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

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 Tissue Plasma Serum Urine Supernatant pellet Homogenate 1X Cell Extraction Dilute in: Sample Diluent NS Buffer PTR Standard 1100 to 17.19 1400 to 21.88 Curve: pg/mL pg/mL 25 October 2016

Cell Cell Culture Tissue Plasma Serum Urine Supernatant pellet Homogenate 1X Cell Extraction Dilute in: Sample Diluent NS Buffer PTR Standard 1100 to 17.19 1400 to 21.88 Curve: pg/mL pg/mL 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

1 2 3 4 5 6 7 8 9 10 11 12 A Standard 1400 1400 1,400 to 21.88 pg/mL B 700 700 C 350 350 D 175 175 87.5 87.5 E 43.75 43.75 F 21.88 21.88 G H 25 October 2016

1 2 3 4 5 6 7 8 9 10 11 12 A Standard 1400 1400 1000 1000 1,400 to 21.88 pg/mL B Jurkat+LPS+IFNg 700 700 500 500 1,000 to 15.625 µg/mL C 350 350 250 250 D 175 175 125 125 87.5 87.5 62.5 62.5 E 43.75 43.75 31.25 31.25 F 21.88 21.88 15.625 15.625 G H 25 October 2016

1 2 3 4 5 6 7 8 9 10 11 12 A Standard 1400 1400 1000 1000 1,400 to 21.88 pg/mL B Jurkat+LPS+IFNg 700 700 500 500 1,000 to 15.625 µg/mL C Negative 350 350 250 250 Sample diluent D 175 175 125 125 87.5 87.5 62.5 62.5 E 43.75 43.75 31.25 31.25 F 21.88 21.88 15.625 15.625 G NS NS H 25 October 2016

Verifying the validity of the measure 25 October 2016

OD STD Jurkat 2.836 2.881 1.029 0.989 1.553 1.602 0.579 0.579 0.833 0.863 0.329 0.327 0.442 0.442 0.186 0.183 0.250 0.260 0.125 0.129 0.154 0.162 0.088 0.090 0.104 0.120 0.070 0.070 0.051 0.056 25 October 2016

OD STD Jurkat 2.836 2.881 1.029 0.989 1.553 1.602 0.579 0.579 Subtract mean of the blank to 0.833 0.863 0.329 0.327 all samples and 0.442 0.442 0.186 0.183 standard points 0.250 0.260 0.125 0.129 0.154 0.162 0.088 0.090 0.104 0.120 0.070 0.070 Mean 0.051 0.056 0.053 25 October 2016

Corrected OD STD Jurkat 2.783 2.828 0.975 0.936 1.499 1.549 0.526 0.525 Subtract mean of the blank to 0.780 0.809 0.275 0.274 all samples and 0.389 0.389 0.133 0.130 standard points 0.197 0.207 0.071 0.076 0.101 0.109 0.035 0.037 0.050 0.067 0.017 0.017 Mean 0.051 0.056 0.053 25 October 2016

Corrected OD CV: Coefficient of STD Jurkat Variation 2.783 2.828 0.975 0.936 SD: Standard Deviation 1.499 1.549 0.526 0.525 SD 0.780 0.809 0.275 0.274 20% CV 0.389 0.389 0.133 0.130 Mean OD 0.197 0.207 0.071 0.076 0.101 0.109 0.035 0.037 0.050 0.067 0.017 0.017 25 October 2016

Corrected OD CV: Coefficient of STD Jurkat Variation Mean SD CV 2.783 2.828 0.975 0.936 2.805 0.956 0.022 0.020 1% 2% SD: Standard Deviation 1.499 1.549 0.526 0.525 1.524 0.526 0.025 0.000 2% 0% 0.780 0.809 0.275 0.274 0.795 0.275 0.015 0.001 2% 0% 0.389 0.389 0.133 0.130 0.389 0.131 0.000 0.001 0% 1% 0.197 0.207 0.071 0.076 0.202 0.073 0.005 0.002 2% 3% 0.101 0.109 0.035 0.037 0.105 0.036 0.004 0.001 4% 3% 0.050 0.067 0.017 0.017 0.058 0.017 0.008 0.000 14% 0% 25 October 2016

Creating the standard curve 25 October 2016

Bioassay like ELISA have a sigmoidal curve Good Fit Bad Fit 4 4 3 3 OD450 OD450 2 2 R² = 0.9609 R² = 0.9996 1 1 0 0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Analyte concentration (pg/ml) Analyte concentration (pg/ml) 4 parameters model (4PL) Linear regression Software of your reader, Excel GraphPad PRISM 25 October 2016

Excel Normal scale STD 1400 2.805 3 700 1.524 2.5 2 350 0.795 OD450 1.5 y = 0.002x + 0.0432 175 0.389 R² = 0.998 1 0.5 87.5 0.202 0 0 500 1000 1500 43.75 0.105 Human PD-L1 (pg/ml) 21.88 0.058 Linear regression pg/ml 25 October 2016

Excel Log/log scale STD 1400 2.805 10 700 1.524 1 350 0.795 1 10 100 1000 10000 OD450 175 0.389 y = 0.002x + 0.0432 0.1 R² = 0.998 87.5 0.202 43.75 0.105 0.01 Human PD-L1 (pg/ml) 21.88 0.058 Linear regression pg/ml 25 October 2016

GraphPad PRISM Excel Log/log scale Log/log scale 10 1 0 O D (4 5 0 n m ) 1 1 1 10 100 1000 10000 OD450 0 .1 y = 0.002x + 0.0432 0.1 R² = 0.998 0 .0 1 1 0 1 0 0 1 0 0 0 H u m a n P D - L 1 (p g / m L ) 0.01 Human PD-L1 (pg/ml) 4 parameters model (4PL) Linear regression 25 October 2016

Calculating the concentration of your samples 25 October 2016

pg/mL pg/mL µg/mL OD Jurkat Value Value 478 433 1000 0.956 263 500 0.526 230 137 250 0.275 117 66 55 125 0.131 37 62.5 0.073 30 18 15 32.25 0.036 8 15.625 0.017 7 4PL Linear regression 25 October 2016

pg/mL pg/mL µg/mL OD pg/mL Jurkat Value Value 1400 478 433 1000 0.956 700 263 500 0.526 230 350 137 250 0.275 117 175 66 55 125 0.131 87.5 37 62.5 0.073 30 43.75 18 15 32.25 0.036 21.88 8 15.625 0.017 7 4PL Linear regression 25 October 2016

pg/mL pg/mL µg/mL OD pg/mL Jurkat Value Value 1400 478 433 1000 0.956 700 263 500 0.526 230 350 137 250 0.275 117 175 66 55 125 0.131 87.5 37 62.5 0.073 30 43.75 18 15 32.25 0.036 21.88 8 15.625 0.017 7 4PL Linear regression 25 October 2016

pg/mL pg/mL µg/mL OD Corrected Corrected Jurkat Value Value Value Value 478 433 478 433 1000 0.956 x2 x2 460 263 526 500 0.526 230 x4 x4 548 468 137 250 0.275 117 x8 x8 440 66 528 55 125 0.131 x16 x16 592 480 37 62.5 0.073 30 18 15 32.25 0.036 8 15.625 0.017 7 4PL Linear regression 25 October 2016

pg/mL pg/mL µg/mL OD Corrected Corrected Jurkat Value Linearity Value Value Linearity Value 478 433 478 433 1000 0.956 x2 x2 460 106% 263 526 110% 500 0.526 230 x4 x4 108% 548 468 137 115% 250 0.275 117 x8 x8 101% 440 66 528 110% 55 125 0.131 x16 x16 112% 123% 592 480 37 62.5 0.073 30 18 15 32.25 0.036 8 15.625 0.017 7 4PL Linear regression 25 October 2016

500 450 PD-L1 Concentration (pg/ml) 400 350 300 250 200 150 100 50 0 1000 500 250 125 62.5 Jurkat (µg/mL) 25 October 2016

25 October 2016

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