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National Center for Emerging and Zoonotic Infectious Diseases
Division of Scientific Resources / Scientific Products and Support Branch
Presenter
Jason Goldstein PhDImmunochemistry Team Leader
Scientific Products and Support Branch
BLI Studies of Anthrax Toxin and Development of Diagnostic Antibodies
• Anthrax is caused by infection with Bacillus anthracis, a spore‐forming, rod‐shaped bacterium
• Spores are highly resistant to environmental conditions and gain entry via open wound (cutaneous) or ingestion (gastrointestinal disease or inhalation anthrax) which can progress into systemic infection leading to shock, respiratory failure and death
• In the U.S. bioterrorism attacks of 2001, inhalation anthrax had a 45% fatality rate despite antibiotic treatment and aggressive supportive care of the patients
• Development of a safe and effective vaccine for anthrax is vital to the health and safety of those at risk of exposure and an essential component of any bioterrorism defense strategy
• Need exists for rapid point‐of‐care diagnostic enabling early diagnosis and treatment
• Toxins are key targets and anti‐toxin mAbs can serve as diagnostic and therapeutic tools for those at risk of acquiring disease
Anthrax Toxin Model
Maldonado‐Arocho et al. (2009). "Anthrax Toxin". Microbial Toxins: Current Research and Future Trends.
•Microbiological•PCR based•Immunofluorescent (DFA; IHC)•Immunoassay (ELISA; MSD; Luminex)•Indirect Enzymatic Assay•Diagnostic POC device
Diagnostics
Begin with blocked Ab‐coated magnetic beads
Incubate LF in plasma (matrix) with beads (1h)
Wash beads to remove matrix (4 min)
Incubate beads with buffer and substrate(2‐20h)
Analyze supernatant by mass spectrometry
Mass‐Spec Lethal Factor Assay
<4 h Total Time for 1stResponse
Detection and Quantification of Anthrax Lethal Factor in Serum by Mass SpectrometryAnne E. Boyer et.al. Anal. Chem. 2007, 79, 8463‐8470
*1 ng LF
1000 1400 1800 2200 2600 3000
Mass (m/z)
7357.0
0
100
% In
tens
ity
1232.5
1589.8 2804.2*
No Toxin
1000 3000
8030.6
0
100%
Inte
nsity
1402.1
2804.2
Detection of Lethal Factor ActivityMass/chargeLF
SKARRKKVYPYPXENFPPSTARPT
SKARRKKVYPYPXENFPPSTARPT
2804.21232.5
1589.8
Detection and Quantification of Anthrax Lethal Factor in Serum by Mass SpectrometryAnne E. Boyer et.al. Anal. Chem. 2007, 79, 8463‐8470
Lethal Factor
• LF is a zinc‐dependent endoprotease known to target the amino‐terminus of the mitogen‐activated protein kinase kinase (MAPKK) family of response regulators
• 4 domains with specific functions
• 90kDa highly immunogenic in mice
• Immunization BalbC (10ug IV)
• Sp2 fusion and HAT selection stable parent hybridomas
White Light ImageM12002 LFG2 Parent 4C2
Automated Clone Selection
Anti‐IgG‐FITC ImageM12002 LFG2 Parent 4C2
Automated Clone Selection
Screen of anti‐LF Hybridoma SupsLOADr‐LF‐biotin
ASSOChybridomasup (1‐7)
DISSOCPBS
ASSOChybridomasup (8‐15)
DISSOCPBS
BLI Screen for anti‐LF mAb
SA sensor‐15ug/ml LF‐undiluted media
4C2:1C1
LFG2:4B10
4C2:1B11D1:1A5
3D1:1B2
2C6:1C3
3E4:1G9 anti‐PA3C3:1C6
1F4
1C8
1G6
2A6
4C2:1C1
4C2:1B1
2C6:1C3
3D1:1F4
3F3:2A6
1D1:1G63C3:1C8
3E4:1G9 anti‐PA
Panel of 10 Novel anti‐LF mAbs
4C2:1C1
4C2:B1
0
100000
200000
300000
400000
500000
600000
700000
0 200 400 600 800 1000 1200 1400 1600 1800 2000
MSD
Cou
nts
ng/mL Anti‐LF mAB
MSD Analysis
4C2:1C1
4C2:1C1*
4C2:1B1
NIH LF 9
3D1:1F2
1D1:1G1
2C6:1F4
LFG2:4B10
NIH LF 6
LFG2:3D10
3C3:1D3
PAG1 3C6
r‐LF Coated 100 ng/well
From Conrad Quinn MVBDB/DBD/NCIRD
Primary mAbassociation
Primary mAbassociation
Primary mAbassociation
Primary mAbassociation
Primary mAbassociation
Primary mAbassociation
Primary mAbassociation
2C6:1B4 mAbassociation
2C6:1F4 mAbassociation
3C3:1A3 mAbassociation
2C6:1H4 mAbassociation
2C6:1BF5 mAbassociation
3C3:1D3 mAbassociation
3C3:1A1 mAbassociation
Epitope Binning with High Affinity mAb Clones
SA sensor‐15ug/ml b‐r LF‐300nM purified IgG
Response to LF 4C2:1C1 4C2:1B1 3C3:1F5 1D1:1A5 2C6:1C3 3D1:1B2 3D1:1C2
4C2:1C1 2.035 0.2903 0.1981 0.4615 0.4064 0.3304 0.3218 0.20914C2:1B1 1.8202 0.3788 0.2777 0.4969 0.4361 0.3624 0.3635 0.2713C3:1F5 0.5113 1.5511 1.3692 0.2643 0.4439 0.3634 0.3626 0.2651D1:1A5 1.3584 1.3192 1.3127 0.5042 0.2598 0.3541 0.3617 0.25142C6:1C3 0.5816 1.5962 1.4197 0.5703 0.4898 0.2414 0.2377 0.15023D1:1B2 0.5973 1.6132 1.4967 0.5779 0.5188 0.254 0.2656 0.15223D1:1C2 0.5504 1.5259 1.3973 0.5481 0.4829 0.2487 0.2547 0.1512LFG2‐4B10 1.6967 1.3832 1.4946 0.5418 0.2598 0.3952 0.3748 0.2749
2C6:1B4 2C6:1F4 2C6:1H4 2C6:1F5 3C3:1C6 3C3:1A1 3C3:1A32C6:1B4 0.634 0.1734 0.2533 0.177 0.2116 0.4747 0.4374 0.56672C6:1F4 0.6966 0.1826 0.2562 0.1753 0.2249 0.5101 0.4641 0.58692C6:1H4 0.5438 0.2183 0.3404 0.2327 0.2817 0.5077 0.472 0.58152C6:1F5 0.5961 0.1836 0.2685 0.1844 0.2355 0.4744 0.4292 0.56743C3:1C6 0.5435 0.3128 0.5724 0.362 0.4493 0.276 0.2363 0.30523C3:1A1 0.4914 0.2888 0.5733 0.353 0.425 0.2804 0.2467 0.32043C3:1A3 0.5125 0.2662 0.5473 0.3306 0.4047 0.2215 0.2091 0.2676LFG2‐4B10 1.6444 0.3262 0.5676 0.3732 0.4247 0.447 0.3972 0.4972
Primary mAb Secondary mAb
Epitope Binning Chart
Lethal Toxin (LTx)
•LTx toxin of B. anthracis are binary combinations of protective antigen (PA) with lethal factor (LF)
•Protective Antigen (PA) is the cell binding component of toxin and is responsible for bringing the catalytic LF into host cells
•PA63 forms an oligomer comprising up to seven molecules of PA63 and is responsible for toxin internalization
• Combines with LF which can trimerize form lethal toxin (LTx) complex >700kDa
LTx
FreePA63
Free LF
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
0 500 1000 1500 2000 2500
MSD
Cou
nts
ng/mL Anti‐LF mAb
MSD Analysis
PAG1 3C6
4C2:1C1
4C2:1C1*
4C2:1B1
1D1:1G1
LFG2 4B10
LFG2:3D10
NIH LF 6
3D1:1F2
2C6:1F4
NIH LF 9
3C3:1D3
r‐LTx Coated 100 ng/wellFrom Conrad Quinn MVBDB/DBD/NCIRD
0
100000
200000
300000
400000
500000
600000
700000
800000
0 500 1000 1500 2000 2500
MSD
Cou
nts
ng/mL Anti‐LF mAb
LTx Coated
4C2:1C1
3D1:1F2
2C6:1F4
3C3:1D3
0
100000
200000
300000
400000
500000
600000
700000
0 500 1000 1500 2000
MSD
Cou
nts
ng/mL Anti‐LF mAB
LF Coated
4C2:1C1
3D1:1F2
2C6:1F4
3C3:1D3
From Conrad Quinn MVBDB/DBD/NCIRD
4C2:1C1
3D1:1F2
2C6:1F43D1:1F23C3:1D3
Antibody Response to LTx Complex by BLI
LTx Toxin Build
PA63 LF
mAb Properties for Diagnostic
1. Sensitivity: mAb recognition of LF antigen at low levels
2. Immunoprecipitation (IP): LF component recognized in LTx by mAb‐bound to magnetic protein‐G beads for enzymatic analysis
3. Enhance Proteolytic Activity: Enhancement of LF catalytic activity throughmAb/epitope Interaction
4. Specificity: Detection LF epitope w/wo LTx confirmation
Immunoblot Detection Predicts Linear Epitope for anti‐LFG2:4B10 mAb
LFG2:4B10
LFG2:3D10
LFG2:4B10 Increases Rate of Catalysis for LF
From Anne Boyer CCB/DLS/NCEH
mAb Epitope Maps to Accessible Loop Proximal to LF Catalytic Pocket
Peptide Loading25ug/ml
mAb LFG2:4B10 Kon mAb LFG2:4B10 Koff
PBSbaseline
LF predicted sequence (7aa)
non‐specific peptide
Epitope PhageDisplay Peptide (7aa)
Epitope Peptide Binding to anti‐LFG2:4B10 mAb
Multivalancy Monoclonal LF Toxin Capture
LTx
FreePA63
Free LF
PA‐2mAb
LFmAb
PA‐1mAb
From Conrad Quinn MVBDB/DBD/NCIRD
Anthrax Toxin Detection POC Diagnostic
Label Free (BLI) Study of LF Toxin Complex
LOADmAb anti‐PA13E4:1G9
B
ASSOCPA63
DISSOCPA63 ASSOC
LF DISSOC
LF
ASSOCanti‐LFLFG2:4B10
DISSOC
SATURATIONanti‐LF
LFG2:4B10
ASSOCanti‐PA23C6:2E9
DISSOC
Titration of anti‐LF mAb
500nM
166nM
LTx Build
56nM18nM6nM
ASSOCanti‐LFLFG2:4B10
DISSOC
Sensor Location Sensor Info
Baseline Loc.
Assoc. (Sample) Loc. Sample ID Dissoc. Loc. Conc. (nM) Response KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error Rmax Rmax Error kobs(1/s) Req
Req/Rmax(%) Full X^2 Full R^2
A7 AVR1162‐biotin A7 AVR1674 A8 500 0.4466 4.79E‐09 2.92E+04 1.51E+03 1.40E‐04 3.99E‐05 0.4073 0.0046 1.48E‐02 0.4034 99 0.075145 0.829905
B7 AVR1162‐biotin B7 AVR1674 B8 166 0.2577 1.40E‐09 1.14E+05 3.14E+03 1.60E‐04 1.95E‐05 0.2553 0.0013 1.91E‐02 0.2532 99.2 0.007403 0.95758
C7 AVR1162‐biotin C7 AVR1674 C8 56 0.2156 3.43E‐10 1.65E+05 4.79E+03 5.65E‐05 2.42E‐05 0.2291 0.002 9.30E‐03 0.2278 99.4 0.007753 0.969468
D7 AVR1162‐biotin D7 AVR1674 D8 18 0.1048 1.69E‐09 1.88E+05 1.26E+04 3.18E‐04 3.84E‐05 0.1319 0.0052 3.70E‐03 0.1205 91.4 0.003061 0.959953E7 AVR1162‐biotin E7 AVR1674 E8 6 0.0022 5.91E‐07 1.09E+04 1.40E+06 6.43E‐03 3.62E‐03 0.5062 64.6666 6.49E‐03 0.0051 1 0.003588 0.22081
F7 AVR1162‐biotin F7 AVR1674 F8 56 0.187 2.00E‐09 1.71E+05 1.75E+03 3.42E‐04 8.80E‐06 0.1948 0.0006 9.92E‐03 0.188 96.5 0.000623 0.99599
G7 AVR1162‐biotin G7 AVR1674 G8 0 ‐0.0131H7 AVR1162‐biotin H7 AVR1674 H8 0 ‐0.0281
Sensor Location Sensor Info
Baseline Loc.
Assoc. (Sample) Loc. Sample ID Dissoc. Loc. Conc. (nM) Response KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error Rmax Rmax Error kobs(1/s) Req
Req/Rmax(%) Full X^2 Full R^2
A7 AVR1162‐biotin A7 AVR1674 A8 500 0.4466 8.23E‐10 9.78E+04 4.32E+03 8.05E‐05 2.57E‐05 0.3841 0.0026 4.90E‐02 0.3835 99.8 0.240155 0.961715
B7 AVR1162‐biotin B7 AVR1674 B8 166 0.2577 8.23E‐10 9.78E+04 4.32E+03 8.05E‐05 2.57E‐05 0.2537 0.0026 1.63E‐02 0.2524 99.5 0.240155 0.961715
C7 AVR1162‐biotin C7 AVR1674 C8 56 0.2156 8.23E‐10 9.78E+04 4.32E+03 8.05E‐05 2.57E‐05 0.2544 0.0047 5.56E‐03 0.2507 98.5 0.240155 0.961715
D7 AVR1162‐biotin D7 AVR1674 D8 18 0.1048 8.23E‐10 9.78E+04 4.32E+03 8.05E‐05 2.57E‐05 0.1744 0.0072 1.84E‐03 0.1668 95.6 0.240155 0.961715
Affinity Constant for anti‐LFG2:4B10 mAb
LOCAL
GLOBAL
KD=0.823 nM
LTx Build
Anti‐LF Binding and Saturation
ASSOCanti‐PAPAG2 3C6:2E9
DISSOC
Titration of anti‐LF mAb
1000nM333nM
111nM
37nM
12nM
4nM
Sensor Location Sensor Info Baseline Loc.
Assoc. (Sample) Loc. Sample ID Dissoc. Loc. Conc. (nM) Response KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error Rmax Rmax Error kobs(1/s) Req
Req/Rmax(%) Full X^2 Full R^2
A7 AVR1162‐biotin A11 AVR1046 A12 1000 1.1425 4.26E‐09 3.15E+04 1.79E+03 1.34E‐04 3.22E‐05 1.083 0.0081 3.16E‐02 1.0784 99.6 0.394374 0.779291
B7 AVR1162‐biotin B11 AVR1046 B12 333 1.2508 6.40E‐10 8.66E+04 4.59E+03 5.55E‐05 3.06E‐05 1.1754 0.0086 2.89E‐02 1.1732 99.8 0.44155 0.81798
C7 AVR1162‐biotin C11 AVR1046 C12 111 1.2515 4.31E‐10 2.05E+05 7.51E+03 8.84E‐05 2.37E‐05 1.1891 0.007 2.28E‐02 1.1845 99.6 0.257847 0.919521
D7 AVR1162‐biotin D11 AVR1046 D12 37 0.8698 5.68E‐10 2.72E+05 2.46E+03 1.54E‐04 7.60E‐06 0.8698 0.0023 1.02E‐02 0.8566 98.5 0.010612 0.99679
E7 AVR1162‐biotin E11 AVR1046 E12 12 0.5084 1.52E‐11 2.81E+05 3.84E+04 4.27E‐06 7.41E‐05 0.5407 0.0418 3.38E‐03 0.54 99.9 0.103913 0.900787
F7 AVR1162‐biotin F11 AVR1046 F12 4 0.1117 5.61E‐11 7.90E+05 1.10E+05 4.43E‐05 7.22E‐05 0.1238 0.0101 3.20E‐03 0.1221 98.6 0.006723 0.907865
G7 AVR1162‐biotin G11 AVR1046 G12 0 1.2911
H7 AVR1162‐biotin H11 AVR1046 H12 0 ‐0.0117
Sensor Location Sensor Info
Baseline Loc.
Assoc. (Sample) Loc. Sample ID Dissoc. Loc. Conc. (nM) Response KD (M) kon(1/Ms) kon Error kdis(1/s) kdis Error Rmax Rmax Error kobs(1/s) Req
Req/Rmax(%) Full X^2 Full R^2
A7 AVR1162‐biotin A11 AVR1046 A12 1000 1.1425 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 1.0448 0.0055 2.01E‐01 1.0445 100 2.341059 0.981921
B7 AVR1162‐biotin B11 AVR1046 B12 333 1.2508 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 1.1564 0.0058 6.71E‐02 1.1553 99.9 2.341059 0.981921
C7 AVR1162‐biotin C11 AVR1046 C12 111 1.2515 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 1.1851 0.0065 2.24E‐02 1.1816 99.7 2.341059 0.981921
D7 AVR1162‐biotin D11 AVR1046 D12 37 0.8698 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 0.8876 0.0087 7.51E‐03 0.8796 99.1 2.341059 0.981921
E7 AVR1162‐biotin E11 AVR1046 E12 12 0.5084 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 0.719 0.016 2.48E‐03 0.6995 97.3 2.341059 0.981921
F7 AVR1162‐biotin F11 AVR1046 F12 4 0.1117 3.35E‐10 2.01E+05 5.88E+03 6.74E‐05 1.53E‐05 0.3387 0.021 8.72E‐04 0.3125 92.3 2.341059 0.981921
Affinity Constant for anti‐PA2 3C6:2E9 mAb
LOCAL
GLOBAL
KD=0.335 nM
Study Conclusions
• Novel Panel of anti‐LF mAbs identified and characterized
• BLI studies using Octet technology were employed in discovery process under several binding orientations
• Distinguish mAb specificity between LF and protein complex LTx
• Data generated from BLI correlated with traditional binding studies
• Stepwise binding and kinetics of antigen‐Ab complex determined with Octet
• Immunodiagnostic tools available for improved detection of early Anthrax infection
For more information please contact Centers for Disease Control and Prevention
1600 Clifton Road NE, Atlanta, GA 30333Telephone, 1‐800‐CDC‐INFO (232‐4636)/TTY: 1‐888‐232‐6348E‐mail: cdcinfo@cdc.gov Web: www.cdc.gov
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
Immunochemistry Team
National Center for Emerging and Zoonotic Infectious Diseases
Division of Scientific Resources / Scientific Products and Support Branch
Uzma AnsariKiosy LinAmanda LyonsR. Lee PittsXiaoling TangStacey Sweat
Scientific Products and Support Branch
Dennis Bagarozzi Jr.Curtis Taylor
Meningitis and Vaccine Preventable Diseases Branch/NCIRD
Conrad QuinnNazia Kamal
Clinical Chemistry Branch/NCEH
Anne Boyer
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