Inter-laboratory comparison of Cry1Ab toxin quantification in MON 810 maize by enzyme-immunoassay

Figures & data

Table 1. Maize samples analysed in the study.

Sample code	Description	Source
A	Lyophilised and homogenised leaf sample – MON 810	Lab 2
B	Lyophilised and homogenised leaf sample – isogenic line	Lab 2
C	Lyophilised and homogenised leaf sample – isogenic line	Lab 2
D	Lyophilised and homogenised leaf sample – MON 810	Lab 2
F	Dried, frozen and homogenised leaf sample – MON 810	Lab 4
L	Lyophilised and homogenised leaf sample – MON 810	Lab 4

Table 2. Instrumentation and experimental setup in the participating laboratories.

	Lab 1	Lab 2	Lab 3	Lab 4
Sample preparation
Homogenisation	Bag (Bioreba AG, Reinach, Switzerland)			Mortar + pestle
Buffer
Joint protocol	PBST	PBST	PBST	PBST
Own protocol	BBST	–	PBST	Abraxis buffer
Measurement
ELISA kit
Joint protocol	Agdia	Agdia	Agdia	Agdia
Own protocol	Agdia	–	Agdia	Abraxis
ELISA microplate reader	Synergy HT (BioTek Instruments, Winooski, VT, USA)	VersaMax (Molecular Devices, Sunnyvale, CA, USA)	MR5000 (Dynatech Laboratories, Chantilly, VA, USA)	iEMS MF (LabSystems, Helsinki, Finland)
Controlling software	KC4 v3.4 (BioTek Instruments, Winooski, VT, USA)	SoftMax Pro v.5.0 (Molecular Devices, Sunnyvale, CA, USA)	MikroWin 2000 (Mikrotek Laborsysteme, Overath, Germany	Ascent v.4.2 (LabSystems, Helsinki, Finland)
Standard curve fitting
Joint protocol	Sigmoid	Sigmoid	Sigmoid	Sigmoid
Own protocol	Sigmoid	–	Sigmoid	Sigmoid + linear

Table 3. Analysis of normality of data distribution by the Shapiro–Wilk test by sample, laboratory and protocol.

Sample	Lab 1		Lab 2		Lab 3		Lab 4
	JP ^a	Own protocol	JP^a	Own protocol	JP^a	Own protocol	JP^a	Own protocol
A	Normal SW-W = 0.9352 p=0.2401	Normal SW-W = 0.9551 p=0.7456	Non-normal SW-W = 0.8900 p=0.0080	n.a.	Normal SW-W = 0.8971 p=0.0608	Normal SW-W = 0.9219 p=0.2658	Normal SW-W = 0.9210 p=0.2941	Normal SW-W = 0.9337 p=0.2254
	Normal SW-W = 0.9344 p=0.0645		n.a.		Normal SW-W = 0.9275 p=0.0532		Normal SW-W = 0.9289 p=0.0577
	Non-normal ^b SW-W = 0.8716 p=0.0000008
D	Normal SW-W = 0.9341 p=0.1665	Normal SW-W = 0.9243 p=0.4293	Non-normal SW-W = 0.8804 p=0.0049	n.a.	Normal SW-W = 0.9582 p=0.3797	Normal SW-W = 0.9333 p=0.2751	Normal SW-W = 0.9394 p=0.4905	Non-normal SW-W = 0.8353 p=0.0243
	Normal SW-W = 0.9838 p=0.9148		n.a.		Normal SW-W = 0.9408 p=0.0954		Normal SW-W = 0.9317 p=0.0681
	Non-normal ^b SW-W = 0.9163 p=0.00003
F	Non-normal SW-W = 0.7566 p=0.0231	n.a.	n.a.	n.a.	Non-normal SW-W = 0.8787 p=0.0249	Non-normal SW-W = 0.8788 p=0.0248	Normal SW-W = 0.9639 p=0.7063	Non-normal SW-W = 0.9020 p=0.0017
	n.a.		n.a.		Normal SW-W = 0.9464 p=0.1351		Normal SW-W = 0.9494 p=0.1633
	Non-normal ^b SW-W = 0.9020 p=0.0017
L	Normal SW-W = 0.8877 p=0.3065	n.a.	n.a.	n.a.	Normal SW-W = 0.9511 p=0.5076	Normal SW-W = 0,9262 p=0.3416	Normal SW-W = 0.9756 p=0.6311	Normal SW-W = 0.8102 p=0.0724
	n.a.		n.a.		Normal SW-W = 0.9464 p=0.1351		Normal SW-W = 0.9445 p=0.1441
	Non-normal^b SW-W = 0.9048 p=0.0003
^aJoint protocol.
^bInter-laboratory analysis of data distribution was determined for data obtained by the JP in each laboratory.

Figure 1.  Analytical standard curves obtained in Lab 1 (▪, solid line), Lab 2 (•, dashed line), Lab 3 (

, dotted line) and Lab 4 (▾, short dotted line) in the standardised JP. Combined sigmoid standard curves among immunoassays performed on several microplates are created as optical densities (ODs) normalised between maximal (OD_max) and minimal (OD_min) values obtained on the same microplate. Spiked concentrations of the Cry1Ab toxin standard were 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.56, 1, 1.6, 2.7, 4.4, 6.8, 10, 20 and 50 ng/ml.

Figure 2.  Analytical standard curves obtained in Lab 4 in buffer (▪, solid line) and in isogenic leaf extract (•, dashed line) in the standardised JP. Spiked concentrations of the Cry1Ab toxin standard were 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.56, 1, 1.6, 2.7, 4.4, 6.8, 10, 20 and 50 ng/ml.

Table 4. Cry1Ab toxin concentrations in GM maize samples determined in different laboratories using the standardised JP.

Sample	Cry1Ab concentration (µg/g dry weight)^a
	Lab 1	Lab 2	Lab 3	Lab 4	Inter-laboratory average
A	12.17±1.77 (−2.9%) bA	11.66±0.75 (−7.0%) bA	8.34±1.40 (−33.5%) aA	19.67±1.59 (+56.9%) cA	12.54±3.95
D	17.32±0.97 (+13.4%) cD	14.32±0.88 (−6.2%) bD	10.53±1.05 (−31.0%) aD	24.44±0.89 (+60.1%) dD	15.26±4.58
F	19.74±1.13 (−28.9%) aF	n.a.	29.18±3.96 (+5.1%) bF	29.03±1.35 (+4.5%) bF	27.76±4.29
L	49.05±2.15 (−32.5%) aL	n.a.	55.67±6.66 (−23.4%) aL	83.37±7.10 (+14.8%) cL	72.64±17.73
Note: Percentage data in parentheses indicate relative differences from the corresponding IA. Different lowercase letters after each detected concentration range indicate significant differences at 0.01 significance level. Different lower index capital letters refer to the given sample set. The significance levels for a given sample set is unrelated to the other sample sets.
^aDetermined concentration values are listed as mean±SD.

Table 5. Relative SD and SE in Cry1Ab toxin determination in GM maize samples determined in different laboratories using a standardised JP.

		Lab 1	Lab 2	Lab 3	Lab 4
Sample	Type of error^a	%SD (%SE)	%SD (%SE)	%SD (%SE)	%SD (%SE)
A	Triplicate	0.8 − 6.5 (0.4–3.7)	0–4.4 (0–2.5)	1.4–2.9 (0.8–1.7)	0.1–1.4 (0.1–0.8)
	Plate-to-plate	5.0 (3.6)	0.6 (0.4)	2.7 (1.6)	4.6 (3.2)
	Within laboratory	14.5 (3.2)	6.4 (1.2)	16.8 (4.0)	8.1 (2.1)
	Inter-laboratory	31.6 (3.5)
D	Triplicate	1.2–7.0 (0.7–4.1)	0–4.7 (0–2.7)	0.3–4.2 (0.2–2.4)	0.8–1.8 (0.4–1.0)
	Plate-to-plate	3.3 (2.3)	4.6 (2.7)	2.3 (1.3)	4.3 (3.0)
	Within laboratory	5.3 (1.2)	6.1 (1.2)	9.9 (1.9)	3.6 (1.0)
	Inter-laboratory	29.9 (3.2)
F	Triplicate	0.4–0.6 (0.3–0.4)	n.a.	0.6–3.7 (0.3–2.2)	0.9–2.5 (0.5–1.4)
	Plate-to-plate	–	n.a.	6.2 (3.6)	4.0 (2.3)
	Within laboratory	5.7 (2.3)	n.a.	13.6 (3.2)	4.6 (1.1)
	Inter-laboratory	15.5 (2.4)
L	Triplicate	0.6–2.4 (0.3–1.4)	n.a.	0.8–10.5 (0.5–6.1)	0.6–7.7 (0.4–5.5)
	Plate-to-plate	–	n.a.	2.8 (1.6)	6.6 (3.8)
	Within laboratory	4.4 (1.8)	n.a.	12.0 (2.9)	8.5 (1.5)
	Inter-laboratory	22.5 (3.0)
^aSample SD and SE, WLSD and WLSE, and ILSD and ILSE.

Table 6. Relative D and SE in Cry1Ab toxin determination in GM maize samples determined in different laboratories using a standardised JP.

Sample	Cry1Ab concentration (µg/g dry weight)^a
	Lab 1	Lab 2	Lab 3	Lab 4
A	13.07±1.08 (+7.5%) n.d. (p=0.1661)	n.a.	15.93±3.39 (+91.7%) s.d. (p<0.0001)	5.1±1.57 (–74.1%) s.d. (p<0.0001)
D	17.05±1.72 (–1.5%) n.d. (p=0.5828)	n.a.	21.89±5.24 (+107.8%) s.d. (p<0.0001)	5.00±1.52 (–79.5%) s.d. (p<0.0001)
F	n.a.	n.a.	39.64±3.96 (+35.9%) s.d. (p<0.0001)	11.86±3.28 (–59.1%) s.d. (p<0.0001)
L	n.a.	n.a.	80.41±29.17 (+44.4%) s.d. (p=0.0022)	26.28±5.13 (–68.5%) s.d. (p<0.0001)
Note: Percentage data in parentheses indicate relative differences from the corresponding average concentration value detected in the same laboratory by the JP. n.d., not different statistically from the corresponding concentration value detected in the same laboratory by the JP. s.d., statistically different from the corresponding concentration value detected in the same laboratory by the JP. Corresponding probability values are listed in parentheses below each concentration value (differences are significant if p<0.01).
^aDetermined concentration values are listed as mean±SD.

Table 7. Relative SD and SE in Cry1Ab toxin determination in GM maize samples determined in different laboratories using their modified (own) protocols.

		Lab 1	Lab 2	Lab 3	Lab 4
Sample	Type of errors^a	%SD (%SE)	%SD (%SE)	%SD (%SE)	%SD (%SE)
A	Triplicate	2.9–10.2 (1.6–5.9)	n.a.	2.1–4.7 (1.2–2.7)	4.1–15.6 (2.4–9.0)
	Plate-to-plate	–	n.a.	4.2 (3.0)	35.3 (24.9)
	Within laboratory	8.3 (2.8)	n.a.	21.3 (5.5)	30.9 (7.3)
	Inter-laboratory	n.a.
D	Triplicate	2.0–5.1 (1.2–3.0)	n.a.	1.7–7.0 (1.0–4.0)	5.9–10.9 (3.4–6.3)
	Plate-to-plate	–	n.a.	3.0 (2.1)	35.6 (25.2)
	Within laboratory	10.1 (3.4)	n.a.	9.8 (2.4)	30.5 (8.8)
	Inter-laboratory	n.a.
F	Triplicate	n.a.	n.a.	2.8–7.8 (1.6–4.5)	2.7–8.4 (1.6–4.8)
	Plate-to-plate	n.a.	n.a.	11.6 (8.2)	35.2 (24.9)
	Within laboratory	n.a.	n.a.	13.2 (3.8)	27.7 (11.3)
	Inter-laboratory	n.a.
L	Triplicate	n.a.	n.a.	13.5–19.6 (7.8–13.9)	3.3–18.2 (1.9–10.5)
	Plate-to-plate	n.a.	n.a.	46.4 (32.8)	13.5 (9.6)
	Within laboratory	n.a.	n.a.	36.3 (10.9)	19.5 (4.6)
	Inter-laboratory	n.a.
^aSample SD and SE, WLSD and WLSE, and ILSD and ILSE.