HORMONAL ASSAYS BY BAMIDELE OLUBAYODE DEPARTMENT OF PHYSIOLOGY

HORMONAL ASSAYS BY BAMIDELE OLUBAYODE DEPARTMENT OF PHYSIOLOGY COLLEGE OF HEALTH SCIENCES BOWEN UNIVERSITY, IWO, NIGERIA.

INTRODUCTION • Hormonal assay is the test carried out on sample of blood to detect and measure the level of a hormone. • It is performed to confirm hormonal abnormalities and also during treatment and after treatment of hormonal abnormalities.

Measurement of Hormone Concentration in the Blood • Hormones are present in the blood in extremely minute quantities. • Some concentrations are as low as one billionth of a milligram (1 picogram) per milliliter. Therefore, it was very difficult to measure these concentrations by the usual chemical means.

History of Hormonal Assay • The history of hormone assays begins earlier, with a variety of in vivo biological tests. • Later, colourimetric assays were found and used to assay for cortisol. Since 1970, immunoassays have become increasingly routine and gained dominance within the clinical laboratory for 40 years. • After the 40 years, a new technology, tandem mass spectrometry (TMS), is starting to emerge as the method of choice

Hormonal Assay Techniques • Bioassay • Immunoassay ü Radioimmunoassay ü Enzyme-linked immunosorbent assay (ELISA) • Tandem mass spectrometry

BIOASSAY • It is defined as the estimation of potency of an active principle in a unit quantity of preparation • It can be otherwise defined as detection and measurement of the concentration of a substance in a preparation using a biological methods.

Importance of Biological Assay • . Chemical assay is too complex and not sensitive enough to measure e. g insulin. • It is used to measure the pharmacological activity of new or chemically undefined substances • It is for biological standandization of drugs obtained from natural sources as these cannot be obtained in pure form e. g oxytocin, vasopressin, insulin e. t. c • It is used to measure drug toxicity and unwanted effects.

Principles of Bioassay • This is to compare the test substance with the international standard preparation of the same. • To find out how much test substance is required to produce the biological effect as produced by the standard,

Examples • The early bioassays included the chick cockscomb test. • Androgen-like material was applied to the cockscomb of a chick, and the increase in size was used as a measure of testosterone. • Another qualitative test used the Xenopus toad, which ovulated in response to human chorionic gonadotrophin injections, and was used as an early pregnancy test.

Types of Bioassay • Quantal assay: The response is in the form of all or none i. e either no response or maximum response. • Graded assay: the response is proportional to the dose and response may lie between no response and maximum. • Bioassay types can either be direct or indirect.

Immunoassay • (a) Radioimmunoassay (RIA): • The development of the radioimmunoassay (RIA) by Yalow and Berson in the 1950 s transformed endocrine studies, providing a technique that is sensitive, and precise. • They were awarded a Nobel Prize in 1977. Owing to their selfsacrifice, they did not patent their findings.

Principles of Radioimmunoassay • An antibody that is highly specific for the hormone to be measured is produced. • A small quantity of this antibody is mixed with a quantity of fluid from the animal containing the hormone to be measured • Then, mixed simultaneously with an appropriate amount of purified standard hormone that has been tagged with a radioactive isotope. • There must be too little antibody to bind completely both the radioactively tagged hormone and the hormone in the fluid to be assayed

Principles of Radioimmunoassay • The natural hormone in the assay fluid and the radioactive standard hormone compete for the binding sites of the antibody. • In the process of competing, the quantity of each of the two hormones, the natural and the radioactive, that binds is proportional to its concentration in the assay fluid. • After binding has reached equilibrium, the antibody-hormone complex is separated from the remainder of the solution, and the quantity of radioactive hormone bound in this complex is measured by radioactive counting techniques

Principles of Radioimmunoassay • The decrease in the amount of radiolabeled hormone bound to specific antibody in the presence of the natural hormone is measured in order to determine the amount of natural hormone present in the test sample. • To make the assay highly quantitative, the RIA procedure is also performed for "standard" solutions of untagged hormone at different concentration levels. Then a "standard curve" is plotted.

Source: Poojar Pawar

Limitations of RIA • It is expensive and involved hazards of preparing and handling the radioactive antigens. • It requires specially trained persons. • Laboratory requires licence to handle radioactive materials

Enzyme-linked immunosorbent assay (ELISA) • ELISA is a common laboratory techniques which is used to measure almost any protein, including hormones.

Why is it called Enzyme-linked immunosorbent assay ? • Antigen/antibody of interest is absorbed on to plastic surface (sorbent). • Antigen is recognised by specific antibody (immuno). • This antibody is recognised second antibody(immuno) which has enzyme attached (enzyme-linked). • substrate reacts with an enzyme to produce product usually, coloured

History of Elisa • In 1977, Peter Perlmann and Eva Engvall at Stochkolm University in Sweden and Anton Schuur and Bauke Van Weemen in Netherlands independently published papers that synthesized the knowledge of radioimmunoassay into methods to perform Elisa.

Principles of Elisa • This test combines the specificity of antibodies with the sensitivity of simple enzyme assays. • It is performed on plastic plates that each have 96 small wells. • Each well is coated with an antibody (AB 1) that is specific for the hormone being assayed. • Samples or standards are added to each of the wells, followed by a second antibody (AB 2) that is also specific for the hormone but binds to a different site of the hormone molecule.

Principles of Elisa • A third antibody (AB 3) is added that recognizes AB 2 and is coupled to an enzyme that converts a suitable substrate to a product that can be easily detected by colorimetric or fluorescent optical methods. • Because each molecule of enzyme catalyzes the formation of many thousands of product molecules, even very small amounts of hormone molecules can be detected. • ELISA methods use excess antibodies so that all hormone molecules are captured in antibody-hormone complexes.

Principles of Elisa • Therefore, the amount of hormone present in the sample or in the standard is proportional to the amount of product formed.

Adapted from: Saba Ahmed

Advantages of Elisa • It does not employ radioactive isotopes, • It is cost-effective • It is an accurate method for assessing hormone levels

Tandem Mass Spectrometry (TMS) • The reduction of mass spectrometers from the size of a double bedroom to a desk top instrument has permitted the introduction of TMS to the clinical laboratory. • It is a technique to break down selected ions (precursor ions) into fragments (product ions). The fragments then reveal aspects of the chemical structure of the precursor ion. • TMS using quadrupoles is only useful for the measurement of small molecules such as steroids, drugs and intermediary metabolites.

Tandem Mass Spectrometry (TMS) • More sophisticated instruments are necessary for peptide hormone measurement. • It is remarkable that, despite it being less than 10 years since the first reports of the use of TMS in endocrinology. • TMS is presently a sophisticated technique that needs skilled staff, but it will undoubtedly become a more friendly technique in the future.

Tandem Mass Spectrometry (Source: Wikipidia)

Three Quadrupoles (Quad 1, Quad 2, and Quad 3) are lined up in a row. Precursor ions are selected in Quad 1 and sent to Quad 2 for dissociation (fragmentation). The generated product ions are sent to Quad 3 for mass scanning.

In the QTOF, precursor ions are selected in the Quadrupole and sent to the Collision Cell for fragmentation. The generated product ions are detected by time-offlight (TOF) mass spectrometry.

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REFERENCES • Guyton and Hall. Textbook of Medical Physiology. 9 th Edition, W. B Saunders company, London, Toronto. Philadelphia. • The Endocrinologist. The magic of measuring hormones. Special feature, page 6 -13.