Evaluation of a Multiplex Electrochemiluminescence Assay for Detection of Anti-Pneumococcal Antibodies in the Diagnosis of Selective Polysaccharide Antibody Deficiency

Abstract

Streptococcus pneumoniae can be responsible for severe infections, especially in patients with primary antibody deficiencies like selective anti-polysaccharide antibodies deficiency (SPAD). The reference method recommaned by the World Health Organization for assessment of anti-pneumococcal capsular polysaccharides (PCPs) IgG antibodies is a standardized serotype-specific ELISA (WHO-SSA), but this manual method is time-consuming and limit the number of evaluated PCPs. We aim to evaluate the performance values of a multiplex assay based on electrochemiluminescence (ECL-plex). A panel of 164 sera from 82 patients sampled before and 4-8 weeks after immunization by the 23-valent pneumococcal polysaccharide vaccine (PPV23) were assessed by the reference WHO-SSA (7 to 13 serotypes) and by an 18-plex ECL assay (18 serotypes). All patients had normal serum Ig/subclasses levels and were classified as good (n = 43) or poor responders (n = 39, i.e. SPAD patients) according to the American Academy of Asthma, Allergy and Immunology's (AAAAI) current guidelines. We observed excellent correlations between the two methods for anti-PCPs titers against 7 serotypes (r = 0.88 [95% CI: 0.87-0.90], n = 124 sera) and 13 serotypes (r = 0.87 [0.87-0.89], n = 40 sera). Using the AAAAI's guidelines for interpretation, the test performance of the 18-plex ECL assay for SPAD diagnosis showed a sensitivity of 95% and specificity of 84%, positive and negative predictive values of 84% and 95%, respectively. The percentage of agreement was 89% between the SSA and the 18-plex ECL assay. The 18-plex ECL assay is a reliable, rapid, and simple method for evaluating anti-PCPs response and screening for SPAD diagnosis.

Keywords: ECL; Electrochemiluminescence; Encapsulated bacterial infections; Primary immunodeficiency; Selective anti-polysaccharide antibody deficiency; Specific antibody deficiency.

Fig. 1

Graphical abstract illustrating the distribution of polysaccharides used on different Meso Scale Discovery^® plates, the schematic workflow of the ECL assay, the generation of 4-PL calibration curves based on the 007SP calibrant to determine anti-PCPs concentrations for each serotype, the interpretation of these concentrations for patient global anti-PCPs response and diagnosis of SPAD

Fig. 2

Spearman correlation and Demings’s regression curve between WHO-SSA and 18-plex ECL assay for pooled 7 serotypes n = 1,089 (a), and Bland Altman analysis illustrating excellent equivalence between WHO-SSA and ECL-assay. Plotted red function represents ideal test x = y

Fig. 3

Spearman correlation for 18-plex ECL assay and mean results obtained from 4 samples provided in an external quality assessment (WHO-SSA and Luminex-Based Multiplex Immunoassays) (n = 4 for 18 serotypes). Empty dots represented underestimated anti-serotype 5 antibodies. Plotted red function represents ideal test x = y. EQA: External Quality Assessment

Fig. 4

ROC curves for 1.3 µg/mL threshold reliability of the ECL-assay for 7 serotypes (serotypes 4, 6B, 9 V, 14, 18 C, 19 F and 23 F) and 13 serotypes (7 serotypes plus serotypes 1, 3, 5, 6 A, 7 F and 19 A)

Fig. 5

Assessment of specific responses to PCPs in the 18-plex ECLA assay in SPAD patients and controls grouped according to serotype. For each serotype, a good response was considered according to the AAAAI’s composite criteria taking account of pre- and post-PPV23 antibody levels. A “good response” was defined if (i) the post-immunization anti-body titer was upper 1.3 µg/mL and (ii) it achieved a 4-fold increase relative to the pre-immunization value or achieved a 2-fold increase if the pre- immunization value was already greater than 1.3 µg/mL. If one of the 2 criteria was absent, it was considered as a “poor response”