Improving National Detection Capabilities – German ...httpAssets...•Research on the potential military use of ricin • Assessination of bulgarian dissident G. Markov (1978) •

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





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






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


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

©G

.Lei

nz, A

lber

ding

k B

oley




• Ricin can be produced easily from the plant (“ricin recipes” in open sources)

©G

.Lei

nz, A

lber

ding

k B

oley




• 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 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 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



• Very high toxicity is per se a challenge for detection technology and demands for detection down to a few pg/mL




• 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


Ricin, a toxin from R. communis

Ricinus communis

Toxicity of ricin (LD50 ): 3 µg/kg i.v. and 1-20 mg/kg orally


Ricinus communis

„Ricin D“

Ricin (RCA60)hybrid B chain!

A B „Ricin E“

Ricin (RCA60)A B


Rutenber et al., 1994


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


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

]



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

]



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



• 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






• 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



• 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:




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:


• 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)





• 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

Documents

Improving National Detection Capabilities – German ...httpAssets...•Research on the potential military use of ricin • Assessination of bulgarian dissident G. Markov (1978) •