1
Characterization and Validation Supplement
2
Characterization of ELLA
3
The precision profile experiment is designed to identify the extremes at which the assay
4
demonstrates acceptable performance (LLOQ –lower limit of quantitation and ULOQ – upper
5
limit of quantitation). The lowest standard (0.188 µg/mL) demonstrated intra- and inter-assay
6
precision of 5.1% and 28.5% respectively, which did not meet the criteria for the assay
7
quantitation range (<15% intra-assay and <25% inter-assay %CV). The standards evaluated
8
between 0.375 and 6 µg/mL demonstrated intra-assay precision ranging from 1.7% to 6.0% and
9
inter-assay precision of 2.9% to 9.5% (Table 1S). Therefore, this precision profile supports a
10
quantitation range between 0.375 µg/mL (LLOQ) and 6 µg/mL (ULOQ), with 0 µg/mL and 24
11
µg/mL (saturation absorbance) serving as bottom and top anchor points of the curve for
12
validation and downstream application of this method. In addition, the precision profile was
13
used to determine an acceptable positive control range, buffer blank maximum optical density,
14
and highest standard minimum optical density. This data is presented in Table 2S.
15
The extraction efficiency experiment is designed to evaluate the efficiency of the
16
procedure used to extract the SBA from the soybean seed sample. In all of the extraction
17
experiments, the only quantifiable results (above the established lower limit of quantitation of
18
0.375 µg/mL) were generated from the first extracts of each individual sample, with a total of
19
four sequential extractions being performed. The extraction efficiency results showed that 92%
20
to 100% of the SBA was extracted in the initial extraction. Based upon these data a single
21
extraction, as described for this method, is appropriate to evaluate SBA levels in soybean seed
22
and the validation target was established as ≥89%, which is 5% less than the average %
23
extraction.
24
The spike and recovery experiments were conducted to determine the expected range of
25
recoveries possible with the ELLA method to describe the accuracy of the method across three
26
spike levels. Recovery at the three spike levels (0.5, 1.5 and 3 µg/mL) ranged from 69 to 95% of
27
the spiked concentration.
28
measurement was between 5.3 to 9.5%. Based upon the data generated during characterization,
29
the validation target was 64% to 100% recovery (± 5% of characterization data).
Across all spike levels the average precision of the recovery
30
Dilutional parallelism experiments were conducted to evaluate the lowest dilution level
31
that could provide results within the quantitation range to be used in further matrix effects
32
experiments. In addition, the accuracy of the ELLA assay was demonstrated by determining the
33
percent relative difference of each dilution to the average sample concentration value. During
34
characterization four dilution levels were tested (1:10, 1:20, 1:40 and 1:80 sample extract to
35
buffer ratio). The highest dilution level tested (1:80) was out of the quantitation range. For the
36
purposes of this assay, as the lowest dilution level tested (1:10) was within the quantitation
37
range, no additional dilution levels were evaluated. The data was summarized by calculating an
38
average concentration across all dilutions, then the individual dilution values were compared to
39
the across dilution average and converted to a percentage. The range of observed values was 69
40
to 125%. Based upon the data generated during method characterization, the validation target
41
was 64% to 130% (± 5% of characterization data).
42
The matrix effects experiments determined to what extent the other components present
43
in soybean seed influence the ability of the ELLA analysis to accurately quantitate SBA in the
44
presence of the matrix. To evaluate the largest potential matrix effect all experiments were
45
conducted at the lowest tested dilution level (1:10 soybean extract to buffer ratio). Across all
46
three plates and four spike levels (0.5, 1, 2 and 4 µg/mL), an overall average of 104% recovery
47
was observed. Individual spike recovery values ranged from 79% to 132% recovery, with % CV
48
at each recovery level ranging from 6.6% to 15%. Based upon these data, the target range for
49
validation (± 5% of characterization data) was established at 74% to 137% recovery.
50
A summary of the data-based validation acceptance criteria obtained through the characterization
51
experiments is presented in the main manuscript text, Table 3.
52
53
Table 1S. Assay Precision Profile determines Intra- and Inter-Assay variability and establishes the Lower and Upper Limit of
Quantitation (LLOQ and ULOQ).
54
55
56
57
58
At a concentration of 24µg/mL, the assay is near saturation and this is considered the anchor point. The precision of the anchor point was
evaluated but did not need to conform to the evaluation criteria. With the exception of the lowest standard (0.188 µg/mL) all other standards used
in the precision profile conformed to an intra-assay %CV lower than 15% and an inter-assay %CV lower than 25%. Based upon these data, the
0.188 µg/mL standard did not demonstrate measurement precision reflective of what would be expected of a quantifiable standard. Based upon
these results, the LLOQ was defined to be 0.375 µg/mL.
Std Conc.
(μg/mL)
24
6
3
1.5
0.75
0.375
0.188
Average:
Intra-Assay
%CV
27.8
5.6
3.4
1.7
2.7
6.0
5.1
4.1
Intra Criteria %CV
Based on
Concentration
n/a
15%
15%
15%
15%
15%
15%
requirement <15%
Inter-Assay
%CV
30.3
6.1
4.8
2.9
5.7
9.5
28.5
9.6
Inter Criteria
%CV Based on
Concentration
n/a
25%
25%
25%
25%
25%
25%
requirement <25%
59
60
61
62
Table 2S. The precision profile was used to determine the acceptable value range for the positive control
concentration, and the maximum buffer blank optical density and minimum highest standard optical
density. The highest standard considered for this assessment was required to be within the limits of
quantitation, for this reason the optical densities of the 6 μg/mL standard were evaluated.
Positive control range
0.922-1.887 μg/mL
Maximum buffer blank
optical density
0.035 AU
Minimum highest standard
(6μg/mL) optical density
0.767 AU
63
AU- Absorbance Units
64
ELLA Validation Data
65
Table 3S. Validation Data for Extraction Efficiency
Extraction 1
Extraction 2
Extraction 3
Extraction 4
%
Extraction
Efficiency
66
Sample #3
(μg/mg)
2.459
0.105
0.075
0.085
92
89
90
Buffer Spike Theoretical
Value µg/ml
3.000
1.500
0.500
1
2
3
Buffer Spike
- Actual
Value µg/ml
2.810
1.431
0.419
Buffer Spike
%Recovery
94
95
84
Sample
(µg/ml)
2.122
1.110
0.384
% Recovery of
Sample vs.
Buffer Spike
(actual value)
76
78
92
Table 5S. Validation Data for Matrix Effects
Spike #
68
Sample #2
(μg/mg)
2.393
0.147
0.061
0.077
Table 4S. Validation Data for Spike and Recovery
Spike#
67
Sample #1
(μg/mg)
3.185
0.122
0.071
0.072
Buffer Spike Theoretical Value
µg/ml
1
2
3
4
5
*Average of three
4.800
3.600
2.400
1.200
0.600
Buffer Spike Actual Value
µg/ml
Buffer Spike
%Recovery
Average
Sample
concentration
(µg/ml)*
5.129
4.006
2.219
1.139
0.660
107
111
92
95
110
3.810
2.804
1.926
1.094
0.628
%Recovery of
Sample
(average) vs.
Buffer Spike
(actual value)
74
70
87
96
95
69
70
71
Table 6S. Validation - Dilutional Parallelism
Sample #1
Sample #2
%Relative to
%Relative to
Average
Average
1:10
77%
81%
Dilution
1:20
95%
103%
Dilution
1:25
110%
104%
Dilution
1:40
117%
112%
Dilution
Sample #3
%Relative to
Average
Sample #4
%Relative to
Average
Sample #5
%Relative to
Average
Average
of all
Samples
77%
85%
82%
80%
99%
99%
100%
99%
105%
104%
106%
106%
119%
113%
111%
114%