Experiments in immunology

Experimental immunologists study responses to simple antigens through immunization, the deliberate induction of an immune response in animal or human subjects.[1] The immunized subject is then monitored for antibody response. This often involves the analysis of antiserum samples that contain specific antibodies against the immunizing antigen, and blood lymphocytes from lymphoid organs for examination of T cell-mediated responses.

However, some antibodies found in antiserum are cross-reactive. This means that they bind antigens that have no apparent relationship to the immunogen, and are problematic when antiserum is used to detect antigens that bind specifically to these antibodies. This can be resolved by absorption of these antibodies with their cross-reactive antigen, leaving only the antibodies that are specific to the immunogen of interest.

B cell immune responses are measured by analyzing antibodies produced through the humoral immune response. Serum is isolated from a clotted blood sample, and the circulating antibody that accumulates in the plasma is assayed by methods that include the following:

• Affinity chromatography, by which antibodies of interest are isolated from solution by the specific binding of antigens anchored to a solid matrix.
• Radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA),[2] which are direct binding assays for antibodies that use pure preparations of known antigens to create a protein standard. The concentration of the unknown antigen can then be determined by comparison with this standard.
• Competitive inhibition assays, which determine the concentration of a particular antigen in an unknown sample based on how successfully it competes with a labelled reference antigen in binding to antibodies bound to a plastic well.
• Precipitin reactions,[3] which determine the absolute and relative amounts of antigen and antibody by measuring the amount of precipitate formed when antibodies mix with soluble macromolecular antigens.

These experiments are used to determine the specificity, amount, isotype and affinity of the antibodies produced. Antibodies are highly specific for their particular antigens. They are therefore relatively easy to isolate and study, and make highly efficient probes in a range of biological experiments.[4]

In studying lymphocytes, the first step is to isolate them so that they can be studied in vitro. Lymphocytes can be isolated from blood, or from lymphoid organs such as the thymus, bone marrow, lymph nodes, and mucosal-associated lymphoid tissues.[1] Methods include the following:

• Density centrifugation, which isolates a population of peripheral blood mononuclear cells (PBMCs) by separating the solution into layers of differing densities. PBMC layers contain mononuclear cells that have been depleted of red blood cells, leukocytes and granulocytes.
• Biopanning, which isolates cell populations from solution. Cells of interest are bound to antibody-coated plastic surfaces, and unwanted cells are removed by treatment with specific antibody and complement.
• Fluorescence-activated cell sorter (FACS) analysis, which detects and counts lymphocytes passing through a laser beam. The FACS is a flow cytometer that separates labelled cells based on differences in the light scattering and fluorescence of cells.[5]

Upon isolation, the lymphocytes are characterized in terms of specificity, frequency and function. Frequently used assays include the ELISPOT, which measures the frequency of T cell response.[1] It is similar to the ELISA assay in that antibodies bound to plastic wells are used to bind the cytokines secreted by T cells.

Findings from the experiments thus described can be used to manipulate the immune response and develop treatments to combat immunological diseases. For example, adoptive transfer[6] is used to study the effects of lymphocytes in the absence of other lymphoid cells. Ionizing radiation kills lymphoid cells while leaving other tissues of the body intact, and this eliminates immune function. The body is thus prepared for restoration of immune function by adoptive transfer of new lymphocytes.

Another method is hematopoietic stem-cell transfer,[7] used for studying lymphocyte development. Hematopoietic cells are killed by ionizing radiation, and the hematopoietic system is replaced by transfusion of donor bone marrow or purified hematopoietic stem cells. The resulting individuals are termed radiation bone marrow chimeras.

The abovementioned methods have been researched extensively, but there remain many areas in immunology that have yet to be covered, ranging from autoimmune diseases to transplant rejection. There is still room for progress, and only the future will tell what developments are yet to come.

• Experiments
• Immunology
• Antibodies
• Lymphocyte