Fab molecules

Figure 21.11 Fab antibody fragments containing free thiols can be activated with Ellman s reagent to form a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol group may be coupled to the activated Fab molecule to produce an immunotoxin complex.

The complex of a peptide derived from HIV (a model antigen) and an Fab molecule, shown in Figure 5-27, illustrates some of these properties. The changes in structure observed on antigen binding are particularly striking in this example. 

Effector function can also be eliminated by removing the Fc portion of the molecule to produce a Fab molecule (e.g., abeiximab). However, Fab molecules... 

Thus, a highly substituted Fab molecule is only slightly less active than an IgG-POase conjugate but has only half the diameter of the latter. 

A new Experimental Design Chart must be used for this Indirect Immunocytochemistry Block-Between (Table 12.1), because it differs from the chart used previously. Here, the experiment uses cultured cells on coverslips and two 1° antibodies made in mouse, mouse anti-Ag A and mouse anti-Ag B. A new row on the Chart is needed, 4. Block-between, to list two new reagents used to block-between the two 1° antibodies. Normal mouse serum is used at 1 20, which is sufficient to block in most cases. The anti-mouse Fab molecule is used at 20 p.g/ml. Incubations for each of these steps is for 1 h with six rinses after each incubation. Note these blocking incubations must be performed in the order of normal mouse serum first and anti-mouse Fab second. 

Another potential problem occurs if the anti-mouse Fab molecules do not block the mouse species-specific sites. Incubation with the mouse anti-Ag A and antimouse 488 fluorophore (Fig. 12.3a) is then blocked with normal mouse serum IgG (Fig. 12.3b, stippled gray). If there is insufficient anti-mouse Fab to block the mouse antibodies from the first set of incubations, then the second 2° antibody will bind to the first set of antibodies. Specifically, the second 1° antibody, mouse anti-Ag B, binds to the Ag B antigen (Fig. 12.3c). Then the lack of anti-mouse Fab fragments... 

The second approach is very different and involves labeling the 1° antibody before incubation with tissues. In effect, this makes the procedure a direct immunocyto-chemistry approach because no 2° antibody is used. The method uses labeled Fab molecules from anti-species antibodies that bind to the Fc end of the 1° antibody. The labeled Fab reagents needed for this method are available as a commercial kit called Zenon (Molecular Probes/ Invitrogen). 

The 2° antibody incubation uses a species-specific antibody labeled with small gold particles (Nanoprobes, or Aurion) (Fig. 15.2a). Using 2° antibodies Fab molecules labeled with small gold gives better penetration because the size of the... 

One can calculate that the area bound by a Fab molecule (single arm combining site of antibody without Fc) is approx 20 nm. This can represent an antigenic site (epitope). Thus, the number of possible sites on the virus is the surface area of the virion divided by the surface area of the combining site. The surface area of a sphere is 4 j A tt j A therefore, for FMDV 4 jA 3.14 jA 12.52 = 12.56 jA 156.25 = 1962 nm2. By dividing this by 20, the maximum number of Fab sites possible is 98. 

Diagram 2 This indicates that the density of mAb can affect binding, even when the orientation is correct and Fab molecules are present. Full-dilution ranges of mAbs should be performed to allow an assessment of binding properties, since it is possible that lower concentrations of mAb are better spaced to allow capture. This is also dependent on the nature of the antigen. 

Figure 2.1 Two different molecular species will be compatible if Fab — aa and Fab molecules will tend to separate if they have sufficient energy for molecular movement.

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