Immunology is a branch of biomedical science that covers the study of all aspects of the immune system in all organisms. It deals with the physiological functioning of the immune system in states of both health and diseases, malfunctions of the immune system in immunological disorders (auto immune diseases, hypersensitivities, immune deficiency, transplant rejection); the physical, chemical and physiological characteristics of the components of the immune system in vitro, in-situ and in-vivo. Immunology has applications in several disciplines of science.
Even before the concept of immunity was developed numerous early physicians characterized organ that would later prove to be part of the immune system. The key primary lymphoid organs of the immune system are the thymus and bone marrow and secondary lymphatic tissue such as spleen, tonsils, lymph vessels, lymph nodes, adenoids, skin and liver. When health condition warrant, immune system organs including the thymus, spleen, portions of bone marrow, lymph nodes and secondary lymphatic tissue can be surgically excised for examination while patients are still alive.
Many components of the immune system are actually cellular in nature and not associated with any specific organ but rather are embedded or circulating in various tissues located through out the body.
CELLS AND ORGANS OF THE IMMUNE SYSTEM
The cell of the lymphoid system respond specifically to immunological stimuli. During embryonic life primitive lymphocytes are derived from haemopoietic stem cells in the yolk sac, liver and bone marrow along with the cells of the myeloid and erythriod series.
The lymphoid stem cells which originate from the bone. Marrow, differentiate to form two distinct lymphocyte subpopulations. One set of populations called T-cells, require to be differentiated in the thymus before they start functioning as immunologically competent cells. The cells of the other subpopulation, B-cells are independent of thymus and are differentiated in the bursa of fabricus, a lymphoid portion of the hindgut birds, however, human B-cell are differentiated in the bone marrow.
PRIMARY LYMPHOID ORGANS of the human immune system are thymus and bone marrow, from where lymphocytes are developed throughout the life span of an individual. Secondary lymphoid organ, are tissues rich in lymphocytes through which mature lymphocytes are constantly migrating and are being activated for a specific immune response. These secondary lymphoid organ include lymph nodes, white pulp of the spleen and miccosa associated lymphoid tissue (MALT) eg tonsils, peyers patehes and appendix. T-cells and B-cells occupy area specific for their development.
LYMPHOCYTE MARKERS: These are T-lymphocyte cells that are found to have many surface receptors. They can be identified by the use of monoclonal antibodies raised against the receptors. These cells are called CD (cluster of differentiation) markers because one marker may bind with several monoclonal antibodies forming a cluster. Thus, the sheep red cell receptor marker is CD2 and is present in all T-cells. And CD4 is expressed primarily by T-helper inducer cells and are thought to interact with class II HLA molecules on the surface of antigen presenting cells. T-cells can be divided into various subsets based on their functions and surface receptors. The main subsets are:-
(i) T-helper cells (T4):- These T-cells are responsible for producing lymphokines that positively regulate or help B-cells in the process of antibody production especially against haptens. B.cells cannot produce antibodies to haptens until the hapten carrier has interacted with helper T-cells. T-cells also help effector T-cells in cell mediated immune response. A large number of T-cells have CD4 receptors on their surface and also CD3, TCR ∂ β
(ii) T- suppressor cells (Ts) some T-lymphocytes are able to negatively regulate or suppress the immune responses of both T-and B-lymphocytes. These cells have CD8 surface markers. Overactivity of Ts cells can result in the state of immunodeficiency leading to many intracellular opportunistic infections such as that by pneumocystis carinii.
(iii) T- inducer cells. Generation of T-suppressor cells is induced by T-inducer cells. They also have a CD4, CD3, TCRỵ8 marker on the surface.
(iv) T-cytoxic cells (Tc) some T-cells are lethal or cytoxic for cells that carry specific antigen on their surfaces eg vinus infected cells, tumor cells or grafted (transplanted) cells. The Tc cells may also mediate the graft- versus host (GVH) reaction. A large number of Tc-cells have CD8, CD3, TCR ∂ β surface markers.
(v) T- delayed hypersensitivity cells (TDH) these T-cells secrete lymphokines which produce an inflammatory response in delayed allergic reaction or in defence against infection caused by bacteria, fungi, and parasites all TDH have CD4 surface marker. They also have CD16, CD56 but not CD3.
These are single specific antibodies that are the same because they are made up by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies have high affinity and avidity for antigen to bind to the same epitope. Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance, they can serve to defect or purify that substance.
PRODUCTION-HYBRIDOMA CELL TECHNIQUE
Monoclonal antibodies are typically made by fusing cells (myeloma and spleen) from mouse that has been immunized with the desired antigen. A technique for the production of a pure, specific antibody by such a clone of cells is called a hybridoma technique. The two types of cells used for the hybrid are;-
1. Multiple Myeloma Cells: multiple myeloma is a malignant tumour of antibody producing plasma cells, usually of no determined specificity. This multiple myeloma cells selected for this technique can multiply indefinite. They must lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) because myeloma cells does not synthesize this enzyme which is necessary for the synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is disputed. This pathway are by passed by exposing cells to aminopterian (a folic acid analogue, which has the ability to inhibit dihydrogolate reductase, DHFR) they become fully auxotrophic for nucleic acids requiring supplementation (briclone) to survive.
2. Splenic cells of a Mouse Immunized with the Specific Antigen
A mouse is immunized with the antigen for which the antibody is to be produced, the mouse responds by producing antibodies to many determinants or epitopes on the antigen injected. The mouse spleen is removed, minced and prepared as a single cell suspension. This should include the B-cell population, which produces antibodies to the injected antigen. These B-cells cannot multiply indefinitely but posses the enzyme hypoxanthine guanine phosphoribosy transferase (HGPRT) and can grow in hypoxanthine, ammopterin and thymidine medium (HAT) this culture medium is selective for fused hybridoma cells. Unfused mycloma cells cannot grow because they lack HGPRT and this cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the mycloma partner has traits that make it dimilar to caner cell.
FUSION OF SPLENIC B-CELLS WITH MULTIPLE MYELOMA CELLS
When these two types of cells are suspended in a HAT medium in the presence of polyethelyne glycol (PEG) a spontaneous fusion occurs between some cells resulting in a hybrid with the hybrid cells are capable of growing indefinitely in HAT medium and produce antibodies to the desired antigen. The hybrids formed by the fusion will be the only cells surviving indefinitely. This is the hybridoma technique.
This is a laser based, biophysical technology used in cell counting, sorting, biomarker defection and protein engineering. It operates by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of physical and or chemical characteristics of up to thousands of particles per second.
Flow cytometry in routinely used in the diagnosis of health disorders, especially blood cancers but has many other applications in basic research, clinical practice, and clinical trials. If physically sort particles based on their particles, so as to purify population of interest.
Modern flow cytometers are able to analyze several thousand particles per second in real time. A flow cytometer is similar to microscope except that instead of producing an image of the cell. Flow cytometers offers an automated quantification of large numbers of cell parameters. To analyze solid tissue, a single-cell suspension must first he prepared.
COMPONENTS OF FLOW CYTOMETER
1. Flow cell –Made up of liquid stream (sheath fluid) this component carries and aligns the cells so that they pass in single file through the light beam for sensing.
2. Measuring System- Commonly used for measurement of impedance (or conductivity) and optical systems such as lamps (mercury, xenon); high power water-cooled lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon (488nm), red- HeNe (633nm) green- HeNe, HeCd (UV); diode laser (blue, green red, violet) resulting on light signals.
3. Detector and analogue –To-digital conversion (ADC) system –which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer.
4. An Amplification System –Linear or logarithmic
5. A Computer for analysis of the signals.
The process of collecting data from samples using the flow cytometer is called acquisition. Acquisition is mediated by a computer physically connected to the flow cytometer and the soft were which handles the digital inferface with the cytometer. The software is capable of adjusting parameters (ie voltage, compensation etc) for the sample being tested and also assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly.