Lymphocyte Proliferation Assay Introduction Lymphocyte proliferation assays measure the functional capability of lymphocytes to respond to antigenic or mitogenic

stimulation. Hence, the assays are a more direct test of immunocompetence than a total lymphocyte count. Indeed, they are the most frequently used in vitro method to assess the cellmediated response to a nutrition intervention. Investigators report a decrease in the response to mitogens in both children and adults with protein-energy malnutrition (Miller, 1978: McMurray, 1984). Decreases in cell-mediated responses occur early in the development of protein-energy malnutrition, so that the lymphocyte proliferation is said to represent a sensitive measure of nutritional status. Principle of the Assay Lymphocyte proliferation assay is used to determine lymphocyte activation and the cell-mediated immune responses. With the help of T cells, B cells are stimulated to undergo proliferation when B cells meet their specific antigens. T cells will proliferate when they are activated by antigen-presenting cells and cytokines. The proliferation of both B and T cells leads to clonal expansion and the initiation of the specific immune responses. Cells that in proliferation processes increase their synthesis rate of protein and DNA. Increase in DNA synthesis is measured by adding [3H] thymidine, (radioisotope-labeled DNA precursor) to the cell culture medium. The amount of tritium taken up by the dividing cells is correlated to the level of cellular proliferation. Cells undergoing proliferation are metabolically active and also increase their cellular level of dehydrogenase and their NADH and NADPH product. NADH and NADPH levels of can be measured by their ability to reduce yellow colored MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) to intracellular purple formazan. Purple products results can be solubilized and quantified by spectrophotometric means. MTT and [3H] thymidine incorporation are two common methods used to measure cell proliferation.

Principle of the Assay

Objective of the Assay Lymphocyte proliferation assay is an assay that measures the ability of lymphocytes placed in short-term 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. This proliferative response of lymphocytes to antigen in vitro happens only if the patient has been immunized to the specific antigen, either by having recovered from infection with the microorganism containing that antigen, or by having been vaccinated. Precautionary Measures Taken During the Test The cells must be kept in a sterile condition. To protect the cells from contamination, a few precautions must be taken: 1. Do not open plates or tubes containing cells and medium or boxes that contain sterilized tips to open air. The plates, tubes, pipette tips, and pipettes must be opened in a closed flow hood. 2. Do not let your cells come in contact with anything that is not sterile, such as a pipette taken outside the hood or an end of a pipette that has come in contact with finger. Another precautionary measure that must be taken is the condition of blood. Make sure that the blood is not clotted or being transferred using inappropriate tubes. Besides that, incubation conditions must meet all the requirements for the cell to proliferate such as the temperature, CO2 level and humidity. Furthermore, the conditions and duration for blood storage prior to isolation of PBMCs in important and must be take precautions.

Expected Result of the Assay Yellow MTT is reduced to purple formazan in the mitochondria of living cells.

The absorbance of this colored solution can be quantified by measuring at a certain wavelength and is usually between 500 and 600 nm by spectrophotometer. The absorption maximum depends on the solvent used. The reduction takes place when mitochondrial reductase enzymes are active, therefore, conversion can be directly related to the number of viable cells. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of cells can be deduced, through the production of a dose-response curve.

CLINICAL LAB TECHNOLOGY IMMUNOLOGY HLD 31103 ASSIGNMENT 2

Tittle: Radioimmunoassay and Enzyme-linked Immunosorbent Assay (ELISA)

Name: Muhammad Fattah bin Fazel I.D number: 12171211070 Lecturer: Mr. Azmil Semester: 5