Determination of the Secretor Property

Principle
Certain blood group substances occur in soluble form in a large proportion (78%) of individuals in secretions such as saliva and gastric juice (see Table 611). These individuals are termed secretors (they possess the Se gene) and secrete antihuman globulin (ABH)soluble antigens. These water-soluble blood group substances are readily detected in very minute quantities because they have the property of reacting with their corresponding antibodies and thereby neutralizing or inhibiting the capacity of the antibody to agglutinate erythrocytes possessing the corresponding antigen. The reaction is termed hemagglutination inhibition and provides a means of assaying the relative activity or potency of these water-soluble blood group substances.

Materials
Saliva Human polyclonal anti-A and anti-B serum Anti-H lectin from Ulex europaeus Test tubes Pipettes Saline 2% to 5% washed group A, B, and O cells

Procedure
1. Collect about 2 to 3 mL of saliva in a test tube. 2. Centrifuge at 900 to 1000 g for 8 to 10 minutes. 3. Transfer supernatant to a clean test tube, and place stoppered tube in a boiling water bath for 10 minutes. This inactivates enzymes that might otherwise destroy blood group substances. 4. Recentrifuge at 900 to 1000 g for 8 to 10 minutes. 5. Collect clear supernatant into a clean tube.

6. Dilute saliva with an equal volume of saline (undiluted saliva contains nonspecific glycoproteins that can inhibit antisera and lead to incorrect results). 7. Add one drop of diluted antiserum to an appropriately labeled tube (anti-A, anti-B, anti-H). For dilution, titrate anti-H, anti-A, and anti-B, testing against appropriate cells at immediate spin. Select the dilution giving 2+ agglutination, and prepare a sufficient quantity to complete the test. 8. Add one drop of supernatant saliva to each tube. Mix and incubate at room temperature for 8 to 10 minutes. 9. Add one drop of the appropriate indicator cells (A, B, or O cells) to the properly labeled tube. 10. Mix and incubate at room temperature for 30 to 60 minutes. 11. Centrifuge. 12. Observe for macroscopic agglutination.

Control
1. One drop of saline is used in place of dilute saliva. Test in parallel with the saliva. 2. Test saliva from a known secretor and a nonsecretor in parallel with test saliva.

Interpretation
1. Nonsecretor: Agglutination of red blood cells (RBCs) by antiserum-saliva mixture; control tube positive. 2. Secretor: No agglutination of RBCs by antiserum and saliva mixture; control tube positive. The antiserum has been neutralized by the soluble blood group substances or antigens in the saliva, which react with their corresponding antibody. Therefore, no free antibody is available to react with the antigens on the reagent RBCs used in the testing. This negative reaction is a positive test for the presence of ABH-soluble antigens and indicates that the individual is a secretor.

ABH Substances in Saliva

ABO Group Secretors A B O AB Nonsecretors A, B, O, and AB

Much None None Much

None Much None Much

Some Some Much Some

None

None

None