A, B, and C are mixed together. Some of the horse raddish peroxidase leaves C and conjugates with the complex formed

of serum acts directly on parathyroid glands lo conlrol parathormone secretion. FUNCTIONS OF HORMONES. OVERVIEW All the functions can be classified under few headings (see Table No. 6.1.3 - previous page). ESTIMATION OF HORMONES AND HORMONAL STATUS Because of the fact that most hormones occur in the blood in exlremely small quantities, conventional chemical analysis (colorimetric estimation) are usually of no avail. It is only in the tast two and half decades, newer and newer technics have been developed and as a result, an explosive amount of informations has been gathered in recent times. The well known modern melhods of hormone estimation are given below. 1 .Radioimmunoassay (RIA). Originally developed by Bershon and Yalow in around 1960, for which they were subsequently awarded a Nobel Prize, the melhod is as follows: For peptide hormones: The peplide hormone is isolated, purified and repealedly injected into an animal's (which belongs to a different species) body. In the recipient animal's body, because of the fact, that the injected hormone is a foreign protein, antibody development begins and attains high liter with time. Blood of this animal is now col-lected, the antibodies isolaled and purified, and stocked. This anlibody can combine with the peptide hormone in question, selectively and with great affinity. Let this sample be called, A Again, a sample of pure peptide hormone in question is obtained and radiolabelled. Let this sample be called B. Such purified specific anybodies (A) and radiolabelled peptide hormones (B) are readily available from big commercial firms, as well as, for India, from 'BARC' (Bhava Atomic Research Center). The anybodies are now mixed with the patient's serum and radiolabelled hormone. If the palient's serum (sample C) contains excess of the hormone, then, on mixing A, B and C, large amount of radiolabelled hormone will remain un combined. Reverse will be the case, when the serum contains too little amount of the hormone. (These anlibodies can combine with the hormones of both the sources, viz, B & C.). Now the uncombined hormone is separated from the combined ones by cenlrifugation (or by eleclrophoresis). In the supernatant solution, thus obtained (containing uncombined hormone) the intensity of the radioactivity is measured by a suitable counter. From the result thus obtained, estimation of the hormone in the patients serum can be made In short, both the radiolabelled hormone (acting as antigen) and the hormone in patient's serum (also acting as antigen) compete with each other and try lo bind with Ihe antibodies. If serum contains more hormone then more radiolabelled hormone will be unbound. The bound hormone, being heavier particles, can be precipitated and removed. With the introduction of RIA, there has been a revolutionary change in the clinical endocrinology. Diagnosis of many endocrmological disorders, today, can be made with impunity. Steroid hormones and Ihyroxine Steroid hormones and thyroid hormones do not elicit antibodies when injected into a different species of animal (not 'antigenic). However, in the blood, these hormones circulate as bound with some proteins. Thus, there are CBG (cortisol binding globulin), TBG (thyroxine binding globulin) and so on, destined to bind cortisol and thyroxine respectively. These proteins are highly specific and elicit antibodies ('antigeinc). Therefore, such proteins (which bind hormones) are used as antigen and the rest of the procedure remains same. However, one inherent flaw of the RIA is this : it measures the amount of immunologically similar molecules. Now, it is possible that a 'prohormone' or a fragment of a hormone is immunologically same as the hormone in question and yet does not otherwise (biologically) behave as the hormone. In such instances, the RIA value will not reflect the biologically active amount of the hormone. Almost all the peplide hormones and many of the steroid and other non-protein hormones, today can be measured by the RIA technic. Because of the simplicity of the procedure, this melhod is extremely popular in clinical endocrinology. 2. ELISA (enzyme linked immuno sorbent assay) technic So far as the principle is concerned, it resembles closely with that of the RIA However, it does not require a counter for measuring the radio activity (inslead only a spectrophotometer to measure the color is sufficient) and therefore, this technic can be used in smaller towns of India. In short, ultimately, instead of measuring the radioactivily (as in RIA), one has to measure the intensity of the color of the final product. Three samples, viz, A, B, C will be required A conlains the slocked highly, purified antibody (obtainable from commercial firms, in the form of 'kit'), B is the serum of the patient containing the hormone which acts here as the an-tigen (whose concentration is to be measured), and C is a purified antigen (ie, a purified specimen of the hormone which is chemically identical in nature with the hormone in B), but conjugated with horse raddish peroxidase. A, B, and C are mixed together. Some of the horse raddish peroxidase leaves C and conjugates with the complex formed by A and B (recall, in the mean time, the antigen of B combines with the antibodies in A so that a complex of A and B is formed). The complex of A and B is now isolated, and washed to remove extraneous materials finally the complex of A & B is stained with a suitable dye, called ABTS (diammonium 2, 2' azino bis (3 - ethyl benzothiozolene 6 sulfonate)] in presence H20