Capture-ELISA: a new assay for the detection of immunoglobulin M isotype antibodies using Chlamydia trachomatis antigen.
We present here a new method of IgM antibody-capture enzyme-linked immunosorbent assay (IgM-Capture-ELISA) for the diagnosis of recently acquired infections with Chlamydia trachomatis. For this analysis, plates were coated with goat IgG anti-human Fc mu. The capture of serum IgM antibodies was revealed indirectly by the sequential addition of biotinylated chlamydial proteins and peroxidase-conjugated streptavidin. In chlamydial extracts, cysteine-rich proteins are preferential antigenic targets for the humoral response. 3-(N-maleimidopropionyl)-biocytin (MPB), which binds biotinylated moieties to sulfhydryl groups, was used for the labeling procedure. The preservation of the antigenic specificity of labeled proteins was controlled by a blotting of these proteins, which were, respectively, probed either with specific IgM antibodies or with streptavidin. This analysis revealed that, after labeling, recognized epitopes are more particularly present on the major outer membrane protein (MOMP) of Chlamydia trachomatis. The validation of IgM-Capture-ELISA was assessed by using 170 selected sera from patients suspected of being infected by Chlamydia. Results were respectively compared to conventional indirect immunofluorescence assays (MIF-IgM assays) and to Western blotting. Sixteen sera were found to possess IgM antibodies against Chlamydia trachomatis with IgM-Capture-ELISA. Among these 16 sera, 14 and 15 were, respectively, positive with MIF-IgM assays and in Western blotting. Data obtained with IgM-Capture-ELISA reveal the absence of false-positive results in sera containing rheumatoid factor, which has been shown to interfere in the two other methods. IgM-Capture-ELISA value was then confirmed using sera from patients consulting for genital or pulmonary diseases, from patients with confirmed chlamydial infections, and from patients with other pathologies. IgM-Capture-ELISA appears as an alternative simple semi-quantitative assay for the detection of early chlamydial infection.
Poussin
M
M
Laboratoire de Bactériologie-Immunologie Générale, CHU d'Amiens, France. mathilde.poussin@u-picardie.fr
Fuentes
V
V
Corbel
C
C
Prin
L
L
Eb
F
F
Orfila
J
J
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D002690:Chlamydia Infections / Q000097:blood / Q000276:immunology / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000097:blood*; D007075:Immunoglobulin M / Q000097:blood*; D051379:Mice
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M