Species-specific monoclonal antibody to a 43,000-molecular-weight membrane protein of Mycoplasma pneumoniae.

A murine immunoglobulin G1 monoclonal antibody was produced that binds to a protease-sensitive, periodate-insensitive epitope on a 43,000-molecular-weight Mycoplasma pneumoniae membrane polypeptide. The 43,000-molecular-weight polypeptide appeared to be a major antigenic component of M. pneumoniae, as determined by immunoblot analysis. This monoclonal antibody reacted with 33 different clinical isolates of M. pneumoniae, but not with normal-flora Mycoplasma species or 18 other microorganisms potentially inhabiting the normal or diseased human respiratory tract. This apparent species-specific monoclonal antibody may have application for the detection of M. pneumoniae antigen in clinical specimens.     

The simultaneous direct detection of Mycoplasma pneumoniae and Legionella pneumophila antigens in sputum specimens by a monoclonal antibody immunoblot assay

An immunoblot (Western) assay was developed employing a species-specific monoclonal antibody to a 43 kDa Mycoplasma pneumoniae membrane polypeptide and a species-specific monoclonal antibody to 29 kDa Legionella pneumophila outer membrane protein. This assay could simultaneously detect these two different antigens directly in sputum. The 43 kDa M. pneumoniae antigen was detected by this assay in each of three M. pneumoniae culture-confirmed sputum specimens. In addition, the 29 kDa L. pneumophila antigen was detected in three of three L. pneumophila culture-confirmed sputum specimens. Neither of these two specific antigens were detected in induced sputum specimens from ten normal individuals.     

Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a monoclonal antibody immunoblot assay

Throat swabs from patients with pharyngitis and sputum specimens from patients with atypical pneumonia were tested for the presence of a Mycoplasma pneumoniae polypeptide with a molecular weight of 43,000 with the use of an M. pneumoniae species-specific monoclonal antibody in an immunoblot assay. This 43,000-dalton polypeptide was detectable in 33 of 33 throat swabs from patients with pharyngitis that were positive for M. pneumoniae by conventional culture as well as a culture-amplified enzyme immunoassay. The 43,000-dalton polypeptide was also detected in three of three M. pneumoniae culture-positive sputum specimens. It was not detected in 3 sputum specimens culture-confirmed for Legionella pneumophila, 10 sputum specimens from normal persons, or 25 throat swabs also from normal persons. This immunoblot assay could be completed within five hours and may be an alternative method for detecting M. pneumoniae antigen directly in sputum or throat swab specimens.     

ELISA for detection of Mycoplasma pneumoniae antigens using monoclonal antibodies

Seven monoclonal antibodies directed against major antigens of Mycoplasma pneumoniae were selected for the development of an antigen detection assay. Three of these were directed to the 170,000-dalton adhesin of M. pneumoniae. The test was an antigen-capture enzyme immunoassay using the different monoclonal antibodies for capture of antigen and a polyclonal rabbit antiserum as detection reagent. With three of the monoclonal antibodies a detection limit of approximately 2 ng M. pneumoniae protein was obtained, as determined by titration of M. pneumoniae organisms in buffer. The detection limit of the assays was only slightly less when the other four monoclonal antibodies were used. In artificially infected nasopharyngeal aspirates the detection limit was approximately 10 times lower. The fact that no significant differences in the detection limit of the assays were recorded using monoclonal antibodies directed against different antigens indicates that these antigens were available for reaction with antibodies irrespective of their location in intact M. pneumoniae cells. In the assay there were no significant cross-reactions with a number of bacterial species potentially colonizing the respiratory tract, except for a protein A-positive strain of Staphylococcus aureus. Our test is equally sensitive to another recently described ELISA using polyclonal antibodies. In comparison with other recommended methods such as immunoblot and culture-amplified antigen detection assays, the ELISA is more rapid and less laborious.     

A Mycoplasma genitalium protein resembling the Mycoplasma pneumoniae attachment protein.

In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.     

Absence of Mycoplasma pneumoniae cytadsorption protein P1 in Mycoplasma genitalium and Mycoplasma gallisepticum

Polyclonal and monoclonal antibodies to Mycoplasma pneumoniae protein P1 were nonreactive with whole-cell or soluble preparations of M. genitalium and M. gallisepticum. However, radioimmunoprecipitation performed with hyperimmune rabbit sera raised against each mycoplasma species indicated antigenic cross-reactivity between M. pneumoniae and M. genitalium.     

Relationship of Pre-Existing Antibody to Subsequent Infection by Mycoplasma pneumoniae in Adults

An extensive study of the epidemiological and serological characteristics of Mycoplasma pneumoniae infection was carried out in a military population. There was an increase in the infection rate at Camp Lejeune during the summer months as indicated by a relative increase in isolations, seroconversions, and hospitalizations for M. pneumoniae pneumonia. Twenty-three percent of the trainees who later became infected had detectable, pre-existing antilipid antibody to M. pneumoniae. When the whole organism was used as antigen, a pre-existing complement fixation (CF) titer of 1:4 or greater correlated with resistance to M. pneumoniae disease as defined by the absence of a fourfold rise in CF antibody, shedding of organisms, and clinical illness. Pre-existing antilipid fraction CF antibody titers of 1:16 or greater correlated with protection against mild and severe M. pneumoniae disease. Antilipid CF antibody titers of 1:4 and 1:8 were related to protection against mild disease but were not associated with protection against pneumonia which required hospitalization. The severity of illness was directly related to the CF antibody response in trainees with acute respiratory disease and pneumonia due to M. pneumoniae. The findings provide a basis for the development of a M. pneumoniae vaccine.     

Acute-phase, indirect fluorescent antibody procedure for diagnosis of Mycoplasma pneumoniae infection

An indirect fluorescent antibody (IFA) technique was developed to detect IgG and IgM-specific antibodies to Mycoplasma pneumoniae. The presence of IgM-specific mycoplasma antibody was interpreted as reflecting active infection in patients with atypical pneumonia or other clinically compatible illness. The procedure is suitable for use in routine clinical laboratories, correlated well with complement fixation test results and did not show cross reaction with Legionella pneumophila antibody. The ready availability of an acute-phase procedure for diagnosis of Mycoplasma pneumoniae infection permits therapeutic judgments based on testing of the acute serum sample.     

Application of an indirect immunofluorescence test for detection of Mycoplasma pneumoniae in respiratory exudates.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae and examined the conditions influencing the ability of an indirect immunofluorescence test to detect the specific antigen in respiratory exudates. The antibody did not cross-react with normal human serum or with respiratory exudates from 10 healthy persons. Cross-reactivity of the antibody with species of mycoplasmas other than M. genitalium was fully diminished when absorbed with horse serum and yeast extract, components of the culture medium. Though the absorbed antibody cross-reacted with M. genitalium, the titer was significantly lower than when tested against M. pneumoniae. Two types of antigen-specific fluorescence were observed in clinical specimens: one is large or small fluorescent granular aggregates found in mucus, and the other is fine fluorescent particles diffused on the entire surface of small epithelial cells. Throat smears from 49 patients with serologically confirmed M. pneumoniae infections were examined by our indirect immunofluorescence method. Positive results were obtained in 42 cases, many of which were positive before a rise in serum antibody titer could be demonstrated, indicating that the method is useful for a preliminary diagnosis at an early stage of the infection.     

Host reactions to Mycoplasma pneumoniae infections in guinea-pigs preimmunized systemically with the adhesin of this pathogen

Guinea-pigs developed systemic and local humoral responses after intraperitoneal immunization with the isolated adhesin (168 kDa protein) of Mycoplasma pneumoniae cells. Hilar lymphocytes of these animals showed proliferation after in vitro stimulation with the 168 kDa protein or sonicated M. pneumoniae whole cell antigen. Animals preimmunized and subsequently infected with M. pneumoniae showed increased M. pneumoniae-specific IgG, IgA and adherence inhibiting antibody activities. Nevertheless these animals developed severe lung lesions of lympho-histiocyte infiltrations. Furthermore hilar lymph nodes were depleted of immunocompetent lymphocytes, suggesting a cell transfer of specific stimulable lymphocytes to the inflammation sites.     

Biological activities of monoclonal antibodies to Mycoplasma pneumoniae membrane glycolipids.

A purified preparation of membranes was obtained by using a unique method of treating Mycoplasma pneumoniae with the ATPase inhibitor, diethylstilbestrol. This method was shown to yield highly purified membranes with little or no cytoplasmic contamination. These membranes were used to immunize mice for subsequent productions of monoclonal antibodies (MAbs). Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay with whole-cell M. pneumoniae and lipid extract antigens. Four stable MAbs were obtained and characterized. MAb CP3-46F5 reacted with a protein of a molecular weight of approximately 52,000 as determined by Western blot (immunoblot). MAbs CP3-50C2, CP3-53C5, and CP3-53C8 did not react with any antigens on Western blots but did bind to at least 10 distinct glycolipid bands as determined by orcinol staining on thin-layer chromatograms of M. pneumoniae lipid extracts. The MAbs did not react with similarly prepared lipid extracts from Mycoplasma genitalium, Mycoplasma neurolyticum, and Mycoplasma gallisepticum. These MAbs did not inhibit M. pneumoniae metabolism or attachment to WiDr cell cultures. The anti-glycolipid MAbs recognize determinants specific to M. pneumoniae, unlike polyclonal hyperimmune sera against M. pneumoniae, which cross-react with lipid extracts of M. genitalium.     

Comparison of enzyme-linked immunosorbent assay (ELISA) and complement fixation test for detection of Mycoplasma pneumoniae antibodies.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies against Mycoplasma pneumoniae, performed with commercial antigen and reagents, is compared with the complement fixation test (CF) in a serological study of 209 human sera. Concordant results were usually obtained by CF test and by IgG ELISA in sera from patients with recent M pneumoniae infection. In contrast, when used for an immunological survey of a general population, approximately 27% of the sera negative in the CF test were positive for IgG by the ELISA, and sera with low CF titres were found to have a broad range of IgG titre by the ELISA. This may be due to the greater sensitivity of the ELISA technique and/or to different types of antibody measured by both tests. IgM was detected by ELISA in sera from all patients with recent M pneumoniae infection diagnosed on the basis of clinical findings and by CF assay. Occasionally false-positive IgM antibodies were due to rheumatoid factor (RF); this potential interference necessitates routine testing of IgM antibody positive sera for RF.     

Immunoglobulin M antibody response against Mycoplasma pneumoniae lipid antigen in patients with acute pancreatitis.

Serial serum samples from patients with acute pancreatitis showed a significant increase in antibodies against methanol-chloroform-extracted lipid antigen from Mycoplasma pneumoniae when tested by complement fixation. The antibodies did not react with antigens prepared from other human mycoplasmas or from pancreatic tissue by lipid extraction. The antibodies were predominantly immunoglobulin M (IgM). No correlation with cold agglutinins or cardiolipid complement-fixing antibodies was found. The IgM antibody response seemed to be prolonged: after 3 to 4 weeks the antibodies were still in many cases exclusively IgM. Similar IgM responses were also found in certain cases of acute meningoencephalitis. We postulate that during the disease antigenic components identical or very similar to major determinants in the M. pneumoniae lipid antigen are revealed and elicit the IgM antibody response. Their resemblance to natural antibodies and their possible biological role is discussed.     

ELISA法检测江门地区性病门诊患者生殖支原体抗原

目的应用酶联免疫吸附试验（ELISA）检测性病门诊患者泌尿生殖道生殖支原体（Mg）抗原，探讨江门地区性传播疾病（STD）门诊患者Mg感染状况。方法采用Mg多克隆抗体，标记酶结合物，建立检测Mg抗原的双抗体夹心ELISA法，对性病门诊患者泌尿生殖道分泌物进行MIg抗原检测。结果双抗体夹心ELISA法可检测到5μg／ml蛋白浓度，除肺炎支原体外与其他泌尿生殖道常见支原体和细菌无交叉反应；共检测了174例标本，Mg抗原阳性33例，阳性率为18．97％，其中男性和女性阳性率分别为19．88％和7．69％；男性患者中，非淋菌性尿道炎（NGU）、淋病、前列腺炎患者的Mg抗原阳性率分别为13．43％、62．5％和4．84％。结论STD门诊患者存在Mg感染，应用双抗体夹心ELISA法检测Mg抗原具有一定的灵敏度，且具有快速、简便的特点，适合临床筛查Mg抗原。     

Coreactivity of Legionella pneumophila immune sera in the Mycoplasma pneumoniae complement fixation test

Sheep and guinea pigs were immunized with cellular and extracellular antigen from Legionella pneumophila bacteria. After immunization, the animals developed immunoglobulin G titers against the immunizing agent. The same sera were also tested in a Mycoplasma pneumoniae complement fixation test. All the preimmunization sera from sheep showed positive M. pneumoniae complement fixation tests of varying titers, with significant antibody rises in two of five sheep as a result of the Legionella immunizations. In contrast to the sheep, all the guinea pigs were negative in the M. pneumoniae complement fixation test, both in their preimmunization sera and after completion of the Legionella immunizations. The results obtained with the sheep sera may be explained as a nonspecific booster effect of Legionella bacteria upon previously elicited immune responses.     

Mycoplasmal pneumonia associated with mesangiocapillary glomerulonephritis type II (dense deposit disease)

A 20-year-old man developed pneumonia and glomerulonephritis concomitantly with significantly rising Mycoplasma pneumoniae complement-fixing antibody titres. Renal biopsy showed mesangiocapillary glomerulonephritis type II (dense deposit disease). Attempts to demonstrate mycoplasmal antigen in the glomeruli failed. This is the third of five previously reported cases of glomerulonephritis associated with Mycoplasma pneumoniae and exhibiting dense deposit disease.     

Serological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay.

Antibodies against Mycoplasma pneumoniae antigen obtained by Tween-ether treatment from purified M. pneumoniae were measured by means of enzyme-linked immunosorbent assay (ELISA). Paired sera from 19 patients with pneumonia and from 13 patients with acute pancreatitis with a significant rise in complement fixing antibodies against M. pneumoniae were studied. Single sera from healthy 1-year-old children were used as controls. High levels of IgG and IgM class antibodies were seen in sera from patients with pneumonia while most patients with acute pancreatitis and all the children showed low levels of antibodies. The results indicate that ELISA using Tween-ether treated M. pneumoniae antigen could be used successfully in the specific laboratory diagnosis of M. pneumoniae infection.     

Mycoplasma pneumoniae protein involved in the antibody response in human infection

Antigen from purified Mycoplasma pneumoniae organisms treated with Tween-80-ether was used in a solid phase enzyme immunoassay and compared with the conventional lipid containing complement fixation antigen for measuring antibodies in sera from patients with aseptic or bacterial meningitis or with apparent M pneumoniae infection. In immunoblotting of the enzyme immunoassay antigen, enzyme immunoassay positive sera detected a polypeptide at Mr = 180.000-200.000, while enzyme immunoassay negative sera whether positive or negative in the complement fixation test did not. These results indicate that the enzyme immunoassay antigen containing the high molecular weight polypeptide can be used to measure M pneumoniae antibodies more specifically than the conventional lipid containing complement fixation antigen.     

In Vitro Response of Human Lymphocytes to Mycoplasma pneumoniae

In vitro culture and stimulation of human peripheral lymphocytes were employed to investigate the role of cellular immunity in Mycoplasma pneumoniae disease. Subjects with documented natural infections served as donors. The lymphocyte response to whole M. pneumoniae organisms was determined as incorporation of tritiated thymidine in a semimicro culture system. The range of cellular reactivity stimulated by specific antigen was within the range stimulated by phytohemagglutinin. The difference between responses of subjects with documented infection and serologically negative controls was highly significant. Specific reactivity of peripheral lymphocytes correlated closely with the presence of serum growth-inhibiting antibodies, and both persisted for several years following infection. Serum complement-fixing titers correlated well with lymphocyte stimulability during the first year but antibody, as measured by this technique, tended to disappear in later convalescence. In light of previous studies, which revealed a lack of correlation between humoral antibodies and resistance to reinfection, these results suggest that immunity to M. pneumoniae infection is mediated by circulating small lymphocytes.     

Antibody to Mycoplasma pneumoniae in Nasal Secretions and Sputa of Experimentally Infected Human Volunteers

After experimental infection with Mycoplasma pneumoniae, 42% of 67 volunteers developed a threefold or greater rise in antibody in nasal secretions as measured by radioimmunoprecipitation. Development of an antibody increase in sputum was detected more often, i.e., in 73% of the volunteers. Each of the antibody increases involved immunoglobulin (Ig) A. Twelve rises in IgG antibody were detected in the specimens which exhibited a rise in IgA antibody. In almost every instance the rise in IgA antibody exceeded that seen with IgG antibody. Analysis of the response to experimental challenge with M. pneumoniae of volunteers with different levels of preexisting respiratory tract IgA antibody suggested that this secretory antibody was related to host resistance to M. pneumoniae disease. Further, respiratory tract IgA antibody appeared to be more directly related to host resistance than was antibody in serum.