Panbio Dengue ELISAs

1. Panbio Dengue ELISAs

2. Most appropriate use: Panbio Dengue ELISAs

3. Panbio Dengue ELISAs • Dengue IgM Capture ELISA (E-DEN01M) • For the diagnosis of active dengue • Suitable for use in endemic and non-endemic dengue regions • Recommended for use with the Dengue IgG Capture ELISA in regions where secondary dengue is prevalent • Dengue IgG Capture ELISA (E-DEN02G) • For the diagnosis of secondary dengue • For use in dengue endemic regions • Use with the Dengue IgM Capture ELISA to detect and distinguish primary and secondary dengue with a high degree of confidence • Dengue Duo IgM and IgG ELISA (E-DEN01D) • This kit consists of the Dengue IgM Capture ELISA and the Dengue IgG Capture ELISA packaged together for convenience and cost savings. • Ideal for use in regions where primary and secondary dengue are common. • For the differentiation of primary and secondary dengue.

4. Panbio Dengue ELISAs cont. • Dengue IgG Indirect ELISA (E-DEN01G) • For detecting past or active dengue • Suitable for seroepidemiological studies

5. Positioning • Laboratory testing • Recommend test based on location and patient types • i.e. endemic region v non-endemic region • Is differentiation between primary and secondary important? • Diagnosis of infection v seroepidemiological screening • Emphasize Panbio: No 1 in dengue fever detection • Numerous publications to support this

6. Panbio Dengue IgG Indirect ELISA Specific serum antibodies combine with dengue 1-4 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 dengue antibodies in the test sample

7. Dengue IgG Indirect ELISA Anti-human IgG HRP conjugated Patient’s dengue specific IgG Dengue antigen coated plate

8. Panbio Dengue IgM & IgG Capture ELISAs

9. Dengue IgM Capture ELISA Dengue antigen-Mab complex IgM in patient’s serum Anti-human IgM coated plate Dengue IgG Capture ELISA is essentially the same except anti-human IgG antibodies are coated on the plate and therefore IgG in the patient’s serum is captured.

10. Interpretation: Capture ELISAs IgM positive / IgG negative • Serology indicates the probability that the patient has current or recent primary dengue infection. IgM positive / IgG positive • Serology indicates the probability that the patient has current or recent secondary dengue infection. IgM negative (equivocal) / IgG positive • Serology indicates the probability that the patient has current or recent secondary dengue infection. • In secondary dengue IgM may be negative in up to 75% of patients, depending on the stage of disease when the sample was collected.

11. Interpretation cont: Capture ELISAs IgM equivocal / IgG negative • Serology indicates that the patient may have current or recent primary dengue infection. • If clinical signs and symptoms persist recommend that a second serum sample be collected and tested. IgM positive / IgG equivocal • Serology indicates the probability that the patient has current or recent primary dengue infection. • Equivocal IgG suggests that the patient may have current or recent secondary dengue infection. • Patient should be monitored. • If clinical signs and symptoms persist recommend that a second serum sample be collected and tested.

12. Interpretation cont: Capture ELISAs IgM negative / IgG negative • Serology indicates the probability that the patient does not have current or recent dengue infection. • Note: Serum samples collected early in infection may yield a negative serological test result. • If a differential diagnosis can not be established and clinical signs and symptoms persist, recommend that a second serum be collected and tested. IgM negative / IgG equivocal • Equivocal IgG suggests that the patient may have current or recent secondary dengue infection. • Patient should be monitored. • If clinical signs and symptoms persist recommend that a second serum sample be collected and tested.

13. Interpretation cont: Capture ELISAs IgM positive / IgG positive • Suggestive of an active dengue infection. Could be either primary or secondary. Use the Dengue IgG Capture ELISA if distinction between primary and secondary dengue is necessary. IgM negative / IgG positive • Suggestive of a past infection. Secondary dengue infection cannot be ruled out as IgM levels in secondary dengue may be undetectable. If the patients IgG result is >40 Panbio Units, it is likely that the patient has a secondary infection.

14. High sensitivity High specificity • Some patients exhibit no IgM in a secondary infection. Therefore detection of high IgG levels to improve the sensitivity is recommended Performance: Capture ELISAs Serological sensitivity and specificitya

15. 94.8% specificity Performance: IgM Capture ELISA Cross reactivity study

16. 96.8% specificity Performance: IgG Capture ELISA Cross reactivity study

17. Publications: Panbio Dengue ELISAs • Vaughn et al (1999). Rapid serological diagnosis of dengue virus infection using a commercial capture ELISA that distinguishes primary and secondary dengue infections. Am.J.Trop.Med.Hyg., 60:693-698. • Lam & Devine (1998). Evaluation of capture ELISA and rapid immunochromatographic test for the determination of IgM and IgG antibodies produced during dengue infection. Clin.Diagn.Virol. 10:75-81. • Chew et al. (1998). Evaluation of commercial capture ELISA for detection of immunoglobulin M and G antibodies produced during dengue infection. Clin.Diagn.Lab.Immunol. 5:7-10. • Cuzzubbo et al. (1997). Commercial assays for the serological diagnosis of dengue infection. In Arbovirus Research in Australia 7:56-60. • Phillips et al. (1997). Flavivirus diagnostic assays: the Queensland perspective. In Arbovirus Research in Australia 7:208-211. • Cuzzubbo et al. (1998). Detection of specific antibodies in saliva during dengue infection. J. Clin. Microbiol. 36:3737-3739. • Allwin et al. (1998). Abstract. Assessment of flavivirus-specific antibody cross-reactivity after vaccination against TBE and yellow fever. Eur. Conf. Clin. Vir., Hamburg. Available upon request

18. Commonly Asked Questionsabout Panbio dengue tests... Q In the Panbio Dengue Duo ELISA and Dengue Rapid Tests, why don’t symptomatic donors from endemic areas show elevation of IgG? A IgG Cutoff set above the level in Primary and Past Dengue.

19. Commonly Asked Questionsabout Panbio dengue tests... QHow do the cutoffs of these tests compare with haemagglutination-inhibition assays? AIgG Cutoff = HAI Titre for 2° Dengue (e.g. HAI 1:2560).

20. Commonly Asked Questionsabout Panbio dengue tests... Q Do I need to use both IgM and IgG for the diagnosis of dengue infection? Why can’t I just use IgM? ASome cases of secondary dengue do not show detectable IgM.

21. References • Dengue IgM Capture ELISA Instructions for Use. Rev 08/12/04 • Dengue IgG Capture ELISA Instructions for Use. Rev 20/10/03

22. Panbio Dengue: The complete range ELISA kits • Dengue IgG Indirect E-DEN01G • Dengue IgM Capture E-DEN01M • Dengue IgG Capture E-DEN02G • Dengue Duo Capture IgG & IgM E-DEN01D Rapid kits • Dengue Rapid Strip test R-DEN02D • Dengue Rapid Cassette R-DEN03D

23. www.panbio.com