Post on 13-Mar-2020
Improving National Detection Capabilities – German Activities:1st International Proficiency Test on the Analysis of Ricin
Brigitte G. Dorner, Ph.D.Center for Biological Security, Microbial ToxinsRobert Koch-Institut, Berlin, Germany
Biological Weapons Convention, Expert MeetingGeneva, 26th August 2010
• Australia Group agent
• Biological Weapons Convention effective since 1975
• Chemical Weapons Convention: effective since 1993ricin: a schedule 1 list “toxic chemical”
OPCW regulations on handling and transport of ricin
• CDC: Category B agent
Ricin: a substance of special interest
Dual use character of ricin
Military, terrorist and criminal use of ricin
Selected examples:• Research on the potential military use of ricin• Assessination of bulgarian dissident G. Markov (1978)• Iraq admits the production of ricin (1995)• Ricin findings in islamic militants' camp (2002)• Ricin letters sent to the US White House and Senate (2003/2004)• A number of cases worldwide where the production and possession of ricin
have been documented
© FBI Press Release
Dual use character of ricin
Military, terrorist and criminal use of ricin
Selected examples:• Research on the potential military use of ricin• Assessination of bulgarian dissident G. Markov (1978)• Iraq admits the production of ricin (1995)• Ricin findings in islamic militants' camp (2002)• Ricin letters sent to the US White House and Senate (2003/2004)• A number of cases worldwide where the production and possession of ricin
have been documented
Use of the toxin-producing plant for the industrial production of castor oil• Pharmaceutical products, cosmetics; technical applications (lubricants, nylon)• Castor meal: use as organic fertilizer
© FBI Press Release
Dual use character of ricin
Military, terrorist and criminal use of ricin
Selected examples:• Research on the potential military use of ricin• Assessination of bulgarian dissident G. Markov (1978)• Iraq admits the production of ricin (1995)• Ricin findings in islamic militants' camp (2002)• Ricin letters sent to the US White House and Senate (2003/2004)• A number of cases worldwide where the production and possession of ricin
have been documented
Use of the toxin-producing plant for the industrial production of castor oil• Pharmaceutical products, cosmetics; technical applications (lubricants, nylon)• Castor meal: use as organic fertilizer
Medical applications: ricin as immunotoxin in the treatment of cancer
© FBI Press Release
Ricin as a potential biowarfare agent
• Availability of the ricin producing plant Ricinus communis• Plant is used at an industrial scale for the production of castor oil
(1,300,000 t per year) accessibility to raw material
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Ricin as a potential biowarfare agent
• Availability of the ricin producing plant Ricinus communis• Plant is used at an industrial scale for the production of castor oil
(1,300,000 t per year) accessibility to raw material
• Ricin can be produced easily from the plant (“ricin recipes” in open sources)
©G
.Lei
nz, A
lber
ding
k B
oley
Ricin as a potential biowarfare agent
• Availability of the ricin producing plant Ricinus communis• Plant is used at an industrial scale for the production of castor oil
(1,300,000 t per year) accessibility to raw material
• Ricin can be produced easily from the plant (“ricin recipes” in open sources)• Ricin has quite a high resistance to physico-chemical stress
©G
.Lei
nz, A
lber
ding
k B
oley
Ricin as a potential biowarfare agent
• Availability of the ricin producing plant Ricinus communis• Plant is used at an industrial scale for the production of castor oil
(1,300,000 t per year) accessibility to raw material
• Ricin can be produced easily from the plant (“ricin recipes” in open sources)• Ricin has quite a high resistance to physico-chemical stress• No therapeutic countermeasures available so far
©G
.Lei
nz, A
lber
ding
k B
oley
Ricin as a potential biowarfare agent
• Availability of the ricin producing plant Ricinus communis• Plant is used at an industrial scale for the production of castor oil
(1,300,000 t per year) accessibility to raw material
• Ricin can be produced easily from the plant (“ricin recipes” in open sources)• Ricin has quite a high resistance to physico-chemical stress• No therapeutic countermeasures available so far• Ricin intoxication is not communicable• Potential use for “small-scale operations” in order to produce panic
©G
.Lei
nz, A
lber
ding
k B
oley
• Toxins are active in the absence of the producing organism and its genetic information
detection of nucleic acid not sufficientdetection of protein necessary
Detection of proteotoxins: a challenge
• Toxins are active in the absence of the producing organism and its genetic information
detection of nucleic acid not sufficientdetection of protein necessary
• Very high toxicity is per se a challenge for detection technology and demands for detection down to a few pg/mL
Detection of proteotoxins: a challenge
• Toxins are active in the absence of the producing organism and its genetic information
detection of nucleic acid not sufficientdetection of protein necessary
• Very high toxicity is per se a challenge for detection technology and demands for detection down to a few pg/mL
• Toxins are often produced in different variants or isoforms which differ in their characteristics
e.g. ricin:produced in different highly similar isolectins with different glycosylation pattern and isoelectric point, together with structurally similar agglutinin
Detection of proteotoxins: a challenge
Ricin, a toxin from R. communis
Ricinus communis
Toxicity of ricin (LD50 ): 3 µg/kg i.v. and 1-20 mg/kg orally
Ricin, a toxin from R. communis
Ricinus communis
„Ricin D“
Ricin (RCA60)hybrid B chain!
A B „Ricin E“
Ricin (RCA60)A B
Toxicity of ricin (LD50 ): 3 µg/kg i.v. and 1-20 mg/kg orally
Rutenber et al., 1994
Ricin, a toxin from R. communis
Ricinus communis
94% identity
A A
85% identity
B B
Agglutinin (RCA120)A BA B
„Ricin D“
Ricin (RCA60)hybrid B chain!
A B „Ricin E“
Ricin (RCA60)A B
Toxicity of ricin (LD50 ): 3 µg/kg i.v. and 1-20 mg/kg orally
Rutenber et al., 1994
protein
DNA
1171
.7
933.
6
2258
.4
1728
.3
1171
.7
1012
.8
2258
.3
1309
.7
1728
.2
934.
9
1580
.3
3442
.3
1000
1000
020
0040
000
020
000
6000
1500 2000m/z [D]
inte
nsity
[AU
]
Ricinaufschluss(Trypsin, 372 amol)
Ricinaufschluss(Trypsin, 14.9 amol)
2500 3000 3500
spectrometricmethods
PCR-basedmethods
functionalmethods
immunological methods
Diagnostics of ricin
protein
DNA
1171
.7
933.
6
2258
.4
1728
.3
1171
.7
1012
.8
2258
.3
1309
.7
1728
.2
934.
9
1580
.3
3442
.3
1000
1000
020
0040
000
020
000
6000
1500 2000m/z [D]
inte
nsity
[AU
]
Ricinaufschluss(Trypsin, 372 amol)
Ricinaufschluss(Trypsin, 14.9 amol)
2500 3000 3500
spektrometrischeMethoden
Diagnostics of ricin
1171
.7
933.
6
2258
.4
1728
.3
1171
.7
1012
.8
2258
.3
1309
.7
1728
.2
934.
9
1580
.3
3442
.3
1000
1000
020
0040
000
020
000
6000
1500 2000m/z [D]
inte
nsity
[AU
]
Ricinaufschluss(Trypsin, 372 amol)
Ricinaufschluss(Trypsin, 14.9 amol)
2500 3000 3500
Background of the 1st ricin proficiency test
• 1st Ricin Workshop in the framework of the GHSAG Lab Network (Nov 2007)
• One major outcome of this theoretical workshop was the identification of open questions regarding technical and standardization issues
• Major gap: lack of quality control measures
Background of the 1st ricin proficiency test
• 1st Ricin Workshop in the framework of the GHSAG Lab Network (Nov 2007)
• One major outcome of this theoretical workshop was the identification of open questions regarding technical and standardization issues
• Major gap: lack of quality control measures
• Parallel development: Interest from experts of the OPCW temporary working group “sampling and analysis” to take part in a ricin proficiency test
1st international proficiency test on the analysis of ricin
Framework: Global Health Security Action Group (GHSAG)OPCW Designated Laboratories (Temporary Working Group)
Time:October 2009; Participants’ Workshop: 30 Nov / 1 Dec 2009
17 participants (12 countries)
both from the health and the military sector
Concept of the ricin proficiency test
• Major goal: to obtain information on the status quo of ricin detection (qualitative and quantitative) in expert laboratories
Concept of the ricin proficiency test
• Major goal: to obtain information on the status quo of ricin detection (qualitative and quantitative) in expert laboratories
• Open with respect to methods applied
Concept of the ricin proficiency test
• Major goal: to obtain information on the status quo of ricin detection (qualitative and quantitative) in expert laboratories
• Open with respect to methods applied
• Sample material containing purified ricin in buffer (mg/mL to pg/mL-range, 6 liquid samples)
• Discrimination pure ricin and crude R. communis extract (RCA60 / RCA120)
Concept of the ricin proficiency test
• Major goal: to obtain information on the status quo of ricin detection (qualitative and quantitative) in expert laboratories
• Open with respect to methods applied
• Sample material containing purified ricin in buffer (mg/mL to pg/mL-range, 6 liquid samples)
• Discrimination pure ricin and crude R. communis extract (RCA60 / RCA120)
• No complex matrices, no test for active versus inactive material
Concept of the ricin proficiency test
• Major goal: to obtain information on the status quo of ricin detection (qualitative and quantitative) in expert laboratories
• Open with respect to methods applied
• Sample material containing purified ricin in buffer (mg/mL to pg/mL-range, 6 liquid samples)
• Discrimination pure ricin and crude R. communis extract (RCA60 / RCA120)
• No complex matrices, no test for active versus inactive material
• Extensive paperwork necessary !!!in Germany: Federal Office of Economics and Export Control
notification of OPCW, Ministry of Health, Federal Foreign OfficeFederal Ministry of Economics and Technology
• Laboratories from 2 countries had to decline from participating due to administrative constraints
Preparation of a highly pure ricin standard
crude extractR. communis
affinitychromatography
size exclusionchromatography
ELISA cytotoxicitySDS-PAGE mass spectr.
characterization
1
2
result: purity of ricin > 97%, material is functionally active
extraction
Shipment of samples
Temperature logger
Canada, 8.10.09 / 9:05
Finland, 7.10.09 / 9:10
Australia, 9.10.09 / 11:20
South Africa, 8.10.09 / 17:15
China, 15.10.09 / 18:50
USA, 9.10.09 / 16:45
Results of the 1st ricin proficiency test
• Very useful and informative exercise; results are treated confidentially within the group of participants
Results of the 1st ricin proficiency test
• Very useful and informative exercise; results are treated confidentially within the group of participants
• It was possible to show the advantages and disadvantages of individual methods:
Results of the 1st ricin proficiency test
• Very useful and informative exercise; results are treated confidentially within the group of participants
• It was possible to show the advantages and disadvantages of individual methods:
insufficient assignment
Laboratory P11 Sample1 2 3 4 5 6
chromatographic methodimmunological method 1spectrometric methodmolecular biological methodfunctional methodimmunological method 2
combination of all methods
ricin detectedcompletely correct
colour coding:
Results of the 1st ricin proficiency test
• Very useful and informative exercise; results are treated confidentially within the group of participants
• It was possible to show the advantages and disadvantages of individual methods:
insufficient assignment
Laboratory P11 Sample1 2 3 4 5 6
chromatographic methodimmunological method 1spectrometric methodmolecular biological methodfunctional methodimmunological method 2
combination of all methods
ricin detectedcompletely correct
colour coding:
Results of the 1st ricin proficiency test
• Overall, acceptable performance of the labs, however, deficits could be clearly identified!
Results of the 1st ricin proficiency test
• Overall, acceptable performance of the labs, however, deficits could be clearly identified!
• Example: 4 out of 17 laboratories (23.5%) correctly identified all samples on a qualitative basis11 out of 17 laboratories (65%) correctly identified the crude R. communisextract
Results of the 1st ricin proficiency test
• Overall, acceptable performance of the labs, however, deficits could be clearly identified!
• Example: 4 out of 17 laboratories (23.5%) correctly identified all samples on a qualitative basis11 out of 17 laboratories (65%) correctly identified the crude R. communisextract
• Results’ workshop held at the RKI in November 2009: very productive discussion about optimal ricin detection scheme
Results of the 1st ricin proficiency test
• Overall, acceptable performance of the labs, however, deficits could be clearly identified!
• Example: 4 out of 17 laboratories (23.5%) correctly identified all samples on a qualitative basis11 out of 17 laboratories (65%) correctly identified the crude R. communisextract
• Results’ workshop held at the RKI in November 2009: very productive discussion about optimal ricin detection scheme
• A consensus pure ricin standard, which was measured during the exercise, was distributed among interested participants for their quality assurancepurposes (March 2010)
Results of the 1st ricin proficiency test
• Overall, acceptable performance of the labs, however, deficits could be clearly identified!
• Example: 4 out of 17 laboratories (23.5%) correctly identified all samples on a qualitative basis11 out of 17 laboratories (65%) correctly identified the crude R. communisextract
• Results’ workshop held at the RKI in November 2009: very productive discussion about optimal ricin detection scheme
• A consensus pure ricin standard, which was measured during the exercise, was distributed among interested participants for their quality assurancepurposes (March 2010)
• An exchange of information on the different methods has been initiated on a voluntary basis among participants
Outlook
• For the future, it is absolutely necessary to repeat this kind of exercise, provided appropriate funding is obtained
• Further improvement of qualitative and quantitative results on ricin detection in expert laboratories
Outlook
• For the future, it is absolutely necessary to repeat this kind of exercise, provided appropriate funding is obtained
• Further improvement of qualitative and quantitative results on ricin detection in expert laboratories
• Further tasks: detection of ricin from complex matrices, discrimination ofactive versus inactive material
• Time line of analysis has not been considered in the current proficiency test
• Proficiency tests on different biological toxins: e.g. botulinum toxins
CHEMSTAT, SwitzerlandDr. Werner Luginbühl
Spiez Laboratory, SwitzerlandDipl. Chem. Marc-André Avondet
Acknowledgements
Robert Koch-Institut, GermanyDr. Sylvia WorbsDr. Diana PaulyDr. Martin DornerDipl. Chem. Skadi KullSebastian KirchnerOlena ShatohinaJutta RybarczykJacek Millert