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Labjournal iRIf September

Measuring own blood (tetanus vaccination)

16.09.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	BSA	5 mg/ml
3	Tetanus Antigen	~0.5 mg/ml
4	pos. control (GFP)	0.5 mg/ml
5	BSA	5 mg/ml
6	Tetanus Antigen	~0.5 mg/ml
7	pos. control (GFP)	0.5 mg/ml
8	Tetanus Antigen	~0.5 mg/ml

17.09.2015 (measured)

Tet serum (-+): 024, 466

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
Serum(-)	4	30	900	1:10
Buffer	5	60	300	1x
anti GFP	6	30	900	1.5 µg/ml
Buffer	7	60	300	1x
Serum(-)	8	30	900	1:10
Buffer	9	60	300	1x

Tet serum (+-): 208, 216

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
Serum(-)	4	30	900	1:10
Buffer	5	60	300	1x
anti GFP	6	30	900	1.5 µg/ml
Buffer	7	60	300	1x
Serum(+)	8	30	900	1:10
Buffer	9	60	300	1x

GFP; Tet serum (-+): 287

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti GFP	4	30	900	1.5 µg/ml
Buffer	5	60	300	1x
Serum(-)	6	30	900	1:10
Buffer	7	60	300	1x
Serum(+)	8	30	900	1:10
Buffer	9	60	300	1x

Results

Serum(-),anti GFP,Serum(+):

Slide 024: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(-),anti GFP, serum(+)

Slide 024: binding curves

]

Slide 466: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(-),anti GFP, serum(+)

Slide 466: binding curves

Serum(+),anti GFP,Serum(-):

Slide 208: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(+),anti GFP, serum(-)

Slide 208: binding curves

Slide 216: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(+),anti GFP, serum(-)

Slide 216: binding curves

anti GFP,Serum(-),Serum(+):

Slide 287: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: anti GFP, serum(-), serum(+)

Slide 287: binding curves

Measuring : The DIA Chip

14.09.2015 (stamped)

PDMS Dia-Chip stamp (incubated 30 minutes with 1 mg/ml GFP) was stamped onto plasmaactivated slide. Kept in 10 mg/ml BSA in PBS at 4° until measurement.

16.09.2015 (measured)

Slides were previously blocked with the backside of a PDMS flowcell to avoid airbubble issues due to the hydrophobicity of the stamp.

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	5 ug/ml
Buffer	5	60	300	1x
StrepCy5	6	60	300	5 ug/ml
Buffer	9	60	300	1x

Slide 287: Cy5 signal after iRIf measurement detected in microarray scanner

Slide 303: Quotient picture of the anti-GFP step

Rabbit-serum (GFP immunized/not immunized) on PDITC

14.09.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (bBSA)	0.2 mg/ml
2	neg. control (BSA)	0.2 mg/ml
3	His-GFP desalt	1mg/ml
4	pos. control (bBSA)	0.2 mg/ml
5	neg. control (BSA)	0.2 mg/ml
6	His-GFP desalt	1mg/ml
7	His-mCherry desalt	1 mg/ml
8	His-mCherry desalt	1 mg/ml

15.09.2015 (measured)

Flush protocol: Positive serum (504,424,466). Negative serum (423).

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
StrepCy5	4	30	600	5 ug/ml
Buffer	5	60	300	1x
Serum (+)/(-)	8	30	900	1:30
Buffer	9	60	600	1x
Anti-Rabbit	10	60	300	5 ug/ml
Buffer	11	60	300	1x

*Slide 466 was measured with 10 ug/ml Anti-Rabbit

Results: Serum (-) fromm rabbit that was not immunized to GFP shows no binding to the GFP spot.
Serum (+) from rabbit that was immunized to GFP shows extensive binding to the GFP spot.

Slide 423 (serum(-)): Selections of ROIs

Slide 423 (serum(-)): Binding curves

Slide 424 (serum(+)): Selection of ROIs

Slide 424 (serum(+)): Binding curves

Slide 466 (serum(+)): Selections of ROIs

Measuring own blood (Tetanus vaccination)

13.09.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	BSA	5 mg/ml
3	Tetanus Antigen	~0.5 mg/ml
4	-	-
5	-	-
6	bBSA	200 µg/ml

14.09.2015 (measured)

Tet serum (-): 208, 254
Tet serum (+): 500, 024

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	1.5 ug/ml
Buffer	5	60	300	1x
Serum SIG002 (-)/(+)	6	30	900	1:20
Buffer	7	60	300	1x
Anti Human	8	30	600	5 ug/ml
Buffer	9	60	300	1x
StrepCy5	10	30	600	5 ug/ml
Buffer	11	60	300	1x

Results

Blood Serum(-):

Slide 208: Quotion picture, flushed with 1:20 diluted blood serum (-)

Slide 208: binding curves

]

Slide 254: Quotion picture, flushed with 1:20 diluted blood serum (-)

Slide 254: binding curves

Blood serum(+):

Slide 024: Quotion picture, flushed with 1:20 diluted blood serum (+)

Slide 024: binding curves

Slide 500: Quotion picture, flushed with 1:20 diluted blood serum (+)

Slide 500: binding curves

Measuring own blood serum (Tetanus vaccination)

12.09.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	BSA	5 mg/ml
3	Tetanus Antigen	~0.5 mg/ml
4	-	-
5	-	-
6	bBSA	200 µg/ml

13.09.2015 (measured)

Tet serum (-): 443, 466
Tet serum (+): 423, 424

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	1.5 ug/ml
Buffer	5	60	300	1x
Serum SIG002 (-)/(+)	6	30	900	1:10
Buffer	7	60	300	1x
Anti Human	8	30	600	5 ug/ml
Buffer	9	60	300	1x
StrepCy5	10	30	600	5 ug/ml
Buffer	11	60	300	1x

Results

Blood Serum(-):

Slide 443: Quotion picture, flushed with 1:10 diluted blood serum (-)

Slide 443: binding curves

]

Slide 466: Quotion picture, flushed with 1:10 diluted blood serum (-)

Slide 466: binding curves

Blood serum(+):

Slide 423: Quotion picture, flushed with 1:10 diluted blood serum (+)

Slide 423: binding curves

Slide 424: Quotion picture, flushed with 1:10 diluted blood serum (+)

Slide 424: binding curves

Measuring own blood (Tetanus vaccination)

05.09.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	BSA	5 mg/ml
3	Tetanus Antigen	~0.5 mg/ml
4	-	-
5	-	-
6	bBSA	200 µg/ml

06.09.2015 (measured)

Flush protocol:

Tet serum (-): 443
Tet serum (+): 466

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	1.5 ug/ml
Buffer	5	60	300	1x
Serum SIG002 (-)/(+)	6	30	600	1:4
Buffer	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

Tet serum (-): 208
Tet serum (+): 303

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	1.5 ug/ml
Buffer	5	60	300	1x
Serum SIG002 (-)/(+)	6	60	600	1:6
Buffer	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

Note: the amount of flushed serum

Results

Blood Serum(-):

Slide 208: Quotion picture, flushed with 1:6 diluted blood serum (-)

Slide 208: binding curves

Slide 443: Quotion picture, flushed with 1:4 diluted blood serum (-)

Slide 443: binding curves

Blood serum(+):

Slide 303: Quotion picture, flushed with 1:6 diluted blood serum (+)

Slide 303: binding curves

Slide 466: Quotion picture, flushed with 1:4 diluted blood serum (+)

Slide 466: binding curves

→ for the blood serum after immunization (+) the iRIf signal for tetanus binding is higher than for the blood serum before immunization (-)

→ there are differences to the results of experiment 59a, the overall tetanus signal is much weaker as well as the binding of the blood serum to bBSA

Measuring own blood: Tetanus (-), Tetanus (+)

04.09.15 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	BSA	5 mg/ml
3	Tetanus Antigen	~0.5 mg/ml
4	-	-
5	-	-
6	bBSA	200 µg/ml

Flush protocol:

SIG 003 (+): 012(no serum was flushed over the slide), 315, 426
SIG 002 (+): 215

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	1.5 ug/ml
Buffer	5	60	300	1x
Blood serum	6	30	600	1:10/1:30
Buffer	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

Results

SIG 003:

Slide 012: Quotion picture

Slide 012: binding curves

Slide 315: Quotion picture

Slide 426: Quotion picture

Slide 426: binding curves

→ slide 012 was not flushed with any blood serum → for blood serum SIG 003 only a slight antigen/antibody binding is detectable (slide 426)

SIG 002:

Slide 215: Quotion picture

Slide 215: binding curves

→ slight antigen/antibody binding is detectable

→ no anti GFP/ GFP binding detected

→ only weak binding of the blood serum to bBSA → difference to results of experiment 59a

Specific surface (Ni-NTA) vs. unspecific surface (PDITC): cellfree expressed GFP

04.09.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	cell free expression mix without DNA (KK)
3	cell free expressed HA-GFP-His6-His6 (KK)
4	pos. control (GFP)	0.5 mg/ml
5	cell free expression mix without DNA (KK)
6	cell free expressed His10-GFP-Spy (KK)
7	BSA	5 mg/ml
8	bBSA	200 µg/ml

05.09.2015 (measured)

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	1.5 ug/ml
Buffer	5	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

PDITC:

Slide 216: Quotion picture

Slide 216: binding curves

Slide 254: Quotion picture

Slide 254: binding curves

Ni-NTA:

Slide 102: Quotion picture

Slide 102: binding curves

Slide 502: Quotion picture

Slide 502: binding curves

→ the his tagged, cell free GFP was specificly bound by the Ni NTA surface, whereas there was no detectable amount of cell free GFP bound on the PDITC surfaces

→ the PDITC surface binds all proteins in the spotted cell free expression mix eaqually good, whereas the Ni-NTA surface specificly binds only the his tagged, cell free expressed GFP

Measuring own blood: Tetanus (-), Tetanus (+) Sig002

02.09.2015 (spotted) Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	mCherry	0.5 mg/ml
3	Tetanus Antigen	~0.5 mg/ml
4	BSA	10 mg/ml
5	BSA(+bBSA)	10 mg/ml
6	bBSA	200 µg/ml

03.09.2015 (measured)

Tet (-): 287,429 (1:30 diluted serum) Tet (+): 443, 423 (1:30 diluted serum)

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti-GFP	4	30	600	1.5 ug/ml
Buffer	5	60	300	1x
Serum SIG002 (-)/(+)	6	20	900	1:10
Buffer	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

Slide 287: ROI selection, ROIs were chosen in similar manner for the other slides

Slide 287: Binding curves; serum (-) diluted 1:10

Slide 443: Binding curves; serum (+) diluted 1:10

Slide 429: Binding curves; serum (-) diluted 1:30

Slide 423: Binding curves, serum (+) diluted 1:30

Distinguish between different antigen/antibody interaction (Salmonella) (on PDITC) [finished]

01.09.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	bBSA	0.5 mg/ml
3	Salmonella Antigen (15)	undiluted

Slides (3): 208,215,216 last slide (208) changed protocol: only 1 ug/ml anti-GFP used; anti-Salmonella desalt was mixed 1:1 with anti-Salmonella lysate

02.09.2015 (measured)

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS)	1	60	600	1x
BSA	2	60	600	10 mg/ml
Buffer (TBS)	3	60	600	1x
anti-GFP	4	30	600	1,5 ug/ml
Buffer (TBS)	5	60	300	1x
anti-Salmonella (13, desalt)	6	30	900	undiluted
Buffer (TBS)	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer (TBS)	9	60	300	1x

Note: Background of Slides 208 looked nicest.

Slide 208: ROI selection

Slide 208: Binding curves

Slide 215: Binding curves

Slide 216: Binding curves

Team:Freiburg/Labjournals/irif/September

Labjournal iRIf September

Measuring own blood (tetanus vaccination)

Results

Measuring : The **DIA Chip**

Rabbit-serum (GFP immunized/not immunized) on PDITC

Measuring own blood (Tetanus vaccination)

Results

Measuring own blood serum (Tetanus vaccination)

Results

Measuring own blood (Tetanus vaccination)

Results

Measuring own blood: Tetanus (-), Tetanus (+)

Results

Specific surface (Ni-NTA) vs. unspecific surface (PDITC): cellfree expressed GFP

Measuring own blood: Tetanus (-), Tetanus (+) Sig002

Distinguish between different antigen/antibody interaction (Salmonella) (on PDITC) [finished]

Measuring : The DIA Chip