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Borrelia microtti and Borrelia persica are 2 Iranian strains of Borrelia found in western Asia and responsible for relapsing fever. The outer surface antigens of Borrelia undergo variations which are responsible for the relapsing phenomenon. The fixed flagellar antigen is required for diagnosis as the variant antigens cannot be used in serological methods of diagnosis. The fixed flagellar antigen was purified for the first time from the Iranian strain of Borrelia microtti using detergent treatment and shearing in an omnimixer. Periplasmic flagella were extracted, as confirmed by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band corresponding to 42 kDa. Indirect haemagglutination kits were designed using the pure flagella and the complete sonicate of Borrelia and showed 98% sensitivity and 95% specificity.