Detection of serum antibodies against Mycoplasma gallisepticum and Mycoplasma synoviae by a dot-immunobinding technique.

Abstract

Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.

A Filter Immunobinding Technique for the Rapid Detection and Simultaneous Identification of Avian and Bovine Mycoplasmas.

“…In our previous study [14], reduction of contact time of the primary and secondary sera did not eliminate nonspecific reactions with rabbit antisera. It has been pointed out in the literature [2,7] that mycoplasma organisms readily adsorb medium components on the surface of their cell membrane, and that it is difficult to remove these 11.…”

“…The advantages associated with the FIB are that cutting the nitrocellulose membrane to fit the size of a dot plate is a much easier process than preparing small square pieces as reported in our previous studies [13][14][15], and that lots of reactions can be performed at one time on one membrane.…”

“…We have previously reported the use of the dotimmunobinding (DIB) technique to detect antibodies against Mycoplasma gallisepticum (MG) and M. synoviae (MS) in chicken serum by using small pieces of nitrocellulose membrane [13][14][15]. Antibodies in chickens experimentally or naturally infected with MG and/or MS were simply and specifically detected by this method provided that mycoplasma antigens were fixed on the pieces of the membrane.…”

“…Thus, a rapid diagnostic technique would be preferable rather than the isolation of pathogens. A rapid plate agglutination test to detect antibodies in blood or serum has been widely used as a convenient serological diagnosis of chicken mycoplasmal infection, in which, however, it is known that false positive reactions easily occur [12,16,17].We have previously reported the use of the dotimmunobinding (DIB) technique to detect antibodies against Mycoplasma gallisepticum (MG) and M. synoviae (MS) in chicken serum by using small pieces of nitrocellulose membrane [13][14][15]. Antibodies in chickens experimentally or naturally infected with MG and/or MS were simply and specifically detected by this method provided that mycoplasma antigens were fixed on the pieces of the membrane.…”