ELISA data Human PD-L1 ELISA Kit [28-8] (ab214565) October 2016 - - PowerPoint PPT Presentation

▶
elisa data
SMART_READER_LITE
LIVE PREVIEW

ELISA data Human PD-L1 ELISA Kit [28-8] (ab214565) October 2016 - - PowerPoint PPT Presentation

Dec 26, 2022 368 likes •676 views

Calculating and evaluating ELISA data Human PD-L1 ELISA Kit [28-8] (ab214565) October 2016 Standard ELISA 25 October 2016 One-step ELISA 25 October 2016 Choosing the standard and preparing the plate 25 October 2016 Cell Cell Culture

slide-1
SLIDE 1

Calculating and evaluating ELISA data

Human PD-L1 ELISA Kit [28-8] (ab214565)

October 2016

slide-2
SLIDE 2

25 October 2016

Standard ELISA

slide-3
SLIDE 3

25 October 2016

One-step ELISA

slide-4
SLIDE 4

Choosing the standard and preparing the plate

25 October 2016

slide-5
SLIDE 5

25 October 2016

Plasma Serum

Cell Culture Supernatant

Urine Cell pellet

Tissue Homogenate

Sample Diluent NS 1X Cell Extraction Buffer PTR 1100 to 17.19 pg/mL 1400 to 21.88 pg/mL Dilute in: Standard Curve:

slide-6
SLIDE 6

25 October 2016

Plasma Serum

Cell Culture Supernatant

Urine Cell pellet

Tissue Homogenate

Sample Diluent NS 1X Cell Extraction Buffer PTR 1100 to 17.19 pg/mL 1400 to 21.88 pg/mL Dilute in: Standard Curve:

slide-7
SLIDE 7

25 October 2016

1 2 3 4 5 6 7 8 9 10 11 12

A B C D E F G H

slide-8
SLIDE 8

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

slide-9
SLIDE 9

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

slide-10
SLIDE 10

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

slide-11
SLIDE 11

25 October 2016

Verifying the validity of the measure

slide-12
SLIDE 12

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

slide-13
SLIDE 13

25 October 2016

0.051 0.056

Subtract mean

  • f the blank to

all samples and standard points

0.053

Mean

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 0.104 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

slide-14
SLIDE 14

25 October 2016

0.051 0.056

Subtract mean

  • f the blank to

all samples and standard points

0.053

Mean

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 2.828

STD Jurkat Corrected OD

slide-15
SLIDE 15

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 2.828

SD: Standard Deviation CV: Coefficient of Variation

CV SD Mean OD 20%

STD Jurkat Corrected OD

slide-16
SLIDE 16

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 2.828

SD: Standard Deviation CV: Coefficient of Variation STD Jurkat Corrected OD

slide-17
SLIDE 17

25 October 2016

Creating the standard curve

slide-18
SLIDE 18

Bioassay like ELISA have a sigmoidal curve

25 October 2016

Linear regression 4 parameters model (4PL) Software of your reader, GraphPad PRISM Excel

R² = 0.9996 1 2 3 4 200 400 600 800 1000 1200 OD450 Analyte concentration (pg/ml)

Good Fit

R² = 0.9609 1 2 3 4 200 400 600 800 1000 1200 OD450 Analyte concentration (pg/ml)

Bad Fit

slide-19
SLIDE 19

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 0.5 1 1.5 2 2.5 3 500 1000 1500 OD450 Human PD-L1 (pg/ml)

Linear regression Normal scale Excel pg/ml STD

slide-20
SLIDE 20

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 OD450 Human PD-L1 (pg/ml)

Linear regression Excel Log/log scale pg/ml STD

slide-21
SLIDE 21

25 October 2016

y = 0.002x + 0.0432 R² = 0.998 0.01 0.1 1 10 1 10 100 1000 10000 OD450 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 ) O D (4 5 0 n m )

4 parameters model (4PL) Excel GraphPad PRISM Log/log scale Log/log scale

slide-22
SLIDE 22

25 October 2016

Calculating the concentration of your samples

slide-23
SLIDE 23

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 regression 4PL

7 15 30 55 117 230 433 8 18 37 66 137 263 478

Jurkat Value Value OD µg/mL pg/mL pg/mL

slide-24
SLIDE 24

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 regression 4PL

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 OD µg/mL pg/mL pg/mL pg/mL

slide-25
SLIDE 25

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 regression 4PL

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 OD µg/mL pg/mL pg/mL pg/mL

slide-26
SLIDE 26

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 regression 4PL

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 OD µg/mL pg/mL pg/mL

slide-27
SLIDE 27

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 regression 4PL

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 OD µg/mL pg/mL pg/mL

slide-28
SLIDE 28

25 October 2016

50 100 150 200 250 300 350 400 450 500 1000 500 250 125 62.5

PD-L1 Concentration (pg/ml)

Jurkat (µg/mL)

slide-29
SLIDE 29

25 October 2016

slide-30
SLIDE 30

Contact us!

English technical@abcam.com German wissenschaftlicherdienst@abcam.com French supportscientifique@abcam.com Spanish soportecientifico@abcam.com