Coomb’s Test, Reactions with Incomplete Antibodies
- Incomplete antibodies, unlike the complete ones, are monovalent with one active antigen-binding site. The rest of the antibody active sites can be destroyed by proteolytic enzymes or due to irreversible block by various agents.
- During the interaction of incomplete antibodies with complete antigen no visible immunological reaction proceeds.
- Incomplete (monovalent) or blocking antibodies bind to the antigens but don’t lead to their visible precipitation or agglutination. In fact, these antibodies are regarded to be more stable to heating and chemical treatment.
- Incomplete Abs encompass a variety of Ab molecules that give rise in certain pathological conditions such as rhesus-agglutinins (Abs directed to human rhesus-positive red blood cells), reagins of allergic patients, autoantibodies of patients with systemic lupus erythematosus (SLE) and rheumatoid polyarthritis, antibodies against tumour antigens in cancer patients, anti-infectious antibodies in patients with brucellosis, syphilis or other diseases.
- Incomplete autoantibodies may cause drug-induced leukopenia, hemolytic anaemia or thrombocytopenia.
- In pregnant rhesus-negative women, incomplete antibodies can arise against the rhesus (Rh)-antigens of Rh-positive fetus.
- In general, rhesus Ags elicit the production of two types of antibodies: complete bivalent anti-Rh-agglutinins and incomplete monovalent anti-Rh-Abs unable to cause the agglutination of Rh+ red blood cells.
- The peak of accumulation of incomplete anti-Rh Abs usually occurs in case of the repeated pregnancy with (Rh+) fetus. These antibodies cross the placental barrier and enter the fetal bloodstream. They cause the damage of fetal erythrocytes, thereby developing hemolytic anaemia and resulting in hemolytic disease of newborns.
- For the detection of incomplete antibodies, a special immunological reaction known as the Coombs’ test was designed based on the principle of hemagglutination.
Direct Coomb’s Test
- The direct Coombs test (also called a direct antiglobulin test) reveals the presence of incomplete antibodies, which are fixed upon the patient’s erythrocytes.
- To determine the fixation of agglutinins on patient’s red blood cells, the animal antiglobulin serum containing antibodies against human immunoglobulins is added. This serum is produced by immunization of laboratory animals with human immunoglobulins.
- Being divalent (or complete), the animal antibodies bind to incomplete human antibodies fixed upon erythrocytes. This interaction causes the agglutination of erythrocytes covered with incomplete agglutinins.
- The mechanism of agglutination is based on the fact that one molecule of antiglobulin simultaneously interacts with two molecules of incomplete agglutinins independently bound to the surfaces of two distinct erythrocytes, thus resulting in hemagglutination.
- The direct version of Coombs’ test is used for detection of anti-erythrocyte autoantibodies that may cause hemolytic anaemia in humans.
Indirect Coomb’s Test
- Indirect Coombs’ test (or indirect antiglobulin test) detects free incomplete antibodies in patient serum (e.g., in pregnant women).
- To aim this, patient’s serum containing free incomplete antibodies is taken; next, it is adsorbed upon human erythrocytes of I (0) group like carriers.
- After successful adsorption of monovalent antibodies on the membranes of carrier erythrocytes, animal antibodies against human immunoglobulins are added, and the reaction of hemagglutination occurs.
- Indirect Coombs’ test is successfully used for testing of pregnant women and for testing of blood samples from donor and recipient before blood transfusion for the presence of anti-Rh antibodies.