IMMUNOASSAY TESTSIMMUNOASSAY TESTS

DEFINITIONDEFINITION

Immunoassays are chemical tests used to detect or quantify a specific substance, the analyte, in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. High sensitivity is achieved by using an indicator system (e.g., enzyme label) that results in amplification of the measured product.

Immunoassays may be qualitative (positive or negative) or quantitative (amount measured). An example of a qualitative assay is an immunoassay test for pregnancy. Pregnancy tests detect the presence of human chorionic gonadotropin (hCG) in urine or serum. Highly purified antibodies can detect pregnancy within two days of fertilization. Quantitative immunoassays are performed by measuring the signal produced by the indicator reaction. This same test for pregnancy can be made into a quantitative assay of hCG by measuring the concentration of product formed.

PURPOSEPURPOSE

The purpose of an immunoassay is to measure (or, in a qualitative assay, to detect) an analyte. Immunoassay is the method of choice for measuring analytes normally present at very low concentrations that cannot be determined accurately by other less expensive tests. Common uses include measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury. Qualitative immunoassays are often used to detect antigens on infectious agents and antibodies that the body produces to fight them. For example, immunoassays are used to detect antigens on Hemophilus, Cryptococcus, and Streptococcus organisms in the cerebrospinal fluid (CSF) of meningitis patients. They are also used to detect antigens associated with organisms that are difficult to culture, such as hepatitis B virus and Chlamydia trichomatis. Immunoassays for antibodies produced in viral hepatitis, HIV, and Lyme disease are commonly used to identify patients with these diseases.

DESCRIPTIONDESCRIPTION

There are several different methods used in immunoassay tests.

IMMUNOPRECIPITATION.IMMUNOPRECIPITATION. The simplest immunoassay method measures the quantity of precipitate, which forms after the reagent antibody (precipitin) has incubated with the sample and reacted with its respective antigen to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative.

PARTICLE IMMUNOASSAYSPARTICLE IMMUNOASSAYS. By linking several antibodies to the particle, the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of antibodies that are markers of such diseases, as infectious mononucleosis and rheumatoid arthritis.

IMMUNONEPHELOMETRY.IMMUNONEPHELOMETRY. The immediate union of antibody and antigen forms immune complexes that are too small to precipitate. However, these complexes will scatter incident light and can be measured using an instrument called a nephelometer. The antigen concentration can be determined within minutes of the reaction.

RADIOIMMUNOASSAY (RIA) RADIOIMMUNOASSAY (RIA) is a method employing radioactive isotopes to label either the antigen or antibody. This isotope emits gamma raysare, which are usually measured following removal of unbound (free) radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassay.

ENZYME (EIA) IMMUNOASSAYENZYME (EIA) IMMUNOASSAY was developed as an alternative to radioimmunoassay (RIA). These methods use an enzyme to label either the antibody or antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection of infectious diseases is the enzyme-linked immunosorbent assay (ELISA).

FLUORESCENT IMMUNOASSAY (FIA)FLUORESCENT IMMUNOASSAY (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.

CHEMILUMINESCENT IMMUNOASSAYSCHEMILUMINESCENT IMMUNOASSAYS utilize a chemiluminescent label. Chemiluminescent molecules produce light when they are excited by chemical energy. These emissions are measured by a light detector.

ASSAYS OF HORMONES AND RECEPTORSASSAYS OF HORMONES AND RECEPTORS

HORMONAL ANALYSES.HORMONAL ANALYSES. Most hormone assays performed today are of the competitive-binding variety. For a competitive-binding assay to be of value it must be practical and reliable.

RADIOIMMUNOASSAYRADIOIMMUNOASSAY

The RIA is the conventional prototype of a competitive-binding assay. There are three fundamental components to the RIA - radioactive ("hot") hormone, unlabeled ("cold") hormone (standard or sample), and antibody. Radioisotopes of tritium ( emitter) and iodine (high specific activity emitter) are incorporated into steroid and protein (Tyr or His residues) hormones, respectively; this must be done without significant damage to the immunoreactivity of the hormone. Tracer and standard or unknown sample compete for a limited number of binding sites on the antibody. Amounts of (excess) tracer and antibody for each reaction are held constant, while quantities of standard hormone are increased step-wise. Reactions are allowed to proceed to equilibrium, and free (unbound) hormone is segregated from antibody-hormone complexes. Emission of energy from the bound complex is monitored by radiation detection equipment. As content of standard is increased from 0 (ie., 100% of antibody is bound by tracer), the amount of antibody-bound tracer declines reciprocally - a standard curve is constructed from these data. Reaction tubes containing sample in place of standard are assayed simultaneously. Estimates of mass of hormone within a sample are interpolated from the standard curve (Figure 2-38).

Antibodies belong mainly to the gamma globulin (IgG) class of immunoglobulins. Each Fab arm of the (bivalent) antibody can bind a molecule of ligand. Binding is mediated by weak noncovalent forces (ionic interactions, hydrogen bonding, hydrophobic attractions, van der Waals attraction); therefore, like that of enzyme-substrate binding, the reaction is reversible.

Antisera can be generated by injecting purified hormone into a species of animal that is capable of mounting an immunological reaction to that hormone (ie., do not produce the hormone in a chemical form that is exactly similar). Some small molecules (haptens) are not antigenic on their own (eg., steroid and peptide hormones, prostaglandins) and must first be coupled (at a nonactive site) to an immunogenic carrier (eg., albumin, keyhole limpet hemocyanin) before injection.

Even under the best of conditions of immunization, antisera can contain antibodies (polyclonal) that cross-react with related substances - the development of technology using monoclonal (homogenous) antibodies has helped in this respect. To obtain monoclonal antibodies an animal (eg., mouse) is injected with purified antigen,

spleen cells capable of secreting a single type of antibody (clones) are screened and isolated, and selected cells are fused with myeloma (immortal) cells to produce a hybridoma. Cells maintained in culture provide a continuous source of antibody. A single hybridoma can yield approximately 1000 specific molecules of antibody per second.

A convenient method to separate antibody-hormone complexes from free hormone is to adhere the antibody to a solid phase, such as to the walls of a test tube. The free hormone can then be decanted (a centrifugation step is not required). Because proteins attach nonspecifically to plastic (eg., polyvinyl chloride or polystyrene), tubes can be coated by simply incubating with a solution containing antibody. Remaining unoccupied sites are then filled with an irrelevant protein, such as serum albumin or gelatin. One criticism of antibody-coated tubes is adsorption can mask immunoreactive (Fab) sites: to overcome this problem, protein A, a molecule derived from staphylococcus aureus that binds the Fc tail of IgG, can be coated to the solid phase (this permits extraction of IgG from the fluid-phase reaction mixture). Alternatively, precipitation of hormone-antibody complexes can be achieved using ammonium sulfate, magnetically-activated antibody, or with a second antibody generated against the first antibody (ie., anti-IgG). Adsorption of free (low molecular weight ligand) can be achieved with dextran-coated charcoal.

Radioimmunoassay is a scientific method used to test hormone levels in the blood without the need to use a bioassay. It involves mixing a radioactive antigen (frequently labelled with isotopes of iodine attached to tyrosine) with antibody to that antigen, then adding unlabeled or "cold" antigen in known quantities and measuring the amount of labeled antigen displaced.

Initially, the radioactive antigen is bound to the antibodies. When cold antigen is added, the two compete for antibody binding sites - and at higher concentrations of cold antigen, more binds to the antibody, displacing the radioactive variant. The bound antigens are separated from the unbound ones in solution and the radioactivity of each used to plot a binding curve.

The technique is both extremely sensitive, and specific, if costly, and it is especially useful in diagnosing and treating autoimmune diseases such as Hashimoto's thyroiditis and Systemic Lupus Erythematosus.

The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,

blood banking diagnosis of allergies endocrinology

The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay.

THE TECHNIQUETHE TECHNIQUE

A mixture is prepared of o radioactive antigen

Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125I or 131I are often used.

o antibodies against that antigen. Known amounts of unlabeled ("cold") antigen are added to samples of the

mixture. These compete for the binding sites of the antibodies. At increasing concentrations of unlabeled antigen, an increasing amount of

radioactive antigen is displaced from the antibody molecules. The antibody-bound antigen is separated from the free antigen in the

supernatant fluid, and the radioactivity of each is measured. From these data, a standard binding curve, like this one shown in red, can be

drawn.

The samples to be assayed (the unknowns) are run in parallel. After determining the ratio of bound to free antigen in each unknown, the

antigen concentrations can be read directly from the standard curve (as shown above).

SEPARATING BOUND FROM FREE ANTIGENSEPARATING BOUND FROM FREE ANTIGEN

There are several ways of doing this.

Precipitate the antigen-antibody complexes by adding a "second" antibody directed against the first. For example, if a rabbit IgG is used to bind the antigen, the complex can be precipitated by adding an antirabbit-IgG antiserum (e.g., raised by immunizing a goat with rabbit IgG). The antigen-specific antibodies can be coupled to the inner walls of a test tube.After incubation,

o the contents ("free") are removed; o the tube is washed ("bound"), and o the radioactive of both is measured.

The antigen-specific antibodies can be coupled to particles, like Sephadex. Centrifugation of the reaction mixture separates

o the bound counts (in the pellet) from o the free counts in the supernatant fluid. o Radioimmunoassay is widely-used because of its great sensitivity.

Using antibodies of high affinity (K0 = 108–1011 M−1), it is possible to detect a few picograms (10−12 g) of antigen in the tube.

The greater the specificity of the antiserum, the greater the specificity of the assay.The main drawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive antigen.

Both 125I or 131I emit gamma radiation that requires special counting equipment;

The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4).

Despite these drawbacks, RIA has become a major tool in the clinical laboratory where it is used to assay

plasma levels of: o most of our hormones; o digitoxin or digoxin in patients receiving these drugs; o certain abused drugs

for the presence of hepatitis B surface antigen (HBsAg) in donated blood; anti-DNA antibodies in systemic lupus erythematosus (SLE).

In the RIA, IgG subclasses are quantified as immune complexes after binding of radioactively labelled specific antibody. In case of a 'direct' technique, a radioactively labelled anti-IgG subclass-specific antibody is used. In an 'indirect' technique, an anti-IgG subclass-specific antibody directed against the first antibody. Since working with radioactively labelled reagents requires special precautions and is relatively costly, radio immuno assays have been largely replaced by enzyme-linked immuno assays.

RADIOIMMUNOASSAYRADIOIMMUNOASSAY

ENZYME-LINKED IMMUNOSORBENT ASSAYENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)(ELISA)

The Enzyme-Linked Immunosorbent Assay (ELISA or EIA for short) is a biochemical technique used in immunology to detect the presence of an antibody or an antigen in a sample. It utilizes two antibodies, one of which is specific to the antigen and the other which is coupled to an enzyme. This second antigen gives the assay its "enzyme-linked" name, and will cause a chromogenic or fluorogenic substrate to produce a signal. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen.

Because of its high sensitivity and specificity, this assay allows accurate measurement of very low levels of IgG subclasses. In figure 10, a schematic outline of the ELISA technique is shown. The sensitive ELISA comprises many incubation and washing steps. Because of the need for high dilutions when measuring IgG subclasses in sera, ELISA assays may be less reproducible in comparison with RID and nephelometry. For measurement of IgG and its subclasses in large numbers of samples, ELISA is increasingly being replaced by nephelometry. ELISA may be advocated for measuring IgG subclasses in other body fluids than serum/plasma, e.g. saliva, cerebrospinal fluid and broncho-alveolar lavage fluid.

Brief outline of the method (figure 10): An ELISA is generally performed in wells of microtitre plates.- Wells of the plates are coated with unlabelled monoclonal antihuman IgG subclass-specific antibody and washed (figure 10A);- Test samples, standard-and control sera are introduced in the respective wells and incubated; The IgG subclass to be determined will bind to the solid phase and non-bound IgG is removed by washing (figure 10B); - Enzyme-labelled anti-human IgG antibodies are added to each well and non-bound conjugate is removed by washing (figure 10C); - Plates are incubated with substrate solution; - After incubation, the coloured reaction product is measured photometrically (figure 10D);

- The concentration of the IgG subclasses in the test samples is calculated relative to the values of the calibration curve.

ELISA PROCEDUREELISA PROCEDURE

1.Indirect ELISA2.Sandwich ELISA3.Sompetitive binding ELISA

INDIRECT ELISAINDIRECT ELISA

The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:

1. Apply a sample of known antigen to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile.

2. The plate wells or other surface are then coated with serum samples of unknown antibody concentration, usually diluted in another species' serum. The use of non-human serum prevents non-specific antibodies in the patient's blood from binding to the antigen.

3. The plate is washed, so that unbound antibody is removed. After this wash, only the antibody-antigen complexes remain attached to the well.

4. The second antibodies are added to the wells, which will bind to any antibody-1 remaining. These second antibodies are coupled to the substrate-modifying enzyme.

5. Wash the plate, so that excess unbound antibodies are removed. 6. Apply a substrate which is converted by the enzyme to elicit a chromogenic

or fluorescent signal. 7. View/quantify the result using a spectrophotometer or other optical device.

The enzyme acts as an amplifier: even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules.

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve which is typically a serial dilution of the target.

A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen.

SANDWICH ELISA ASSAYSSANDWICH ELISA ASSAYS

One of the most useful of the immunoassays is the two antibody “sandwich” ELISA. This assay is used to determine the antigen concentration in unknown samples. This ELISA is fast and accurate, and if a purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample. The sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies.

To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate. Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding.

Unlike Western blots, which use precipitating substrates, ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3’5,5’- tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively.

The Sensitivity of the Sandwich ELISA is Dependent on Four Factors:

1. The number of molecules of the first antibody that are bound to the solid phase.

2. The avidity of the first antibody for the antigen. 3. The avidity of the second antibody for the antigen. 4. The specific activity of the second antibody.

The amount of the capture antibody that is bound to the solid phase can be adjusted easily by dilution or concentration of the antibody solution. The avidity of the antibodies for the antigen can only be altered by substitution with other antibodies. The specific activity of the second antibody is determined by the number and type of labeled moieties it contains.

The steps are as follows:

1. Prepare a surface to which a known quantity of antibody is bound. 2. Apply the antigen-containing sample to the plate. 3. Wash the plate, so that unbound antigen is removed. 4. Apply the enzyme-linked antibodies which are also specific to the antigen. 5. Wash the plate, so that unbound enzyme-linked antibodies are removed. 6. Apply a chemical which is converted by the enzyme into a fluorescent signal. 7. View the result: if it fluoresces, then the sample contained antigen.

COMPETITIVE ELISA ASSAYSCOMPETITIVE ELISA ASSAYS

When two “matched pair” antibodies are not available for your target, another option is the competitive ELISA. Another advantage to the competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISAs, below is an example for one such configuration. In order to utilize a competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the immunogen or the primary antibody. The protocol below uses a labeled immunogen as the competitor. Briefly, an unlabeled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards and unknowns. After this reaction is allowed to go to equilibrium, conjugated immunogen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled immunogen. Thus, the more immunogen in the sample or standard, the lower the amount of conjugated immunogen bound. The plate is then developed with substrate and color change is measured

The steps for this ELISA are somewhat different than the first two examples:

1. Unlabeled antibody is incubated in the presence of its antigen. 2. These bound antibody/antigen complexes are then added to an antigen

coated well. 3. The plate is washed, so that unbound antibody is removed. (The more

antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")

4. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.

5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal

RADIO IMMUNOSORBENT TEST (RIST) RADIO IMMUNOSORBENT TEST (RIST)

The antibody content of a patient’s serum can be assessed by the ability of antibody to bind to Antiglobulin, which has been immobilized on a solid surface by adsorption. The solid surface could be polycarbonate tube or nitrocellulose paper discs. This test is usually done for the detection of IgE antibodies in severely allergic patients.

Anti-IgE is raised in rabbits and used in labelled as well as unlabeled form. Unlabeled anti-IgE is adsorbed on to a solid surface (polycarbonate tube) and to this, patient’s serum to be tested for the presence of IgE is added and incubated. After some time excess of serum is removed and the tube is washed with saline to remove excess of serum proteins. To the washed tube, radiolabeled anti- IgE is added. Radiolabeled anti IgE will bind to IgE, which has already complexed with adsorbed anti- IgE. Geiger counter measures radioactivity in the tube and the antibody quantity is detected.

A convenient method to separate antibody-hormone complexes from free hormone is to adhere the antibody to a solid phase, such as to the walls of a test tube. The free hormone can then be decanted (a centrifugation step is not required). Because proteins attach nonspecifically to plastic (eg., polyvinyl chloride or polystyrene), tubes can be coated by simply incubating with a solution containing antibody. Remaining unoccupied sites are then filled with an irrelevant protein, such as serum

albumin or gelatin. One criticism of antibody-coated tubes is adsorption can mask immunoreactive (Fab) sites: to overcome this problem, protein A, a molecule derived from staphylococcus aureus that binds the Fc tail of IgG, can be coated to the solid phase (this permits extraction of IgG from the fluid-phase reaction mixture). Alternatively, precipitation of hormone-antibody complexes can be achieved using ammonium sulfate, magnetically-activated antibody, or with a second antibody generated against the first antibody (ie., anti-IgG). Adsorption of free (low molecular weight ligand) can be achieved with dextran-coated charcoal.

Other analytical systems that exploit the same basic principle as the RIA include the protein-binding assay, radioreceptor assay (RRA), scintillation proximity assay (SPA), enzyme immunoassay (EIA), fluoroimmunoassay (FIA), and chemiluminescent assay (CIA).

Protein-binding and radioreceptor assays are radioligand assays that utilize an endogenous plasma protein (eg., for steroid hormones) or cellular receptor, respectively - instead of an antibody. Protein-binding assays lack the specificity of an immunoassay. The radioreceptor assay has an advantage over the RIA in that it only detects bioactive hormone (ie., antibodies can interact with sites on the hormone molecule not involved in receptor binding). Notwithstanding, it is difficult to isolate abundant quantities of stable receptor for routine analyses. Fortunately, data obtained from RIAs and RRAs are usually comparable.

RADIORECEPTOR ASSAY (RRA)RADIORECEPTOR ASSAY (RRA)

SCREENING FOR ANALYTES USING LABELED RECEPTORS. Introduction :-Receptor binding assays have been commonly used for the assessment of the pharmacological properties of New Chemical Entities (NCE). Due to the introduction of combinatorial chemistry in the pharmaceutical industry, in an attempt to find succesful drug candidates, an enormous increase in NCE requires a concomitant demand for high through-put screening systems. Due to their specific properties receptor assays have been considered valuable analytical tools for the quantitation of highly potent drugs that exert their pharmacological action via a receptor interaction.

The term receptor is exclusively used for proteins which can interact with hormones, neurotransmitters and drugs or poisons yielding or blocking a pharmacological response. Thus therefore antibodies, circulating or membrane-bound proteins e. g. enzymes cannot be considered receptors even if they should have ligand binding properties.

The principle of receptor binding assays is based on the competition between a ligand and an analyte for binding to a certain receptor. After incubation of ligand, analyte and receptor followed by separation of the receptor bound and the free fraction of the ligand by either filtration, centrifugation or dialysis, subsequently

one or both resulting fractions are quantitated. The subsequently acquired data can be used for assessment of the affinity of a NCE for the receptor or for quantitation of a particular receptor binding analyte.

Up till now, all receptor assays have been construed around the use of a (radio) labeled ligand. Furthermore generally receptors present in animal tissue or cultivated cell lines have been used after having undergone only little purification.

Typical receptor densities in commonly used receptor assays range from 10-100 picomole per gram tissue. This subsequently implies that the amount of displaceble labeled ligand in such assays is limited. The use of radioactive ligands in such cases is notwithstanding this attractive because radioactivity can be detected with good sensitivity and limited back-ground signals from such receptor material. Another important reason in favor of use of radioactive labels is that development is easy once a compound has been identified that binds to a particular receptor with high affinity. Replacing 1-6 hydrogen atoms by the same number of tritium atoms yields a product that has a receptor affinity similar to that of the unlabeled ligand. However disadavantages such as the limited shelf-life and the problems encountered with the use of radioactive tracers has stimulated the search for alternatively labeled ligands. Another motivation for such search was also the expectation that alternative labels might improve the sensitivity of the assay with regard to limits of quantitation. Taken into consideration the physical half-life and the counting time of each sample it can be calculated for tritium by way of example that only 1 out of each million labeled molecules is detected.

Almost all approaches with non-radioactive labeled ligands have been with fluorescent labels. The development of fluorescent ligands with a high receptor affinity, if the ligands itself does not have sufficient native fluorescence, is quite difficult and a compromise between affinity and fluorescence properties has always been required. The aforementioned low receptor density implies that the maximal signal is limited and lies close to the limitations of available instrumentation. High amounts of receptor containing material needs to be used per assay in order to sufficiently increase the signal.

Furthermore the currently used receptor containing materials in receptor assays contain large amounts of non-receptor proteins which cause a high fluorescence background.

Traditional receptor assays using radioactive or non-radioactive ligands require a separation step enabling quantitation of bound and or free fractions of the labeled ligand. Procedures used for the separation are dialysis, centrifugation and filtration. The selection depends on available instrumentation and equilibrium dissociation constants of the labeled ligand and of the analyte. It is a requirement that the separation step may not alter the amount of receptor bound ligand.

A newly-developed methodology, SPA, does not require separation of bound from free ligand. Competitive binding of labeled ligand in proximity to antibody- or receptor-coated fluoromicrospheres allows the energy emitted to excite the fluor and produce detectable light that can be measured in a scintillation counter without liquid cocktail. Unbound tracer is too far from the microsphere to enable energy transfer before it is absorbed by the aqueous solution.

In the EIA, FIA ,and CIA, radioactive hormone is replaced by an enzyme-, fluorescein- or luminol-tagged ligand, respectively. Quantification is accomplished with a fluorometer in FIA and a luminometer in CIA. In EIA an extra step is required first - addition of substrate. An example of an enzyme commonly used in enzyme immunoassays is horseradish peroxidase: hydrogen peroxide (substrate) is reduced by this enzyme, and in the process an appropriate hydrogen donor (eg., o-phenylenediamine) is oxidized, causing a change in color of solution - appearance of product is measured by spectrophometric analysis of color reactions (ie., absorbance) to graded concentrations of hormone.

Antibody-excess immunoassays include the immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA). In the IRMA cold ligand is "sandwiched" between an antibody coated to a solid phase and a second radiolabeled antibody raised against a different hormonal epitope (this works best with macromolecular hormones); sensitivity is not mandated by competition, and therefore, reactions can be carried out expeditiously over a wide range of detection. In a sandwich ELISA, hormone is bound to an antibody attached to a solid phase, and then an antibody-enzyme conjugate and substrate are added (Figure 2-39). These methods engender a direct relationship between radioactivity measured in the final complex and concentration of standard or analyte (in contrast to the inverse correlation between bound radioactivity and standard or sample concentrations in an RIA).

Nonradioisotopic procedures, such as ELISAs, are becoming popular because of lowered equipment costs, reduced hazard to users and the environment (ie., associated with handling and disposal of radionuclides), and can be adapted (subjective appraisal of color-

change) for in-the-home or on-the-farm/ranch diagnostics. However, ELISAs tend to be less sensitive than the RIA (Table 2-8).

A reverse hemolytic plaque assay is used to detect secretion of hormone from individual cells (eg., gonadotropes) contained within a heterogeneous population. The concept is that a secretory product of a cell can be measured by specific antibodies in the presence of erythrocytes coated with protein A and added complement. Interaction of hormone with binding sites on the antibody causes stearic alterations in the antibody allowing for fixation of complement by juxtaposed Fc. Complement-induced hemolysis leads to the formation of a clear zone of erythrocyte membrane "ghosts" (ie., a plaque) surrounding the secretory cell (Figure 2-40). The plaque technique is sensitive and areas of lysis can be quantitated.

Receptor analyses. It is technically more difficult to monitor changes in populations of hormonal receptors than to evaluate alterations in patterns of secretion of hormones; yet, knowledge of dynamics of cellular receptors is no less important (eg., in diseases of endocrine resistance). The task of receptor measurement can be accomplished by exposing a constant amount of receptor (eg., tissue homogenate) to increasing concentrations of radioactive hormone. Receptor bound with hormone is separated from free radiolabel and each fraction is counted - the Scatchard plot is a common method of data assessment (Figure 2-41). Receptors not occupied by hormones are generally characterized unless special methods are first used to elute endogenous ligand from its binding site.

PRECAUTIONSPRECAUTIONS

Blood samples are collected by vein puncture with a needle. It is not necessary to restrict fluids or food prior to collection. Blood should be collected in tubes containing no additive. Risks of vein puncture include bruising of the skin or bleeding into the skin. Random urine samples are acceptable for drug assays; however, 24-hour urine samples are preferred for hormones and other substances which show diurnal or pulse variation.

Special safety precautions must be observed when performing RIA methods. Radioactive isotopes are used by RIA tests to label antigens or antibodies. Pregnant females should not work in an area where RIA tests are being performed. Personnel handling isotope reagents must wear badges which monitor their exposure to radiation. Special sinks and waste disposal containers are required for disposal of

radioactive waste. The amount of radioisotope discarded must be documented for both liquid and solid waste. Leakage or spills of radioactive reagents must be measured for radioactivity; the amount of radiation and containment and disposal processes must be documented.

NORMAL RESULTSNORMAL RESULTS

Immunoassays which are qualitative are reported as positive or negative. Quantitative immunoassays are reported in mass units, along with reference intervals (normal ranges) for the test. Normal ranges may be age- and gender-dependent. Positive immunoassay test results for HIV and drugs of abuse generally require confirmatory testing.

Although immunoassays are both highly sensitive and specific, false positive and negative results may occur. False-negative results may be caused by improper sample storage or treatment, reagent deterioration, or improper washing technique.

False-positive results are sometimes seen in persons who have certain antibodies, especially to mouse immunoglobulins (immune cells) that may be used in the test. False-positive results have been reported for samples containing small fibrin strands that adhere to the solid phase matrix. False-positives may also be caused by substances in the blood or urine that cross-react or bind to the antibody used in the test.

PREPARATIONPREPARATION

Generally, no special instructions need be given to patients for immunoassay testing. Some assays require a timed specimen collection, while others may have special dietary restrictions.

AFTERCAREAFTERCARE

When blood testing is used for the immunoassay, the vein puncture site will require a bandage or light dressing to accomplish blood clotting.

RISKSRISKS

Immunoassay is an in vitro procedure, and is therefore not associated with complications. When blood is collected, slight bleeding into the skin and subsequent bruising may occur. The patient may become lightheaded or queasy from the sight of blood.