Direct immunocytochemistry method

Fig. 7.1 Direct immunocytochemistry. In the direct immunocytochemistry method, the 1° antibody binds its antigen and the label is directly attached to it...

To help evaluate possible labels and methods, use Table 9.1. Note that the number ratings in Table 9.1 are based on the author s experience and should only be used to compare methods within this table. Detection resolution is the ability to localize the label to the exact site of the primary antibody. The direct immunocytochemistry method does this and is rated as a detection resolution of 10 (Table 9.1, Fig. 9.1a, arrow). With detection methods that have lower detection resolution the number decreases (Table 9.1, Fig. 9.1a, numbers). 

For direct immunocytochemistry, the label is bound to the 1° antibody. For example, to locate an antigen in cells, the 1° antibody would be a rabbit anti-antigen labeled with fluorophore (Fig. 7.1). Such a procedure with directly conjugated antibodies requires only the 1° antibody and no additional antibodies. Thus, the direct immunocytochemical procedure is the simplest method, and historically, was the first method for immunocytochemistry. 

Direct immunocytochemistry - a method where the label is bound to the 1° antibody, which then binds to the antigen in the cells. 

There are several modifications to the procedure that allow the target to be identified and quantified. Since the topic is immunocytochemistry, the procedures described deal with ELISAs performed on intact cells fixed onto the wells of 96-well plates. There are two forms of the procedure direct and indirect. The direct method calls for linking the enzyme directly to the antibody of interest. Depending on the availability of the antibody and its activity, direct conjugation of the enzyme to the antibody may interfere with specificity or success of binding with the target. The preferred method is the indirect ELISA. The antigen or cell of interest is immobilized onto the well. The primary antibody (often a... 

Nucleic acid immunocytochemistry also differs from nucleic acid cytochemical approaches in that, for the latter, a reagent, such as a stain, reacts directly with the cellular nucleic acids. Such methods lack the exquisite specificity and sensitivity of nucleic acid immunocytochemistry. Table 1 compares these methods. 

Due to limits on space this chapter will deal onfy with those issues and methods that are germane to immunocytochemistry as it is practised in most diagnostic pathology laboratories. The reader is directed to the excellent texts cited in the Further Reading section for more detailed information. 

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