1° antibody controls specificity control methods

The four important H antibody specificity control methods are listed here in the order of preference among experienced users. 

Immunohistochemistry is a powerful method for identification of proteins in cells and tissues, but control procedures are necessary. The specificity of the results depends on two independent criteria the specificity of the antibody and of the method used (Burry 2000). The antibody specificity is best determined by immu-noblot and/or immunoprecipitation. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG (must be the same species as primary antibody at the same concentration), and a positive control, testing the primary antibody with tissues known to contain the antigen. 

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

The method lends itself to additional specificity controls in that the primary specific antibody may be omitted, or another antibody of irrelevant specihcity may be substituted, providing a valuable assessment of the validity of any staining pattern observed. 

Immunocytochemistry harnesses antibodies that are specific reagents and which allow unique detection of proteins and molecules. Using antibodies requires specific methods, labels, and controls. Performing immunocytochemistry experiments requires some basic knowledge of biology. 

Fig. 8.2 1° antibody specificity controls. The specificity of the 1° antibody for the antigen is demonstrated by several methods, (a) Knockout control antigen No. 3 is not present, (b) ColocaMzation of a GFP-tagged protein and a 1° antibody to that protein. 

In experiments with multiple 1° antibody and multiple 2° antibodies, the control must show that (1) each 2° binds with only the correct 1° antibody and (2) each 2° does not bind to other 2° antibodies. Detailed protocols for multiple 1° antibody experiments will vary depending on the number of 1° antibodies, the species used to make the 1° antibodies, and the labeling protocol used. Descriptions of multiple antibody methods in Chapters 11 and 12 will include a discussion of the specific 2° antibody controls needed. 

Plan to add controls that confirm successful blocking steps between two sets of antibodies (Table 12.2). Because of the sequential addition of antibodies, the controls are different from other experiments with the indirect method of immunocyto-chemistry. The first 1° antibody is not eliminated because there are no competing antibodies for the first 1° antibody. Also, the no 1° antibody control for the first 1° antibody was done previously when the Dilution Matrix showed it was bound specifically by the 2° antibody. The controls here test the potential binding of the second 1° antibody and second 2° antibody to the first set of antibodies. 

The classical methods of demonstrating specificity of antibody reactions with antigens, such as immunodiffusion plates, are obsolete. Immunofluorescence is a very sensitive method that requires controls for specificity of equal sensitivity. The most important controls are not just those that demonstrate the presence of an antibody that reacts with the antigen of interest, but more importantly, the absence of antibodies that react with other things. 

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