A SPECIFIC AND ULTRASENSITIVE CHEMILUMINESCENT SANDWICH ELISA TEST FOR THE DETECTION AND QUANTITATION OF PNEUMOLYSIN

• Published in: Journal of immunoassay & immunochemistry Vol. 22; no. 2; pp. 99 - 112
• Main Authors: Cima-Cabal, María Dolores, Méndez, Francisco J., Vázquez, Fernando, García-Suárez, María del Mar, de los Toyos, Juan R.
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis Group 2001
• Subjects: Bacterial Proteins; Enzyme-Linked Immunosorbent Assay - methods; Humans; Luminescent Measurements; pneumolysin; Recombinant Proteins - analysis; Sensitivity and Specificity; Streptococcus pneumoniae; Streptolysins - analysis; Streptolysins - blood
• ISSN: 1532-1819; 1532-4230
• DOI: 10.1081/IAS-100103223

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.