Research paper
Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes

https://doi.org/10.1016/j.jim.2004.11.006Get rights and content

Abstract

Serum IgG levels specific for Streptococcus pneumoniae are currently quantified using the ELISA. However, this method has significant limitations in that a separate test is required for each serotype-specific antibody. Here, we describe a rapid and simple method for the simultaneous quantitation of IgG to nine pneumococcal serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F). Pneumococcal polysaccharides were covalently attached to fluorescent microspheres and these beads were serologically assessed using 89-SF standard serum. The multiplex assay was linear over a 24-fold serum dilution range and comparison of monoplex and nonaplex assays revealed no evidence of bead interference. The nonaplex assay was shown to be specific with <30% heterologous inhibition occurring and assay sensitivity was high with the LOD ranging between 32.3 and 109.7 pg/ml. The assay was shown to have low inter- and intra-assay variability and conjugated microspheres were shown to remain stable over 12 months. When samples were assayed there was a good correlation of the nonaplex assay with the ELISA. Finally, four bead sets coated with meningococcal polysaccharides (serogroups A, C, Y and W135) were added into the nonaplex assay, with no effect on the pneumococcal or meningococcal IgG levels generated. The pneumococcal nonaplex assay will prove to be a valuable tool to aid in the evaluation of pneumococcal capsular polysaccharide-based vaccines.

Introduction

Streptococcus pneumoniae remains a significant cause of worldwide morbidity and mortality. Globally, pneumococcal infections have been estimated to cause 70,000 deaths per year in young children in developing countries (Obaro and Adegbola, 2002). A 23-valent polysaccharide vaccine incorporating 23 of the most common pneumococcal serotypes is licensed for use in the elderly and children over 2 years of age who are at high risk of the disease. However, these vaccines are poorly immunogenic in children under 2 years of age in whom there is a high incidence of invasive pneumococcal disease and this has resulted in the development of a new generation of pneumococcal conjugate vaccines. Prevnar™ (Wyeth Vaccines) is a 7-valent vaccine containing the capsular polysaccharides of the common disease causing serotypes 4, 6B, 9V, 14, 18C, 19F and 23F coupled to the protein carrier CRM 197. Conjugation of polysaccharides to polypeptides leads to the production of a T-cell dependent immune response and resultant immunogenicity in young children and infants (Wuorimaa et al., 2001). Trials of Prevnar™ in Northern California have demonstrated that the vaccine is effective in preventing invasive pneumococcal disease caused by vaccine serotypes (97.4% efficacy) (Black et al., 2000) and Prevnar™ is now widely used in the USA. In 2001, a European licence was granted for use in healthy infants and in 2002, the UK Chief Medical Officer recommended the vaccine for use in children at high risk of pneumococal disease (Chief Medical Officer et al., 2002).

Production of pneumococcal capsular polysaccharide specific antibody is the principal mechanism of resistance to infection caused by S. pneumoniae (Musher et al., 1993) and the concentration of pneumococcal serotype-specific IgG strongly correlates with functional opsonophagocytic activity (Anttila et al., 1999). The main format for pneumococcal serology is the ELISA as it is a specific and sensitive assay, which is well suited to testing many samples against a single analyte. However, evaluation of IgG levels following pneumococcal conjugate vaccination, entails measurement of at least seven analytes and screening by ELISA has become time consuming, costly and requires large sample volumes. Studies have demonstrated the safety and immunogenicity of 9-valent (Dagan et al., 2002) and 11-valent (Capeding et al., 2003) pneumococcal conjugate vaccines. With the inclusion of additional serotypes to vaccines the ELISA will become increasingly unsuitable for this purpose and new methods which can rapidly and reliably determine serotype-specific IgG levels will be required.

Microsphere-based flow cytometric assays permit the simultaneous measurement of many analytes from just a single sample. Studies have demonstrated the ability to effectively multiplex a range of assays such as cytokine detection (de Jager et al., 2003), bacterial pathogen nucleic acid detection (Dunbar et al., 2003) and antibody detection and quantitation (Pickering et al., 2002a, Pickering et al., 2002b, Moss et al., 2004). Multiplexing allows high sample throughput and use of very small sample volumes, whilst still retaining the advantages of a conventional ELISA.

Microsphere-based technology permits transfer of the simple ELISA format into a bead format in which the fluorescent bead is used as the solid support for the target antigen. We recently reported on the development and validation of a microsphere-based meningococcal tetraplex assay for the quantification of anti-meningococcal serotype-specific IgG (Lal et al., 2004). Here, we extend this work to describe the development and evaluation of a nonaplex assay for the quantification of serum IgG against nine pneumococcal serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and the inclusion of the meningococcal tetraplex.

Section snippets

Conjugation of pneumococcal polysaccharide antigens to carboxylated microspheres

The conjugation methods utilised are modifications of previously published methods (Gray, 1979, Pickering et al., 2002a). Pneumococcal polysaccharides (serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F (American Type Culture Collection, Virginia, USA) were conjugated to poly-l-lysine (PLL) (Sigma-Aldrich, Dorset, UK) and using a two-step carbodiimide reaction (Staros et al., 1986), this conjugate was covalently bound to carboxylated microspheres (Luminex, TX, USA). Pneumococcal polysaccharides

Development of a nonaplex assay for the detection of pneumococcal serotype-specific IgG

Each pneumococcal polysaccharide was conjugated to an individual bead set. To assess the conjugations and to determine the range of fluorescence generated, each bead set (monoplex), two bead sets together (biplex) and all nine bead sets (nonaplex) were assayed with seven 4-fold dilutions of 89-SF reference sera. The MFIs generated from the nonaplex assay were similar for all nine bead sets and demonstrated linearity over the seven 4-fold standard dilution range (Fig. 1). The possibility of

Discussion

We have described the development and validation of a nonaplex assay for the simultaneous quantification of IgG against nine pneumococcal serotypes, in a single serum dilution. The main advantage of such an assay is the ability to screen very small volumes of sera against many analytes, which is particularly relevant for analysing paediatric samples. The bead assay requires just 5 μl of serum compared to at least 40 μl in the ELISA. Furthermore, due to the kinetics of bead-based assays, the

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