Evaluation Of Different Substrates Indirect Immunofluorescence Test Results For Bullous Diseases

In order to study different substrates indirect immune-fluorescence reactivity of bullous disease circulation antibodies and screen out substrates thai can be acquired easily and with strong sensitivity, we tested Pemphigus and Bullous Pemphigus sera in indirect immunofluorescence on different kinds of substrates including normal human skin and umbilical cord, esophagus, small intestine, bladder and tongue of guinea pig, small white mouse, large white mouse and rabbit. They provide material resources for molecular research of the target antigens of Pemphigus and Bullous Pemphigus.Methods1) Sera of 10 cases of pemphigus, 5 cases of Bullous Pemphigus, and 3 cases of normal human were put into vessels respectively, and kept refrigerator at 30℃ until used. The esophagus, small intestine, bladder, tongue of guinea pig, small mouse, large mouse and rabbit were cut into 0. 8 x0. 8cm blocks of each tissue, then these fresh specimens were completely covered in OCT mounting medium and immediately frozen in liquid nitrogen, and kept in refrigerator at 70℃ until used. Reagent; FITC-labeled goat anti-human IgG was diluted 1-80.2) Pigmentation method: indirect immunofluorescence technique, fluorescence pigmentation.3) Observing index: serum was diluted 1:10. We observed circulation antibodies deposit and the intensity of fluorescence and came into view fluorescence antibodies binding or positive uniform linear fluorescence at the basementmembrane zone.4) Scoring method: We record 3 scores when the intensity of fluorescence pictures among pemphigus cells and basement membrane zone is very strong and dazzling. We record 2 scores with strong intensity, and record 1 score with less intensity, and mark 0.5 score when the intensity of fluorescence is diminished. Two persons were appointed to accomplish all scoring. The final outcome is the average of the two results.5 ) Statistical analysis: t test.Results1)10 cases of Pemphigus sera were diluted 1:10, rabbit tongue and rabbit esophagus got the highest score in the substrates (both of them score are 22).2) 10 cases of Pemphigus sera were dilute 1:20, rabbit esophagus got the highest score when we took rabbit esophagus and rabbit tongue as substrate.3)5 cases of Bullous Pemphigus sera were diluted 1:10, guinea pig tongue got the highest score in the different kinds of substrates (14 score).4)3 cases of normal human skin sera were diluted 1:10, and different substrates indirect immunofluorescence test results were negative result.5) 10 cases of Pemphigus sera were tested in serial dilution ( starting at 1: 10) for intercelluar antibodies by indirect immunofluorescence by a standard technique. We took rabbit esophagus and normal human skin as substrates, and detect antibody of Pemphigus titer. No apparent reactivity difference between them (P>0.5).Conclusion1) Rabbit esophagus is the most sensitive substrate to the evaluation of indirect immunofluorescerice for circulation pemphigus antibodies. No apparent sensitivity difference was found compare to human skin substrate ( P > 0. 5 ). Rabbit tongue, tongue of guinea pig, esophagus of guinea pig and esophagus of small mouse also can be commonly used for substrates.2 ) Tongue of guinea pig as most sensitive substrate is used to test the antibodies of basement membrane zone, We can also take esophagus of small mouse, bladder of big mouse and rabbit tongue as substrates.The research showed that rabbit esophagus and guinea pig tongue can substitute for normal human skin and monkey esophagus as substrates, and used to bullous diseases research. Because both of these materials can be acquired largely, desmosome component can be picked-up from them. They provide material resources for molecular research of the target antigens of Pemphigus and Bullous Pemphigus.