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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 afterthe 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 additionthey 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 oftest is the subjective nature of its quantification, since it requires a visual

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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

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a control

5. Add 25 ul (4 units) of dengue antigen to each well. Incubate at 4oC 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

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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)