ABSTRACT
A chemiluminescent sandwich ELISA test has been developed for the detection and quantitation of pneumolysin. The test is based on a mouse monoclonal as the capture antibody and on rabbit polyclonal IgGs as detection antibodies, in combination with an anti-rabbit IgG alkaline phosphatase conjugate. The estimated detection limit of the purified recombinant toxin in phosphate-buffered saline with 0.05% Triton X-100 is around 5 pg ml(-1), with averaged intra- and inter-assay variation coefficients of 7% and 13.5%, respectively. The assay has been applied to the quantitation of pneumolysin in pneumococcal isolates, providing, for the first time, a direct measurement of the amount of the toxin produced by different strains, a variation has been found in their pneumolysin content. The test is highly specific as no other purified toxins or human pneumonia- or meningitis-associated bacteria yielded false-positive results. This specific and highly sensitive method could help in the diagnosis of human infections.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Streptolysins/analysis , Bacterial Proteins , Humans , Luminescent Measurements , Recombinant Proteins/analysis , Sensitivity and Specificity , Streptolysins/bloodABSTRACT
Thiol-activated cytolysins share a conserved hydrophobic, Trp-rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY-5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.
Subject(s)
Cell Membrane/metabolism , Conserved Sequence/immunology , Cytotoxins/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Bacteria , Bacterial Proteins , Cytotoxins/metabolism , Epitope Mapping , Erythrocyte Membrane/metabolism , Molecular Sequence Data , Sequence Homology, Amino Acid , Sheep , Streptolysins/immunology , Sulfhydryl Compounds/metabolism , Tryptophan/immunologyABSTRACT
A pneumolysin-based agglutination test which allows an easy, rapid, cost-effective, and accurate (100% specific and 95% sensitive) discrimination between pneumococci and other related human and animal pathogenic bacterial strains has been assayed.