EXPERIMENT NO.

DETERMINATION OF ABH SUBSTANCE IN SALIVA

(Hemagglutination-Inhibition Method)

A. Collection and Preparation of Saliva

1. The patient is asked to rinse his/her mouth very well with

water.

2. 3-5 mL saliva is collected by directly expectorating in to a clean 15 x 100 mm test tube. Saliva is
the fluid that collects into the mouth coming from the sublingual saliva glands. Do not collect
that which is derived into coughing or that from nasopharyngeal are.

3. Remove the bubbles form the collected saliva using swab.

 4. Inactivate the specimen promptly. Place the tube in a boiling
water bath for 10 minutes. The specimen may become cloudy
upon heating.

5. Cool the tube for a few minutes. Centrifuge the saliva to

sediment the precipitates.

6. Collect the supernatant fluid into a separate tube.

7. Do a double serial dilution as follows.

B. Serial Dilution Procedure:

1. Label 12 x 75 mm test tubes numbering them 1 to 5.

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

NSS (mL) 0.2 0.2 0.2 0.2 0.2
Clear saliva 0.2 0.2 from #1 0.2 from #2 0.2 from #3 0.2 from #4
Dilution 1:2 1:4 1:8 1:16 1:32
N.B. Discard 0.2 mL from the 5th tube to make the volume in all tubes equal to 0.4 mL

Transfer 0.2 mL NSS

Transfer 0.2 mL clear saliva

C. Hemagglutination-Inhibition Procedure:

1. Add 1 drop of the appropriate antiserum to each of the tubes.

Mix by gently swirling

2. Incubate at room temperature for 30 minutes to allow neutralization of soluble substance (if
present) by the antiserum.

3. Add 0.2 mL of appropriate red cell suspension (RCS) to each

tube.
 4. Centrifuge for 2 minutes at 2500 rpm.

5. Check each tube for the presence or

absence of agglutination of red cells. Do the
tilt and wiggle technique.

6. Record the results in each tube.

RESULT: Absence of agglutination in any of the tube: SECRETOR, titer = 2