Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides

Figures & data

Table 1. Geometric mean ratios (GMR) with variable coating concentrations.

Serotype	n^a	9 µg/mL vs. 10 µg/mL GMR (%)	90% CI	11 µg/mL vs. 10 µg/mL GMR (%)	90% CI
Ia	132	103.6	(101.6, 105.7)	102.1	(100.1, 104.1)
Ib	129	94.6	(92.7, 96.6)	97.2	(95.2, 99.2)
II	128	98.1	(96.0, 100.3)	97.4	(95.3, 99.6)
III	122	97.7	(95.5, 99.9)	98.8	(96.6, 101.1)
IV	132	95.6	(93.3, 98.1)	98.3	(95.9, 100.8)
V	129	105.7	(103.4, 108.0)	106.1	(103.8, 108.5)

^an indicates the number of total study observations.

Table 2. Results from assay robustness design of experiments.

Serotype	RSD (%)
	Primary Incubation		Secondary Incubation		Residual	Total
	Time intervals evaluated (16 h to 24 h)	Temperatures evaluated (2°C to 8°C)	Time intervals evaluated (60 min to 120 min)	Temperatures evaluated (18°C to 25°C)
Ia	2.3	0.5	0.3	0.0	7.1	7.5
Ib	7.8	0.0	2.6	1.4	13.6	16.0
II	2.6	0.8	1.6	0.0	12.2	12.6
III	1.7	3.2	2.3	1.2	9.9	10.9
IV	5.7	3.8	1.2	0.0	11.2	13.2
V	4.6	1.7	0.0	0.0	12.2	13.2

Table 3. Specificity assessment of the 6-plex GBS IgG dLIA using an immunized serum reference pool.

Competitor 1 µg/mL	% Competition^a of Immunized Human Serum Reference Pool with Serotype-Specific GBS CPS as Competitor
	GBS Ia	GBS Ib	GBS II	GBS III	GBS IV	GBS V
GBS Ia	99	23	−11	14	−13	−9
GBS Ib	5	93	−9	−7	−9	−5
GBS II	−2	−5	93	2	−3	−5
GBS III	−1	−4	8	94	−7	−4
GBS IV	−3	−4	−7	−5	98	−3
GBS V	−6	15	−4	−8	3	94
Pn6B^b	−4	−2	−2	−4	−5	−4
Pn14^c	−2	−2	13	10	−2	−3

^aPercent (%) Competition was calculated as follows: [Baseline sample IgG result (human serum reference pools incubated in buffer only, no GBS CPS) – Serotype-specific IgG result of the human serum reference pool in the presence of GBS CPS competitors/baseline sample IgG result] x 100.

^bPn6B: Streptococcus pneumoniae serotype 6B polysaccharide was tested as a competitor in the 6-plex GBS IgG dLIA to assess assay specificity to other non-related bacteria.

^cPn14: Streptococcus pneumoniae serotype 14 polysaccharide was tested as a competitor in the 6-plex GBS IgG dLIA to assess assay specificity to other non-related bacteria.

Table 4. Assay range based on standard curve bias, dilutional linearity and precision.

GBS serotype	Well concentration (µg/mL)						Assay Range (µg/mL)
	Standard Curve Bias		Dilutional Linearity		Precision
	Lower limit	Upper limit	Lower limit	Upper limit	Lower limit	Upper limit	Lower limit	Upper limit
Ia	3.00E–06	5.24E–03	1.00E–06	1.10E–02	4.00E–06	9.15E–03	4.00E–06	5.24E–03
Ib	9.00E–06	5.67E–03	3.00E–06	7.82E–03	8.00E–06	1.88E–03	9.00E–06	1.88E–03
II	1.40E–05	8.72E–03	8.00E–06	2.08E–02	1.60E–05	1.79E–02	1.60E–05	8.72E–03
III	3.00E–06	4.23E–03	1.80E–05	3.41E–03	3.00E–06	4.48E–03	1.80E–05	3.41E–03
IV	8.00E–06	4.58E–03	3.00E–06	5.07E–03	8.00E–06	5.60E–03	8.00E–06	4.58E–03
V	1.30E–05	7.62E–03	2.10E–05	2.88E–03	6.00E–06	8.74E–04	2.10E–05	8.74E–04

Figure 1. Dynamic ranges of the reference standard curves for the 6-plex GBS IgG dLIA. The reference standard serum dilution profiles for each of the 6 GBS CPS serotypes are shown: (a) Ia, (b) Ib, (c) II, (d) III, (e) IV, and (f) V. Median fluorescence intensity (MFI) signals for the reference standard curves are on the y axis, and specific IgG concentrations (µg/mL) are on the x-axis. The vertical dotted lines indicate the lower and upper assay limits determined from the validation study. Error bars represent the standard deviation from independent reference standard curves (n = 3).

Table 5. Final assay range and lower limit of quantitation (dilution adjusted).

GBS serotype	Dilution adjusted assay range (µg/ml)
	Lower limit	Upper limit^a	LLOQ (µg/mL)
Ia	0.002	262.140	0.002
Ib	0.005	93.950	0.005
II	0.008	436.100	0.022^b
III	0.009	170.650	0.009
IV	0.004	229.075	0.004
V	0.010	43.700	0.010

^aUpper limit of quantitation (ULOQ) was calculated by multiplying the well concentration upper limit by 50,000.

^bSerotype II LLOQ was restricted based on the acceptable precision results (<25% RSD) from the least diluted samples, which account for the highest sample matrix concentration. All other serotype LLOQs were defined by the dilution-adjusted lower limit of the assay range.

Table 6. Intermediate assay precision.

GBS serotype	n^a	Analyst (%RSD)	Day (%RSD)	Bead lot (%RSD)	Residual (%RSD)	Total (%RSD)
Ia	574	7.0	9.0	3.7	13.5	18.2
Ib	494	4.7	7.3	4.4	12.1	15.6
II	676	5.9	9.2	2.6	14.5	18.4
III	446	5.0	7.9	0.0	12.5	15.6
IV	552	4.9	8.2	0.9	12.8	16.0
V	551	5.1	8.2	1.7	14.6	17.7

^an indicates all sample results collected during assay validation using ≥ 2 laboratory analysts, ≥4 days, and 2 microsphere lots.

Figure 2. Proficiency panel performance in the 6-plex GBS IgG dLIA. A proficiency panel with IgG concentrations ranging from low to high was evaluated post-assay validation, quarterly, for >1 year. Each panel shows the reported IgG concentrations (µg/mL; y-axis) across the quarterly timepoints tested (x-axis) for the following GBS serotypes: (a) Ia, (b) Ib, (c) II, (d) III, (e) IV, and (f) V. The geometric mean concentration with 95% CI is shown within a given time point and each black dot represents a proficiency panel sample. Of note, while 44 samples were used for the proficiency panel for all six serotypes, not every sample yielded a reportable result for every serotype. Therefore, the designated samples that constitute each of the six serotypes’ proficiency panel were slightly variable but lie between 41–44 samples.

Figure 3. Microsphere stability for the 6-plex GBS IgG dLIA. The geometric mean (GM) IgG concentration (y-axis) for two independently prepared microsphere lots (red circles, lot A; blue squares, lot B) across 12 months (x-axis) were tested by two laboratory analysts. Each panel depicts stability of the CPS PLL-coated microspheres for the following GBS serotypes: (a) Ia, (b) Ib, (c) II, (d) III, (e) IV, and (f) V. In each panel, the predicted line (black line) represents the linear regression line (or the line of best fit) for all bead lots (A and B) recorded over the 12-month period with 95% confidence intervals shown as the dashed lines.

Table A1 Comparison of the 6-plex GBS IgG dLIA run as multiplex versus single plex.

GBS Serotype	Multiplex versus Single plex
	N^a	GMR^b	90% CI
Ia	28	115.8%	(108.9, 123.2)
Ib	28	103.0%	(96.8, 109.5)
II	28	96.6%	(92.8, 100.5)
III	28	88.4%	(84.1, 92.9)
IV	28	93.6%	(90.9, 96.3)
V	28	109.4%	(106.7, 112.1)

^aN = total number of samples.

^bGMR = geometric mean ratio, expressed as a percent.

Data availability statement

All raw and processed data files are available upon request to the corresponding author. The data are not publicly available due to privacy restrictions.