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Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme‐linked immunosorbent assay (ELISA). Many patients' sera cross‐reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF≥40) titers gave significantly higher ELISA values to M. hyorhinis (P<0.001) and M. pulmonis (P<0.001), which are not parasitic for humans, than those with low CF (CF<20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross‐reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.

Varying levels of cross-reactivity to some mycoplasma species were observed in the sera of patients infected with Mycoplasma pneumoniae and even in normal human sera by enzyme-linked immunosorbent assay (ELISA). The absorption of the patients' sera with M. pneumoniae lysate showed the decrease in ELISA titers not only to M. pneumoniae, but also to other mycoplasma species. These results suggested the existence of cross-reactive antibodies to mycoplasmas in human sera. Cross-reactive antibodies to M. pneumoniae and other mycoplasmas in the patients' sera were also demonstrated by Western blotting technique.The enzyme-linked immunosorbent assay (ELISA) has been used for routine serological diagnosis of Mycoplasma pneumoniae infection because of its high sensitivity and simplicity (4,5,7,19,23,24). But, as high background readings are often observed in the ELISA, the interpretation is often very difficult. It is suspected that the main factor causing background readings is the nonspecific adsorption of serum and the conjugate on the surface of plate wells or on M. pneumoniae antigens.We previously showed by the ELISA that varying levels of cross-reactivity to some mycoplasma species were seen in human normal immunoglobulin G (human normal IgG) prepared from pooled plasma of at least 500 normal human donors and in some human sera not associated with M. pneumoniae infections (21). Furthermore, we also showed that the nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate in M. pneumoniae antigen could be disregarded (21). These results suggested the existence of the cross-reactive antibodies to mycoplasmas that lowered the specificity of the ELISA in serodiagnosis of M. pneumoniae infection.In this study, we investigated by ELISA and immunoblotting technique the characterization of the cross-reactive antibodies found in sera of patients infected with M. pneumoniae.

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