National Center for Emerging and Zoonotic Infectious Diseases Division of Scientific Resources / Scientific Products and Support Branch Presenter Jason Goldstein PhD Immunochemistry Team Leader Scientific Products and Support Branch BLI Studies of Anthrax Toxin and Development of Diagnostic Antibodies
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BLI Studies of Anthrax Toxin and Development of Diagnostic … · 2020-02-22 · • Anthrax is caused by infection with Bacillus anthracis, a spore‐forming, rod‐shaped bacterium
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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.
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
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