27JUN07

Parasitological and serological methods

New tools for improveddiagnosis

Dr. Magdalena RadwanskaFIND

27JUN07

FIND HAT diagnostic projects

• Parasite detection• Improved mAECT• Trapping the parasite on the filter membranes• Isolating the parasite using magnetic beads

• Antigen detection– Novel probes:

• VHH Nanobodies: camel heavy chain antibodies• scFv fragments: single chain variable fragment antibodies• Aptamers: Nucleic acid-derived probes

• Antibody detection– Collection of a large number of antigens followed by screening

and selection of the most promising ones

27JUN07

Parasite detection in HAT today

• Examination of lymph node aspirate, cerebrospinal fluid and blood

• mini Anion Exchange Centrifugation Technique (mAECT)– currently best test on blood– prone to improvement– availability not secured

27JUN07

mAECT production

• Situation– "new model" designed by Institute of Tropical

Medicine Antwerp and produced at Institut National de Recherche Biomédicale Kinshasa with finances from WHO and Belgian Development Cooperation

– production at INRB halted for technical reasons• FIND strategy

– improve design– resume sustainable production at INRB in Kinshasa– set up a quality system

27JUN07

mAECT production

• Institut National de Recherche Biomédicale, Kinshasa, D.R. Cong– National Reference Laboratory for HAT in DRC– Former mAECT production site for DRC

• FIND with ITM Antwerp:– investment in infrastructure, equipment, material and reagents– support for production of 30,000 tests until April 2008– mAECT kits now available at 3 USD/test, package of 10 tests

• mAECT quality system– mAECT handbook with SOPs and fully described quality system– internal quality check at INRB Kinshasa– external quality check and lot release decision at ITM Antwerp

27JUN07

Parasite separation - mAECT

New collector tube

New collector tube holder

New holding rack

Upgraded facilities at INRB-DRC

Trained INRB staff in assembly

Assembly of 30,000 units ongoing

New collector tube

New collector tube holder

New holding rack

Upgraded facilities at INRB-DRC

Trained INRB staff in assembly

Assembly of 30,000 units ongoing

27JUN07

Serodiagnosis of HAT today

Point-of-care tests:

– Antibody detection test for T.b. gambiense• CATT/T.b.gambiense (direct agglutination test)• LATEX/T.b.gambiense (indirect agglutination test)• both based on native, variable antigens• stay positive after successful treatment

– Antibody detection test for T.b. rhodesiense• Not available

– Antigen detection tests for T.b. gambiense and T.b. rhodesiense• Not available

27JUN07

Antigen detection

New tools for antigen detection

27JUN07

Challenges for antigen detection

Images: M. Ferguson

VSG surface coat

VSG molecule

Antigenic variation

27JUN07

New tools for antigen detection

• Antibody-derived probes:– Nanobodies: camel heavy chain VHH antibodies– scFv single chain variable fragment antibodies

• Nucleic acid-derived probes» RNA, and ssDNA aptamers

• Targets: invariable epitopes on VSG’s and ISG’s

27JUN07

scFv Ab fragments and VHH nanobodies

CH1 VHCLVL

CH3

CH2Fc

scFv

VHH

CH2

CH3

Fc

VHH

Nanobody: camel heavy chain VHH Single domain antigen binding fragment (15 kDa)

Single chain variable fragment (scFv)

antibodies

Classic antibody

Camel heavy chain antibody

Unconventional IgG2, IgG3

Monomeric: Diameter 2.4 nm Height 4 nm

Images provided by VIB/VUB Brussels

27JUN07

How VHH nanobodies and scFv are selected

scFvVHH

or

Phage or yeast display system

secretion

27JUN07

VSG coat

Advantages

scFv

VHH

VHH

Classic Ab’s1) Small size probes reach

new hidden epitopes

2) Affinities in nanomolar range

3) Can be easily coupled to various dyes or particles

4) Can be used in ELISA (Enzyme linked Immunosorbent Assay)

Images provided by VIB/VUB, Brussels

27JUN07

Antigen/nucleic acid interaction

• Aptamers– Artificial nucleic acid ligands that can be generated against amino acid,

proteins, drugs– Composed of RNA, single stranded DNA– Size: 6 to 40 kDa– Complex 3D structures– Bind targets in nanomolar range

Antigen

RNA aptamer

Images provided by Darmstadt University

27JUN07

Antigen/nucleic acid interaction

• Advantages:– Aptamers bind to new epitopes– Small size, penetration of the coat– Affinities in nanomolar range– Can be used in immunoassays such as

ALISA (Aptamer linked Immunosorbent Assay)

Images provided by VIB/VUB, Brussels

27JUN07

How aptamers are selected

27JUN07

Antibody detection

New tools for antigen detection

27JUN07

Antibody detection

• Strategy: – Screening of new antigens recognized by T.b. rhodesiense and

T.b. gambiense patients– Incorporate these antigens in point-of-care test format

• Antigen collection: 32 antigens, most recombinant, from diverse academic partners– Univ. Cambridge, Univ. Bordeaux, ILRI Kenya, Univ. Glasgow,

ITM Antwerp, Univ. Leicester, Univ. Califonia, Univ. Wisconsin, Univ. Texas, ICP Brussels, Univ. Geneva

• Serum collection: 40 T.b. gambiense patients from R.D. Congo, 10 T.b. rhodesiense patients from Uganda, 58 negative controls from Benin, R.D. Congo, Belgium

• Screening: Antigens screened with sera in ELISA and in DOT BLOT by private company Microcoat, Germany

27JUN07

Antibody detection test - Antigens being screened

EP17 ++

EP19 ++

EP23 +/-

EP26 +++

EP36 +

EP37 +

EP38 +++

EP39 +/-

Reactivity with clinical samples

27JUN07

List of partners

• Antigen detection– Partners

• Settle Biomedical Research Institute (SBRI), USA• VIB/VUB Free University of Brussels, Belgium• Darmstadt University of Technology

• Antibody detection– Partners

• Cambridge University, UK• Institute of Tropical Medicine (ITM), Belgium• University of Wisconsin, USA• University of Dundee, UK• Christian de Duve institute of cellular Pathology (ICP-TROP), Belgium• University of Geneva, Switzerland• University of California, USA• University of Leicester, UK• University of Texas Southwestern Medical Center at Dallas, USA• University of Bordeaux, France• International Livestock Research Institute (ILRI), Kenya• National Livestock resources research Institute (NALIRRI), Uganda• Microcoat, Germany