Fast and Efficient Soluble Antigen-Antibody Screening ...€¦ · Inna Vainshtein, Scott Kurose,...
1
Hybridoma Primary Screening Therapeutic antibodies, a growing class of novel therapeutics, require rapid generation of anti- bodies to different disease targets. High expenses associated with maintenance of antibody produc- ing B-cells and hybridomas make screening of antibodies a critical part in antibody generation. Robust and reliable ELISA routinely employed in antibody screening is a slow process requir- ing large number of incubation/washing steps. In order to accelerate antibody screening, a homo- geneous AlphaScreen technology was exam- ined. Application of AlphaScreen for hybridoma fusion screens, serum titers, antibody quantifica- tions and competitive ELISAs were evaluated and will be discussed. Overall AlphaScreen technol- ogy proved to be more sensitive than ELISA and allowed for identification of a larger number of hits. Miniaturization and homogeneous nature of the assay significantly shortened assay run time and eased screening operations. In addition, suc- cessful conjugation of our proprietary antibod- ies and proteins to AlphaScreen beads further expanded applicability of this technology in anti- body screening. Fast and Efficient Soluble Antigen-Antibody Screening Using Homogeneous AlphaScreen Assay Inna Vainshtein, Scott Kurose, Justin Vickroy, J. Russell Grove, and Meina Liang Abgenix, Inc., Fremont CA Abstract AlphaScreen Platform in Antibody Binding Assays Antibody Light Chain Detection Antibody Blocking Protein-Protein Interactions Introduction Antibody binding to antigen is the basis for screening assays to identify hybridomas that pro- duce antigen (Ag)-specific antibodies. For soluble antigens, ELISA is the well-accepted method for hybridoma selection. ELISA has proven to be use- ful due to its easy assay development and reliable data generation. However, there has been increas- ing concern that antigens captured on plate sur- face will hinder epitopes and lead to failure of identifying antibodies for these epitopes. In addi- tion, ELISA is a laborious and time consuming process due to its multiple washing steps. For these reasons, an easy solution-based method to assess antibody binding to soluble antigens is in demand for hybridoma screening. AlphaScreen technology provides a homogeneous and sensitive alternative assay to ELISA format. In this poster, we demonstrated the feasibility of this technology for hybridoma screening and antibody character- ization. 1 Assay Formats Donor Beads + Acceptor Beads + bio-Ag + Hybridoma Supernatants Incubate 2-4 hours Read Signal - SA D Bio Ag A anti-hlgG-Fc hybridoma Excitation 680 nm 1 O 2 SA Bio Ag Emission 520-620 nm 250 nm Detection IgG hybridoma D A Ag Ag Ag anti-hlgG-Fc hybridoma plate HRP Antigen Coating Wash x 3 overnight Blocking Aspirate x 1 2 hours Hybridoma Supernatants Wash x 3 2 hours Secondary Antibodies Wash x 3 1 hour Substrate (TMB) Stop solution 0.5 hour Sandwich ELISA requires addi- tional step of antibody coating extending time for 2 hours 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 0 5000 10000 15000 20000 25000 30000 35000 40000 bio - Ag 3.3 nM bio - Ag 5 nM bio - Ag 10 nM AlphaScreen Assay Format ELISA Assay Format 3 Feasibility of AlphaScreen for Hybridoma Primary Screen Antibody [M] Alpha Signal Although a hooking effect is observed, a broad detectable concentration range of 2.5 log would be sufficient for identification of antibodies in hybri- doma supernatants where antibody concentrations are unknown and broad. 4 Hybridoma Primary Screen Neat Supernatants Diluted Supernatants Sample # Alpha Signal Alpha Signal Sample # Due to a hooking effect, dilution of the samples resulted in higher RLU values and higher hit numbers. Due to higher sensitivity of AlphaScreen, primary screens can be per- formed with diluted hybridoma supernatants. 6 Hit Comparison Between ELISA and AlphaScreen 5 AlphaScreen and ELISA Comparison ELISA Solution interaction Solid phase interaction Biotinylated antigen Unmodified antigen Homogeneous, simple and fast Multi-washing steps, long proce- dure High sensitivity Less sensitivity Use less antigen Use more antigen Reagent cost, ~$0.06/well Reagent cost, ~$0.06/well Hooking effect No hooking effect -SA Bio Ag D -SA Bio Ag -SA Bio Ag A anti-hu-kappa anti-hu-lambda hybridoma 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 bio - Ag 10 nM bio - Ag 5 nM bio - Ag 3.3 nM kappa-antibody [M] Alpha Signal Kappa Detection Serum Titers – Beads-Washing Protocol 0 10000 20000 30000 40000 50000 60000 70000 mouse 1 mouse 2 mouse 3 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 Donor beads + bio-Ag 15 min Mouse serum 2 hours Wash x 1 Acceptor beads 2 hours Read signal Antibody Quantification XenoMouse® Serum Dilution Alpha Signal - SA D Bio A hybridoma anti-hlgG-Fc anti-hlgG-Fc 10 -3 10 -2 10 10 0 10 1 10 2 10 3 2000 4000 10 -1 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 - SA D Bio Ag A hybridoma anti-hlgG-Fc binding antibody SA D Bio Protein A A hybridoma Protein B - - Assay Sensitivity for Blocking Protein-Protein Interactions AlphaScreen ELISA Hybridoma Competitor [M] Hybridoma Competitor [M] OD 650 Alpha Signal AlphaScreen assays are more sensitive than ELISA. AlphaScreen was performed with 0.1 nM of binding antibody, ELISA with 0.67 nM of binding antibody. Hit Comparison Between ELISA and AlphaScreen Total 148 samples screened: identified 23 ELISA positives and 55 Alpha positives. All ELISA hits were a subset of Alpha hits. Alpha identified more hits due to higher assay sensitivity. Conclusions AlphaScreen provides a robust and homogeneous alternative assay to ELISA. The advantages for AlphaScreen assay are solution-based, high sensitivity, less antigen required, and high throughput. Feasibility for serum titer, antibody light chain determination, and IgG quan- tification has been demonstrated using AlphaScreen. AlphaScreen can also be used to characterize the ability of antibodies to block protein-protein interactions. In summary, AlphaScreen technology is very useful for antibody screening and characterization. Society for Biomolecular Screening 10th Anniversary Conference and Exhibition September 11-15, 2004 2 Assay Protocols AlphaScreen Assay Protocol ELISA Assay Protocol Total Samples ELISA Hits Alpha Hits ELISA/ Alpha common % of ELISA/ Alpha overlap Target 1 Primary Screen 3760 172 129 79 Confirmation screen 122 ND 77 63% Target 2 Primary Screen 6110 374 338 226 Confirmation screen 263 ND 212 81% Target A Target B Sample # Hits are clearly identified due to tight background. Similar result was observed for lambda detection. AlphaScreen is suitable for antibody light chain detection. Serum contains interfering components for AlphaScreen signal; therefore, serum testing requires a washing step. Antibody [ng/ml] Alpha Signal IgG Standard Curve Assay can be used to determine total IgG (antigen-specific and non-specif- ic) concentrations in hybridoma supernatants Standard curves were generated using purified IgGs Unknown antibody concentrations were calculated according to the stan- dard curves 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 0 2000 4000 6000 8000 10000 12000 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 0.0 0.2 0.4 0.6 0.8 1.0 ELISA Alpha % Inhibition Samples % Inhibition Samples IC 50 = 0.07 nM IC 50 = 9 nM
Transcript of Fast and Efficient Soluble Antigen-Antibody Screening ...€¦ · Inna Vainshtein, Scott Kurose,...
Hybridoma Primary Screening
Therapeutic antibodies, a growing class of novel therapeutics, require rapid generation of anti-bodies to different disease targets. High expenses associated with maintenance of antibody produc-ing B-cells and hybridomas make screening of antibodies a critical part in antibody generation. Robust and reliable ELISA routinely employed in antibody screening is a slow process requir-ing large number of incubation/washing steps. In order to accelerate antibody screening, a homo-geneous AlphaScreen technology was exam-ined. Application of AlphaScreen for hybridoma fusion screens, serum titers, antibody quantifica-tions and competitive ELISAs were evaluated and will be discussed. Overall AlphaScreen technol-ogy proved to be more sensitive than ELISA and allowed for identification of a larger number of hits. Miniaturization and homogeneous nature of the assay significantly shortened assay run time and eased screening operations. In addition, suc-cessful conjugation of our proprietary antibod-ies and proteins to AlphaScreen beads further expanded applicability of this technology in anti-body screening.
Fast and Efficient Soluble Antigen-Antibody Screening Using Homogeneous AlphaScreen Assay
Inna Vainshtein, Scott Kurose, Justin Vickroy, J. Russell Grove, and Meina Liang Abgenix, Inc., Fremont CA
Abstract
AlphaScreen Platform in Antibody Binding Assays
Antibody Light Chain Detection Antibody Blocking Protein-Protein Interactions
Introduction
Antibody binding to antigen is the basis for screening assays to identify hybridomas that pro-duce antigen (Ag)-specific antibodies. For soluble antigens, ELISA is the well-accepted method for hybridoma selection. ELISA has proven to be use-ful due to its easy assay development and reliable data generation. However, there has been increas-ing concern that antigens captured on plate sur-face will hinder epitopes and lead to failure of identifying antibodies for these epitopes. In addi-tion, ELISA is a laborious and time consuming process due to its multiple washing steps. For these reasons, an easy solution-based method to assess antibody binding to soluble antigens is in demand for hybridoma screening. AlphaScreen technology provides a homogeneous and sensitive alternative assay to ELISA format. In this poster, we demonstrated the feasibility of this technology for hybridoma screening and antibody character-ization.
1 Assay Formats
Donor Beads + Acceptor Beads + bio-Ag + Hybridoma Supernatants
Incubate 2-4 hours
Read Signal
-SAD Bio Ag Aanti-hlgG-Fc
hybridoma
Excitation680 nm
1O2
SA Bio Ag
Emission520-620 nm
250 nm
Detection IgG
hybridoma
D A
AgAg Ag
anti-hlgG-Fc
hybridoma
plate
HRP
Antigen CoatingWash x 3 overnight
BlockingAspirate x 1 2 hours
Hybridoma SupernatantsWash x 3
2 hours
Secondary AntibodiesWash x 3
1 hour
Substrate (TMB)Stop solution 0.5 hour
Sandwich ELISA requires addi-tional step of antibody coating extending time for 2 hours
10-13 10-12 10-11 10-10 10-9 10-8 10-70
5000
10000
15000
20000
25000
30000
35000
40000 bio - Ag 3.3 nM
bio - Ag 5 nM
bio - Ag 10 nM
AlphaScreen Assay Format AlphaScreen Assay Format ELISA Assay FormatELISA Assay Format
3 Feasibility of AlphaScreen for Hybridoma Primary Screen
Antibody [M]
Alp
ha S
igna
l
Although a hooking effect is observed, a broad detectable concentration range of 2.5 log would be sufficient for identification of antibodies in hybri-doma supernatants where antibody concentrations are unknown and broad.
4 Hybridoma Primary Screen
Neat Supernatants
Diluted Supernatants
Sample #
Alp
ha S
igna
lA
lpha
Sig
nal
Sample #
Due to a hooking effect, dilution of the samples resulted in higher RLU values and higher hit numbers.
Due to higher sensitivity of AlphaScreen, primary screens can be per-formed with diluted hybridoma supernatants.
6 Hit Comparison Between ELISA and AlphaScreen
5 AlphaScreen and ELISA Comparison
Alpha ELISA
Solution interaction Solid phase interaction
Biotinylated antigen Unmodified antigenUnmodified antigen
Homogeneous, simple Homogeneous, simple and fast
Multi-washing steps, long proce-dure
High sensitivityHigh sensitivity Less sensitivity
Use less antigenUse less antigen Use more antigen
Reagent cost, ~$0.06/well Reagent cost, ~$0.06/well
Hooking effect No hooking effectNo hooking effect
-SA Bio AgD -SA Bio Ag-SA Bio Ag
Aanti-hu-kappa anti-hu-lambda
hybridoma
10-14 10-13 10-12 10-11 10-10 10-9 10-8 10-70
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000bio- Ag 10 nM
bio- Ag 5 nM
bio- Ag 3.3 nM
kappa-antibody [M]
Alp
ha S
igna
l
Kappa Kappa Detection
Serum Titers – Beads-Washing Protocol
0
10000
20000
30000
40000
50000
60000
70000 mouse 1
mouse 2
mouse 3
10-6 10-5 10-4 10-3 10-2 10-1
Donor beads + bio-AgDonor beads + bio-Ag 15 minMouse serum 2 hoursWash x 1Acceptor beads 2 hoursRead signal
Antibody Quantification
XenoMouse® Serum Dilution
Alp
ha S
igna
l
- SAD
BioA
hybridoma
anti-hlgG-Fc anti-hlgG-Fc
10-3 10-2 10 100 101 102 103
2000
4000
10-10
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
- SAD Bio Ag A
hybridoma
anti-hlgG-Fcbindingantibody
SAD Bio Protein A A
hybridoma
Protein B
--
Assay Sensitivity for Blocking Protein-Protein Interactions
AlphaScreenAlphaScreen
ELISA
Hybridoma Competitor [M]
Hybridoma Competitor [M]
OD
650
Alp
ha S
igna
l
AlphaScreen assays are more sensitive than ELISA. AlphaScreen was performed with 0.1 nM of binding antibody, ELISA with
0.67 nM of binding antibody.
Hit Comparison Between ELISA and AlphaScreen
Total 148 samples screened: identified 23 ELISA positives and 55 Alpha positives.
All ELISA hits were a subset of Alpha hits.
Alpha identified more hits due to higher assay sensitivity.
Conclusions
AlphaScreen provides a robust and homogeneous alternative assay to ELISA. The advantages for AlphaScreen assay are solution-based, high sensitivity, less antigen required, and high throughput.
Feasibility for serum titer, antibody light chain determination, and IgG quan-tification has been demonstrated using AlphaScreen.
AlphaScreen can also be used to characterize the ability of antibodies to block protein-protein interactions.
In summary, AlphaScreen technology is very useful for antibody screening and characterization.
Society for Biomolecular Screening10th Anniversary Conference and Exhibition
September 11-15, 2004
2 Assay ProtocolsAlphaScreen Assay ProtocolAlphaScreen Assay Protocol ELISA Assay ProtocolELISA Assay Protocol
Total Samples
ELISAHits
Alpha Hits
ELISA/Alpha common
% of ELISA/Alpha overlap
Target 1 Primary Screen
3760 172 129 79
Confirmation screen
122 ND 77 63%
Target 2 Primary Screen
6110 374 338 226
Confirmation screen
263 ND 212 81%
Target ATarget A
Target BTarget B
Sample #
Alp
ha S
igna
l
Hits are clearly identified due to tight background.
Similar result was observed for lambda detection. AlphaScreen is suitable for antibody light chain detection.
Serum contains interfering components for AlphaScreen signal; therefore, serum testing requires a washing step.
Antibody [ng/ml]
Alp
ha S
igna
l
IgG Standard IgG Standard Curve
Assay can be used to determine total IgG (antigen-specific and non-specif-ic) concentrations in hybridoma supernatants
Standard curves were generated using purified IgGs Unknown antibody concentrations were calculated according to the stan-
dard curves
10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60
2000
4000
6000
8000
10000
12000
10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60.0
0.2
0.4
0.6
0.8
1.0
ELISA
AlphaAlpha
% In
hibi
tion
Samples
% In
hibi
tion
Samples
IC50 = 0.07 nM
IC50 = 9 nM
