Lab.3/ Immunology
Brucella test (Rose Bengal Test):
Brucella antigens are bacterial suspensions for use in slide agglutination test to detect the presence of bacterial agglutinins associated with bacterial infection or previous exposure to a related organism. This slide test is recommended as a screening procedure only to establish the presence or absence of homologous antibody.
Reagents:
Rose Bengal Brucella antigen (0.5% phenol).
Positive control (0.01% sodium azide).
Negative control (0.01% sodium azide).
Specimen collection and preparation:
Collect 5 ml whole blood samples aseptically from the patient.
Allow blood to clot and remove serum as soon as possible to prevent excess hemolysis.
Store serum at 2-8°C until testing can be preformed.
Materials required:
Brucella antigen, Rose Bengal stained.
Positive control.
Negative control.
White glass slide.
Stirring sticks.
Pipettes for dispensing (40mml) serum.
Timer.
Procedure:
Allow reagents and serum samples to reach room temperature for testing.
Shake the antigen bottle gently to insure a uniform suspension.
Place (40 ml) sample serum onto the selected ring of the slide.
Place one drop of the Rose Bengal antigen onto serum sample.
Mix serum sample with Rose Bengal antigen using stirring stick.
Repeat these steps using the positive and negative controls instead of serum sample.
Gently rock the slide for 2 minutes (automatic rotator can also be used).
Observe for agglutination after 2 minutes from beginning of shaking (this is the optimum time limit).
Results:
Negative: No agglutination.
Positive: (Presence of specific antibodies) Agglutination.
Cell-Mediated Immunity:
Although humoral immunity is an important host defense against many bacterial and viral diseases, in many other bacterial infections (especially intracellular infections such as tuberculosis) and viral infections, it is primarily the cell-mediated arm that imports resistance and aids in recovery.
Furthermore, cell-mediated immunity is important in defense against fungi, parasites and tumors and in the rejection of organ transplants.
The constituents of the cell-mediated system include several cell types:
Macrophages: Which present the antigen to T cells.
Helper T cells: Which participate in antigen recognition and in regulation (helper and suppressor) functions.
Natural killer (NK) cells: Which can inactivate pathogens.
Cytotoxic T cells: Which can kill virus-infected cells with or without antibody.
Macrophages and helper T cells produce cytokines that activate helper and cytotoxic T cells, leading to the killing of the pathogens or tumor cells.
Delayed (cell-mediated) Hypersensitivity:
Is a function of T lymphocytes, not antibody.
The macrophage ingests the antigen, processes it, and presents an epitope on its surface in association with class II MHC protein. The helper T (Th-1) cell is activated and produces gamma interferon, which activates macrophages. These two types of cells mediated delayed hypersensitivity.
It can be transferred by immunologically committed (sensitized) T cells, not by serum.
The response is delayed ex. It starts hours (or days) after contact with the antigen and often lasts for days.
In certain contact hypersensitivity, such as poison oak, the pruritic, vesicular skin rash is caused by CD8-positive cytotoxic T cells that attack skin cells that display the plant oil as a foreign antigen. In the tuberculin skin test, the indurations skin rash is caused by CD4-positive helper T cells and macrophages that are attracted to the injection site.
Skin test: (Tuberculin- Type Hypersensitivity)
Delayed hypersensitivity to antigens of microorganisms occurs in many infectious diseases and has been used as an aid in diagnosis. It is typified by the tuberculin reaction.
When a patient previously exposed to mycobacterium tuberculosis is injected with a small amount of tuberculin (PPD [Purified Protein Derivative]) intradermally, there is little reaction in the first few hours. Gradually, however, indurations and redness develop and reach a peak in (48-72) hours. A positive skin test indicates that the person has been infected with the agent, but it does not confirm the presence of current disease. However, if the skin test converse from negative to positive, it suggests that the patient has been recently infected.