Detection of Antigen-Antibody Complexes

in culture; the cells are then screened for the antibody in question. These hybridomas are separately grown, leading to pure antibodies being made. As you can imagine, this is expensive and time-consuming.

What is the purpose of making monoclonal antibodies? Part of the purpose is to turn mouse antibodies into human monoclonal antibodies. Mouse antibodies will be recognized as foreign by human so they will be neutralized by the human immune system. Genes can be manipulated that make a hybrid antibody that will be mostly human in origin. These are used to treat certain cancers by making antibodies against the cancer cell.

Because of the expense, it is necessary to find an alternative way of making hybrid antibodies. Genetically-engineered plants can be used to make plant-related antibodies that can be humanized. These might be cheap enough to be made for use in treating common infectious diseases. Animal-derived monoclonal antibodies are just too expensive to make otherwise. Making these types of antibodies in plants is called making plantibodies.

DETECTION OF ANTIGEN-ANTIBODY COMPLEXES

In vitro assays involve the detection of antibody-antigen complexes in a test tube or other medium outside of the body. When an antigen and antibody can be seen, it is referred to as a precipitin. In this type of reaction, an antibody and antigen are mixed together. If they combine, they precipitate out of solution, leading to a precipitin. Most of these tests are done using polyclonal antibodies, which form a precipitate more easily.

The ratio of antibodies and antigens is important in a precipitation reaction. If there are too many antibodies, they will not combine well to make a precipitate. If there are too many antigens, the amount of precipitation will also decrease. There is an equivalence zone that provides an optimal ratio between antigens and antibodies in the reaction.

A related test is called a precipitin ring test. It can tell the relative amounts of antigen and antibody in a sample. Test tubes are made that have the same amount of antigen in them. Glycerol is added to the antigen; then there is serial dilution of the antiserum. The glycerol allows antigen-antibody complexes to form just at the interface between the

glycerol and water. The highest dilution that produces a visible ring of precipitate is called the titer. The higher the titer, the more antibodies are in the sample. It won’t give an absolute concentration of antibodies, however.

Another test is called an Ouchterlony assay, which uses a matrix made of agar gel rather than water and glycerol testing. The gel is clear and holes are punched into the gel to make wells. Antigen and antibodies are added to adjacent wells with the diffusion of proteins between the wells showing a precipitate between them. It can quickly tell if there is an antigen and antibody connection.

Another related test is the radial immunodiffusion assay. This will actually quantify the concentration of antigen in the serum. Antiserum is mixed with liquid agar and allowed to cool in a plate. Wells are added and the antigen is added to the well. If they are a match, there will be a zone of precipitation. The zone of precipitation is measured and the concentration of antigen is mathematically determined.

There is a similar test called a flocculation test. It is used for antigens that are not already soluble in water, such as lipids. It creates a flocculant instead of a precipitin. Flocculants won’t precipitate in solution but instead forms a foam in the test tube. This is a test used for syphilis testing. The VDRL test is a syphilis test that is essentially a flocculation test.

Another test is a neutralization test, usually done to detect viruses. It makes use of antiviral antibodies that will coat the viral particles that block viral binding. Because viruses will lyse and damage cells, if they are neutralized, this can be seen as an area on a plate containing cells that isn’t damaged by the virus particle. The test makes use of a serial dilution of patient serum to see if the plaques of dead cells go away. The end result is some type of titer so that the highest titer involves a greater concentration of antibodies. It will not be able to detect an active infection unless the test is repeated in a couple of weeks.

A PAGE assay or a polyacrylamide gel electrophoresis assay is done when a patient has too many or too few proteins in the blood. It can tell which proteins are present in the patient’s serum. A related test is an immunoelectrophoresis or IEP test. This test uses a