Table 1.

Detection and quantification of BoNT in 13 PT samples. Paralysis times were determined based on various dilutions of the samples employing the MPN assay. In the first run, samples were diluted 1:1000 and tested for paralysing activity. Depending on the value of the paralysis time, higher or lower dilutions were tested for activity to obtain paralysis times within the range of the calibration curves (Figure 2). Upon serotype identification (Table 2) the paralysis times within the grey fields were chosen for the calculation of the concentration of BoNT except for sample 8. The individual concentration of BoNT/A and B in sample 8 was calculated employing paralysis times obtained upon complete neutralization of the other serotype and listed in Table 2.

Sample	Serotype	Matrix	Dilution Factor of Sample									BoNT Concentration						z (BoNT)	Q (BoNT)	(%)
			10,000	2000	1000	500	200	100	50	40	13	X1 (ng/mL)	X2 (ng/mL)	N	X (ng/mL)	Xa (ng/mL)	D (ng/mL)
			Measured Paralysis Time t1/2 (min)
S1	BoNT/A	Meat extract			142		69/78					44.0	27.0	2	35.5	10.5	25.0	9.3	2.39	239
S2	BoNT/A	0.1% BSA/PBS			120		88/83					17.4	21.8	2	19.6	9.9	9.7	3.9	0.98	98
S3	-	0.1% BSA/PBS			>160		>180			>180	>180			4	-	-	-	-	-	-
S4	BoNT/E	0.1% BSA/PBS			154		116	118	92/82			12.5	18.3	2	15.4	10.9	4.5	1.6	0.42	42
S5	BoNT/A	Meat extract			65/70							277	209	2	243	108	135	4.9	1.25	125
S6	BoNT/B	0.1% BSA/PBS			>180					87/88		10.0	9.0	2	9.5	9.0	0.5	0.2	0.06	5.7
S7	BoNT/A	0.1% BSA/PBS			65/60							277	376	2	327	100	227	8.9	2.27	227
S8A	BoNT/A	0.1% BSA/PBS				138		83/79				22.0 # §	26.3 # §	2	24.2	4.7	19	16	4.11	411
												11.4 * §	8.7 * §	2	10.0	4.7	5.3
S8B	BoNT/B	0.1% BSA/PBS										5.60 §	4.30 §	2	4.95	4.5	0.4	0.4	0.1	10
S9	BoNT/A	0.1% BSA/PBS			>180					121/120		1.08	1.04	2	1.06	0.5	0.6	4.5	1.14	114
S10	BoNT/A	Milk			139		69/85					44.0	19.8	2	31.9	10.3	22	8.2	2.09	209
S11	BoNT/A	Serum			121		74/81					22.6	24.0	2	23.3	9.8	13	5.4	1.37	137
S12	BoNT/A	0.1% BSA/PBS	66/64	36	35							2.617 #	2.942 #	2	2.75	1001	−998	−3.9	−1	−100
												2617 *	2942 *	2	2780	1001	1779
S13	BoNT/A	Milk			59/54							401	562	2	482	112	370	13	3.31	331

X_1,2: reported concentration based on single measurement; X: mean of reported participant’s results; X_a: nominal concentrations as determined by the organizing laboratory using sandwich ELISAs [54]; D: difference between X and X_a; z: z-score $z=\frac{X-X_a}{\sigma }$; Q: Q-score $Q\left(\text{\%}\right)=100\left(\%\right)·\frac{X- X_a}{Xa}$; * correctly calculated X₁ and X₂ based on paralysis times → ^# reporting error; § paralysis times denoted in Table 2 were used for calculation of X_1,2.