes with maleimide (or pyridyl disulfide) derivatives introduces into these molecules active maleimide groups that react with subsequently added antibody molecules carrying thiol groups. All these coupling procedures give rise to heterogeneous populations of enzyme-antibody conjugates, but under certain experimental conditions homogeneous preparations of conjugates composed of one molecule of peroxidase coupled to one molecule of antibody are obtained using either glutaraldehyde or maleimide derivatives as the cross-linking agent.

Enzymes are also linked to antibodies (or antigens) using a number of specifically interacting systems, but only the antigen-antibody and the avidin (or streptavidin)-biotin systems are currently being used. The procedures based on the antigen-antibody reaction require an antibody recognizing the antigen and a second antibody recognizing the enzyme label, both prepared in species A, as well as an antibody prepared in species B and recognizing the immunoglobulins of species A. In a first step, the antibody is allowed to react with the antigen, then the antiimmunoglobulin antibody is added, followed by the addition of the antienzyme antibody, the enzyme and the substrate. These procedures are based on the principles that: 1) enzyme-antienzyme complexes are always catalytically active; 2) the anti-immunoglob-ulin antibody, added in excess, reacts with the antigen-bound antibody only by one of its two active sites and thus can operate as acceptor for the anti-enzyme antibody. These multistep protocols are somewhat shortened by the prior formation of an enzyme/antienzyme soluble immune complex, the most commonly used being the peroxidase-antiperoxidase (PAP) procedure.

Two procedures are used for linking enzymes to antibodies on the basis of the strong interaction between avidin and biotin (dissociation constant 10 15 m). The first requires the preparation of antibodies labeled with biotin and an avidin-enzyme conjugate. Biotinylated antibody is allowed to react with the antigen and this step is followed by addition of enzyme-labeled avidin. In the second method antibodies labeled with biotin, native avidin and enzyme labeled with biotin are used. This protocol involves, in the first step, the interaction of the biotinylated antibody with the antigen: in the second step, the incubation with avidin, followed by the addition of biotin-labeled enzyme and substrate. This technique is based on the principle that avidin possesses four specific binding sites able to create bridges between two proteins labeled with biotin. This assay is somewhat shortened by the prior formation of an avidin-biotinylated-enzyme complex and is often called the ABC procedure. Streptavidin, yielding less background, is being more and more used instead of avidin. Its affinity for biotin is similar to that of avidin.

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