J. Med. Microbiol. - Vol. 36 (1992), 341-346 0 1992 The Pathological Society of Great Britain and Ireland lmmunoblottingpatterns with Mycoplasmapneumoniae of serum specimens from infected and non-infected subjects G. AUBERT, B. POZZETTO, J. HAFID and 0. G. GAUDIN Microbiology Laboratory, Faculty of Medicine Jacques Lisfranc, University of Saint-Etienne, 15 rue Ambroise Par&, 42023 Saint-Etienne Cedex 2, France Summary. Two hundred and ninety-four serum specimens from 248 subjects, whose complement fixation (CF) titres to Mycoplasma pneumoniae were known, were further investigated by IgG immunoblotting. After analysis of M. pneumoniae proteins by SDS- PAGE, nine polypeptides (p) with mol. wts of 180-43 Kda were selected for immunoblotting studies. Antibodies to M. pneumoniae measured by immunoblotting appeared progressively with age; most subjects more than 19 years old gave positive results. For most of the polypeptides, there was an increase in the frequency of band detection when the CF titres were higher. Furthermore, paired serum specimens from 10 patients with M. pneumoniae infection, as demonstrated by a rise in CF antibody titre, were tested for IgG blotting patterns. Generally, p180 (the P1 adhesin of M. pneumoniae), p172 and p84 were shown to be the dominant targets of the immune response to this organism and may have diagnostic value. Introduction Mycoplasma pneumoniae causes a wide range of clinical manifestations, including primary atypical pneumonia. In most cases, the diagnosis of M. pneumoniae disease is confirmed by serology. The most widely used method for serodiagnosis is the comple- ment fixation (CF) test with a M. pneumoniae glycolipid antigen. ' Cross-antigenicities are known to occur between this glycolipid and similar antigens of other origins, resulting in non-specific diagnostic reac- tion~.~-~ The use of blotting methods provided a valuable tool for investigating the response to M. pneumoniae antigens.6-' These studies pointed out the importance of antibodies to some proteins in the course of acute infection, especially those directed to a high-mo1.-wt polypeptide (designated P 1 and known to be the major adhesin of M. pneumoniae) of 160-1 90 Kda, according to different authors.' 2~1 In the present study, we have investigated by immunoblotting the antibody response to M. pneumon- iae antigens of subjects of all ages, with or without active infection as indicated by CF test results. The aims of this work were : (1) to provide epidemiological data on the distribution of antibodies in relation to age, by the immunoblot technique; (2) to evaluate the immune response to different polypeptides of M. pneumoniae; and (3) to define their putative value for improving the diagnosis of M. pneumoniae infection. Received 19 Dec. 1990; revised version accepted 8 July 1991. Materials and methods Selectwn of serum samples Two hundred and ninety-four serum specimens from 248 subjects (152 males, 96 females) were classified diagnostically by the CF test and then analysed by the IgG immunoblotting test. The investigated population comprised 14 hospitalised infants under 1 year old, 73 hospitalised children under 10 years old, 102 hospitalised patients over 10 years old and 59 healthy adult blood donors. For an epidemiological study (see Results, section 2), serum specimens that gave negative results in the CF test (titre < 8) were examined from 191 of the 248 subjects described above. These fell into 10 age-groups and >59 years), each of about 20 subjects, and they comprised 59 healthy adults and 132 hospitalised patients; no clinical data were available for most of these patients. All these serum specimens were collected within the same 6-month period. The remaining serum specimens belonged to se- lected patients with antibody titres to M. pneumoniae of 2 8 by CF test (range 8-1024). These included 10 patients with respiratory infection who exhibited a four-fold or more rise in titre by CF test on paired sera (mean time between the two samples: 20.5 SD 14.9 days). ( < l , 1, 2-5, 6-9, 10-19, 20-29, 30-39, 40-49, 50-59 M. pneumoniae strain and antigenpreparation The strain of M. pneumoniae (C200) was a gift from Dr S. Bossard (Facultk de Mkdecine Rockefeller, 341