Comparison of a single-antigen microimmunofluorescence assay and inclusion fluorescent-antibody assay for detecting chlamydial antibodies and correlation of the results with neutralizing ability

Peterson, Ellena M.; Oda, R; Tse, P; Gastaldi, Cristina; Stone, Scott; Maza, Luis M. de la

doi:10.1128/jcm.27.2.350-352.1989

Cited by 22 publications

(16 citation statements)

References 7 publications

(8 reference statements)

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“…MAbs were purified from ascitic fluid obtained from BALB/c mice as previously described with a protein A column (Bio-Rad Laboratories, Richmond, Calif.) (16). Fractions that had both a high protein content and inclusion immunofluorescent titer (14) to serovar E were pooled and extensively dialyzed. Purified MAb was frozen in aliquots at Ϫ70°C at concentrations from 1 to 0.4 mg/ml in phosphate-buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection

et al. 1997

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It has been previously shown with an in vitro neutralization system that monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of Chlamydia trachomatis, depending on the isotype of the MAb and the host cell used, can either neutralize or enhance the infectivity of this organism. MAbs to variable domain 4 (VD 4) of MOMP have been described that neutralize the infectivity of C. trachomatis when tested in a system in which either the host cell does not have detectable Fc␥RIII receptors or complement is added to block the interaction of the MAb with the receptor. However, if Fc␥RIII receptors are available, immunoglobulin G2b (IgG2b) MAbs to the VD 4 are able to enhance the infectivity of this pathogen. Two MAbs that recognize the sequence TLNPTIA in VD 4 of the MOMP but differ in isotype, E4 (IgG2b) and E21 (IgG1), were used to test whether in vivo the isotype of the MAb modulates the outcome of a vaginal infection in a murine model. A third MAb, CP33 (IgG2b), that recognizes the chlamydial lipopolysaccharide but does not neutralize infectivity of C. trachomatis, was also tested. Elementary bodies (EBs) of C. trachomatis, serovar E (BOUR), were pretreated with the three MAbs and were used to inoculate the vaginas of C3H/HeJ mice which had been pretreated with progesterone. Subsequently mice were monitored over a 5-week period with vaginal cultures. In the groups that were inoculated with EBs pretreated with MAbs directed to VD 4 of MOMP, there was a significant decrease (P < 0.05) in the number of mice infected. Only 30% of the mice were infected in the MAb E4-treated group, and 10% were infected in the MAb E21 group. This was in contrast to the groups inoculated with EBs pretreated with MAb CP33 and control untreated EBs, which resulted in 100 and 79% of the mice infected, respectively. Therefore, in this setting in which EBs were introduced in vivo coated with MAb, there was no enhancement of infection by IgG2b MAbs; rather, the results paralled the in vitro neutralization results, in which cells lacking Fc␥RIII receptors were employed. Mice were also given the MAbs, as well as purified IgG as a control, by intraperitoneal injection before and after intravaginal inoculation with C. trachomatis. Despite relatively high levels of MAbs in serum and detectable levels of MAbs in the vagina at the time of infection, there was only modest protection in animals receiving MAb E21, with 60% of the mice infected in contrast to 90% of the mice receiving MAb E4, MAb CP33, and IgG. However, by the second week of infection compared to controls, there was a significant increase (P < 0.05) in the amount of chlamydiae recovered from the vaginas of mice that had received the two IgG2b MAbs, E4 and CP33. In summary, the presence of IgG2b MAbs directed to surface components of C. trachomatis at certain times during the course of infection may play a role in enhancing the infectivity of this pathogen.

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Section: Methodsmentioning

confidence: 99%

Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection

et al. 1997

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“…The dot blot assay utilizing untreated and heat-treated (56°C, 30 min) elementary bodies was performed as previously described by Zhang et al (30). An inclusion IFA was done as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars

Cheng

Maza

et al. 1993

Infect Immun

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A monoclonal antibody (MAb), C10, that neutralized in vitro the infectivity of serovars C, I, J, and L3 (members of the C and C-related complexes) of Chlamydia trachomatis was identified. Of the 15 major serovars and the mouse pneumonitis strain of C. trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, which were used as nontreated and heat-treated (56°C, 30 min) antigens in a dot blot assay, only serovars C, I, J, and L3 were recognized with both the native and treated antigens. Western blot (immunoblot) results showed that MAb C10 recognized the major outer membrane protein of these four serovars. Overlapping hexameric peptides corresponding to variable domains (VDs) I, II, III, and IV of the major outer membrane protein of C. trachomatis serovar C were synthesized, and peptide screening showed that MAb C10 mapped to the VD I amino acid sequence VAGLQNDPT. Results of an in vitro neutralization assay correlated with those of the indirect immunofluorescence assay, Western blot, and dot blot assay in that only serovars C, I, J, and L3 were neutralized by MAb C10. In vitro competitive neutralization experiments, using a peptide representing VD I of serovar C to compete with C. trachomatis serovar C for MAb C10 binding, revealed that both serological and neutralizing activities of MAb C10 were inhibited by the VD I peptide. In an in vivo toxicity/infectivity assay using serovar L3 pretreated with MAb C10, there was 100o survival of mice infected with a lethal dose at 48 h. In contrast, the control group, consisting of mice injected with the same dose of 13 pretreated with a MAb that does not recognize L3, had no survivors during a 48-h observation period. In summary, since the surface-exposed contiguous epitope recognized by MAb C10 binds neutralizing antibodies that are subspecies specific for the C and C-related complexes, it should be considered for inclusion in the development of a chlamydial vaccine.

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“…Routine diagnosis of chlamydial infections is based on the isolation of the microorganism in tissue culture, antigen detection assays or detec-dia-infected tissue culture cells and fluorochrome-or enzyme-labeled, immunoglobuline class-specific second antibodies [8][9][10]. Heterogeneous antigens do not allow the determination of distinct antibody specificities, and Western blots, using whole cell lysates of EBs, separated by SDS-PAGE [11] are not useful in a routine laboratory and lack sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Occurence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen

Brade¹,

Brunnemann²,

Ernst³

et al. 1994

FEMS Immunology &Amp; Medical Microbiology

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An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia-specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occurring in patient sera. These mAbs recognized Chlamydia-specific epitopes [alpha 2-->8-linked disaccharide of 3-deoxy-D-manno-octulosonic acid (Kdo) or the trisaccharide alpha Kdo-(2-->8)-alpha Kdo-(2-->4)-alpha Kdo] or those shared between chlamydial and Re-type LPS (alpha Kdo, alpha 2-->4-linked Kdo disaccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.

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Comparison of a single-antigen microimmunofluorescence assay and inclusion fluorescent-antibody assay for detecting chlamydial antibodies and correlation of the results with neutralizing ability

Cited by 22 publications

References 7 publications

Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection

Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection

Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars

Occurence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen

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