Monovalent antibody

Antibody affinity from the Latin, affinis = connected with, having things in common. In immunohistochemistry, antibody affinity determines the strength of binding of a monovalent antibody, such as Fab fragment, to one epitope, i.e., how tightly an antibody binds to its particular antigen. 

Unless all these factors are taken into account, it is usually more appropriate to refer to the results of a flow cytometric analysis in terms of antibodies bound per cell rather than antigens per cell. Stoichiometry will be even less certain with multivalent IgM antibodies the usually low monovalent affinity and strong role of avidity in the binding of IgM antibodies make them of limited value for antigen quantitation. Theoretically, the most precise alternative would be the use of directly labeled monovalent antibody fragments, which would avoid problems of variable stoichiometry. However, in addition to the inconvenience of producing suitable labeled monovalent antibody fragments, the increased off rate of monovalently bound antibody may make analysis more difficult. 

Whereas the word affinity describes the strength of a reaction of a monovalent antigen (haptene) with a monovalent antibody (antigen docking unit), the word avidity is used to describe the total tendency of an antibody to bind an antigen, particularly that of antibodies... 

Lingwood CA, Hakomori S Selective inhibition of cell growth and associated changes in glycolipid metabolism induced by monovalent antibodies to glycolipids. Exp. Cell Res. 1977 108 385-391. 

The relative insensitivity of ATP formation and hydrolysis in contrast to the strong inhibition of the Pi-ATP exchange by preincubation of chloroplasts with monovalent antibodies against CFi is shown in Fig. 1. 

Step 1 Mixing of unconjugated primary antibodies with labeled monovalent Fab fragments... 

Primary antibodies Labeled monovalent Fab fragments Labeling complexes and unbound Fab fragments... 

Step 2 Absorption of unbound labeled monovalent Fab fragments with excess serum from the same species as the primary antibody... 

Dilute the resultant primary antibody Fab fragment complexes in staining buffer containing 10 20% normal serum from the same species as the primary antibody to give the concentration of about 1 10 pg/ml of the primary antibody, and incubate for 15 30 min at room temperature to block unbound labeled monovalent Fab fragments. ... 

This resultant mixture of primary antibody Fab fragment complexes containing blocked labeled monovalent Fab fragments can be stored for a few days at +4°C until use. 

For double immunolabeling with primary antibodies from the same host species, it is not necessary to resort to labeling with monovalent Fab fragments when primary antibodies from the same host species are different isotypes (subclasses) of IgG, for instance such as mouse IgGl and mouse IgG3. In these cases, isotype-specific antibodies may be used to distinguish between the two primary antibodies... 

Another approach to this persistent problem relies on haptenylation of primary antibodies. Hapten (e.g., biotin, digoxigenin or any fluorophore) can be covalently bound to the antibody via A-hydroxysuccinimide esters (NHS-ES) (see Sect. 2.1), or conjugated employing monovalent IgG Fc-specific Fab fragments (see Sect. 2.2). Haptenylated primary antibodies can be subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (Fig. 8.5). Fluorophore-labeled primary antibodies can be directly visualized in a fluorescent microscope. 

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