| Objective:To prepare rabbit polyclonal antibody against human FXâ…¢A subunit by immunologic method, and to identify antibody specifility and activity. Applying the antibody, FXâ…¢deficiency and FXâ…¢binding epitopes were determined in a patient with acquired FXâ…¢inhibitor. The preparation of antibody provides a basis for studying the mechanism of acquired FXâ…¢deficiency and clinical diagnosis.Methods:1. Prepare polyclonal antibody by immunizing rabbit.2. Identify the specifility of polyclonal antibody with dot blot, and determine the activity with antibody-neutralizing/urea-lysis test.3. To detect FXâ…¢deficiency with western blot and to analyse FXâ…¢binding epitopes with dot blot.Results:1. (1)On PVDF memberane, the spot of purified FXâ…¢protein or spot of synthesized polypeptideâ…¡, when the membrane was incubated with rabbit anti-human FXâ…¢serum, both spots showed positive signal. However, no positive signal was detected on spot of synthesized polypeptideâ… .(2)In the antibody-neutralizing test, plasma fibin clots were formed following regular method, and anti-serums with different volume were added into reactive system(0ul,10ul,40ul,80ul). Then the urea-lysis test(5M urea) was carried out. The results showed that the size of fibin clot was related to the amount of anti-serum.2. (1)On PVDF membrane, which was loaded with purified human FXâ…¢protein, normal plasma, FXâ…¢deficiency plasma(case 1 or case 2), repectively. And FXâ…¢was probed with anti-serum, there was positive signal on the location of 82.5KD molecular size, and normal plasma, while case 1 plasma only gave a weak signal, no signal was detected on case 2 plasma.(2)There was a positive signal on PVDF membrane, where polypeptideâ…¡was spoted, and was incubated with case 1 serum or rabbit serum; howver, there was not any signal on PVDF membrane where it was incubated with case 2 serum or rabbit serum.Conclusions:1. Rabbit polyclonal antibody against human FXâ…¢A subunit had been successfully prepared by immunizing rabbit, the antibody can not only bind purified human FXâ…¢and transglutaminase active site, but also inhibits the activity of FXâ…¢in vitro.2. The antibody can be applied to detect FXâ…¢deficiency and determing FXâ…¢binding epitopes. We conclude that the active site of transglutaminase on FXâ…¢is the binding epitope of FXâ…¢inhibitor in case 2, but not in case 1. |
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