Practical Endocrinology PDF

Endocrinology Practical Part Methods of hormonal assay: 1. Anatomical method: It is carried out by macroscopical microscopical examination of the endocrine gland to determine the its site and structure in the animal body. 2. Surgical removal method: It is carried out through removal of endocr...

Endocrinology Practical Part Methods of hormonal assay: 1. Anatomical method: It is carried out by macroscopical microscopical examination of the endocrine gland to determine the its site and structure in the animal body. 2. Surgical removal method: It is carried out through removal of endocrine gland and studying the effect of its absence on animal physiology (eg. Removal of the testes in cock results in regression of combs, wattles, and ear lobes). 3. Replacement or substitution method: It is carried out through injection of gland extract to animal whom gland was surgically removed. It was observed that the atrophied organ will develop again. 4. Isolation of active hormone method: It is carried out by extraction of hormone from the gland or blood and purified and then analyzed to determine its chemical structure to confirm its physiological activity. 5. Injection of antisera of specific hormone method: It is very important for determination of hormone concentration.  6. Immunological methods (Immunoassay). An immunoassay is the test that uses antibody and antigen complexes as a mean of generating measurable results. An immunoassay is an analytical method which uses antibodies as reagent to quantitate specific analytes. General principal: The immunoassay is a technique which includes the binding of antigen with an antibody.  Methods of immunoassay: 1- Isotopic immunoassay: based on competition for an antibody between radioactive indicator and unlabeled antigen in the test sample. Examples: Radioimmunoassay Immunoradiometric assay  Methods of immunoassay: 2- Non-isotopic immunoassay Differ from the isotopic immunoassay in: Type of label used. The methods of end point detection. Examples: ELISA (Enzyme – linked immunosorbent assay). Fluroimmunoassay.  (A) Radioimmunoassay (RIA) Radioimmunoassay (RIA): is an in vitro technique used to measure concentrations of antigens (for example, hormone levels in the blood) without the need to use a bioassay (lab. animals' method). Principle: RIA are assays that are based on the measurement of radioactivity associated with antigen-antibody reaction to estimate a ligand (unknown hormone). The reaction is as follows: Ag + Ag* + Ab → Ag Ab + Ag*Ab + Ag* Where Ag = Antigen to be measured in sample, Ag*= Radiolabeled Antigen The technique The technique: Add buffer. Add known amount of unlabeled antigen to the mixture (these compete for the binding sites of antibodies). Add the radioactive antigens to the mixture. Add fixed amount of antibody to the tubes. Radioactive antigen is displaced from the antibody molecules by the unlabeled antigen. Precipitate Ag-Ab complexes with secondary antibody. The antibody-bound Ag separated from the free antigen in the supernatant fluid and the radioactivity of each is measured. From the data, a standard binding curve can be drawn and the concentration of unknown Ag in sample can be read directly from the standard curve. The following substances are detected by RIA: Hormones: insulin, GH, Thyroxin, Estrogen Serum Protein: IgE antibodies Some metabolites: cAMP, folic acid Drugs: Digoxin, Digitoxin, Morphine Microbial agents and antibiotics: Hepatitis B surface antigen in a donated blood Advantages: High specificity: Immune reactions are highly specific. High sensitivity: Immune reactions are highly sensitive. Disadvantages: Radiation hazards. Requires specially trained person. Labs require special license to handle radioactive materials.  (B) ELISA (enzyme-linked immunosorbent assay) ELISA is a widely used method for measuring the concentration of a particular molecules (e.g. hormone, drug). In a fluid such as serum or urine. The requirements of the test: The antibodies fixed to a solid surface; such as the inner surface of a test tubes. A preparation of the same antibodies coupled with an enzyme which produces a colored product from colorless substrate. The technique: The technique: The tubes are filled with sample solution (unknown Ag) to be assayed. Any Ag molecules present bind to the immobilized antibody molecules. The antibody-enzyme conjugate is added to the reaction mixture. This antibody binds to any antigen molecules that were bond previously, creating antibody-antigen-antibody sandwich. After washing away any unbound conjugate, the substrate solution is added. After a set interval, the reaction is stopped, and concentration of colored product formed is measured in a spectrophotometer. The intensity of the color is proportional to the concentration of bound Ag. Advantages: High sensitivity. High specificity. Easy handling with multiple samples. No dealing with radioactivity.