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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