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Test Results
Test Results
Comparison of ELISA Protocol
MicroFluere®
- Use 20 µL or less
- Fewer steps
| MicroFluere® | Incubation (min) | Step |
| Capture | 60 | #1 |
| Blocking | 30 | #2 |
| Wash | 0 | #3 |
| Standard | 15 | #4 |
| Detection | 15 | #5 |
| HRP | 5 | #6 |
| Wash | 0 | #7 |
| Wash | 0 | #8 |
| Wash | 0 | #9 |
| Substrate | 5 | #10 |
| TOTAL | 130 | 10 |
Capture antibody coating
90 minutes
90 minutes
Actual assay: 40 minutes
Traditional
- Use 100 µL or more
- More steps
| Traditional | Incubation* (min) | Step |
| Capture | 540 | #1 |
| Wash | 0 | #2 |
| Wash | 0 | #3 |
| Wash | 0 | #4 |
| Blocking | 60 | #5 |
| Wash | 0 | #6 |
| Wash | 0 | #7 |
| Wash | 0 | #8 |
| Standard | 120 | #9 |
| Wash | 0 | #10 |
| Wash | 0 | #11 |
| Wash | 0 | #12 |
| Detection | 120 | #13 |
| Wash | 0 | #14 |
| Wash | 0 | #15 |
| Wash | 0 | #16 |
| HRP | 20 | #17 |
| Wash | 0 | #18 |
| Wash | 0 | #19 |
| Wash | 0 | #20 |
| Substrate | 20 | #21 |
| TOTAL | 880 | 21 |
Capture antibody coating
Overnight + 60 minutes
Overnight + 60 minutes
Actual assay: 280 minutes
Summary Performance Data Comparison
| Nb | Manufacturer | Kit Name | Analyte | Manufacturer Advertised Dynamic Range (pg/ml) | Manufacturer Dynamic Range (pg/ml) |
| 1 | Thermo Fisher | AAAV9 | AAV9 affinity ligand | 250-10000 | 62.5-8000, 125-16000 |
| 2 | R&D Systems | DY266 | Human CXCL10/IP-10 | 31.2-2000 | 18.75-9600 |
| 3 | R&D System | DY202 | Human IL-2 | 15.6-1000 | 18.7-9600 |
| 4 | R&D Systems | DY206 | Human IL-6 | 9.6-600 | 9.6-9600 |
| 5 | R&D System | DY210 | Human IL-8/CXCL8 | 31.2-2000 | 18.7-4800 |
| 6 | R&D System | DY208 | Human TNF-alpha | 15.6-1000 | 18.75-9600 |
| 7 | R&D System | DY293B | Human VEGF | 31.2-2000 | 37.5-1200 |
| 8 | R&D System | DY7360-05 | HIV-1 Gag p24 | 15.6-1000 | 21.7-9322 |
| 9 | R&D System | DY3689 | Rat TIM-1/KIM-1/HAVCR | 15.6-1000 | 11.3-9000 |
| 10 | R&D System | DY564 | Rat VEGF | 15.6-1000 | 12.5-9000 |
| 11 | R&D System | DY247 | Human IL-15 | 15.6-1000 | 21.7-9106 |
| 12 | R&D System | DY240 | Human TGF-beta 1 | 31.2-2000 | 37.5-9600 |
| 13 | R&D System | DY212 | Human GDNF | 31.2-2000 | 75-9600 |
| 14 | R&D System | DY285 | Human IFN-gamma | 15.6-1000 | 37.5-9600 |
| 15 | Sino Biologica | SEKB10395 | Human IL-6 | 2.34-150 | 1.56-1600 |
| 16 | Sino Biological | KIT11725A | Human IFN-gamma | 23.44-1500 | 23.44-12000 |
| 17 | Sino Biological | N/A | SARS COV-19 N-Protein | N/A | 23.39-XXX |
| 18 | Sino Biological | N/A | SARS COV-19 S-Protein | N/A | 390-XXX |
| 19 | Sino Biological | N/A | SARS COV-19 IgG antibody | N/A | 1000-XXX |
Substrate: QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit from Thermo Fisher
MicroFluere® Result Vs. Analytical Test Result of the Kit AAV9 Ligand Leakage
Normalized at lowest concentration
Normalized at highest concentration
| MicroFluere® | Kit’s Manual | Note | |
| Assay time | 40 minutes | 200 minutes | excluding washing and handling time |
| LOD | ≤0.125 ng/mL | ≤1 ng/mL | – |
| Linear range | 0.0625 – 4 ng/mL | 0.25 – 4 ng/mL | – |
| Dynamic range | 0.0625 – 8 ng/mL (MF 1000X Dec.) 0.125 – 16 ng/mL (MF 1250X Dec.) |
0.25 – 16 ng/mL | – |
Human CXCL10/IP-10 Quantikine ELISA Kit
31.2-2000 pg/mL
- R-square: 0.99823
- Dynamic range: 75-9,600 pg/mL
- Sample/reagent volume: ≤20µL
- Assay time: 40 minutes
Human IL-2 DuoSet ELISA
15.6-1000 pg/mL
- R-square: 0.99844
- Dynamic range: 18.7-9,600 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Human IL-6 DuoSet ELISA
9.4-600 pg/mL
Human IL-8/CXCL8 DuoSet ELISA
31.2-2000 pg/mL
- R-square: 0.99941
- Dynamic range: 18.75-4,800 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Human TNF-alpha DuoSet ELISA
15.6-1000 pg/mL
- R-square: 0.99867
- Dynamic range: 18.75-9,600 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
- R-square: 0.99867
- Dynamic range: 18.75-4,800 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Human VEGF DuoSet ELISA
31.2-2000 pg/mL
- R-square: 0.99867
- LOD: 12.5 pg/mL
- Dynamic range: 12.5-9,000 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
HIV-1 Gag p24 DuoSet ELISA
15.6-1000 pg/mL
- R-square: 0.99986
- LOD: 21.1 pg/mL
- Dynamic range: 21.7-9,322 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Rat TIM-1/KIM-1/HAVCR DuoSet ELISA
15.6-1000 pg/mL
- R-square: 0.99081
- LOD: 11.3 pg/mL
- Dynamic range: 11.3-9,000 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Rat VEGF DuoSet ELISA
15.6-1000 pg/mL
- R-square: 0.99784
- Dynamic range: 37.5-1200 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Human IL-15 DuoSet ELISA
15.6-1000 pg/mL
- R-square: 0.99838
- LOD: 21.7 pg/mL
- Dynamic range: 21.7-9,106 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Human TGF-beta 1 DuoSet ELISA
31.2-2000 pg/mL
- R-square: 0.99529
- Dynamic range: 37.5-9600 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Human GDNF DuoSet ELISA
31.2-2000 pg/mL
- R-square: 0.97313
- Dynamic range: 75-9,600 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Human IFN-gamma DuoSet ELISA
15.6-1000 pg/mL
- R-square: 0.9976
- Dynamic range: 37.5-9,600 pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
MicroFluere® Vs. SEKB10395 kit – Human IL-6
| Conc. (pg/mL) | Zero standard subtracted (FL) |
| 0 | 0 |
| 1.5625 | 121 |
| 3.125 | 200 |
| 6.25 | 485 |
| 12.5 | 1103 |
| 25 | 1912 |
| 50 | 3790 |
| 100 | 7753 |
| 200 | 15599 |
| 400 | 29655 |
| 800 | 53358 |
| 1600 | 91553 |
| 3200 | 133184 |
| 6400 | 146631 |
| Concentration (pg/mL) | Zero standard subtracted OD |
| 0 | 0 |
| 2.34 | 0.033 |
| 4.69 | 0.072 |
| 9.38 | 0.154 |
| 18.75 | 0.310 |
| 37.5 | 0.612 |
| 75 | 1.184 |
| 150 | 2.113 |
| MicroFluere® | SEKB10395 | Note | |
| Assay time | 35 minutes | 200 minutes | excluding washing and handling time |
| LOD | 1.93 pg/mL | 2.34 pg/mL | Zero standard + 3β |
| Linear range | 1.56 – 1600 pg/mL | 2.34 – 150 pg/mL | – |
HRP -conjugated detection antibody – Human IFNg
Conc. (pg/mL) | A | B | C | Avg | STD | CV% |
0 | 1723 | 1352 | 1371 | 1482 | 208.9282 | 14% |
5.85938 | 1841 | 1613 | 1573 | 1675.667 | 144.5729 | 9% |
11.71875 | 2254 | 2024 | 1690 | 1989.333 | 283.5936 | 14% |
23.4375 | 2831 | 2094 | 2458 | 2461 | 368.5092 | 15% |
46.875 | 3209 | 2936 | 2489 | 2878 | 363.4873 | 13% |
93.75 | 4617 | 4293 | 3677 | 4195.667 | 477.499 | 11% |
187.5 | 7180 | 6929 | 7280 | 7129.667 | 180.8323 | 3% |
375 | 11235 | 11274 | 9543 | 10684 | 988.3274 | 9% |
750 | 18098 | 17855 | 15684 | 17212.33 | 1329.14 | 8% |
1500 | 34187 | 35273 | 31240 | 33566.67 | 2086.835 | 6% |
3000 | 64935 | 61310 | 58076 | 61440.33 | 3431.357 | 6% |
6000 | 104089 | 103277 | 96657 | 101341 | 4076.73 | 4% |
12000 | 131987 | 137376 | 123600 | 130987.7 | 6942.157 | 5% |
24000 | 148590 | 145788 | 136413 | 143597 | 6377.319 | 4% |
| MicroFluere® | KIT11725A | Note | |
| Assay time | 35 minutes | 200 minutes | excluding washing and handling time |
| LOD | 6.9 pg/mL | 15.1 pg/mL | Zero standard + 3β |
| Linear range (4PL range) | 23.44 – 6000 pg/mL (23.44-12000 pg/mL) | 23.44 – 1500 pg/mL (NA) | – |
COVID-19 N-protein ELISA
15.6-1000 pg/mL
061920
LOD: 0.02839 ng/mL
R2: 0.99785
COGS of reagent: $25/plate
LOD: 0.02839 ng/mL
R2: 0.99785
COGS of reagent: $25/plate
Note: The reagent cost are calculated based on retail price.
COVID-19 S-protein ELISA
- R-square: 0.99794
- Dynamic range: 390-XXX pg/mL
- Sample/reagent volume: ≤20 µL
- Assay time: 40 minutes
Mouse TGF-β1 ELISA Experiment at Prof. Tae-Hwa Chun’s Lab
31.2 – 2,000 pg/mL
Dynamic range = ~50 pg/mL – 15,000 pg
Human IFN-alpha 2/IFNA2 DuoSet ELISA
3.1-200 pg/mL
Human IL-10 DuoSet ELISA
31.2-2000 pg/mL
Human TGF-beta 1 DuoSet ELISA
15.6-1000 pg/mL
Compatibility tested commercial ELISA readers
- EnSpire 2300 Multimode Plate Reader (PerkinElmer)
- Infinite F200 Fluorescent Microplate Reader System (Tecan)
- Synergy Neo2 Hybrid Multi-Mode Reader (BioTek Instruments)
- Synergy H1 Hybrid Multi-Mode Reader (BioTek Instruments)
- Synergy HT (BioTek Instruments)
- GloMax®-Multi+ Detection System (Promega)
- Varioskan Flash (Thermo Fisher Scientific)
Intra- assay CVs
Plate 1
Conc. (pg/mL) | Rep 1 | Rep 2 | Rep 3 | Avg | STD | CV% |
0 | 399 | 365 | 412 | 392 | 24 | 6.2% |
3.125 | 511 | 464 | 464 | 480 | 27 | 5.7% |
6.25 | 585 | 550 | 461 | 532 | 64 | 12.0% |
12.5 | 661 | 667 | 704 | 677 | 23 | 3.4% |
25 | 923 | 1027 | 911 | 954 | 64 | 6.7% |
50 | 1452 | 1510 | 1536 | 1499 | 43 | 2.9% |
100 | 2287 | 2619 | 2607 | 2504 | 188 | 7.5% |
200 | 4638 | 4672 | 4234 | 4515 | 244 | 5.4% |
Plate 2
Conc. (pg/mL) | Rep A | Rep B | Rep C | Avg | STD | CV% |
0 | 450 | 379 | 369 | 399 | 44 | 11.1% |
3.125 | 454 | 439 | 401 | 431 | 27 | 6.3% |
6.25 | 592 | 471 | 474 | 512 | 69 | 13.5% |
12.5 | 763 | 587 | 680 | 677 | 88 | 13.0% |
25 | 1008 | 993 | 872 | 958 | 75 | 7.8% |
50 | 1710 | 1581 | 1514 | 1602 | 100 | 6.2% |
100 | 2957 | 2334 | 2665 | 2652 | 312 | 11.8% |
200 | 4702 | 4807 | 5020 | 4843 | 162 | 3.3% |
Inter- assay CVs
Conc. (pg/mL) | Plate 1 | Plate 2 | Avg | STD | CV% | ||||
Rep 1 | Rep 2 | Rep 3 | Rep A | Rep B | Rep C | ||||
0 | 399 | 365 | 412 | 450 | 379 | 369 | 396 | 32 | 8.1% |
3.125 | 511 | 464 | 464 | 454 | 439 | 401 | 456 | 36 | 7.9% |
6.25 | 585 | 550 | 461 | 592 | 471 | 474 | 522 | 60 | 11.6% |
12.5 | 661 | 667 | 704 | 763 | 587 | 680 | 677 | 58 | 8.5% |
25 | 923 | 1027 | 911 | 1008 | 993 | 872 | 956 | 62 | 6.5% |
50 | 1452 | 1510 | 1536 | 1710 | 1581 | 1514 | 1551 | 89 | 5.7% |
100 | 2287 | 2619 | 2607 | 2957 | 2334 | 2665 | 2578 | 244 | 9.5% |
200 | 4638 | 4672 | 4234 | 4702 | 4807 | 5020 | 4679 | 258 | 5.5% |
