Epitopes controls

If a monoclonal antibody was generated by immunization with a full-length native protein rather than a peptide, then the immunized mouse will generate antibodies that recognize both linear and conformationally dependent epitopes. Only a small subset of these monoclonal antibodies will likely be useful for clinical use on formalin-fixed, paraffin-embedded tissue (FFPE) samples. Those that are useful tend to have epitopes that are linear the epitopes are not dependent on the protein s three-dimensional conformation (see Chapter 16). Therefore, for antibodies generated in response to immunization with full-length proteins, the peptides that serve as positive controls will be linear stretches of amino acids derived from the native protein sequence, as listed in protein databases. 

A process control is just that, a control for the stain process. While the concept can be extended to the primary epitope also, this may not be practical in all cases. Purified protein epitopes are very expensive, and adding a dilution series of primary epitope to a control slide would create a unique and possibly... 

Fig. 8.10 Titers of antibodies at day 50 induced by plant-derived CTB-2L21 recombinant protein. Balb/c mice were intraperitoneally immunized with leaf extract from CTB-2L21 transgenic plants. Animals were boosted at days 21 and 35. Each mouse received 20 pg of CTB-2L21 recombinant protein. Individual samples of mouse serum were titrated against 2L21 synthetic peptide,VP2 protein and a control peptide (amino acids 122-135 of hepatitis B virus surface antigen). Titers were expressed as the highest serum dilution to yield twice the absorbance mean of preimmune sera. M1-M6 mice 1 to 6 2L21 epitope from the VP2 protein of the canine parvovirus CTB cholera toxin B VP2 protein of the canine parvovirus that includes the 2L21 epitope.

As a consequence, any nonspecific adsorption of protein to the interface will also cause an attenuation in electrochemistry. Hence, the antifouling layer is required to ensure the attenuation of the electrochemistry associated with the presence of antibodies is due to a specific interaction with the binding epitope on the molecular wire rather than any nonspecific effects. The ability of this layer to resist nonspecific adsorption of proteins, as well as preventing electroactive species reaching the electrode, was demonstrated by Liu and Gooding [131] previously and is demonstrated in the current paper via controls that show there is little attenuation in the ferrocene electrochemistry if a different antibody is used or if the biotin epitope is absent from the interface. 

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