Endocrine Methodologies:Rate of development of any branch of science frequently is limited by available method and techniques. Often in endocrinology, introduction of new methods have lead to quantum leaps in design, amount of information and understanding of the subject. The types of scientific question that are asked are depended on the methodology. Many discoveries are only the confirmation done earlier and scientifically proved by experiment later.Classical techniques: prevalent for many years. Although many modern techniques are available, but now the modern techniques are best understood and used extensively.Eg: the most classical earlier technique is removal of gland and their function is studied. And by replacement of gland by extracting the fractions of gland and each hormonal ation is studied. Histological techniques are also involved in classical techniques i.e. studying the histological morphology of endocrine glands. In later times, the quantitative techniques are also used by measure the particular amount secreted and determine the quality of the hormones. Radio immunological essay, autoradiography, histochemistry, immunohistochemistry, recently – genetic engineering techniques.Castration is extensively used in order to improve the flavour of meat.Histological and cytological studies:Descriptive endocrinologists are used H and C studies for descriptive study of glands by observing in light microscope. By using the strained sections , they are able to describe the gland and it is called plain histology. By using tri and tetrachrome staining techniques, they are able to differentiate the different cells in a given section. Eg: in pituitary, different cells produce different hormones. Plain history lead to histophysiology and at the same time there is a development of histochemistry or cytochemistry for identification of substances present in it. Enzyme histochemistry is also used for localization of enzymes. “Histoenzymology” adding substrate, cofactor to enzymes)eg: Testis (steroidogenic silk)Limitations of these techniques:1) Not quantitative 2) Very laborious.Later these techniques are metamorphosised to electro microscopic studies. The main advantage here is in the order to explain the functional status of substances present in it. And also to differentiate the different cells in a gland. Electro microscopy is of two types.Scanning E.M- surface structure and the different cell types and Transmission E.M- but still it constitutes the morphological techniques only with out any quantitative but eventhough they provide to use powerful in consumption with quantitative Bio-chemical methods.Immunohistochemistryto overcome the specificity of the eaction, immuno-histochemistry technique is developed. Although histological or histochemical stains are used for histological study, they lack specificity of a particular group. Immuno histochemistry is used to localise proteins hormones etc., IHC, combines histochemistry and immunology disciplines. To define IHC, identification of the tissue constituent in situ by antigen- antibody reaction tagged by a visual label. The visual marker is a frozen dye, enzyme, fluorescent dye radioactive marker. Maximum signal strength with minimum background or non specific stain.The pre requisite is that an anti serum should be prepared where it should contain an antibody which is against the action of the protein. The antibody is raised against the antigen in rabbit. Purified rat prolactin (protein) is injected for rabbit, the rabbit immunity system respinds and produced antibody and the blood is collected where it contains a high concentration of rabbit anti rat prolactin IgG . the antibody produced is an immuno globulin molecule. For eg: when rabbit IgG is injected to a goad it produces Goat anti rabbit IgG resulting in a secondary antibody- “it is produced against rabbit IgG”. This secondary antibody of a particular species reacts with all the antibodies of a particular species like FSH IgG, LH IgG, ACTH IgG. Mainly there are 2 methods: 1) Antibody conjugated to a fluorescent dye. 2)Antibody conjugated to an enzyme.I) Antibody against teh peptides are conjugated to dyes is to localize hormone producing cells, it gets deposited only these cells producing antibody. The specificity is determined by prior treatment with non-specific antibody.ii) antibody conjugated to an enzyme is peroxidase is allowed to interact to tissue slides, when substrate is added to an enzyme containing slides it produces a color compound which can be easily identified in ordinary microscope. By the use of electron microscope and immunograph method, the site of th eenzyme activity can be traced, which gives evidence of hormones under investigation.Applications of IHC:Direct methodIndirect method- it is preferred more.Direct method: It was the first to be developed. The labelled primary antibody is applied to the sections and indentifies the antigenic sites. Indirect method: this is more sensitive than the direct one. Because the same number of primary antibody bound to the tissue antigens will be more intensively labelled in comparison with 1) same or may be visualised by low concentration of primary antibody. 2) same section is again injected with secondary antibody in order to get a clear visualisation.Two methods used in immunocytochemistry arePeroxidase- antiperoxidase (PAP):Developed by sternberher which had a great impact on Immunocytochemistry. (Antibody is against the peroxidase). It consists of different reagents in a series. First layer is rabbit primary rabbit against the specific antigen. Second layer consists of unlabelledanti-rabbit IgG after incubation of teh first and it is obtained from goat. IIIrd layer consists of Rabbit-antihorse radish peroxidase.IV layer consists of horse radish peroxidaseIn order to increase the specificity, many antibodies against antigen are placed one next to another.Horse radish peroxidase is produced by histochemical method by hydrogen peroxidase (H2O). Sternberger modified this method where in combination with anti peroxidase with peroxidase forming a simple complex of 2- peroxidase and 3antibody. This complex is seperated from the unreached antibody and peroxidase before . this acts as a third layer. There is a high ratio of peroxidase 2 to primary antigen method. The increased amount of level allows the primary antibody is highly diluted which has the ability to producing unwanted staining.Alkaline Phosphatase and Gluose phosphate is also used as an enzyme instead of peroxidase. In the immunofluorescence technique, the application of second layer is anti-fluorescent conjugated anti rabbit ϒ globulin obtained from goat. Fluorescence can be seen in the areas where antigen-antibody reaction takes place using uv light of fluorescent microscope. Utility of Immunohistochemistry:For experimental study of basic science in research purpose site of its production, storage etc.The latest technique supported the immunohistochemistry is insitu hybridisation using m-RNA probes.It has got immense utility in diagonostic purposes. Eg: for tumor, foreign organism, abnormal cellular constituents, specific antigen marker, lymphocyte, abnormal hormone production.BIO ASSAYSOne of the earliets method used in endocrinology for quantifying the hormones, storage product etc. The physiological activity of the hormone is deducted by bio assay.Avidin- Biotin method:Avidin is a basic glycoprotein which has a high affinity for small water soluble vitamin called Biotin. Biotin can be conjugated to a variety of biological molecules including antibodies, mainly biotin can be attached to a single molecule of protein, called as Biotinylated protein. Biotinylated protein can bind to more than one molecule of Avidin but recently streptanilin is used. The tissue sections are treated with primary antibody then with biotinylated secondary antibody and then with streptavidin HRP complex and the enxyme action is triggered by H2O2.Bio Assay:It is a played a mojor role in the early phases of Endocrinology. The basis of bioassay relies on measurement of Physiological response to a hormone.Radio Immuno Assay (RIA):Antibody having a fixed number is allowed to react with labelled antigen (labelled hormone at a known cone and fixed) and Unlabelled antigen (known concentration with varying concentration) mix well and incubated. After incubation how much labelled antigen is bound to antibody is measured by scintilation counter. Thyroxine RIA:Antibodies against the pure thyroxine is obtaines by injecting pure hormone.A reaction mixture containing fixed antibodies and fixed and known amount of labelled thyroxine prepared in buffer solution.Unlabelled thyroxine is added to this mixture and different concentrations of unlabelled thyroxine are selected starting from 0 concentration. These varied concentrations are used in the experiment.The mixture of antibody, lab thyroxine and unlabelled thyroxine are incubated for several hours in order to allow the competition between labelled and unlabelled hormones.In these tubes containing unlabelled hormone at equilibriium only a small portion of unlabelled hormone bound when tubes contain little amount of labelled hormone.Once th binding equillibrium is reached, the antibody bound and unbound are seperated. Saturated RIA is used for this purpose for the seperation of antibody bound and free ones because free thyroxine(both labelled and unlabelled) is soluble in NH4SO4. Thyroxine bound to antibody are precipitated. The supernatent containing unbound hormone is discarded. The amount of radioactivity is measured in scintillation counter. (Solid and liquid ones are used.)The counts of various standards resulting from this procedure are plotted on graph shows the amount of antigen bounded to an antibody.Once the standard curve is available, unknown sample’s blood sample compound sample.Here as the concentration of unlabelled hormone increases, the amount of bound radio active hormone decreases. Seperating techniques are different for different hormones.Procedures: Method of seperation of free hormone from the bound one. 1) Testing the Reliability: This is done in order to find out the recovery test measures the accuracy. Fixed amount of sample volume is added to various standards and then estimated. The aspected value is “mathematical sum of standard cone concentration + concentration of sample”. If variation is recorded, data is not valid. Infrassay is conducted by using various replicates. Advantages: Sensitivity and precision for quantification of hormones. Eg: A typical hormone of RIA measures Picograms (pg).It is easy to perform and have it is established and easy and very less expensive.It is used with various hormones.Disadvantages: Non or unspecific to bind to other hormones. (one hormone mimics to the other hormone in blood serum).Priority of procedures are used in order to overcome these problems.Need for appreciable amount of purified hormones for generating the antisers.Applications:It is a basic tool in basic and applied research because of precision and sensitivity of hormones.It has got clinical application for in teh defeciency of hormones or oversection of hormones can be deducted. (Hypersection of TSH or deficiency of Iodine leads to Goitre)Later the advancement of competitive RIA, two step RIA became important since convetional RIA provided the measurement of hormones and for diagonosis of thyroid and steroid hormone diseases. Hence 2 step RIA developed. Cooperative Immunoassay (CIA)- to enhance the specificity of hormone. To develop a monoclonal antibody, it involves use of monoclonal antibodies, the assay is 100 fold more sensitive than the competitive RIA.ELISA or EIA:“Enzyme linked immunosaorbant assay” or “Enzyme Immunoassay”.There are 3 types of ELISA Competitive EIAThe double antibody methodIndirect method. Competitive EIA: Enzyme linked and not linked antigens compete to bind to antibody. Coated to a solid phase competitive binding unbound antigen are removed by washing later enzyme is provided. By keeping the linked antigen constant and by adding unlinked antigen, a standard graph is constructed. And an unlinked antigen is replaced by unknown sample. The concentration of a sample is measured by superimposing absorbed values in a graph. An antibody which binds specifically to antigen which supports the solid surface.Method: A fixed amount of antibody is added to wells in the bottom of the cellulose plates. First a standard curve is constructed. A fixed amount of enzyme linked labelled antigen added where as enzyme linked antigen is fixed. And wells are incubated for different periods depending upon the antigen is estimated. The unbound antigen is washed off and reporter enzyme is assayed for activity is seen. The enzyme activity results in teh formation of colour compound. And the intensity of the colour is measured in absorption spectra. The absorbance obtained in the assay is proportional to the enzyme linked antigen to antibody is obtained. And the graph is plotted against absorbance to the antigen concentration, a smooth curve is obtained. For deducting the concentration in unknown samples, some assays are repeated and the unlinked antigen by samples.Antibody against the hormone under the hat are coated to the wells, and unknown samples are added. Final volume. Repeat the same process is repeated to the absorbants value of unknown samples are superimposed on graph to obtain concentration of antigen on wells. The Double Antibody Method: This is typically used for large antigens such as proteins. The method uses two typical antibodies which are typically to bind antibody. The antibody 1 is added to solid support and the solution may contain antigen that allowed to react in the well and washed off. The antibody 2 is linked to a receptor enzyme and it is added and allowed to react with an already bound antigen. Then the reporter enzyme activity is conducted by providing substrate and measures the absorbance under specific conditions, the amount of antibody is bounded to specific antigens. Indirect Method: Sometimes it is required to quantify the amount of antibody in a solution in order to after production of successful antibody or deduction of antibody in the fluid of body under pathological conditions. Desired Antigen is attached to the solid surface and the desired antibody is added and the reaction is allowed in the well and unbound antibody is washed off. The solutions containing 2o enzyme linked antibody to 1o antibody is allowed to react in the well and unbound antibody is washed off and the bound antibody is measured by its colour intensity.It is used in deduction of Diabetes and mainly used in deduction of AIDS.RADIO RECEPTOR ASSAY (RRA):The receptor does not differentiate between the labelled and unlabelled antigen so thre exists a competition to the receptor sites. The chemical procedure used for this this is same as that of RIA. Technique:Isolation of receptors by cell disruption, differential centrifugation by means of this receptors can be seperated.Hormones binding to the receptors are as follows:Receptors have high affinity to the hormone.Receptors are present in such a low concentration that are capacity for binding the hormone is low.Hormones bind to some extent to other components other than the cell receptors.The non receptor binding site in a cell fraction has a reality low affinity for hormone but for their abundance, teh binding will be more.The primary aim of the technique is seperate identification of high affinities and low capacity receptor binding end low affinity and high capacity binding will be found by the hormones. This is done by incubating various fractions of radio labelled and unlabelled hormone for the saturation of receptors.In an assay of nuclear receptors, one series of tubes containing nuclear receptors are used and in one series of tubes containing a constant amount of nuclei and various concentrations of radio-labelled hormone is incubated for a time to reach the equillibrium.[Nuclear receptor + Radio labelled hormone] (fixed) (100 receptors) (Various concentrations) mgIn a second series of tubes containing same amount of nuclei, then various concentrations of radio-labelled hormone and then added an unlabelled hormone 100 times more than the laelled hormone. [100 receptors + 1mg ϒ labelled hormone + 100mg of unlabelled hormones]And after equillibrium is reached, the nuclei is seperated by ultracentrifugation. And the supernatent and n pellet is seperated. The nuclei and ϒi-hormone represents both the hormone bound to the receptors and other cell components in 1st series. In 2nd sereis represents the hormone binding to non receptor components and the receptor hormone binds to teh low affinity and high capacity hormone is attached. A graph of bound Vs free hormone illustrates the binding between and non specific binding in the 2nd series of tubes gives the variance between free hormones and bound ones. Specific binding increases as the hormone concentration increases and the hormone receptor becomes saturated.RRA is essentially invitro test, where it measures the activity and not specific structure. The specificity can be attended by non specific binding and hence it is used as a research tool and hence it is not used in clinical diagnostic test. However, it provides the information in addition to obtained in RIA, ELISA. This may give the instant of endocrine diseases.Advantages:Understanding the biological activity and measuring the physiologically active species of hormones. (T3 and T4)Studying the structure and function analogs of peptide hormones.It is useful for estimating the hormone antibodies in Diabetes mellitus and Graves disease.