Enzyme-linked immunosorbent assay for quantitative determination of capsular polysaccharide production in Streptococcus pneumoniae clinical isolates

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Biotechnology and Applied Biochemistry (Impact Factor: 1.36). 06/2006; 44(Pt 2):101-8. DOI: 10.1042/BA20060007
Source: PubMed


A simple, specific, sensitive and reproducible ELISA has been developed to quantify the level of CPS (capsular polysaccharide) production in supernatants of Streptococcus pneumoniae cell cultures. CPSs from Strep. pneumoniae have been widely used as vaccine antigens. The quantification method is based on two type-23F serotype-specific polyclonal antibodies: IgG, purified from sera of mice immunized with a pneumococcal type-23F CPS conjugate, used in the coating step, and a serotype-specific rabbit serum as the second antibody. Solutions of purified type-23F CPS were used as standards. The relationship between A(492) and type-23F CPS concentration was linear over the range 1-310 ng/ml (r=0.989), with 1 ng/ml as the lower limit of sensitivity. The specificity of ELISA was assessed because purified type-19F CPS and cell-wall polysaccharide samples were not detected after their evaluation by the ELISA described in the present study. Repeatability and intermediate precision of the assay were good, the coefficients of variation being 3 and 10% respectively. This ELISA allowed selection of an appropriate vaccine strain, for a natural polysaccharide vaccine, among several 23F pneumococcal clinical isolates and constituted a valuable analytical tool for Strep. pneumoniae fermentation and CPS purification follow-up.

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Available from: Tania Carmenate, Jun 10, 2015
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    • "The results of strain screening showed that growth performance and the ability to produce capsular polysaccharide varied considerably among the nine S. pneumoniae serotype 1 strains evaluated, which corroborates the results obtained for other pneumococcal serotypes (Macleod and Krauss 1950; Cruz-Leal et al. 2006; Gogola et al. 2012) and confirms that strain selection cannot be neglected in process development for pneumococcal polysaccharide production. S. pneumoniae is a member of the lactic acid bacteria, so named because their main metabolic product is lactic acid. "
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    ABSTRACT: Streptococcus pneumoniae is a human pathogen largely transmitted by aerosols. Vaccines are the main strategy against this pathogen, and the capsular polysaccharide (PS) is its major antigen. S. pneumoniae serotype 1 is associated with large outbreaks and epidemics of invasive diseases. The aims of this work were to screen serotype 1 strains to identify the best PS1 producer, evaluate three peptones for PS1 production, investigate the effects of culture medium components using a design of experiments (DoE), a statistic tool for optimization, and propose a new medium/cultivation strategy. After flask cultivation of nine strains, two that produced high PS1 and biomass values were chosen for further evaluation in the bioreactor, and ST595/01 was chosen as the best PS1 producer strain. Among the peptones tested (Casamino acids, Soytone, and Phytone), the highest PS1 production (298 mg/L) was reached with Phytone. Next, DoE (2(4-1)) was performed to evaluate the effects of yeast extract (YE), Phytone, L-asparagine (Asn), and L-glutamine (Gln), yielding the following results: Phytone presented positive effects (p < 0.05) for maximum production of biomass, PS1, acetate, and lactate; YE showed positive effects for biomass and acid production (p < 0.05); Gln exerted a minor positive effect on PS1 yield factor on glucose (p < 0.1); and Asn presented only an effect on acetate production (p < 0.1). Hence, a new culture medium was formulated based on Phytone, YE, and glucose, and batch and fed-batch cultivations were evaluated. The fed-batch cultivation showed almost 2 times the biomass and 2.5 times the PS1 production as the batch culture, and 8-10 times higher PS1 production than has been previously reported.
    Applied Microbiology and Biotechnology 08/2015; DOI:10.1007/s00253-015-6928-z · 3.34 Impact Factor
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    • "In relation to growth kinetics, all strains showed comparable maximum OD 600 around 3.1, with the exception of ST366/08, whose maximum OD 600 = 2.5 (Fig. 5A). Besides, a diversified production of PS14 free in supernatant among the strains (Fig. 5B) was observed, which corroborate the results obtained for other pneumococcal serotypes by Macleod and Krauss [26] and Cruz-Leal et al. [24]. For the majority of strains, it was observed that the release of PS14 to the culture broth started when the cell growth reached the maximum concentration. "
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    ABSTRACT: Streptococcus pneumoniae is a major cause of mortality in underdeveloped countries, where more than one million people die from pneumococcal disease every year. Vaccines are the most efficient method for preventing the infection and are based on the capsular polysaccharide (PS) protection. The serotype 14 is the most frequent in pediatric infections worldwide. This study aimed to establish a quantification protocol for PS present in culture broth samples of S. pneumoniae serotype 14 (PS14) and use this protocol for selection of the best PS14 producer strain. Phenol-sulfuric, HPSEC, competitive ELISA, and sandwich ELISA methods were tested for PS14 quantification. Sandwich ELISA was the method with the best reproducibility and sensitivity and the least susceptible to interferences. The quantification limit and detection limit of this method were 0.99 and 0.57 ng/mL, respectively. Statistical analysis was performed to calculate the coefficient of variation (CV) intraassay (1-3% intraplate and 2-6% interplate) and interassay (11-15%) and the reproducibility in different days (CV<20%). The sandwich ELISA allows us to select, among six strains evaluated, the strain 5287 as the best PS14 producer (11.68 mg PS14/biomass) and it was shown to be the best choice for measurement of pneumococcal polysaccharides in culture broth samples.
    Analytical Biochemistry 12/2011; 421(1):250-5. DOI:10.1016/j.ab.2011.11.029 · 2.22 Impact Factor
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    ABSTRACT: The capacity of Streptococcus pneumoniae to produce capsular polysaccharide (CPS) is essential for virulence. The CPS biosynthesis proteins CpsB, CpsC, and CpsD function to regulate CPS production via tyrosine phosphorylation of CpsD. This mechanism of regulating CPS production is important for enabling S. pneumoniae to cause invasive disease. Here, we identify mutations affecting the attachment of CPS to the cell wall. These mutations were located in cpsC, such that CpsC functioned independently from CpsD tyrosine phosphorylation. These mutants produced WT levels of CPS, but were unable to cause bacteremia in mice after intranasal challenge. This finding suggests that cell-wall attachment of CPS is essential for invasive pneumococcal disease; production of WT levels of CPS alone is not sufficient. We also show that cpsB mutants, which lack the phosphotyrosine-protein phosphatase, produced less CPS than the WT strain, but attached substantially more CPS to their cell wall. Thus, the phosphorylated form of CpsD promotes attachment of CPS to the cell wall.
    Proceedings of the National Academy of Sciences 06/2006; 103(22):8505-10. DOI:10.1073/pnas.0602148103 · 9.67 Impact Factor
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