30 Saliva Testing for ABH Substances

Principle

Approximately 80% of all individuals possess the Se gene that governs secretion of water-soluble ABH antigens into all body fluids with the exception of the CSF. These secreted antigens can be demonstrated in saliva by inhibition tests with ABH and Lewis antisera.

Specimen

1. Collect 5-10 ml. of saliva in a small beaker. (to encourage salivation, the patient may chew wax, paraffin, or a clean rubber band, but not gum or anything else that contains sugar or protein).
2. Transfer saliva to a clean test tube
3. Centrifuge saliva for 8-10 minutes at 3400 rpm.
4. Transfer supernatant to a clean test tube and place in a bath of boiling water for 8-10 minutes to inactivate salivary enzymes
5. Re-centrifuge at 3400 rpm for 8-10 minutes, remove supernatant fluid and discard the rest. Dilute the supernatant fluid with an equal volume of saline
6. Refrigerate if testing is to be done within several hours. If testing is not to be done on same day of collection, freeze the sample and store at -20O C.

Reagents

1. Polyclonal anti-A, –B, -A,B. Note some monoclonal reagents may not be appropriate for use; therefore, appropriate controls are essential.
2. Anti-H lectin from Ulex europaeus obtained commercially or prepared by saline extraction of Ulex europaeus seeds.
3. A1 and B cells

Preparation of Antisera and Selection of Blood Grouping Reagent Dilution

1. Prepare doubling dilutions of anti-A, anti-B and anti-H with saline.
   1. Use 0.5ml of saline in each labeled tube (dilutions 1:1 to 1:512).
   2. Add 0.5ml of antisera to tubes 1 and 2.
   3. Mix contents of tube 2 and transfer 0.5 ml to the next tube (1:4).
   4. Continue this process with a clean pipet until the last tube is reached.
   5. These tubes will be master dilutions and the last tube will have 1.0 ml in it.
2. Group A1, B and O red cells (2-5 % washed cell suspensions).
3. Determine which dilution is to be used in the procedure.
   1. Label tubes appropriately to be used with the three antisera and coordinating indicator cells. (A1, B, or O cells)
   2. To one drop of each dilution add one drop of saline suspended indicator cells.
   3. Centrifuge and examine macroscopically.
   4. Select the highest dilution that gives 2+ agglutination.
   5. This is the dilution that will be used in the inhibition testing.

Procedure

1. Label tubes for patient and controls.
2. Add one drop of diluted antisera to each appropriate tube (anti-A, anti-B and anti-H) for patient and controls (known secretor, non-secretor and saline).
3. Add one drop of the saliva/controls to appropriate tubes.
4. Mix the contents of the tubes.
5. Incubate for 8-10 minutes at room temperature.
6. Add 1 drop of 2-5% washed indicator cells to appropriate tubes (group A, B or O cells)7.             
7. Mix the contents of the tubes.
8. Incubate for 30-60 minutes at room temperature.
9. Centrifuge each tube and inspect macroscopically for agglutination.

Interpretation

1. Inhibition is indicated by a lack of agglutination (indicating that the saliva had the antigen and neutralized the antibody in the antisera and therefore there was no antibody to react with the antigen on the indicator cells).
2. The failure of antibody in the saline control tube to agglutinate indicator red cells invalidates the results of saliva testing (indicates the reagents are too dilute-repeat with appropriate dilution).