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Various methods of detection are available for measuring antibodies specific to M. pneumoniae. We examined over 600 sera of patients with clinical diagnosis of M. pneumoniae infection using complement fixation (CF) test. 91 gave positive reaction and were therefore selected for a comparative analysis with other methods such as metabolic inhibition (MI), mycoplasmocidal (MC) and radioimmunoprecipitation (RIP) methods. MC and RIP test gave antibody titers comparatively higher than MI and CF. Comparative studies on the sensitivity of the various methods considered were also carried out on 6 paired sera collected from the same patients at 10 days intervals. The increase in antibody titers were much higher when MC and RIP tests were considered. When RIP and MC methods were compared, the first appeared more sensitive in all cases studied.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann has been used for the detection of antibodies against Mycoplasma pneumoniae. These organisms, grown in flat-bottom wells of microtiter plates, were used as antigen. Preliminary results suggest that ELISA is specific and that its sensitivity is somewhere in between those of the metabolic inhibition and radioimmunoprecipitation methods. Unlike other serological methods, such as metabolic inhibition and mycoplasmacidal tests, in which the presence of antibiotics in sera may give false results, ELISA provided results that were not influenced by antibiotics. Furthermore, ELISA offers the possiblity of measuring not only immunoglobulin M, but also complement-independent antibodies, especially those of the immunoglobulin A class. Theoretical aspects concerning the different sensitivities of some serological reactions are discussed.

Many studies of the interaction between phagocytes and mycoplasmas have given controversial results. This is probably due both to the small size of the microorganisms and their ability to attach to the cell membrane, making it difficult to distinguish between adsorption and ingestion. To overcome these difficulties we took advantage of a phenomenon we noted occurring concomitantly with phase-contrast microscope-monitored phagocytosis of heat-killed C. albicans, i.e., a reduction of [3H]uridine uptake by macrophages from culture medium. This approach allowed us to measure the ability of mouse peritoneal macrophages and the macrophage-like P 388 D 1 continuous cell line to phagocytose Mycoplasma pneumoniae and Acholeplasma laidlawii. Live, UV-killed and specific antiserum-opsonized mycoplasmas were tested. A. laidlawii was ingested under all the conditions mentioned above, while live M. pneumoniae was not phagocytosed unless UV-killed. Phagocytosis of UV-killed M. pneumoniae was directly verified by transmission electron microscopy studies. Data obtained with opsonized M. pneumoniae indicated no ingestion by mouse peritoneal macrophages and incomplete phagocytosis with P388 D 1 macrophages, suggesting that different responses by different types of phagocytes can be observed. In spite of a lack of information concerning the biological meaning of the inhibition of macrophage RNA metabolism during phagocytosis, our data suggest that this phenomenon may be used to study the phagocytosis of microorganisms which are difficult to visualize.

The enzyme-linked immunosorbent assay (ELISA) has been used for detection of antibodies against Mycoplasma pneumoniae. The results of ELISA and its sensitivity compared with other serological methods, such as complement fixation (CF), metabolic inhibition (MI), mycoplasmacidal test (MC), and radioimmunoprecipitation (RIP) are reported. ELISA and MC showed greater sensitivity than CF and MI, while RIP showed serum titer two- to 16-fold higher. ELISA was specific as determined using other human mycoplasma. A simplified method based on the determination of ELISA antibody end-point titer by a single serum dilution has been proposed. ELISA presented several advantages: sensitivity, rapidity, and low cost and, if adequately standardized, could become a reliable method for the serodiagnosis of M. pneumoniae infection.