Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples.: Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination

Abstract

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1M EDTA pH 7.5 at 90 degrees C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.

Community-acquired pneumonia in the childhood: analysis of the diagnostic methods

“…Our proposed Dot-ELISA proved to be a practical alternative procedure for bacterial antigen detection, in view of its execution without the need for reading equipment, and economy of pneumococcal omniserum and other bacterial antiserum, that may be used at 1:200 dilution [6][7][8]. Positivities obtained by Dot-ELISA are higher than that obtained by CIE and LA, when pleural fluid, serum, and/or urine samples are employed in the tests, despite the similar values of sensitivity.…”

“…Paired samples of pleural fluid, serum and urine from 550 children with clinical diagnosis of bacterial pneumonia were assayed by counterimmunoelectrophoresis (CIE), latex agglutination (LA), and dot-enzyme-linked immunosorbent assay (Dot-ELISA) for antigen detection according to previously standardized procedures [4][5][6][7][8]. Anti-Streptococcus pneumoniae omniserum against 90 serotypes (pneumococcal omniserum) from Seruminstitut, Copenhagen, Denmark [9] and anti-Haemophilus influenzae type b produced according to routine procedure [10] were employed in the immunological methods.…”

“…The pellet was dissolved in 0.5 mL of sterile distilled water plus 0.5 mL of 0.1 M sodium EDTA pH 7.5, followed by heat-treatment (waterbath,100°C/10 min). After new centrifugation (2,500 rpm/15 min), the supernatant was stored until use in the immunological tests [4][5][6][7][8].…”

“…Immunological assays have been employed to detect bacterial antigens of S. pneumoniae and/ or H. influenzae type b, the most common bacteria causing pneumonia. The polysaccharide antigens have been detected in serum, urine, and/or pleural fluid samples from infants and children with suspected bacterial pneumonia [4][5][6][7][8].…”

Rapid detection of Streptococcus pneumoniae in community-acquired pneumonia

“…ELISA-based rapid tests allow a diagnosis of pneumococcal pneumonia to be made with a sensitivity of >70% by the detection of capsular antigen in serum [15] or pleural fluid [16]. However, this method is not widely used and is not yet commercially available.…”