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Tony Rook
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Please find the link for the following protocol:



Lymphocyte proliferation assay (LPA) measures the ability of lymphocytes placed in shortterm tissue culture to undergo a clonal proliferation when stimulated in vitro by a foreign
molecule, antigen or mitogen. CD4+ lymphocytes proliferate in response to antigenic peptides in association with class II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). This proliferative response of lymphocytes to antigen in vitro occurs only if the patient has been immunized to that antigen, either by having recovered from an infection with the microorganism containing that antigen, or by having been vaccinated. Therefore, some normal individuals may not respond to a given antigen, but most people will respond to at least one of several common microbial antigens.

Antigen-specific T-cell proliferation is a major technique for assessing the functional capacity of CD4+ lymphocytes to respond to various stimuli. In the AIDS Clinical Trials Group (ACTG) it is used to measure improvements in immunological function following antiretroviral therapy, to measure the development of anti-HIV immune responses following
the administration of an HIV-vaccine, and to detect the presence of immune responsesagainst specific opportunistic pathogens [1, 2]. This assay has been used in numerous adult and pediatric ACTG protocols.

Almost everyones lymphocytes can be stimulated to proliferate nonspecifically by stimulating them in vitro with the mitogens phytohemagglutinin (PHA) or pokeweed mitogen (PWM), or the antibody anti-CD3. However, these substances provide strong
stimuli that are not antigen specific, and usually do not discriminate as well as antigens in reflecting different levels of immunodeficiency. Antigen-specific T-cell proliferation can be
measured in vitro using such antigens as cytomegalovirus (CMV) antigen, tetanus toxoid (TT), varicella zoster virus (VZV) antigen, and HIV-1 antigens (e.g., gp120 and p24), if an individual has been previously exposed to these agents.

Our consensus method involves isolating peripheral blood mononuclear cells (PBMCs), placing 100,000 of the cells in each well of a 96-well plate with or without various stimuli, and allowing the cells to proliferate for six days at 37°C in a CO2 incubator. The amount of proliferation is detected on the sixth day by adding radioactive 3H (tritiated) thymidine for six hours, which is incorporated into the newly synthesized DNA of the dividing cells. The amount of radioactivity incorporated into DNA in each well is measured in a scintillation counter and is proportional to the number of proliferating cells, which in turn is a function of the number of lymphocytes that were stimulated by a given antigen to enter the proliferative response. The readout is counts per minute (cpm) per well.