ABSTRACT
Recombinant antibodies such as Fab and scFv are monovalent and small in size, although their functional affinity can be improved through tag-specific immobilization. In order to find the optimum candidate for oriented immobilization, we generated Fab and scFv fragments derived from an anti-pneumolysin monoclonal antibody PLY-7, with histidine and cysteine residues added in diverse arrangements. Tagged antibody fragments scFv-Cys7-His6, His6-scFv-Cys7, and Fab-Cys7 lost considerable affinity for the antigen; however, Fab-His6, Fab-Cys1, and scFv-His6-Cys1 were able to detect immobilized antigen, revealing that the position and number of histidine and cysteine residues are involved differently in the reactivity of antibody fragments. Random and orientated immobilizations were carried out using conventional polystyrene and commercial surface-pretreated ELISA plates. The best orientation performance was obtained with Fab-Cys1-biotin on streptavidin-coated plates with increased signal levels of 62%, while oriented immobilization of Fab-His6 and scFv-His6-Cys1 on nickel- and maleimide-coated plates failed to improve the ELISA sensitivity.
Subject(s)
Immunoglobulin Fragments/chemistry , Streptolysins/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Streptolysins/chemistryABSTRACT
A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Pneumococcal Infections/diagnosis , Streptolysins/urine , Adolescent , Adult , Aged , Aged, 80 and over , Bacterial Proteins/urine , Female , Humans , Male , Middle Aged , Pneumococcal Infections/urine , Sensitivity and SpecificityABSTRACT
BACKGROUND: Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia. METHODS: To examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice. RESULTS: In bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues. CONCLUSION: The data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival.
Subject(s)
Immunologic Factors/immunology , Lung/immunology , Lung/pathology , Pneumonia, Pneumococcal/immunology , Pneumonia, Pneumococcal/pathology , Streptolysins/immunology , Animals , Bacterial Proteins/immunology , Disease Models, Animal , Mice , Survival RateABSTRACT
Streptococcus pneumoniae susceptibility to 12 antimicrobial agents was assessed using isolates collected from patients with invasive and non-invasive infections in a Spanish medical center. Two hundred and thirty-six invasive and 478 non-invasive pneumococcal isolates obtained between 1998 and 2004 were tested. Penicillin non-susceptible isolates were more likely to exhibit resistance to cephalosporins, macrolides, chloramphenicol, and tetracycline when compared to penicillin-susceptible isolates. Penicillin resistance was present in 8.1% of the invasive and 18.6% of the non-invasive isolates. Overall, antimicrobial resistance was greater in non-invasive versus invasive isolates in adults. Serogroups included in the 7-valent and 23-valent formulation accounted for approximately 92.8 and 88.3% of the invasive isolates in children 2 years old or younger and the elderly, respectively. The proportion of isolates not susceptible to penicillin, erythromycin, and/or tetracycline decreased significantly over the surveillance period. Local epidemiological data assisted in the clinical determination of treatment protocols and effective prevention strategies.
