Targeted Modulation of Complement Activation in Lupus Nephritis Using a Bispecific CATCH- Construct

This folder contains a dataset for the SNF funded project “Targeted Modulation of Complement Activation in Lupus Nephritis Using a Bispecific CATCH- Construct”

It contains a set of 4 experiments showing binding properties of 1F5 to C1q and binding of 5C6 to Factor H.

Experiment1_raw.txt
Experiment2_raw.txt
Experiment3_analysis.txt
Experiment3_raw.txt
Experiment4_raw.txt

Experiment1 meta data:
ELISA assay: 5C6 catching of FH from NHS
– coating with column A: neutravidin, B: neutravidin + bio 5C6_cyclic 10µg/ml, C: normal human IgG, D: HSA 
– Addition of NHS in given dilution  as source for FH 1/50, 1/100, 1/200, 1/800, 1/2400, 
– Incubation 1h  at RT
– Detect goat anti-FH (CompTech A237), Anti-goat HRP #P0449 Dako, TMB
Given is OD at 405nm

Experiment2 meta data:
TRIFMA assay: 1F5 binding to C1q-coating
– coating with 2ug/ml human C1q in PBS 
– Addition of rFabs (with murine constant region) in TBS-Tween hiSalt (1M NaCl) at given dilution. Column A: rFab 34A4, column B: negative control Fab “no21” binding to CD40L, column C: 1F5
– Incubation 1h  at RT
– Detect with Biotin Goat anti-mouse kappa light chain BioRad, Ref 105008, 0.25ug/ml
Given are counts after development with Europium-Strp

Experiment3 meta data:
TRIFMA assay: 1F5 binding to C1q-surface after competition with excess fluid phase C1q.  
– coating with 2ug/ml human C1q in PBS
– Preincubation of rFab (0.5ug/ml) with fluid-phase C1q 1h at 37°C in TBS/Tw  hiSalt (1M Nacl) (line1) or TBS/Tw (line2) in molar ratio Column A 1:0, Column B 1:10, Column C and 1:1. Column D blank 
– addition of 
– Incubation 1h at RT
– Detect with Biotin Goat anti-mouse kappa light chain BioRad, Ref 105008, 0.25ug/ml
Given are counts after development with Europium-Strp
In “Experiment3_analysis.txt” percent signal compared to 1:0 (no addition of fluid-phase C1q) is given.

Experiment4
TRIFMA assay: C1q binding to normal human IgG surface in the presence of 1F5 
– coating with 5ug/ml human IgG in coating buffer
– addition of NHS 1/100 with various dilutions of rFab 1F5 or control rFab 34A4 as indicated 
– Incubation 1h at RT
– Detect with Biotin rabbit anti-C1q (Dako)
Given are counts after development with Europium-Strp