Abstract: 【Abstract】 Objective To optimize indirect immunofluorescence on salt-split skin （IIF-SSS）, and to evaluate its performance in detection of bullous pemphigoid （BP） antibodies. Methods Normal human foreskin and non-foreskin skin tissues were used to prepare salt-split substrates under 3 different experimental conditions: traditional group rotated at 4 ℃ for 48 - 72 hours, low-temperature immersion group soaked at 4 ℃ for 48 - 72 hours, room-temperature immersion group soaked at 25 ℃ （range: 23 - 27 ℃） for 24 hours. Serum samples were obtained from 20 patients with bullous pemphigoid （BP） in Hospital of Dermatology, Chinese Academy of Medical Sciences between August 2019 and August 2020, and subjected to IIF on the intact skin or salt-split substrates by using a multiple dilution method. Paired-sample t test was used for comparisons of means between two paired samples. Results No dermal-epidermal separation was observed in the substrates prepared in the low-temperature immersion group at 48 - 72 hours, while dermal-epidermal separation occurred in the lower lamina lucida of the foreskin and non-foreskin substrates in the room-temperature immersion group and the traditional group. For the 20 patients with BP, the reciprocal end-point titers （M ［Q1, Q3］） detected with the salt-split non-foreskin skin and salt-split foreskin in the room-temperature immersion group, and with the salt-split non-foreskin skin in the traditional group were 5 120（2 560, 17 920）, 1 280（640, 2 560）, 1 280（640, 2 560） , respectively. Moreover, 19 （95%） patients with BP showed that the reciprocal end-point titers detected with the substrates in the room-temperature immersion group were 1 - 5 times those in the traditional group （t = 8.04, P＜0.001）, suggesting that the performance of salt-split skin in the room-temperature immersion group was superior to that in the traditional group in the detection of BP antibodies; however, there was no significant difference in the reciprocal end-point titers of BP antibodies between the salt-split foreskin in the room-temperature immersion group and salt-split non-foreskin skin in the traditional group （t＜0.001, P＞0.05）. The reciprocal end-point titers in 20 BP sera detected by conventional IIF on the intact non-foreskin skin and foreskin were 320 （160, 640） and 480 （160, 1 120）, respectively; the reciprocal end-point titers detected by IIF on the salt-split foreskin and non-foreskin skin in the room-temperature immersion group, as well as on the salt-split non-foreskin skin in the traditional group, were all consistent with or 1 - 7 times higher than those detected by conventional IIF（t = 6.47, 14.83, 5.26, respectively, all P＜0.001）. Conclusion The soaking method at room temperature 25 ℃（23 - 27 ℃） for preparing salt-split substrates has advantages of short duration and simple procedure, and the sensitivity of IIF-SSS using the substrates prepared by this method is equal or superior to the traditional salt-split method for detecting BP antibodies.

Key words: Pemphigoid, bullous, Diagnosis, Fluorescent antibody technique, indirect, Temperature, Salt-split skin, Optimization of method