Immunology Techniques

Antibody Staining
There are two types of antibody staining: direct staining and indirect staining. Direct staining is faster but costlier than indirect staining. Direct staining uses a monoclonal antibody that has been previously conjugated to a fluorochrome (ie, FITC or PE). The cells are simply incubated at 4C for a halfhour with the antibody, then washed several times to remove weakly or nonspecifically bound antibodies. The cells are then ready for microscopy or flow cytometry. Indrect staining uses a monoclonal antibody with no bound label. The sample is incubated with this primary antibody, then washed, then the sample is incubated with a secondary antibody. The secondary, fluorescent antibody binds the general class of the unlabeled primary antibody. Generally, antibodies to non-human mammals are of poorer quality than those that bind human cells, due to a lack of regulatory rules governing their production. Properly titering an antibody will quantify its quality. It is imperative to not use too little antibody that not enough cells of interest are bound. And it is crucial to not saturate your sample with so much antibody that the antibodies bind promiscuously. Since 3g per 100L (106 cells) is the initial concentration where the nonspecific binding component of all IgGs becomes detectable, this is a convenient starting concentration for titering. Regardless of how specific is your antibody, its Fc end will promiscuously bind any cells with Fc receptors; and dead cells are notorious for soaking up antibodies. Thus it is imperative to use an isotype control as a negative control for your antibody. An exception is with routine immuno-phenotyping where positive staining may be distinguishably bright enough. An isotype control is of the same immunoglobulin isotype (subclass) as the experimental antibody. It will allow you to ascertain how much background stain is due to irrelevant stickiness such as dead
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cells and Fc receptors. This isotype control will be as similar as possible to your experimental antibody but have a different specificity. For example, consider if you stained your human cells with 10g/mL of a murine IgG2a antibody (conjugated with six fluorescein molecules per antibody) that is specific to a T lymphocyte CD3 protein. An appropriate isotype control would be another murine IgG2a antibody, at the same concentration, with the same fluorescein conjugation ratio, but with a specificity for any antigen unlikely to be found on a human blood cell.

Hybridoma Technology
Hybridoma are cells that have been engineered to produce a desired antibody in large amounts. To produce monoclonal antibodies, B-cells are removed from the spleen of an animal that has been challenged with the relevant antigen. These Bcells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of the desired antibodies. They have to be selected and subsequently cloned by limiting dilution. Supplemental media containing Interleukin-6 (such as briclone) are essential for this step.The production of monoclonal anti-bodies was first invented by Cesar Milstein, Georges J. F. Khler and Niels Kaj Jerne in 1975. Selection occurs via culturing the newly fused primary hybridoma cells in selective-media, specifically media containing 1x concentration HAT for roughly 10-14 days. After using HAT it is often desirable to use HT containing media. Cloning occurs after identification of positive primary hybridoma cells. Clone by limited dilution. While some may believe that IL-6 is essential for this step, it is not necessary to add that expensive supplement, rather use 50% heat-inactivated FBS for the first week. Add 10% FBS DMEM to the clone culture plate after screening for single colony wells.

Mixed-Lymphocyte Reaction
The mixed-lymphocyte reaction (aka the mixed-leukocyte reaction, or MLR) is an in vitro method for assaying TH cell proliferation and for generating a population of CTLs. When allogeneic (different MHC haplotype) lymphocytes are cultured together, TH cell populations expand. WIthin another 48 hours, an expanding CTL population is generated. The total proliferation of lymphocytes from the allogeneic strains is measured by adding [3H]-thymidine to the culture medium and monitoring its uptake (uptake occurs during each cell division). However, it is unclear from [3H]-thymidine uptake how much each individual population has proliferated. The one-way mixed-lymphocyte reaction (aka the oneway mixed-leukocyte reaction or one-way MLR) resolved this issue. One population the stimulator is first inactivated (via mitomycin c or lethal xirradiation) before being added to the MLR well. These inactivated cells merely provide foreign alloantigens to the responder population. Within 24-48 hours, the responder T cells have begun proliferating and within another 48 hours an expanding population of functional CTLs has been formed. CD4 (TH) cells, dendritic cells and certain accessory cell types are all critical for the MLR to function.

Cell-Mediated Lympholysis Assay

The cell-mediated lympholysis assay (aka CML) assays CTL ability to lyse target cells. The target population (meant for lysis) is incubated in Na251CrO4, thus labeling the cells intracellularly with chromium-51 (aka 51Cr). The 51Cr cannot diffuse back out of the cells, so the only way 51Cr can be released back into the supernatent is if the target cells are lysed. When activated CTLs are incubated for 1-4 hours with washed target cells (to remove unabsorbed Na251CrO4) the amount of 51Cr in the supernatent correlates directly to the extent of target cell lysis by the CTLs. Thus, the specificity of CTLs for allogeneic cells tumor cells, virus-infected cells and artificially modified cells can be assayed. CTLs will typically only lyse cells of the same MHC Class I haplotype.