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.     

Demonstration of multiple antigenic determinants on Mycoplasma pneumoniae attachment protein by monoclonal antibodies.

Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage, using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum against M. pneumoniae or monoclonal antibodies. Five distinct bands with intact antigenic determinants were detected by the antiserum, of which two bands were each reactable with two monoclonal antibodies. A sequential binding assay suggested that these monoclonal antibodies recognized different antigenic sites of each band. These results demonstrate the existence of multiple antigenic sites on the attachment protein and describe procedures that should prove useful for identifying those antigenic sites critical to the specific attachment of M. pneumoniae.     

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.     

Binding sites of attachment-inhibiting monoclonal antibodies and antibodies from patients on peptide fragments of the Mycoplasma pneumoniae adhesin.

The adherence protein (P1 protein) of Mycoplasma pneumoniae was purified by electroelution and cleaved with cyanogen bromide. The resulting peptides were separated by two-dimensional electrophoresis. Spots reacting in Western immunoblots with two attachment-inhibiting monoclonal antibodies were isolated, and the amino-terminal ends of these peptides were microsequenced. The two monoclonal antibodies had different binding sites. One was associated with the amino-terminal region of the whole P1 protein beginning at amino acid position 237, and the other was associated with amino acid position 702, which was localized approximately in the middle of the P1 amino acid sequence. Serum samples from three M. pneumoniae-infected patients were tested by Western blotting against the cyanogen bromide peptide pattern. All three serum samples reacted with peptide fragments beginning at amino acid position 702, but the serum of only one patient also had antibodies against the oligopeptides beginning at amino acid position 237. These results indicate that the corresponding epitopes of the P1 protein are also immunogenic if they are presented at the surface of the infecting organism.     

Inhibition of lipopolysaccharide mitogenicity with characterized anti-lipid A monoclonal antibodies.

Antibodies to lipid A were raised in mice immunized with non-hydrolysed Bordetella pertussis microorganisms, coated with lipid A isolated from the same bacteria. Anti-lipid A activity of immune sera was measured by radioimmunoassay. Four hybrid cell lines that secrete antibodies directed against the hydrophobic region of B. pertussis lipopolysaccharide were produced by cell fusion between myeloma cells and spleen cells from immunized C3H/He-PAS mice. Differences were observed in the potency of the isolated monoclonal antibodies to inhibit B cell proliferation induced by lipopolysaccharide (LPS) or by lipid A, suggesting a selective recognition of effector sites present on the hydrophobic region of LPS.     

An ELISA to detect monoclonal antibodies specific for lipid determinants of Mycoplasma pneumoniae

An enzyme-linked immunosorbent assay (ELISA) was developed for analysis of monoclonal antibodies directed towards lipid determinants of Mycoplasma pneumoniae. Chloroform-methanol lipid extracts as well as chromatographed lipid fractions of M. pneumoniae were bound to polyvinylchloride microtiter wells. Of 293 clones positive to M. pneumoniae as detected by a whole cell ELISA, a total of 78 clones produced antibodies which selectively bound to lipid extracts of M. pneumoniae. The simplicity and reproducibility of the assay permit rapid screening for detection of antibodies directed against non-protein cellular antigens.     

Smooth muscle antibodies in Mycoplasma pneumoniae infection.

Paired sera from forty-five cases of Mycoplasma pneumoniae (MP) infection associated with acute lower respiratory tract illness were examined by immunofluorescence for antibodies to smooth muscle. Twenty-five (56%) of these cases had smooth muscle antibodies (SMA) of IgM class. A significant (greater than or equal to 4-fold) increase in titre of these antibodies was demonstrated in fifteen of thirty-five patients with a significant rise in titre of MP antibodies. SMA of IgG class occurred in eleven of forty-five cases (24%), but a 4-fold rise in antibody titre was found only in two cases. Three of forty-five sera (7%) from healthy donors contained SMA of IgM class and eight sera (18%) SMA of IgG class. MP antigen did not absorb SMA. Liver tests were performed in twenty-nine patients. In eighteen patients SGPT values were moderately or slightly elevated. There was no correlation between the occurrence of increased levels of transaminases and the presence of SMA in serum. In a patient with active chronic hepatitis, who had had a high titre of SMA exclusively of IgG class for 2 years, SMA of IgM class appeared transiently in association with an acute respiratory illness due to MP.     

Effects of lipopolysaccharide on macrophages analyzed with anti-lipid A monoclonal antibodies and polymyxin B

Six monoclonal antibodies (mAb) to the lipid A region of bacterial lipopolysaccharide (LPS), obtained from mice immunized with lipid A-coated Bordetella pertussis cells (mAb 3.E8, 2.21, 2.37, 2.41) or with lipid A covalently coupled to keyhole limpet hemocyanin (mAb R1 and R7), were examined for their potential to inhibit in vitro activities of LPS on macrophages. mAb R7 was inactive in vitro, but the five other mAb inhibited efficiently some in vitro activities of LPS. mAb R1, 2.21 and 3.E8 reduced the LPS-induced secretion of tumor necrosis factor (TNF) and interleukin 1 (IL 1) by macrophages, but did not modify the binding of LPS to macrophages. On the other hand, mAb 2.37 and 2.41 reduced LPS binding to macrophages and subsequent IL1 secretion, but did not modify TNF production. This is in agreement with our previous finding that IL1 and TNF productions can be selectively triggered by synthetic analogs of lipid A substructures (Lasfargues and Chaby, Cell. Immunol. 1988. 115: 165). The pattern of in vitro inhibition of LPS activities (LPS binding to macrophages and production of TNF and membrane IL 1) by polymyxin B was different from those of the two groups of anti-lipid A mAb mentioned above. These observations suggest the presence on lipid A of four functionally distinct substructures.     

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.     

Binding activity of a murine anti-lipid A monoclonal antibody.

In this report we briefly describe an immunoglobulin G3 monoclonal antibody, 2G6/1H11, which binds purified lipid A from Salmonella minnesota and a lipid A precursor molecule derived from Salmonella typhimurium. 2G6/1H11 does not bind well to purified whole S. typhimurium lipopolysaccharide (LPS), S. minnesota LPS, LPS preparations from a series of S. minnesota rough mutants, or intact S. typhimurium bacteria. Thus, there are antigenic determinants in purified lipid A which are not exposed when lipid A is presented as part of the whole LPS molecule or intact bacteria.     

IgE antibodies to Mycoplasma pneumoniae in asthma and other atopic diseases

IgE antibodies to Mycoplasma pneumoniae antigens were measured by RAST assay in 152 patients with asthma and other IgE and non-IgE mediated diseases. Five patients with asthma and/or atopic dermatitis had highly elevated Mycoplasma RAST binding ratios, p < 0.001. For these five patients the ratios ranged from 1.78 to 4.74. These increased ratios persisted at least two to 16 months in three of four individuals who were evaluated sequentially. RAST inhibition studies using Mycoplasma pneumoniae, Mycoplasma control, Streptococcus MG and viral antigens showed the specificity of binding for M. pneumoniae in these five patients indicating the presence of IgE antibodies to M. pneumoniae.     

Murine monoclonal antibodies to Klebsiella pneumoniae protect against lethal endotoxemia and experimental infection with capsulated K. pneumoniae.

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopoly-saccharide (LPS) from Klebsiella pneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 10A5) were directed against the lipid A fraction. Only one (2A4) recognized the O antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2.     

Demonstration of antibodies to Mycoplasma pneumoniae attachment protein in human sera and respiratory secretions.

Antibodies specific to the attachment protein of Mycoplasma pneumoniae were demonstrated in sera and respiratory secretions of human patients. The results indicate that the attachment protein is a major immunogen.     

Monoclonal antibodies to salmonella lipopolysaccharide: functional analysis of anti-lipid A antibodies.

We have produced monoclonal antibodies (MoAb) to the Rb core and lipid A regions of Salmonella lipopolysaccharide (LPS) and have assessed their ability to inhibit LPS-mediated mitogenic responses in vitro, and to protect against LPS toxicity and lethal Salmonella infection in vivo. Monoclonal antibodies RC-8 and RC-16 were specific for LPS Rb core determinants, and MoAb LA-1, LA-2, LA-3, LA-4 and LA-5 were specific for lipid A. Anti-lipid A MoAb LA-2, LA-3 and LA-5 were found to abrogate mitogenic responses of C3H/HeN spleen cells to smooth S. typhimurium LPS (S LPS) and to rough S. minnesota R595 LPS (Re LPS). Monoclonal antibody LA-5 was effective in extending the median length of survival of C3H/HeN mice challenged with a lethal dose of either S LPS or Re LPS. Antibody LA-2 could extend the median length of survival of C3H/HeJ mice challenged with Re LPS but not with S LPS, and failed to extend significantly the length of survival of S LPS-challenged C3H/HeN and DBA/2 mice. Neither 20 micrograms of anti-Rb core or anti-lipid A MoAb nor 200 micrograms of anti-lipid A MoAb were able to protect C3H/HeN or BALB/c mice, respectively, against lethal infection with S. typhimurium SR-11. These results suggest that the importance of anti-lipid A antibodies in host defence may lie more in their ability to neutralize pathological effects of LPS, than in their ability to protect against bacterial infection.     

The effect of immunological adjuvants on the relative affinity of anti-protein antibodies.

Inbred mice of a strain (B1OD2 new) known to produce either no detectable antibody or antibody of low affinity to two protein antigens administered in saline, were immunized with human serum transferrin (HST) in one of nine adjuvants. Such immunization increases the level and relative affinity of anti-HST antibody. The adjuvants used varied in the degree to which they augmented these parameters of the antibody response--that is, FCA and FIA were capable of inducing high levels of high affinity antibody, whereas other adjuvants elicited lower levels of high affinity antibody. The possibility is discussed that substances with adjuvant activity may effect antibody production at two stages: (1) at the stage of antigen selection of cells for proliferation and (2) at the stage or proliferation of antibody producing cell precursors.     

Effect of Mycoplasma pneumoniae on poliovirus replication.

Results are presented which indicate that different species of mycoplasma may have varying effects on the replication of different viral types.     

Direct radioimmunoassay for the detection of IgM antibodies against Mycoplasma pneumoniae

A direct solid-phase radioimmunoassay is described for detecting IgM-class antibodies against Mycoplasma pneumoniae. The assay achieves a prelminary separation of IgM from other serum proteins by immunoadsorption to anti-IgM-coated wells in microtitre plates. The IgM is then tested for antigen specificity by measuring its ability to bind radiolabelled M. pneunoiae. Positive results are confirmed by retesting sera after treatment with 2-metcaptoethanol. The assay is specific for IgM-class antibodies and specific for M. pneumoniae. It is not affected by competitive inhibition from IgG and avoids the use of density gradient centrifugation or gel filtration to separate IgM from other immunoglobulins. It is sensitive, reproducible, rapid, simple and requires very little serum.     

High frequency of anti-protein Z antibodies in patients with anticardiolipin antibodies

Protein Z (PZ) is a vitamin K-dependent protein involved in the down-regulation of coagulation by forming a complex with the protein Z-dependent protease inhibitor. The complex inhibits the activated factor X on phospholipid surface. Presence of anti-PZ (aPZ) antibodies was first described in women with pathological pregnancies but the significance of aPZ antibodies in other pathological situations was poorly studied. In this work we analyzed the frequency of aPZ antibodies in a series of 86 consecutive patients with anticardiolipin (aCL) antibodies and studied the association of aPZ with other antiphospholipid (aPL) antibodies [lupus anticoagulant (LAC) and anti-ß2GP-1 antibodies] and the clinical signification of these aPZ antibodies in term of thrombosis or fetal loss. Anti-PZ antibodies (IgG and IgM) were detected using commercially available ELISA assays. The frequency of aPZ antibodies was 40.7% in the patient group versus 6.8% in a group of 59 healthy volunteers (p?<?0.0001). The frequency of aPZ antibodies significantly increases (p?<?0.05) in patients with a double or triple positivity of aPL antibodies and a higher frequency of aPZ antibodies was observed in patients with LAC (57.7%) than in patients without LAC (25.6%, p?=?0.02). There were no significant differences in aPZ antibodies frequency between patients with and without thrombotic events. Interestingly, among the 8 women with recurrent foetal losses, aPZ antibodies were observed in 7 cases, in agreement with previous observations suggesting that aPZ antibodies may be associated with obstetrical complications.     

Protein serotyping of Streptococcus pneumoniae based on reactivity to six monoclonal antibodies

Six monoclonal antibodies to proteins of Streptococcus pneumoniae were tested in a dot blot assay for reactivity with 499 clinical isolates of pneumococci. Forty-four percent of the isolates reacted with at least one of the antibodies. Nineteen patterns of reactivity were identified and each designated as a provisional protein serotype. Protein serotyping identified pneumococcal strains independently of their capsular type and made it possible to differentiate strains within most capsular types. A protein serotyping system provides a new dimension to the phenotypic identification of S. pneumoniae and may eventually provide a basis for assessing the population structure of these organisms.