Immunology/Experimental Methods in Immunology

Stem Cells

The growth of stem cells in the bone marrow is the basis for cellular immunity. In vitro mimicry of this growth can be achieved by filling a semisolid medium with stromal cells. By adding different growth factors and cytokines, as well as stem cells of various differentiation level, the influence of chemical mediators of hematopoiesis can be understood. HSCs can be taken from a donor and injected into a person who has a defective or absent hematopoietic system. As little as 10% of the donor's bone marrow is removed and injected into the recipient, and the HSCs will find their way to the bone without direction, replenishing the hematopoietic system of the recipient. There are several types of stem cell grafts:

autologous--donor is the recipient themself; the recipient can freeze HSCs prior to chemotherapy or radiation therapy for cancer; additionally, genetically engineered autologous HSCs can be injected back into the patient
syngeneic--donor is genetically identical to recipient (identical twins)
allogeneic--donor is genetically different from recipient
- this can lead to graft-vs.-host disease (GVHD)
- GVHD is based on MHC/HLA types, and so a bone marrow "match" occurs when an allogeneic member of the population is found to have relatively similar MHC/HLA types relative to the recipient

Knock-Out Mice

Genetic factors that produce certain cytokines (or certain cell types themselves) can be studied through the use of knock-out mice. In these mice, a certain gene is inactivated and the animal is allowed to grow. The resulting phenotype is compared against other knock-out mice, in an attempt to piece together the genetic basis of different cellular functions and phenotypes. The process of gene knockout has become fine-tuned in recent years. Basically, a gene library is searched for a candidate gene to be knocked-out. A full mouse genome with the candidate gene knocked-out is developed and grown in a preliminary stem cell, and it is injected into a mouse stem cell via electroporation. Besides making the candidate gene inoperable, the altered genome will usually have two factors:

A gene to make the cultured preliminary stem cell resistant to a certain antibiotic. Because not all of the stem cells will incorporate the altered genome, an antibiotic is applied to the treated cells, and only those that survive have therefore been injected with the altered genome (including both the antibiotic resistance gene and the knocked-out gene sequence)
A gene to make the phenotype of a fully-altered knockout mouse obvious. For example, the altered mouse genome might include a gene to make the knockout mice pure white in color. The stem cells, before being treated, are collected from a black mouse. Thus, when the black fur mother mouse is injected with the treated stem cells, she will give birth to baby mice with varied coat colors, from black to white and grey in between. The nearly-white progeny are then cross-bred with each other, and the process is repeated until mice with pure white coats are created. These mice are most likely to have both the pure white genotype and the knocked-out gene. Thus, linked phenotypes are a marker for linked genotypes.

Several genes effect hematopoiesis, and have been developed via gene knockout. These include:

GATA-2--regulates lymphoid and myeloid cell production, as well as RBC (erythrocyte) production
Ikaros--regulates lymphoid cell production
Oct-2--regulates differentiation of naive B cells into plasma cells

Hematopoietic Stem Cell Concentration

Irradiation of a mouse can wipe out the HSCs in the bone marrow, destroying the mouse's ability to produce red blood cells and leukocytes. These cells can be replaced with the bone marrow cells of an immunologically identical mouse, although in the injected replacement cells very few cells are actually HSCs. In order to concentrate the amount of actual HSCs in a sample, mice can be irradiated and replacement bone marrow cells are injected with fluorescent immunoglobulins that bind to mature RBCs and WBCs. A flow cytometry method can now be used to weed out these labelled cells, and the process can be repeated until only those that are most likely undifferentiated remain. The HSCs can be found in a subset of cells that contain the CD34 receptor on their surface; the CD34+ subpopulation is almost totally HSC with few differentiated cells.

Monoclonal Antibodies

A given antigen can have many epitopes, each one reacting with the immune system to create antibodies specific for each of the epitopes. When a given antigen is given to an experiment animal, the animal will produce antigens for each of the epitopes of the antigen, forming a polyclonal antibody response. Plasma B cells are then isolated from the animal's spleen, and these cells are fused with special cancerous (and therefore immortal) myeloma B cells, which do not generate any specific antibody themselves. The myeloma cells lack a certain enzyme, hypoxanthine-guanine phosphoribosyltransferase, or HGPRT, and therefore cannot grow under certain conditions (namely in the presence of HAT medium). The product of these B cell/myeloma cell hybridizations are known as hybridomas, and they secrete monoclonal antibody indefinitely. After hybridization, there will be three types of cells: 1) spleen B cells, 2) myeloma cells, and 3) successfully formed hybridomas. In order to select for the hybridomas, the mixture is grown on HAT medium (killing any unhybridized myeloma cells) and the spleen B cells (as they are not cancerous and will die of old age) are allowed to die. Only those cells that can live a very long time (myeloma component) and contain HGPRT (plasma cell component) are able to survive the selection process. This results only in hybridomas, each secreting a different antibody. The mixture of polyclonal hybridomas are now separated via differential dilution in order to isolate the hybridomas for each specific epitopes, finally resulting in functional, immortal, monoclonal B cells.

Monoclonal antibodies (mAbs) are incredibly useful. They can be radiolabelled or labelled with fluorescent molecules in order to detect proteins, such as cancer proteins in a tissue. The use of fluorescent antibodies has also allowed scientists to image cellular components, increasing the understanding of the cell's structure as well as its genetics. Additionally, the anticancer properties of immunotoxins result from a mAb bound to a certain toxin, which becomes localized to the cancer epitope that matches the mAb. Antibodies can also be formed with enzymatic function (abzymes), which hold the promise of cleaving proteins at specific points in order to study proteomes more successfully.

Affinity Chromatography

A column of beads contains monoclonal antibodies. A mixture of proteins (potential antigens) is then passed through the column, and the antigen in question binds with the monoclonal antibodies. The column is then washed, removing any antigen that is not captured by the monoclonal antibodies. The pH is then adjusted in order to break the Ab/Ag interactions, and the column is rinsed; the Ag in question will be eluted in this last wash.

Radioimmunoassay (RIA)

A radioimmunoassay is a scientific method used to test hormone levels in the blood without the need to use a bioassay. It involves mixing a radioactive antigen (frequently labelled with isotopes of iodine attached to tyrosine) with antibody to that antigen, then adding unlabeled or "cold" antigen in known quantities and measuring the amount of labeled antigen displaced.

Initially, the radioactive antigen is bound to the antibodies. When cold antigen is added, the two compete for antibody binding sites - and at higher concentrations of cold antigen, more binds to the antibody, displacing the radioactive variant. The bound antigens are separated from the unbound ones in solution and the radioactivity of each used to plot a binding curve.

Once this standard curve has been created, a sample can be taken from a patient. The sample is then added to the Ab/radioactive Ag mixture. The sample is allowed to mix with the radioactive Ag, and the unlabelled Ag (often a hormone being tested for in the body) will displace some of the radioactive Ag. The mixture is then "rinsed," and the amount of remaining radiolabelled antigen is compared with a standard curve, allowing an easy determination of unlabelled (patient-derived) Ag/hormone levels.

The technique is both extremely sensitive, and specific, if costly, and it is especially useful in diagnosing and treating autoimmune diseases such as Hashimoto's thyroiditis and Systemic Lupus Erythematosus.

Enzyme-Linked ImmunoSorbent Assay (ELISA)

A sample of antigen is applied to the surface of a well, and the spaces between the sample are filled with proteins that do not bind antibody. A large amount of antibody, linked to a certain enzyme that can create a color change, is added to the sample under conditions that allow only Ab/Ag binding; there must be enough Ab added to react with every possible Ag molecule in the sample--if you have more Ag than Ab, the extra Ag will not be detected by the test. The well is then washed, and the color-changing reaction is activated. The level of color change can be measured by spectrometry, and the concentration of antigen can be determined via comparison of color change with a standard curve.

Sandwich (capture) ELISA--for Antigens of very low concentration, such as cytokines, binding to the well plate can be difficult. This method uses an Ab specific for one of the epitopes on the Ag, attaching these bottom antibodies to the plate well, where they bind the added small concentration of Ag. A second type of antibody, one that binds to a different epitope on the Ag, is added to the well. Thus, the Ag is sandwiched between a bottom "holding" antibody and an upper, "detector" antibody that is linked to a color-changing enzyme.

Competitive Inhibition Assay

Similar to the RIA, in this test a fixed amount of Ab is attached to the surface of a well. A known amount of radiolabelled Ag is added to the well, and the sample (ostensibly containing unlabelled Ag) is also added to the well. There must be enough labelled Ag added to react with every Ab, otherwise the test will not be accurate. This is because the unlabelled Ag must compete with the labelled Ag, and to truly compete every Ab binding site must be potentially filled with the labelled Ag. The well are rinsed, and the amount of unlabelled Ag that "out-competed" the labelled Ag is compared to a standard curve (thus, the amount of remaining labelled Ag is inversely proportional, via some relationship in the std curve, to the amount of Ag in the sample.

Blood Typing

The fact that each antibody has at least two (and, in the case of dimers and pentamers, more than two) identical binding sites for an epitope allows agglutination of antigens. Hemagglutination is the use of this property to test for blood types; anti-A Ab anti-B Ab, and a mixture of anti-A and anti-B Ab are added to aliquots of a blood sample, and the clumping (agglutination) indicates the blood type.

Coombs Test

The Coombs Test (also Coombs' Test) is a blood test used to determine whether there are red blood cell antibodies, which leads usually to hemolysis, especially in Rh disease. Coombs antibody is an anti-human globulin. It was first described in 1945 by Cambridge immunologists Robin Coombs, Arthur Mourant and Rob Race. The test is also used in screening blood prior to blood transfusion.

Two types of the test exist:

Indirect Coombs test - also known as the indirect antiglobulin test (IAT). This is used in the matching of blood products. It detects immunoproteins present on red blood cell membranes, by adding a polyspecific antiserum which contains antibodies specific for human immunoglobulins and complement to agglutinate the cells.
Direct Coombs test - also known as the direct antiglobulin test (DAT). It detects antibodies capable of attaching to normal red blood cells, by incubating normal red blood cells in the serum, washing the cells, and then using a polyspecific antiserum which contains antibodies specific for human immunoglobulins and complement to agglutinate the cells. The DAT is used to determine if the patient has immune-mediated hemolysis (antibody-mediated destruction of red blood cells), as occurs in Rh disease.

The Coombs Test is used to detect the presence of agglutinated red blood cells in a patient's blood. If positive, the interpretation is that an antigen-antibody reaction has taken place in vivo. The Coombs test is sometimes referred to as the Direct Antiglobulin Test. The indirect antiglobulin test is also called an antibody screen. In this test, a few drops of patient serum is placed in a small test tube with a drop of reagent red blood cells and checked to see if there is an antigen-antibody reaction. If there is such a reaction, it means the patient's serum contains antibodies to the known antigen on the reagent red blood cells.

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