Parasitological and serological methods. New tools for improved diagnosis. Dr. Magdalena Radwanska FIND 27JUN07

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1 Parasitological and serological methods New tools for improved diagnosis Dr. Magdalena Radwanska FIND

2 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

3 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

4 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

5 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

6 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

7 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

8 Antigen detection New tools for antigen detection

9 Challenges for antigen detection VSG molecule VSG surface coat Antigenic variation Images: M. Ferguson

10 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

11 scfv Ab fragments and VHH nanobodies Classic antibody VL CL CH1 VH scfv Fc CH2 CH3 Single chain variable fragment (scfv) antibodies Camel heavy chain antibody VHH VHH Monomeric: Diameter 2.4 nm Height 4 nm Unconventional IgG2, IgG3 Images provided by VIB/VUB Brussels Fc CH2 CH3 Nanobody: camel heavy chain VHH Single domain antigen binding fragment (15 kda)

12 How VHH nanobodies and scfv are selected or Phage or yeast display system secretion scfv VHH

13 Advantages VSG coat Classic Ab s VHH VHH scfv 1) 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

14 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

15 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

16 How aptamers are selected

17 Antibody detection New tools for antigen detection

18 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

19 Antibody detection test - Antigens being screened Reactivity with clinical samples EP17 ++ EP19 ++ EP23 +/- EP EP36 + EP37 + EP EP39 +/-

20 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