Manual of basic techniques for a health laboratory - libdoc.who.int

332 Manual of basic techniques for a health laboratoryImmunofluorescenceFluorescent dyes such as fluorescein isothiocyanate and tetra-methylrhodamineisothiocyanate can be coupled to antibodies without destroying their specificity.Fluorescence occurs when molecules that have been excited to a higher energystate return to their normal energy state. The excess energy is released in the formof light. A fluorescent microscope, which is a modified light microscope, is used tovisualize the light emitted.Two types of immunofluorescence technique can be used.Direct immunofluorescenceDirect immunofluorescence is used when testing for an antigen. In this technique afluorescent dye is linked to the isolated portion of an antiserum containing antibodiesdirected against a specific component of cells or tissue. The antiserum isapplied directly to the tissue specimen. The antigen and antibody react, then thetissue specimen is washed.The tissue specimen is examined under the microscope and fluorescence is seenwhere the antibody is attached to the antigen (Fig. 11.5).Indirect immunofluorescenceIndirect immunofluorescence is used to determine whether antibodies are presentin a patient’s serum. The serum is applied directly to an appropriate tissue specimencontaining an antigen directed against the specific antibody under investigation.The antigen and antibody react, then the tissue specimen is washed. Ananti-immunoglobulin with a fluorescent label is added and the tissue specimen isthen incubated before being washed again.The labelled anti-immunoglobulin will attach to any antibody already bound to theantigen in the tissue specimen and shows up under the microscope as areas offluorescence (Fig. 11.6). The indirect method is more sensitive than the directmethod because it is amplified in the sense that each unlabelled antibody can bebound by two labelled antibodies.11.2.2 Secondary binding testsThe secondary binding tests enable visible manifestations following the primaryreaction to be observed. In these tests one can actually see the effects of the bindingevent without the aid of an additional label. These tests include agglutination,precipitation, complement-dependent reactions and neutralization methods. Theagglutination and precipitation methods are routinely employed for diagnostic purposes.These methods are briefly described below.FFFFFIncubatethenwashFFFFFAntigen(cell or tissue specimen)Fluorescence-labelledantibodiesFig. 11.5 Principle of direct immunofluorescenceFluorescence