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BioAssay: AID 829

Complement C1s ELISA

The classical pathway mediates specific antibody responses. The classical pathway is initiated by the binding of antibodies to cell surface antigens. Subsequent binding of the antibody to complement C1q subunits of C1 result in catalytically active C1s subunits. The two activated C1s subunits are then able to catalyze the assembly of the C3 convertase (complement C4b2a) from complements C2 and C4.(Ref. 1) ..more
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 Related BioAssays
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AID: 829
Data Source: PCMD (C1s ELISA)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-10-11
Modify Date: 2008-10-07

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeComment
538Complement factor C1sScreeningdepositor-specified cross reference
787Complement factor C1s IC50 from mixture screenConfirmatorydepositor-specified cross reference
Description:
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Scott L. Diamond, University of Pennsylvania
MLSCN Grant: X01-MH076406-01

The classical pathway mediates specific antibody responses. The classical pathway is initiated by the binding of antibodies to cell surface antigens. Subsequent binding of the antibody to complement C1q subunits of C1 result in catalytically active C1s subunits. The two activated C1s subunits are then able to catalyze the assembly of the C3 convertase (complement C4b2a) from complements C2 and C4.(Ref. 1)

Assay

The high-throughput screen on mixture plates for complement factor C1s inhibitors and single compound IC50 determination of active compounds has been reported earlier (AID 538 and AID 787). In this assay, we use ELISA, which can determine the activity of the classical pathway as a whole, to test the activity of hits from the C1s mixture screen.

Materials

Human Serum was purchased from Complement Technology, Inc. (Cat# NHS). Chicken ovalbumin (OVA) was from Sigma-Aldrich (Cat# A5378). Anti-human C3 HRP conjugated antibody was from MP Biomedicals (Cat# 55237). Gelatin veronal buffer (GVB++) was form Sigma-Aldrich (Cat# G6514). Development solution was BD OptEIA# TMB Substrate Reagent Set (Cat# 555214). Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X) was from Invitrogen (Cat# 14190-136). 96-well MaxiSorp clear plates were from Nunc (Item# 460984). Mg2+ -EGTA GVB++: 25uL 1M MgCl2 + 550uL 0.1M Mg.Mg2+ EGTA in 5mL GVB++. EDTA-GVB++: 550uL 0.5M EDTA in 5mL GVB++.

Assay

The plates were coated with chicken ovalbumin (OVA) at 4deg C overnight. After washing and blocking for another hour, anti-OVA antibody was added to the plates to form antigen-antibody complex. The plates were then washed again and human serum mixed with compounds was added to the plates. The plates were incubated at 37deg C for 1 hour. This was followed by washing and addition of HRP conjugated anti-human C3 antibody to the plates. After incubation at room temperature for 1 hour, the plates were once again subjected to washing and BD OptEIA# TMB Substrate Reagent was added along with 2N sulphuric acid as stop solution. Optical density was measured at 450nm.
Protocol
Protocol

1. Coat assay plates with chicken ovalbumin (OVA) (1mg/ ml in PBS, 50uL per well) at 4deg C overnight.
2. Wash assay plates three times with 300uL PBS-0.05% Tween-20 at room temperature.
3. Block assay plates by treating with 200uL of 1% BSA-PBS for 1 hour at room temperature.
4. Wash assay plates three times with 300uL PBS-0.05% Tween-20 at room temperature.
5. Add 50uL Anti-OVA (1:2000 dilutions in the blocking buffer) and incubate for 1 hour at room temperature.
6. Serial dilute single compounds at 20x concentration in DMSO (8 two-fold dilutions from 1 mM to 7.8125 uM) in a 96-well Greiner V-bottom plate.
7. Wash assay plates three times with 300uL PBS-0.05% Tween-20 at room temperature.
8. For columns 1 to 11 (positive control/compound), dilute human serum 1:10 in Mg2+ -EGTA GVB++, take 57uL diluted serum and 3uL compound from the compound plate, mix them in the mother plate. Transfer 50uL mix per well from the mother plate to the assay plate and incubate for 1 hour at 37deg C.

For column 12 (Blank), dilute human serum 1:10 in EDTA GVB++, take 57uL diluted serum and 3uL DMSO from the compound plate, mix them in the mother plate. Transfer 50uL mix per well from the mother plate to the assay plate and incubate for 1 hour at 37deg C.
9. Stop the complement reaction by adding 200uL of ice-cold mixture of 10mM EDTA-PBS.
10. Wash three times with 300uL PBS-0.05% Tween-20 at room temperature.
11. Incubate with 50uL HRP anti-human C3 antibody (1:4000 dilutions in the blocking buffer) for 1 hour at room temperature.
12. Wash four times with 300uL PBS-0.05% Tween-20 at room temperature.
13. Add 100uL of development solution (TMB solution from BD biosciences).
14. Stop the reaction by adding 50uL of 2N Sulphuric acid.
15. Measure OD at 450 nm on Envision.

Data analysis

IC50 plates contained compounds (Human serum in Mg2+ -EGTA GVB++) in columns 2-11, control (Human serum in Mg2+ -EGTA GVB++, no compound) in column 1, and blank (Human serum in EDTA-GVB++) in column 12. Each column 2-11 contained 8 two-fold dilutions of a single compound, ranging in concentration from 50 uM to 0.39 uM. Percent activity was calculated for each dilution of each compound from the signal in optical density units (OD) and the mean of the plate controls and the mean of the plate blanks using the following equation:

% Activity = 100*((signal-blank mean)/ (control mean-blank mean))
Comment
Activity scoring

IC50 scores were calculated as follows:

Score = IC50 value



Activity Outcome

IC50 values were determined as described in protocol above. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >50 uM.

Activity outcome is reported as follows:

IC50 >50 uM in IC50 determinations = inactive

Analysis of screening results

Results of retesting the compounds that gave percent inhibition >20 in the primary HTS (AID 538), and verified as active compounds in the second dose response test (AID 787) were as follows:

Hits from primary screen (>20% inhibition) = 184
Hits active in IC50 = 22 (12 % retest rate)
Hits active in ELISA IC50 = 0


Reference:

1.http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Cell_Signaling_Enzymes/Complement_Proteins.html

Contributors

This assay was submitted to the PCMD by Scott Diamond, assay was conducted by Chun-Hao Chiu, and data were submitted by Nuzhat Motlekar and Andrew Napper, all of the University of Pennsylvania. Our thanks to Yuko Kimura for help with the assay.

Correspondence

Please direct correspondence to Andrew Napper (napper@seas.upenn.edu).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierString
2IC50*FloatμM
3Number of IC50 determinationsInteger
4IC50 minimum concentrationFloatμM
5Percent Activity at IC50 min concentrationFloat%
6IC50 maximum concentrationFloatμM
7Percent Activity at IC50 max concentrationFloat%
8OD at 0.3906 uM (0.3906μM**)FloatAU
9OD at 0.78125 uM (0.78125μM**)FloatAU
10OD at 1.5625 uM (1.5625μM**)FloatAU
11OD at 3.125 uM (3.125μM**)FloatAU
12OD at 6.25 uM (6.25μM**)FloatAU
13OD at 12.5 uM (12.5μM**)Float
14OD at 25 uM (25μM**)FloatAU
15OD at 50 uM (50μM**)FloatAU
16Control MeanFloatAU
17Control Standard DeviationFloatAU
18Control percent CVFloat%
19Blank MeanFloatAU
20Blank Standard DeviationFloatAU
21Blank percent CVFloat%
22Signal-to background ratioFloatratio
23Z-factorFloat

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View All Data
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