Conjugation methods in immunofluorescence.

We have described methods of labeling antibody preparations with FITC, TMRI, and RBI. The degree of labeling with FITC can be precisely controlled by using well-defined conjugation procedures and FITC of a known degree of purity. Our experience shows that relatively high F/P ratios of the order of 20 to 25 mug/mg are desirable for antibacterial conjugates. Many commercial preparations of rhodamine isothiocyanate are of very poor quality and are unsatisfactory for use in conjugate preparation. Therefore, one should analyze the rhodamine isothiocyanate product before preparing immune conjugates. Our experience indicates that very satisfactory conjugates of immune IgG or pure antibody can be prepared with TMRI of about 60% purity by using a dye-protein ratio of 20 mug/mg. The optimal dye-IgG ratio for labeling with RBI appears to be about two times that for labeling with TMRI because of the lower specific absorbance and fluorescence emission of RBI. Rhodamine conjugates may be preferred to FITC conjugates in certain situations where tissue autofluorescence interferes with the observation of the yellow-green emission of FITC. Furthermore, mixed rhodamine and FITC conjugates of different specificity can be used to great advantage in double-staining techniques that allow simultaneous screening for two antigenically different organisms on a single microscope slide.