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
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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
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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
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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