HEMAGGLUTINATION INHIBITION TEST AND DENGUE BLOT Purpose To establish the presence of dengue virus infection in clinically suspect

patients Background : Test to measure antibodies such as hemagglutinatlon inhibition (Hl), complement fixation (GF), and neutralization tests exploit biological markers of dengue viruses. There are reliable tests for measuring antibodies, but they do not distinguish IgM from lgG. For this reason these tests usually require paired samples (acute and convalescent) to make a diagnosis. More recent serologic techniques such as ELISA, fluorescent antibody and dot blot tests have been developed to measure antibody class individually. Hl test has become the World Health Organization standard test for the serologic confirmation and serologic classification of dengue infection. The assay depends on the ability of antibodies to inhibit viral glycoprotein-dependent agglutination of goose or human type O red blood cells. Serum specimens for testing must be treated to remove non-specific inhibitors of agglutination by acetone or kaolin extraction followed by red blood cell adsorption. The endpoint of the titration is the highest dilution of serum that inhibits agglutination of a standard amount of antigen. Four fold or greater changes in Hl titer of paired serum are considered diagnostic for recent infection. Interpretation of the Hl test is based on the titer and the time after onset of symptoms. In primary infections, detectable Hl antibody generally appears after the fifth day and rises slowly over a period of weeks, the titer generally not exceeding 1/640. In the ease of secondary or tertiary infections, there is an anamnestic response, which results in a rapid elevation of the titer within a few days of onset. Titers of 1/2560 to 1/20480 are frequently attained in convalescent samples and may persist for several weeks. Dengue blot (dot blot) tests, which do not require the equipment necessary for conventional serology, are simple and rapid. A limitation of these tests, however, is that they are not useful for testing large number of samples, since individual strips of paper or membrane must be handled for each serum tested. In addition they are expensive. The advantage of these tests is that they can be used to distinguish IgG from lgM and to aneasure titer of IgG antibodies semi quantitatively. Dot blot tests specific for anti dengue IgM have been modeled after lgM-capture ELISA in order to avoid the interfering effect of IgG antibody from previous infections. This can be accomplished by the use of nitrocellulose membrane coated with anti-human IgM antibodies. A dot blot test for lgG antibody has potential to replace more cumbersome serologic tests such as the Hl and lgG ELISA tests. The problem with this type of test is the subjective nature of its quantification, since it requires a visual

judgment of color intensity. At a dilution of 1: 1000 the test is not sensitive enough to detect low tittered lgG antibody from previous infection, so single serum sample is sufficient for identification of recent infection. EQUIPMENT AND MATERIALS Hemagglutination inhibition test: 96-well microtiter plate Micropipette lncubator Serum Dengue antigen Goose erythrocyte

Dengue Blot: Plate Towel paper Membrane with dot-blotted dengue antigen Conjugate Substrate Aquadest Washing buffer Diluent buffer

PROCEDURE: Hemagglutination inhibition test: 1. Extract serum sample with kaolin to remove non-specific inhibitor 2. Absorb the sample with goose erythrocyte to remove agglutinin 3. Make twofold dilution of the sample starting at 1/10 until 1/10240 dilution 4. Put diluted sample into well nos. 1 - 11. Well no.12 is filled with serum diluted 1 : 1 and serves as

a control 5. Add 25 ul (4 units) of dengue antigen to each well. Incubate at 4 oC overnight 6. On the next day add 25 ul of goose erythrocyte suspended in VAD (pH 6.2). Incubate the plate at 36oC for 45 minutes. 7 . Positive result will show intact erythrocytes accumulating on the bottom of the wells.

Dengue Blot: 1. Washing buffer preparation : 10 ml of 20 x concentration washing buffer + 190 ml aquadest (can be stored for a week) 2. Diluent buffer: 20 ml of washing buffer + 1 gram of non-fat skimmed milk. Mix well (should be freshly made) 3. Sample preparation : 5 ul of serum buffer + 500 ul of diluent buffer 4. Put the membrane into the wells 5. Fill the wells with 450 ul of diluent buffer 6. Add 50 ul of diluted sample, cover the plate with paper, and stand it at room temperature for 60 minutes 7. Suck the fluid from the wells, wash the membrane with washing buffer 3 times (3 minutes each time) L Conjugate preparation: 6 ul conjugate + 3 ml of diluent buffer 9. Fill the wells with 250 ul of conjugate, incubate at room temperature for 60 minutes 10. Take the fluid out of wells, wash the membrane with washing buffer as described above 11' Substrate preparation : 1,5 ml of substrate A + 1,5 ml of substrate B (prepare it just before use) 12. Add 250 ul of substrate solution into the wells 13. cover the plate, let it stand at room temperature for 30 minutes 14. Suck the fluid and add aquadest into the wells 15. Examine the development of colored dot on the membrane

ASSESMENT

students will be evaluated for the final score. Evaluation will include: 1. Pretest (will be held just before commencing practical class) 2. Performance and activity during laboratory work 3. Score of laboratory work report 4. Post test (will be held at the end or after practical class)