1. serology & panbio product overview may 2005

2. The Immune system The human immune system responds from attacks from outside the body. Consists of: –Skin, tears, saliva, mucous –Thymus –Spleen –Lymph system –Bone marrow –White blood cells –Antibodies –Complement system –Hormones

3. Antibodies Antibodies, also called immunoglobulins The part of the immune response that is mounted when a germ (antigen) invades the body. Bind to the antigen (Ag) enabling the immune cells to recognise the foreign invaders and therefore remove & destroy them. There are 5 classes of antibodies produced by the body- IgG, IgA, IgM, IgE and IgD. –IgA, IgM and IgG are the main antibodies formed in response to viral or bacterial infection and are the antibodies tested for by Panbio kits.

4. IgA Antibodies Predominant Ab in seromucous secretions (saliva, tracheobronchial secretions). Critical first line defence system that protects against invasion by microorganisms. Indicative of acute infection. Important marker for mucosal infections such as Pertussis and Mycoplasma. IgA dimer

5. IgM Antibodies First antibodies to appear after primary antigenic stimulus. Marker of acute phase of an infection. Disappear usually within 1-3 months after infection.

6. IgG Antibodies Major antibody of secondary (anamnestic) responses. Provide life long protection. Indicator of past exposure or infection and immune status. Marker of active infection in paired sera.

7. Antibody Response Curve

8. © 2001 Panbio Limited. All Rights Reserved. Dengue immune response

9. Immunological tests & techniques There are a number of tests that diagnose disease based on the detection of antibodies. Some common techniques are listed below. –Immunofluorescence (IFA) –Neutralisation –Hemagglutination Inhibition (HAI) –Complement Fixation (CFT) –Enzyme-linked Immunosorbent assay (ELISA) –Rapid immunochromatographic assays

10. Immunofluorescence (IFA) Method for identification of antigens in tissue sections & on cells, or for identifying antibodies to them. –Direct - Fluorescent Ab is incubated with cells or tissue section. The Ab binding to Ag is visualised by UV light. –Indirect - Cells or tissue are incubated with test serum Ab, which is then visualised by the addition of a second layer fluorescent anti-antibody. Still used as a reference method for diagnosis of many diseases Panbio offers many IFA kits – based on the indirect IFA procedure

11. Panbio IFA kits Diluted patient serum samples are applied to cultured cells containing inactivated antigens provided on paint delineated wells on glass microscope slides. During incubation, specific antibody forms a complex with the antigens in the cells. Washing removes nonspecific antibody and other unreacted serum proteins Fluorescein-conjugated goat anti-human IgG, IgM or IgA is applied to the wells of the glass slide. The conjugate combines with the specific human antibodies, if present, during the incubation period. The slides are viewed by fluorescence microscopy. A positive antibody reaction is denoted by bright green fluorescence at the antigen sites.

12. Panbio IFAs Procedure will vary slightly depending on the kit

13. ELISA Enzyme-linked immunosorbent assay Panbio kits are of two types Standard Indirect ELISA majority of Panbio kits Ag coated onto plate binds specific Ab in patient’s serum. The bound Ab is detected using a labelled Ab. The reaction is visualised by a colour change. Capture ELISA Anti-human IgG or IgM coated onto plate captures patient’s Ab. Specific Ag bound to the plate also or added individually then binds to specific patient Ab. The bound Ag is then detected by a labelled monoclonal Ab. The reaction is visualised by a colour change.

15. Panbio Indirect ELISA Specific serum antibodies combine with antigens attached to the polystyrene surface of the microwells Washing removes residual serum Peroxidase-conjugated anti-human specific immunoglobulin is added The colourless substrate, tetramethylbenzidine/hydrogen peroxide (TMB / H2O2) is hydrolysed to a blue chromogen Stopping the hydrolytic reaction with acid turns the TMB yellow Colour development indicates the presence specific antibodies in the test sample

16. Panbio Capture ELISA Serum antibodies combine with the anti- human IgM or IgG coated on the plate. Simultaneously the peroxidase conjugated MAb and antigen supplied form complexes when incubated together. Washing removes residual serum The antigen-Mab complexes bind if antigen-specific antibodies have been captured. The colourless substrate, tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) is hydrolysed to a blue chromogen Stopping the hydrolytic reaction with acid turns the TMB yellow Colour development indicates the presence of specific antibodies in the test sample

17. Calculation of results On completion of reading the assay, the Panbio Units for each sample must be calculated. This is done as follows: 1.Calculate the average absorbance of the triplicates of the cut-off calibrator. This is the cut-off value. In some Panbio kits a calibrator is supplied rather than a cutoff sample. In this instance the average absorbance of the triplicates of the calibrator should be calculated and this figure multiplied by the calibration factor supplied on the specification sheet. 2. Panbio units can be calculated by dividing the sample absorbance by the cut-off value (calculated in step (1) above) and multiplying by 10. Panbio Units = 10 XAbsorbance of sample Mean absorbance of cut-off

18. Example Sample A Absorbance = 0.949 Sample B Absorbance = 0.070 Mean absorbance of cut-off = 0.302 Sample A0.949 / 0.302 X 10 = 31.4 Panbio Units Sample B0.070 / 0.302 X 10 = 2.3 Panbio Units

19. Interpretation: IgM ELISA* Panbio UnitsResultInterpretation <9NegativeNo evidence of recent infection (see Note 1) 9 - 11EquivocalSuggest samples be retested (see Note 2) >11PositiveSuggestive of a recent infection Note 1: If specific IgM antibodies are not detected and a recent infection is suspected, this can be confirmed by testing a further specimen 7-14 days later. Note 2: If specimen remains equivocal following repeat testing then the specimen may be tested by an alternate method or another patient specimen obtained and tested. *General interpretation supplied. May vary depending on disease (i.e. Dengue)

20. Interpretation: IgG ELISA* Panbio UnitsResultInterpretation <9NegativeNo evidence of IgG antibodies (See Note 1) 9 - 11EquivocalSuggest samples be retested (see Note 2) >11PositiveSpecific IgG antibodies present. Suggestive of recent or past exposure. Note 1: If specific IgM and IgG antibodies are not detected and a recent infection is suspected, this can be confirmed by testing a further specimen 7-14 days later. Note 2:If specimen remains equivocal following repeat testing then the specimen may be tested by an alternate method or another patient specimen obtained and tested. *General interpretation supplied. May vary depending on disease (i.e. Dengue)

21. Features/benefits: Panbio ELISAs Breakapart wells –Minimise wastage Consistent procedures –Decreases chances of error –Easier for programming automated instruments Short assay times ( 1 hr 10 min Indirect; 2 hr 10 min Capture) –Provide results faster Ready-to-use colour coded reagents –Decreases chances of error preparing reagents –Leads to shorter overall assay time – results generated faster Compatible with standard microplate technology –Suitable for use on a wide range of automated instruments

22. IgM/IgA ELISAs & Absorbent Panbio IgM & IgA ELISA kits contain Absorbent (goat anti-human IgG). Absorbent has two functions:- 1.Remove competing IgG that can cause false negative results. 2.Remove Rheumatoid Factor (RF) that can cause false positive results.

23. Competing IgG and false negs IgG Antigen IgM Without Absorbent Anti-human IgG Immobilised IgG IgM Antigen With Absorbent

24. False positives and rheumatoid factor Antigen IgG RF (IgM or IgA) Anti-human IgM HRP

25. Panbio Rapid Immunochromatographic tests Dengue Duo Cassette Dengue Duo IgM & IgG Rapid Strip

26. Procedure: Dengue Duo Cassette

27. Procedure: Dengue Rapid Strip

28. Advantages of rapid tests Fast (results in less than 30 minutes) Simple (3 step procedure)