Characterization of Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-Antigens in Human Sera

Abstract

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies.

Keywords: Enzyme-Linked Immunosorbent Assay (ELISA); Generalized Modules for Membrane Antigens (GMMA); antibodies; human sera; iNTS; non-typhoidal Salmonella; vaccine.

Figure 1

(A) Layout of a 96-well plate used in ELISA assay allowing analysis of 70 different sera (A1-F10); high and low control (HC and LC, respectively) highlighted by red rectangle (positions F11-F12); standard curve in 10 serial dilution points (ST1 to ST10) in duplicate highlighted by blue rectangle and four blank wells (B) (G1, H1, G12, H12); (B) a representative ELISA standard curve obtained by fitting a 5 parameter logistic (5 PL) curve to the individual values obtained at different experimental dilutions tested for the curve. One ELISA unit is defined as the reciprocal of the dilution of the standard serum that gives an absorbance value equal to 1 (red circle); (C) high control and low control against both serotypes have been set as the average value of 12 replicates.

Figure 2

Upper and Lower Limit Standard Curve Accuracy (ULSCA/LLSCA). Average RE% (black line) and respective upper (red line) and lower (green line) 90% confidence interval for each standard curve point have been plotted in function of nominal concentration. Black horizontal dashed lines represent the acceptance range [+25%; −25%] of RE%. Vertical blue dashed lines represent the values of LLSCA and ULSCA.

Figure 3

Anti-STm EU/mL (A) and anti-SEn EU/mL (B) in the function of the serum dilution tested to assay linearity. The two repeats of each single serum performed by each operator are represented by full symbols (for operator 1) and empty symbols (for operator 2), repeats on different days are shown in red circles for day 1, blue squares for day 2, and green triangles for day 3.

Figure 4

ELISA units/mL obtained in precision test performed probing 40 human single sera: (A) 20 against STm OAg and (B) 20 against SEn OAg in 2 independent replicates, performed on 3 different days and by two operators. The two repeats of each single serum performed by each operator are represented by full symbols (for operator 1) and empty symbols (for operator 2), repeats on different days are shown in red circles for day 1, blue squares for day 2, and green triangles for day 3. Geometric means from all repeats of each serum tested against both STm and SEn OAgs are represented by the black lines.

Figure 5

Heterologous specificity results: (A) for S. Typhymurium and (B) for SEn, respectively. ELISA units reduction compared to undepleted control sera expressed as a percentage for each individual sera after incubation with homologous and heterologous competitors at selected concentration of 20 µg/mL. STm: S. Typhimurium; SEn: S. Enteritidis; Sflex: S. flexneri.