Alternative Pathway ELISA_orthogonal screening
The alternative complement pathway does not require antibody for its activation. A variety of antigens such as bacterial lipopolysaccharide and components of viruses and other pathogens have the ability to activate this pathway. The complement component C3 is spontaneously cleaved into C3a and C3b fragments. If C3b binds to bacteria and other foreign materials, the factor B bound to C3b would be cleaved by Factor D. The fragment Ba floats away, while Bb stays associated with C3b. The resulting C3bBb molecule is the alternative pathway C3 convertase1. ..more
BioActive Compounds: 52
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Scott Diamond, University of Pennsylvania
MLSCN Grant: MH076406-01
The alternative complement pathway does not require antibody for its activation. A variety of antigens such as bacterial lipopolysaccharide and components of viruses and other pathogens have the ability to activate this pathway. The complement component C3 is spontaneously cleaved into C3a and C3b fragments. If C3b binds to bacteria and other foreign materials, the factor B bound to C3b would be cleaved by Factor D. The fragment Ba floats away, while Bb stays associated with C3b. The resulting C3bBb molecule is the alternative pathway C3 convertase1.
The high-throughput screen for inhibitors reported here used ELISA to measure the amount of C3, which is related to the activity of the whole alternative pathway. The MLSCN compound library was screened as mixtures of 10 compounds per well. Active compounds will be confirmed by single compound IC50 determination.
Human Serum was purchased from Complement Technology, Inc. (Cat# NHS). Lipopolysaccharides (LPS) was from Sigma-Aldrich (Cat# L7136). Anti-human C3 HRP conjugated antibody was from MP Biomedicals (Cat# 55237). Gelatin veronal buffer (GVB++) was from 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). SpectraPlate-384HB clear high protein-binding plates were from PerkinElmer (Item# 6007500). Mg2+ -EGTA GVB++: 25uL 1M MgCl2 + 550uL 0.1M Mg.Mg2+ EGTA in 5mL GVB++. EDTA-GVB++: 550uL 0.5M EDTA in 5mL GVB++.
The plates were coated with Lipopolysaccharides (LPS) at 4degC overnight. After washing and blocking for another hour, mixed human serum with the compounds was added to the plates, incubated at 37degC 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 using Envision (Perkin Elmer) plate reader.
200 plates containing 64,000 compounds from the MLSCN library were arranged in two 10 x 10 grids of 100 plates each. Sets of ten plates were pooled as follows to give 20 mixture plates per 100 single compound plates:
Plate 1_2_3_4_5_6_7_8_9_10 pooled to mixture plate 1-10
Plate 11_12_13_14_15_16_17_18_19_20 pooled to mixture plate 11-20
Plate 21_22_23_24_25_26_27_28_29_30 pooled to mixture plate 21-30
Plate 31_32_33_34_35_36_37_38_39_40 pooled to mixture plate 31-40
Plate 41_42_43_44_45_46_47_48_49_50 pooled to mixture plate 41-50
Plate 51_52_53_54_55_56_57_58_59_60 pooled to mixture plate 51-60
Plate 61_62_63_64_65_66_67_68_69_70 pooled to mixture plate 61-70
Plate 71_72_73_74_75_76_77_78_79_80 pooled to mixture plate 71-80
Plate 81_82_83_84_85_86_87_88_89_90 pooled to mixture plate 81-90
Plate 91_92_93_94_95_96_97_98_99_100 pooled to mixture plate 91-100
Plates were also pooled 'vertically':
Plates 1, 11, 21, 31, 41, 51, 61, 71, 81, 91 pooled to mixture plate 1-91
Plates 2, 12, 22, 32, 42, 52, 62, 72, 82, 92 pooled to mixture plate 2-92
And so on until mixture plate 10-100.
For example, the single compound in well A3 in Plate 1 is mixed with 9 compounds in well A3 in mixture plate 1-10, and also with 9 different compounds in well A3 in mixture plate 1-91.
The concentration of each mixture was 2.5 mM in DMSO (250 uM per compound). Pintool transfer into the HTS assay gave a final mixture concentration of 50 uM in dilute human serum (5 uM per compound).
1. Coat assay plates with Lipopolysaccharides (LPS) (40ug/ml in PBS, 20uL per well) at 4 degC, overnight.
2. Wash assay plates four times with 70uL PBS-0.05% Tween-20 at room temperature.
3. Block assay plates by treating with 60uL 1%BSA-PBS for 1 hour at room temperature.
4. Wash assay plates four times with 70uL PBS-0.05% Tween-20 at room temperature.
5. Prepare two intermediate plates:
(a) For serum plate, column 3 to 24 (positive control/compound), add dilute human serum 1:15 in Mg2+ -EGTA GVB++ 15 uL; column 1 to 2 (Blank), add dilute human serum 1:15 in EDTA GVB++ 15 uL.
(b) For intermediate plate, column 3 to 24 (positive control/compound), add dilute human serum 1:15 in Mg2+ -EGTA GVB++ 10uL; column 1 to 2 (Blank), add dilute human serum 1:15 in EDTA GVB++ 10 uL.
6. Pintool compound 4 times from 20uL compound plate to 10uL mix plate. Each time it would transfer 110 nL compound. Then use evolution to transfer 12 uL serum from 15 uL serum plate to the intermediate plate and mix well.
7. Transfer 20 uL dilute human serum and compound mixture from intermediate plate to assay plate using Evolution. Incubate assay plate at 37 degC for one hour.
8. Stop complement reaction by adding ice cold 40 uL of 10 mM EDTA-PBS.
9. Wash assay plates four times with 70 uL PBS-0.05% Tween-20 at room temperature. 10. Incubate with 20 uL HRP anti-human C3 antibody (1:4000 dilutions in the blocking buffer) for 1 hour at room temperature.
11. Wash assay plates four times with 70uL PBS-0.05% Tween-20 at room temperature.
12. Add 40 uL development solution (TMB solution from BD biosciences).
13. Stop reaction by adding 20 uL of 2N Sulphuric acid.
14. OD 450 measurement on Envision.
Mixture percent inhibition---Data were analyzed in IDBS ActivityBase. Each HTS plate contained compound mixtures (50 uM in dilute human serum) in columns 3-22, controls (dilute human serum 1:15 in Mg2+ -EGTA, no compound mixture) in columns 23 and 24, and blanks (dilute human serum 1:15 in EDTA GVB++) in columns 1 and 2. HTS percent inhibition was calculated for each compound mixture 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:
% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))
Assignment of percent inhibition to individual compounds---Percent inhibition results from the mixture HTS were retrieved in SARgen (IDBS) together with the identity of the 10 individual compounds within each mixture. As each compound was present in two mixtures the data were rearranged using a custom Excel macro to align both percent inhibition values associated with each compound. Compounds that gave >30% inhibition in both mixture locations were selected as hits and will be retested individually in dose-response.
Activity scoring is complicated by the fact that the two percent inhibition values associated with each compound in fact represent two different mixtures of 10 compounds. Thus simple averaging of the two values is not meaningful. Instead each compound was assigned a percent inhibition score based on the lower of the two percent inhibition values. This system appropriately scores hits showing activity in both mixtures, but avoids assignment of an erroneously high score to inactives that shared one location with an active compound.
The activity score reported here is based on percent inhibition observed in the primary HTS (see above):
Percent inhibition scores were calculated from the lower of the two percent inhibition values associated with each compound as follows:
(1) For percent inhibition between 0 and 100, score = 0.4 * percent inhibition
(2) For negative percent inhibition, score = 0
Compounds that gave percent inhibition >30 in both locations in the primary HTS were judged to be hits and these 51 compounds will be put through follow-up IC50 testing.
Activity outcome is reported as follows:
(1) Percent inhibition > 30 in both locations = active
(2) Percent inhibition < =30 in one or both locations = inactive
This assay was submitted to the PCMD by Scott Diamond, assay development was done by Chun-Hao Chiu and Yuko Kimura, HTS was done by Chun-Hao Chiu and data were submitted by Chun-Hao Chiu and Nuzhat Motlekar, all of the University of Pennsylvania.
We would like to thank Wen-Chao Song (University of Pennsylvania) for providing us with the ELISA assay for screening the Alternative Complement Pathway.
Please direct all correspondence to Dr. Andrew Napper (firstname.lastname@example.org)
** Test Concentration.
Data Table (Concise)