Qualification of an enzyme-linked immunosorbent assay for quantification of anti-Vi IgG in human sera

Abstract

Effective vaccines against Salmonella Typhi, targeting the Vi capsular polysaccharide, have been developed and are being introduced into routine immunization in endemic countries. Vi conjugated vaccines are also being tested in new multi-component vaccine formulations. Simple, high-throughput and cost-effective assays to quantify Vi-specific IgG in clinical sera are needed. In this study we present the development and qualification of a new anti-Vi ELISA with continuous readout, which expresses results as ELISA Unit/mL (EU/mL). We have qualified the assay in terms of precision, linearity and specificity, demonstrating performance in line with a commercially available anti-Vi ELISA. We have also calibrated the assay against the 16/138 anti-Vi international standard and established conversion factor between EU/mL and international units/mL, to allow comparability of results across studies. In summary, this new assay met all the suitability criteria and is being used to evaluate anti-Vi responses in clinical studies.

Keywords: Vi; antibodies; enteric fever; enzyme - linked immunosorbent assay (ELISA); human; typhoid; vaccine.

Figure 1

(A) ELISA layout: 1:100 diluted samples are assayed in triplicates on different plates. Up to 70 samples can be tested on the same layout. If necessary, the same layout can be assessed 1:4000 and 1:160000 fold diluted, making 1:40-fold serial dilution from the previous one. One sample layout can result from three to nine 96 well plates. (B) Sigmoidal Logistic 5PL interpolation of sample on the standard curve.

Figure 2

ELISA Assay Parameters (A) ELISA precision and dilution linearity. A total of 24 repeated measurements of EU/mL from independently handled samples, by two operators on three different days. Single repeats of each operator are represented by circles symbols (for operator 1) and star symbols (for operator 2), repeats on different days are shown in orange for day 1, green for day 2 and blue for day 3. Geometric means and geometric standard deviations from 24 repeats are represented by the grey line for each of the tested samples. (B) Homologous and Heterologous specificity.

Figure 3

Anti-Vi polysaccharide IgG WHO international standard (16/138) was tested in 32 independent replicates by two operators (circles and stars for operator 1 and 2, respectively) in three different days (repeats of Days 1 (orange) and 2 (green) are 4, while in Day 3 (blue) they are 8). Geometric means and geometric standard deviations from 24 repeats are represented by the grey line for each of the tested samples.

Figure 4

Commercial ELISA vs customised ELISA. A total of 24 repeated measurements of EU/mL from single independently handled samples, by two operators on three different days. Single repeats of each operator are represented by circle symbols (for operator 1) and star symbols (for operator 2). Repeats on different days are shown in orange for day 1, green for day 2 and blue for day 3. On each assay results are expressed in their respective arbitrary units (EU/mL for customized ELISA and U/mL for commercial ELISA). Geometric means and geometric standard deviations from 24 repeats are represented by the grey line for each of the tested samples.