Calculating and evaluating ELISA data - Abcam · 25 October 2016 15.625 0.017 32.25 0.036 62.5...
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Calculating and evaluating ELISA data Human PD-L1 ELISA Kit [28-8] (ab214565) October 2016
Transcript of Calculating and evaluating ELISA data - Abcam · 25 October 2016 15.625 0.017 32.25 0.036 62.5...
Calculating and evaluating
ELISA data
Human PD-L1 ELISA Kit [28-8] (ab214565)
October 2016
25 October 2016
Standard ELISA
25 October 2016
One-step ELISA
Choosing the standard and preparing the plate
25 October 2016
25 October 2016
Plasma SerumCell Culture
SupernatantUrine
Cell
pelletTissue
Homogenate
Sample Diluent NS1X Cell Extraction
Buffer PTR
1100 to 17.19
pg/mL1400 to 21.88
pg/mL
Dilute in:
Standard
Curve:
25 October 2016
Plasma SerumCell Culture
SupernatantUrine
Cell
pelletTissue
Homogenate
Sample Diluent NS1X Cell Extraction
Buffer PTR
1100 to 17.19
pg/mL1400 to 21.88
pg/mL
Dilute in:
Standard
Curve:
25 October 2016
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
25 October 2016
21.88 21.88
43.75 43.75
87.5 87.5
175 175
350 350
700 700
1400 1400
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Standard
1,400 to 21.88 pg/mL
25 October 2016
Jurkat+LPS+IFNg
1,000 to 15.625 µg/mL
Standard
1,400 to 21.88 pg/mL
15.625 15.625
31.25 31.25
62.5 62.5
125 125
250 250
500 500
1000 1000
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
21.88 21.88
43.75 43.75
87.5 87.5
175 175
350 350
700 700
1400 1400
25 October 2016
Negative
Sample diluent
NS NS
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Standard
1,400 to 21.88 pg/mL
21.88 21.88
43.75 43.75
87.5 87.5
175 175
350 350
700 700
1400 1400
Jurkat+LPS+IFNg
1,000 to 15.625 µg/mL
15.625 15.625
31.25 31.25
62.5 62.5
125 125
250 250
500 500
1000 1000
25 October 2016
Verifying the validity of the measure
25 October 2016
0.070 0.070
0.088 0.090
0.125 0.129
0.186 0.183
0.329 0.327
0.579 0.579
1.029 0.989
0.051 0.056
0.104 0.120
0.154 0.162
0.250 0.260
0.442 0.442
0.833 0.863
1.553 1.602
2.836 2.881
STD Jurkat
OD
25 October 2016
0.051 0.056
Subtract mean
of the blank to
all samples and
standard points
0.053
Mean0.070 0.070
0.088 0.090
0.125 0.129
0.186 0.183
0.329 0.327
0.579 0.579
1.029 0.989
0.104 0.120
0.154 0.162
0.250 0.260
0.442 0.442
0.833 0.863
1.553 1.602
2.836 2.881
STD Jurkat
OD
25 October 2016
0.051 0.056
Subtract mean
of the blank to
all samples and
standard points
0.053
Mean0.017 0.017
0.035 0.037
0.071 0.076
0.133 0.130
0.275 0.274
0.526 0.525
0.975 0.936
0.050 0.067
0.101 0.109
0.197 0.207
0.389 0.389
0.780 0.809
1.499 1.549
2.783 2.828
STD Jurkat
Corrected OD
25 October 2016
0.017 0.017
0.035 0.037
0.071 0.076
0.133 0.130
0.275 0.274
0.526 0.525
0.975 0.936
0.050 0.067
0.101 0.109
0.197 0.207
0.389 0.389
0.780 0.809
1.499 1.549
2.783 2.828SD: Standard Deviation
CV: Coefficient of
Variation
CVSD
Mean OD20%
STD Jurkat
Corrected OD
25 October 2016
0.008 0.000
0.004 0.001
0.005 0.002
0.000 0.001
0.015 0.001
0.025 0.000
0.022 0.020
0.058 0.017
0.105 0.036
0.202 0.073
0.389 0.131
0.795 0.275
1.524 0.526
2.805 0.956
14% 0%
4% 3%
2% 3%
0% 1%
2% 0%
2% 0%
1% 2%
Mean SD CV
0.017 0.017
0.035 0.037
0.071 0.076
0.133 0.130
0.275 0.274
0.526 0.525
0.975 0.936
0.050 0.067
0.101 0.109
0.197 0.207
0.389 0.389
0.780 0.809
1.499 1.549
2.783 2.828SD: Standard Deviation
CV: Coefficient of
VariationSTD Jurkat
Corrected OD
25 October 2016
Creating the standard curve
Bioassay like ELISA have a sigmoidal curve
25 October 2016
Linear regression4 parameters model (4PL)
Software of your reader,
GraphPad PRISMExcel
R² = 0.9996
0
1
2
3
4
0 200 400 600 800 1000 1200
OD
45
0
Analyte concentration (pg/ml)
Good Fit
R² = 0.9609
0
1
2
3
4
0 200 400 600 800 1000 1200
OD
45
0
Analyte concentration (pg/ml)
Bad Fit
25 October 2016
0.058
0.105
0.202
0.389
0.795
1.524
2.805
21.88
43.75
87.5
175
350
700
1400
y = 0.002x + 0.0432
R² = 0.998
0
0.5
1
1.5
2
2.5
3
0 500 1000 1500
OD
45
0
Human PD-L1 (pg/ml)
Linear regression
Normal scale
Excel
pg/ml
STD
25 October 2016
0.058
0.105
0.202
0.389
0.795
1.524
2.805
21.88
43.75
87.5
175
350
700
1400
y = 0.002x + 0.0432
R² = 0.998
0.01
0.1
1
10
1 10 100 1000 10000
OD
45
0
Human PD-L1 (pg/ml)
Linear regression
Excel
Log/log scale
pg/ml
STD
25 October 2016
y = 0.002x + 0.0432
R² = 0.998
0.01
0.1
1
10
1 10 100 1000 10000
OD
45
0
Human PD-L1 (pg/ml)
Linear regression
1 0 1 0 0 1 0 0 0
0 .0 1
0 .1
1
1 0
H u m a n P D - L 1 (p g / m L )
OD
(4
50
nm
)
4 parameters model (4PL)
ExcelGraphPad PRISM
Log/log scaleLog/log scale
25 October 2016
Calculating the concentration of your samples
25 October 2016
15.625 0.017
32.25 0.036
62.5 0.073
125 0.131
250 0.275
500 0.526
1000 0.956
Linear regression4PL
7
15
30
55
117
230
433
8
18
37
66
137
263
478
Jurkat Value Value
ODµg/mL pg/mL pg/mL
25 October 2016
15.625 0.017
32.25 0.036
62.5 0.073
125 0.131
250 0.275
500 0.526
1000 0.956
Linear regression4PL
21.88
43.75
87.5
175
350
700
1400
7
15
30
55
117
230
433
8
18
37
66
137
263
478
Jurkat Value Value
ODµg/mL pg/mL pg/mL pg/mL
25 October 2016
15.625 0.017
32.25 0.036
62.5 0.073
125 0.131
250 0.275
500 0.526
1000 0.956
Linear regression4PL
21.88
43.75
87.5
175
350
700
1400
7
15
30
55
117
230
433
8
18
37
66
137
263
478
Jurkat Value Value
ODµg/mL pg/mL pg/mLpg/mL
25 October 2016
15.625 0.017
32.25 0.036
62.5 0.073
125 0.131
250 0.275
500 0.526
1000 0.956
480
440
468
460
433
592
528
548
526
478
Linear regression4PL
7
15
30
55
117
230
433
x2
x4
x8
x16
8
18
37
66
137
263
478
x2
x4
x8
x16
Jurkat Value
Corrected
Value Value
Corrected
Value
ODµg/mL pg/mL pg/mL
25 October 2016
15.625 0.017
32.25 0.036
62.5 0.073
125 0.131
250 0.275
500 0.526
1000 0.956
480
440
468
460
433
592
528
548
526
478
Linear regression4PL
112%
101%
108%
106%
123%
110%
115%
110%
7
15
30
55
117
230
433
x2
x4
x8
x16
8
18
37
66
137
263
478
x2
x4
x8
x16
Jurkat Value
Corrected
Value Linearity Value
Corrected
Value Linearity
ODµg/mL pg/mL pg/mL
25 October 2016
0
50
100
150
200
250
300
350
400
450
500
1000 500 250 125 62.5
PD
-L1
Co
nc
en
tra
tio
n (
pg
/ml)
Jurkat (µg/mL)
25 October 2016
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