1. Classical blot procedure No predilution of the serum Original presentation Automation (preanalytic) Avoiding difficult interpretation Analytic programme Avoiding interference and variations due to test conditions or blood samples Less hand on time Easy comparison between positive and cut-off Economical Profiling in one step - one strip Single test The unique system having an internal cut-off control

2. Procedure

3. Interpretation

4. The positive control DOT must be positive (coloured) in all cases The negative (cut-off) control DOT gives the extent of non-specific antibody binding of the sample in the test. The central DOTS, coated with the specific antigens, give the results when compared to the Negative DOT. The colour intensity depends of the titer of the specific antibody in the tested sample. To be positive, the intensity of the colour must be higher than the Negative DOT. When the intensity of the colour is below the Negative DOT, the result is negative. When the intensity of the colour is equal to the intensity of the Negative DOT, the result is considered negative. Interpretation

5. OUR DOTS PROGRAMME The detection of HEp-2 positive patients requires further identification steps of the autoimmune markers. Our full DOTS programme, entirely automatisable, allows the identification of all the autoantibodies correlating with systemic autoimmune diseases... SYSTEMIC AUTOIMMUNE DISEASES Systemic Lupus Erythematosus (SLE) Mixed Connective Tissue Disease (MCTD) CREST Syndrome Progressive Systemic Sclerosis (PSS) Rheumatoid Arthritis (RA) and related overlap... and polyautoimmune syndromes... Nucleosomes DOT Nucleosomes Nucleosomes + Histones DOTNucleosomes, Histones. ENA DOTSm, Sm/RNP, SSA, SSB, Jo-1, Scl-70. Nucleosomes + ENA DOTNucleosomes, Sm, Sm/RNP, SSA, SSB, Jo-1, Scl-70. ENA Screen DOTSm, Sm/RNP, SSA, SSB, Jo-1, Scl-70, PMScl, CENP-A/B. ENA TOTAL DOTNucleosomes, Histones, Sm, Sm/RNP, SSA, SSB, Jo-1, Scl-70. Lupus Screen DOTNucleosomes, Histones, Sm, Ribosomes Cytoplasmic DOTM2, Jo-1, Ribosomes Cytoplasmic Profile DOTM2, Jo-1, Ribosomes, PL7, PL12 A full DOTS strategy for systemic diseases

6. ENA (Sm, Sm/RNP, SSA, SSB, Jo1, Scl70)

7. Nucleosomes + Histones Lupus Screen (Nucleosomes, Histones, Sm, Ribosomes)

8. Nuclear and Cytoplasmic Antigens ANTIGENSDESCRIPTIONTYPE/SOURCE Nucleosomes dsDNA wrapped around a corehistones octamer. Heterogenous mixture of pure native poly-nucleosomes composed of about 7 to 28 mononucleosomes. Contain Histones H2a, H2b, H3-H4, and traces of H1. Purified from bovine thymus chromatin. Histones Mixture of H1, H2a, H2b, H3-H4. Sm Core proteins of snRNP particles contains mainly D protein, E, F, G subunits are detectable. BB’ proteins are not. Purified from bovine thymus.

9. ANTIGENSDESCRIPTIONTYPE/SOURCE Sm/RNP snRNP particles. Contains essentially 68kD, A, BB’, C and D proteins. snRNA is detectable. Purified from bovine thymus. SSA Ro 60kD protein.Human recombinant produced in BSf9 cells. SSB La 50kD protein.Purified from bovine thymus. CENP-A Centromere protein A.Human recombinant produced in BSf9 cells. CENP-B Centromere protein B.Human recombinant produced in BSf9 cells. Scl70 DNA Topoisomerase IPurified from bovine thymus. PCNA Proliferating cell nuclear antigen Human recombinant produced in BSf9 cells. Nuclear and Cytoplasmic Antigens

10. ANTIGENSDESCRIPTIONTYPE/SOURCE PM-Scl Polymyositidis-Scleroderma antigen. Human recombinant produced in BSf9 cells. Jo1 Histidyl-tRNA synthetase.Purified from bovine thymus. Ribosomes Ribosomal P0, P1, and P2 proteins. rRNA is detectable. Purified from bovine thymus. PL7 Threonyl-tRNA synthetase.Human recombinant produced in BSf9 cells. PL12 Alanyl-tRNA synthetase.Human recombinant produced in BSf9 cells. Nuclear and Cytoplasmic Antigens

11. Cytoplasmic Antigens HEp2 cytoplasmic patterns have to be characterized and differentiated PBC M2 complex Pyruvate DH, 2-OXO-ADH,  Ceto glutarate DH Polymyositidis Jo-1 PL-7 PL-12 Histidyl-t RNA Synthetase Treonyl-t RNA Synthetase Alanyl-t RNA Synthetase SLE-neurologic SLE RibosomesAcid Phosphoproteins P0, P1, P2

12. Profiling the liver autoimmune diseases with our full liver dots programme AUTOIMMUNE LIVER DISEASES Primary Biliary Cirrhosis (PBC) Autoimmune Hepatitis type I (AIH I) Autoimmune Hepatitis type II (AIH II) Post infectious autoimmune hepatitis... HOW HELPING DIAGNOSIS WITH SEROLOGY ? F-ActinSpecific AIH type I marker LKM1Specific AIH type II marker LC1An AIH type II sometimes present in LKM1 negative patients SLASoluble Liver Antigen remains one of the rare but classical markers of AIH M2 PDHSpecific PBC marker OUR FULL DOTS PROGRAMME : You have to cover a large panel of liver autoimmune pathologies characterized by serological markers... but detection and differentiation are frequently uneasy... 1M2 PDH IgG (H+L) DOT 2M2 PDH - LKM1 - LC1 DOT 3M2 PDH - LKM1 - LC1 - SLA - F-Actin DOT 4F-Actin DOT 1 234

13. Liver markers ANTIGENSDESCRIPTIONTYPE/SOURCE F-Actin In vitro polymerized Actin filaments. Prepared from purified G- Actin (rabbit skeletal muscle). SLA Soluble Liver Antigen.Purified from rat liver. M2 Cluster of mitochondrial inner membrane enzyme complexes (PDHC + AGDC + BCOADC). Purified from bovine thymus and spleen. LKM1 Cytochrome oxydase P4502D6 (Liver - Kidney Microsome type 1 antigen). Purified from bovine thymus and spleen. LC1 Formiminotransferase cyclo- deaminase (Liver Cytosol type 1 antigen). Human recombinant produced in Baculovirus infected Sf9 cells.

14. Primary Biliary Cirrhosis AMA Pyruvate DH 95% 2-OXO-Acid DH 50%  CETO glutarate DH 40% 5% of the PBC are PDH negative The E2 sub-units of the 3 complexes Are the specific PBC EPITOPES

15. The Actin confusion SMA..........non F-Actin..........F-Actin remain a « mix-up » Only anti-F(filamentous) Actin antibodies are the specific markers of AIM type I. Anti-SMA (desmin, reticulin, vimentin, actin...) do not correlate with AIH type I. TISSUEHEp2 SMA positive patients are not necessarily F-Actin positive. F-Actin appears as actin cables crossing the cells. Specific thorn (spines) reaction in the intertubular area (rat kidney section).

16. AUTOIMMUNE SYSTEMIC VASCULITIS : Wegener disease Rapid Progressive Glomerulonephritis Goodpasture syndrome OUR AUTOIMMUNE SYSTEMIC VASCULITIS STRATEGY GIVES YOU IN ONE RUN A CORRECT CLASSIFICATION USING THE MOST RELIABLE IFA, DOTS AND ELISA KITS... THE INFAILLIBLE STRATEGY : Vasculitis is a complex family of diseases... DETECTING and DIFFERENTIATING among the different vascular pathologies those with specific markers is a PRIORITY allowing an urgent and efficient THERAPY... 2ANCA IFA Screening DOT MPO and PR3 detection 3Vasculitis DOT MPO, PR3, GBM detection 1 GBM-Goodpasture DOT GBM detection MPO, PR3 and GBM ELISA Quantification and follow-up Profiling your patients with autoimmune systemic vasculitis 1 23

17. Vasculitis markers ANTIGENSDESCRIPTIONTYPE/SOURCE MPO Myeloperoxydase antigen (p-ANCA). Purified from human neutrophils. PR3 Proteinase 3 antigen (c-ANCA). Purified from human neutrophils. GBM Collagen IV (alpha 3 chain). Human recombinant produced.

18. We are also concerned with less prevalent autoimmune diseases ARE CONCERNED : Biermer’s pernicious anaemia Atrophic gastritis Stiff Man neurologic syndrome WE SUGGEST YOU : The DOT method is particularly adapted to the detection of rare antibodies remaining convenient and economical 1 Intrinsic Factor DOT 2 Intrinsic Factor and Parietal cells (K + H + ATPase) DOT 3 GAD I and GAD II DOT 123

19. Biermer’s anaemia markers ANTIGENSDESCRIPTIONTYPE/SOURCE Intrinsic factor Vit B12 - binding protein. Purified from porcine stomach. Parietal cells H + K + ATPase (  -  subunits) Purified from porcine gastric mucosa.

20. Neurologic markers ANTIGENSDESCRIPTIONTYPE/SOURCE GAD I Glutamate Decarboxylase (67 kD Isoform). Human recombinant produced in BSf9 cells. GAD II Glutamate Decarboxylase (65 kD Isoform). Human recombinant produced in BSf9 cells.

21. Food intolerance is today a major concern… frequently mistaken for allergy ! HOW CAN SEROLOGY DIFFERENTIATE FOOD INTOLERANCE FROM ALLERGY ? ALLERGY involves IgE antibodies INTOLERANCE involves IgA and IgG antibodies INTOLERANCE PATHOLOGIES CONCERN MAINLY : Gluten Cowmilk proteins Soya Gluten ELISA Gliadin IgA Gliadin IgG tTransglutaminase IgA DOT Gliadin + tTg IgA Milk proteins and Soya ELISA Beta Lactoglobulin IgG Soya IgG DOT Beta Lactoglobulin IgG + Soya IgG

22. Cœliac disease markers ANTIGENSDESCRIPTIONTYPE/SOURCE Gliadin Unfractionated wheat gliadin. Contains , ,  and  gliadin. Purified from wheat gluten. tTg tissue Transglutaminase. Human recombinant produced in BSf9 cells.