Evaluation of the Specificity of Pneumococcal Polysaccharide Enzyme-Linked Immunosorbent Assay and the Effect of Serum Adsorption Based on Standard Pneumococcal Serogroup- or Serotype-Specific Rabbit Antisera |
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| Authors: | Hans-Christian Slotved Christina Guttmann Charlotte Demuth Pedersen Jasper Neergaard Jacobsen Karen Angeliki Krogfelt |
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| Affiliation: | Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Copenhagen, Denmark |
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| Abstract: | Worldwide, Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality, especially in infants and elderly people. Pneumococcal capsular polysaccharides are well characterized, and more than 90 different serotypes have been identified. Serotype-specific antibodies against the capsular polysaccharide are produced during infection. Detection of antibodies against pneumococci by enzyme-linked immunosorbent assay (ELISA) is performed according to WHO guidelines, using antigens provided by ATCC. However, testing the ELISA for specificity is challenging due to the difficulty in obtaining human naïve serum with pneumococcal antibodies as well as human serum with antibodies against a single serotype. The application of well-defined serotype-specific sera produced in animals to evaluate the specificity of the ATCC antigens and the effect of adsorption with cell wall and 22F polysaccharides has not been performed before, to our knowledge. In this study, the specificity of ATCC antigens (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) was tested by using commercial serotype-, serogroup-, and pool-specific pneumococcal rabbit antisera.Worldwide, Streptococcus pneumoniae (pneumococcus) is a significant cause of morbidity and mortality, especially in infants and elderly people. Pneumococcal infections range from mild upper respiratory tract diseases and otitis media to pneumonia, bacteremia, and meningitis (4). Pneumococcal strains possess a polysaccharide capsule, and more than 90 different varieties (serotypes) have been identified (11, 18). Serotype-specific antibodies against the capsular polysaccharides provide protection against the corresponding serotypes. Four vaccines have been developed, two of which are currently used, including a 23-valent polysaccharide (Pneumovax) for the elderly and for children over the age of 2 years and a 7-valent protein-conjugated vaccine (Prevenar) for children under 2 years of age (9). In 2009, a PCV-10 vaccine (PhiD-CV; GlaxoSmithKline) and a PCV-13 vaccine (Wyeth) are expected to be licensed and used (9).Detection of antibodies against pneumococci is performed by an enzyme-linked immunosorbent assay (ELISA) according to WHO guidelines. Antigens are provided by ATCC, with the exact compositions being unknown (16; www.vaccine.uab.edu).The ATCC antigens used in the WHO ELISA are believed to be serotype specific; however, very few studies have been performed to investigate the actual specificity of the antigens (15, 17). These studies indicate that cross-reactions between serotypes occur, resulting in diagnostic challenges. Furthermore, the antibody responses in human sera seem to differ between immunized and naturally infected subjects (15, 17). Adsorbing the human sera by using pneumococcal cell wall polysaccharides (C-Ps) and 22F polysaccharide was shown to improve the specificity of the WHO ELISA (3). However, due to the widespread colonization/infection of humans by different pneumococcal serotypes, it is uncertain if individuals have been exposed to the bacterium and to what extent. Thus, it is almost impossible to obtain a human serum naïve of pneumococcal antibodies as well as human serum with antibodies against a single serotype. The use of alternative, well-defined serotype-specific sera produced in animals to evaluate the specificity of the ATCC antigens and the effect of adsorption with C-Ps and 22F polysaccharide has not been tested previously, to our knowledge. Thus, the specificity of the ATCC antigens was evaluated in this study by using commercial pneumococcal rabbit antisera specific to capsular polysaccharides (13). Furthermore, the effect of serum adsorption with C-Ps and/or 22F polysaccharide on the pneumococcal titer was determined. |
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