Monoclonal antibodies with enzyme

This chapter provides protocols for the most convenient methods of labelling mouse IgG monoclonal antibodies with enzymes (alkaline phosphatase and peroxidase), a fluorescent molecule (fluorescein), biotin, and DIG. A general protocol for the evaluation of the labelled monoclonal antibody is also given. 

Bonfante et al. (73) used monoclonal antibodies and enzyme-gold complexes to reveal pectins and cellulose at the interface between the fungal wall and the host plasma membrane in AM roots (Fig. 6), and additional wall components have been investigated with other molecular probes (74-76). These studies indicate that the interface is an apoplastic space of high molecular complexity where the boundaries of the partners are defined. The examination of other endomycorrhizal systems has demonstrated that their interface is morphologically similar but different in composition. Cellulose and pectins are present at the interface... 

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. 

The reader, doubting that this method is sufficiently rigorous, may think that it is not possible to discover a radically new determinant by testing monoclonal antibodies with antigens that are already well known. In any case, after having found an active sequence, it is possible to modify it chemically or enzymically so as to create an original structure. If this modification increases the immune response, we arrive closer to the authentic specificity of the monoclonal antibody being studied. This is how an unknown determinant may be discovered. Whatever may be, monoclonal antibodies have served to point out the differences between embryonic and adult cells, between normal and cancerous cells. In this chapter we present a few characteristic examples and a brief survey of certain experimental techniques. 

The detection of ricin to confirm exposure is difficult, not least because the toxin is metabolized and eliminated rapidly. The most commonly used method is the enzyme-linked immunosorbant assay (ELISA) which can be applied to environmental and biological samples and allows ricin detection for up to 48 h after exposure with a detection limit of approximately 200 pg/ml (Griffiths et al, 1986 Leith et al, 1988). More recently, an ELISA that uses monoclonal antibodies with distinct specificities for ricin A and B chains has been developed (Shyu et al, 2002a) which offers high specificity but is too slow to be useful for rapid diagnosis. The same group has now developed a sensitive and rapid... 

Pollart S.M., Smith T.F., Morris E.C., Gelber L.E.. Platts-Mills T.A.E. and Chapman M.D. (1991) Environmental exposure to cockroach allergens analysis with monoclonal antibody-based enzyme immunoassays. J. Allergy Clin. Immunol., 87, 505-510. 

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

Figure 1. Transverse section of barley leaf epidermal cells taken perpendicular to the long axis of the cells and anticlinal to the leaf surface. The section has been labeled by the EMSIL technique (see Methods) utilizing purified C. sativus endopolygalacturonase and monoclonal antibody EPG-4, which is specific for this enzyme, in order to localize the substrate of the enzyme at the typical site penetrated by the fungal pathogen. Bar = 1 pm. Inset Comparable cell wall region as in Fig. 1 but labeled with monoclonal antibody JIM 5 to localize non-esterified pectin. Bar = 1 pm. Note the identical labeling patterns obtained with either method.

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