Immunodetection of pneumolysin in human urine by ELISA.
An ELISA test has been employed for the detection of pneumolysin (PLY) in urine from 14 pneumococcal pneumonia patients and from 11 healthy adult volunteers. The urines of all the 11 healthy adult volunteers developed signals around the mean of the blanks, whereas all the pneumococcal pneumonia patient urines rendered signals at least five times this mean. Chemiluminescent Western blot analyses of these urines, carried out with the PLY-specific rabbit polyclonal IgG preparation used in ELISA, were negative. The 30-kDa filtrates of three high-signal urines were ELISA negative, suggesting that this ELISA test mainly detected high molecular weight forms in urine rather than free PLY-derived antigenic fragments. The urine sample, which rendered the highest ELISA signal, was then concentrated by filtration through a 10-kDa filter. When this concentrate was subjected to Western blot with the ELISA-capture monoclonal antibody, a major band was developed. Its relative molecular mass was similar to that of recombinant PLY and its peptide mass fingerprinting showed peptides corresponding to amino acid stretches from the four domains of the PLY molecule. When the pool of PLY-negative urines was sham-contaminated with purified recombinant pneumolysin, a conspicuous matrix effect was observed; nevertheless, this ELISA test was still reproducible and highly sensitive, detecting pneumolysin in the order of picograms per milliliter. A comparison was also made between this PLY-ELISA and the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in analysing bacterial isolates. On the basis of the minimum number of pneumococci examined, both tests were shown to have similar potency, but strain-dependent discrepancies were observed. This ELISA could provide an alternative to the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in the diagnosis of pneumococcal pneumonia.
Area de Microbiología, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain.
María Dolores
MD
Cima-Cabal
Francisco J
FJ
Méndez
Fernando
F
Vázquez
Carlos
C
Aranaz
Javier
J
Rodríguez-Alvarez
José María
JM
García-García
Ana
A
Fleites
Jaime
J
Martínez González-Río
Luis
L
Molinos
Dolores
D
de Miguel
Juan R
JR
de los Toyos
D000328:Adult; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D011018:Pneumonia, Pneumococcal / Q000382:microbiology / Q000652:urine*; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000276:immunology*; D013301:Streptolysins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000652:urine*
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae