Indirect ELISA of Com. Vs Inhouse fgf23 by Burozumab
1.
2.
3.
4.
Coating: Full-length fgf23 (w/o His tag, (Abcam): 2 µg/mL
Coating inhouse fgf23 (uncleaved sumo his tag): 2 µg/mL
1st Ab: Burosumab(AMW1) 9 µg/mL (3 serial dilution down)
2nd Ab: Goat anti-Human (H+L) HRP (1:10,000)
1st Thawing
Commercial fgf23
0.900
1.000
0.800
0.900
In-house fgf23
0.800
0.700
0.700
450
0.600
450
0.500
0.600
0.500
0.400
0.400
0.300
0.300
0.200
0.200
0.100
0.100
0.000
1E-005
0.000
1E-005
0.0001
0.001
0.01
0.1
1
Concentration
Commercial fgf23. EC50 = 0.0466
10
0.0001
0.001
0.01
0.1
1
10
Concentration
In-house sumo fgf23. EC50 = 0.0562
ELISA analysis
1.
2.
3.
4.
2nd Thawing:
start making
aliquot
Coating: Full-length fgf23 (w/o His tag, (Abcam): 2 µg/mL
Coating inhouse fgf23 (uncleaved sumo his tag): 2 µg/mL
1st Ab: Burosumab(AMW1) 9 µg/mL (3 serial dilution down)
2nd Ab: Goat anti-Human (H+L) HRP (1:10,000)
IgG std.120509
Inhouse
truncated fgf23
0.800
1.600
0.700
1.400
0.600
1.200
0.500
1.000
450
450
std.120509
CommercialIgGfull-length
fgf23
0.400
0.800
0.300
0.600
0.200
0.400
0.100
0.200
0.000
1E-005
0.000
0.0001
0.001
0.01
0.1
<Plate Layout Settings>
Concentration
Commercial fgf23. EC50 = 0.096
1
10
1E-005
0.0001
0.001
0.01
0.1
<Plate Layout Settings>
Concentration
Inhouse fgf23. EC50 = 0.051
1
10
ELISA analysis
1.
2.
3.
4.
Coating: Full-length fgf23 (w/o His tag, (Abcam): 2 µg/mL
Coating inhouse fgf23 (uncleaved sumo his tag): 2 µg/mL
1st Ab: Burosumab(AMW1) 9 µg/mL (3 serial dilution down)
2nd Ab: Goat anti-Human (H+L) HRP (1:10,000)
3rd Thawing
IgG std.120509
Commercial
full-length fgf23
IgG std.120509
Inhouse
truncated fgf23
1.100
1.200
1.000
1.000
0.900
0.800
0.800
450
450
0.700
0.600
0.600
0.500
0.400
0.400
0.300
0.200
0.200
0.100
1E-005
0.000
0.0001
0.001
0.01
0.1
Concentration
<Plate Layout Settings>
Commercial fgf23. EC50 = 290
1
10
1E-005
0.0001
0.001
0.01
0.1
Concentration
<Plate Layout Settings>
Inhouse fgf23. EC50 = 0.055
1
10
ELISA analysis
1.
2.
3.
4.
3rd Thawing:
take from the
aliquots
Coating: Full-length fgf23 (w/o His tag, (Abcam): 5 µg/mL
Coating inhouse fgf23 (uncleaved sumo his tag): 2 µg/mL
1st Ab: Burosumab(AMW1) 9 µg/mL (3 serial dilution down)
2nd Ab: Goat anti-Human (H+L) HRP (1:10,000)
IgG std.120509
Inhouse truncated fgf23
1.400
1.600
1.200
1.400
1.000
1.200
0.800
1.000
450
450
IgG std.120509
Commercial
full-length fgf23
0.600
0.800
0.400
0.600
0.200
0.400
0.000
0.200
-0.200
1E-005
0.000
0.0001
0.001
0.01
0.1
Concentration
<Plate Layout Settings>
Commercial fgf23. EC50 = 7000
1
10
1E-005
0.0001
0.001
0.01
0.1
Concentration
<Plate Layout Settings>
Inhouse fgf23. EC50 = 0.1
1
10