Agglutination Assays

PAGE test to separate the proteins and antisera to detect the exact proteins that are present in the sample.

The IEP can detect monoclonal antibodies in excess, such as is seen in multiple myeloma. Patients with multiple myeloma make too many monoclonal antibodies in a disease that is cancerous to the individual. A protein peak in a certain spot will indicate that a given antibody is being made in excess.

The Western Blot test is one that is used after protein gel electrophoresis. The PAGE test is done to separate proteins and then the proteins are immobilized on a nitrocellulose membrane. Antibodies and the membrane are mixed together along with a second antibody that is coupled to something that fluoresces, an enzyme, or something that changes color. This identifies particular antigenic proteins by the presence of a color change or fluorescence.

Antibodies in general will also activate complement proteins. These complement proteins will opsonize bacteria in order to facilitate phagocytosis, while others will cause gram-negative bacterial cell lysis. The fact that this can happen can be used to detect the presence of certain antibodies in the patient’s serum. Red blood cells can also be used in a related test called the complement fixation test, which can detect antibodies against fungi, intracellular pathogens, and viruses, which cannot easily be cultured.

If there is an antibody and antigen mixed together, all of the complement will be fixed and there won’t be enough complement to lyse the RBCs when these are added along with RBC antibodies. If the antibodies are present, the cells won’t lyse but if the antibodies are absent, the cells will lyse. This is how the complement fixation test is performed.

AGGLUTINATION ASSAYS

Agglutination tests clump cells together or antigen-coated latex beads together. It can detect antibodies against red blood cells or against bacteria. These are easy to do and are done on a slide or on a microtiter plate, which is an array of wells used to look for agglutination. There can be direct agglutination assays or indirect agglutination assays. Direct assays look at bacterial cells that agglutinate themselves. Indirect assays use

antibodies or antigens connected to latex beads that also can agglutinate. IgM antibodies are best detected because they easily agglutinate. The rheumatoid factor test looks for agglutination of beads in rheumatoid arthritis patients.

Agglutination tests are done in parts of the world where culturing of bacteria is expensive and difficult. It isn’t as accurate as bacterial culturing but is inexpensive and quick to do. It is also limited by the fact that it sometimes takes weeks for seroconversion or the presence of enough antibodies to occur. Agglutination tests work for antibody and antigen testing.

Red blood cell agglutination is called hemagglutination. The direct Coomb’s test is a hemagglutination test. This is also called a direct antihuman globulin test, which looks for antibodies that do not agglutinate. It is used in looking for antibodies to the child from the mother when the child has neonatal jaundice. It is also used to look for transfusion reactions, syphilis, infectious mononucleosis, and Mycoplasma pneumonia.

Coomb’s reagent is an antiserum that contains antihuman IgG antibodies. It will agglutinate red blood cells that have antibodies already attached to them. Without the reagent, binding and agglutination does not usually occur. Indirect Coomb’s testing screens for antibodies made against red blood cell antigens. It is used to detect antibodies that aren’t bound in the serum and is done before giving a blood transfusion.

Some of this kind of testing can be done to detect viral infections. This can be done to check for the presence of influenza viruses, which can agglutinate red blood cells in a direct hemagglutination assay. It will also detect rubella and mumps virus infections. As in related testing, a titer can be determined to see what the concentration of the virus is in the serum. This only works for agglutinating viruses.

The test can be modified in order to measure how many antiviral antibodies are in the patient’s serum. If there are antibodies in the patient’s serum, the virus will be neutralized and won’t agglutinate red blood cells. The titer involved is the highest dilution that will block agglutination of red blood cells.

Blood typing involves an agglutination assay. The patient’s blood is mixed with antibodies to the different red blood cell antigens. If the antigen is on the red blood cells, there will be agglutination. This type of test is done when cross-matching blood prior to