Common Methods for Testing Hormones and Drugs Radioimmunoassay (RIA) Enzyme Immunoassay (EIA) Fluorescence Polarization Immunoassay

say (FPIA) Thin Layer Chromatography High Performance Liquid Chromatography (HPLC) Gas Chromatography Mass Spectrometry (GCMS) Enzyme Immunoassay an unknown amount of antigen is affixed to a surface a specific antibody is applied over the surface so that it can be bind to the antigen Antibody is linked to an enzyme and substrate is added that the enzyme can convert to some detectable signal, most commonly a color change in a substrate Radioimmunoassay (RIA) competition between endogenous and radio labeled hormone for binding sites on a limited amount of antibody Separation of antibody bound and free radio ligands is by double antibody precipitation or by using solid phase second antibody procedure amount of labeled analyte bound to antibody is inversely proportional to amount of unlabeled analyte present in the serum Users radioactive isotopes of iodine (125I , 131I) and tritium (3H) as labels IRMA does not require a quantity of purified antigen because the antigen cannot be labeled, obviates problem that may be caused by iodination of labile antigen a known quantity of an antigen is made radioactive, frequently by leading it with gamma-radioactive isotopes of iodine attached to tyrosine Radio-labeled antigen is mixed with a known amount of antibody for that

antigen and the two chemically bind to one another serum from a patient containing of that same antigen is added the unlabeled (cold) antigen from the serum completes with the radio labeled antigen (hot) for antibody binding sites as concentration of cold antigen is increased, more binds to the antibody displaces the radio-labeled variant, reducing the ratio of antibody-bound radio-labeled antigen to free radiolabeled antigen the bound antigen are separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma scintillation counter using known standards, a binding curve can be generated which allows the amount of antigen in the patient's serum to be derived. Fluorescence Polarization Immunoassay fluorescein-labelled drug competes with unlabeled drug for antibody sample excited with plane polarized light (490nm) Fluorescein emits plane polarized light (520nm) small, free drug-fluorescein, rotates faster leading to less emission larger, antibody-drug-fluorescein, rotates slower and produces more emission drug in sample competes fro antibody with fluorescein bound drug more drug in the sample, less fluorescein labeled drug bound to antibody, lower emission of plane polarized light higher drug concentration results in lower light emission values Advantages: available for a variety of drugs rapid turnaround times, sensitivity, ease of operation Disadvantages: background interference in serum sample (requires blank

measurement) Liquid Chromatography liquid chromatography (LC) is a separation technique in which the mobile phase is liquid can be carried out either in a column or a plane in liquid chromatography, a mixture of molecules dissolved in solution (mobile phase) is separated into its constituent parts by passing through a column of tightly packed solid particles (stationary phase) Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC) High Performance Liquid Chromatography (HPLC) a form of liquid chromatography to separate compounds that are dissolved in solution a form of liquid chromatography that utilizes smaller column size, smaller medium inside the column, and higher mobile phase pressures an advanced form of liquid chromatography used in separating the complex mixture of molecules in chemical and biological systems in order to understand better the role of individual molecule HPLC has four basic components: a solvent delivery system to provide the driving force for the mobile phase a means by which the sample can be introduced into the column the column a detector, a recorder is used to display the results and an integrator performs the calculations HPLC instruments consists of a reservoir of mobile phase, a pump, an injector, a separator column, and a detector Compounds are separated by injecting a plug of sample mixture onto the column

The liquid sample is introduced into a sample loop of an injector of a syringe. When the loop is filled, the injector can inject the sample into the stream by placing the sample loop in line with the mobile phase tubing the column is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane by a liquid (mobile phase) at high pressure components in the mixture pass through the column at different rates due to differences in partitioning behavior between the mobile liquid phase and the stationary phase the separation occurs because each component in the mixtures interacts differently with the stationary phase molecules that interact strongly with the stationary phase will move slowly through the column, while the molecules that interact less strongly will move rapidly through the column this differential rate of migration facilitates the separation of molecules solvents must be degassed to eliminate the formation of bubbles the pumps provide a steady high pressure with no pulsating, and can be programmed to vary the composition of the solvent during the course of the separation the presence of analytes in the column effluent is recorded by detecting a change in refractive index, UV-VIS absorption at a set wavelength, fluorescence after excitation with a suitable wavelength, or electrochemical response Mass spectrometers can also be interfaced with liquid chromatography to provide structural information and help identify the separated analytes subclasses of HPLC (based on the polarity of the mobile and stationary phase) Normal Phase Liquid Chromatography (NPLC) the stationary phase is more polar than

the mobile phase (e.g toluene as the mobile phase, silica as the stationary phase) Reversed Phase Liquid Chromatography (RPLC) (e.g water-methanol mixture as the mobile phase and C18, octadecylsilyl as the stationary phase) allows analysis to be done in shorter time achieves higher degree of resolution separation of constituent is more complete allows stationary column to be reused a number of times without requiring that they be regenerated results of analysis are more reproducible permits both instrumentation and quantitation to be automated

inside of a small diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column) Gas Chromatography Mass Spectrometry (GCMS) method that combines the features of gas liquid chromatography and mass spectrometry used together, a much finer degree of substance identification is made that either unit used separately composed of two major building blocks: the gas chromatography and the mass spectrometer utilizes a capillary column which depends on the column's dimensions (length, diameter, film thickness) as well as the phase properties (e.g. 5% phenyl polysiloxane) the difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column molecules take different amounts of time (called retention time) to come out of (elute from) the gas chromatograph mass spectrometer is allowed to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. Each molecule is broken down into ionized fragments fragments are detected using their mass to charge ratio an accurate identification of a particular molecule can be made by performing it in gas chromatography and mass spectrometry the mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame Ionization Detector) detects multiple molecules that happen to take the same amount of time to travel through the column (e.g. have the same retention time)

Thin Layer Chromatography (TLC) a widely employed laboratory technique similar to paper chromatography instead of using stationary phase of paper, involves a stationary phase of a thin layer of absorbent like silica gel, alumina, or cellulose on a flat, inert substrate compared to paper, it has the advantage of faster runs, better separations, and the choice between different absorbents for even better resolution and to allow for quantification, High Performance TLC can be used * HPLC column * TLC plane Gas Chromatography a separation technique in which the mobile phase is a gas is always carried out in a column, which is typically packed or capillary based on partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium) the stationary phase is adhered to the