In the more common protocol for immunofluorescence detection

In the more common protocol for immunofluorescence detection of cellular proteins, an investigator uses two antibodies. The first binds specifically to the protein of interest. The second is labeled with fluorochromes for easy visualization, and it binds to the first antibody. In principle, one could simply label the first antibody and skip one step. Why use two successive antibodies?

Solution

Antibodies have Fc and Fab parts. The Fab part binds with the epitope of the antigen. Diversity in Fab is responsible for innumerable number of antbodies. The part of the antibody which binds with epitope is called paratope. to produce antibodies which specifically bind to a protein , we must be able to produce specific antibodies by techniques like monoclonal antibody production. the gene which codes for that specific antibody is inserted into a yeast and antibodies are produced in bulk. these antibodies bind specifically to the protein of interest. Now another antbody which is labelled with flouroscent dye is used. it specifically binds to Fc fragment of the monoclonal antibody which is now bound to the protein with its Fab fragment.The Fc fragment of second antobody is tagged with flouroscent dye. uing two antibodies increases the sensitivity and specificity of the test.