Agglutination Tests

Agglutination tests are based on the presence of agglutinating antibodies in patient sera that can react with specific antigens to form visible clumps. In the agglutination tests, the antibody - antigen reaction can be either a direct or passive agglutination reaction. In direct agglutination tests the agglutinating antibodies directly react with antigens on the surface of bacterial cells (brucella and tularemia febrile agglutinin tests), or erythrocytes (direct hemagglutination test) to form visible clumps of particles. In passive hemagglutination tests, agglutinating antibodies react with antigens that have been passively coupled to carrier particles such as erythrocytes (indirect or passive hemagglutination test) or gelatin (TP-PA) to form visible clumps.


Brucella and Tularemia are tested for by a standard tube agglutination test adapted to run in microtiter plates. In both procedures, the bacterial cells are incubated in microtiter wells along with patient sera and controls. If antibodies to either Brucella or Tularemia are present, an antigen-antibody complex forms and results in a lattice in the well (#1 in the image left). If no specific antibodies are present, no clumping occurs and the antigen settles out to form a button (#2 in the image left).

Treponema pallidum Particle Agglutination (TP-PA) is based on the agglutination of colored gelatin particle carriers sensitized with T. pallidum antigen. Patient sera are incubated with sensitized particles in microtiter wells and unsensitized gelatin particles in control wells. Patient sera containing specific antibodies will react only with the antigen sensitized colored gelatin particles to form a smooth mat of agglutinated particles in the microtitration tray (+ or ++). A compact button formed by the settling of the non-agglutinated particles in the microtiter wells containing sensitized particles indicates lack of specific antibody in patient sera (-).  If agglutinaition is seen with both sensitized and unsensitized particles, nonspecific agglutination is indicated. In such cases, patient serum may or may not contain T. palllidum specific antibodies and results are reported as Inconclusive for TP-PA.

Treponema pallidum Particle Agglutination test

The only type of hemagglutination test used by the Serology Branch at this time is passive microhemagglutination (PHA). It is used to test for antibodies to a specific antigen (Fraction 1A envelope protein) of the plague (Yersinia pestis) bacterium. The F1 antigen is adsorbed on the surface of glutaraldehyde-stabilized sheep red blood cells. When patient sera and sensitized cells are added together in a microtiter well, clumping or lattices occur in the presence of F1-specific antibodies (#1 at right), or in their absence, no agglutination occurs and a compact button forms by the settling of the sheep erythrocytes (#2 at right).

Last updated May 7, 2014