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  • 1. Pharmacy 2203: Immunoassay Methods 1 I. Immunoassay Methods Objectives: 1. Describe the basic components of an immunoassay. 2. Outline how a typical competitive binding assay and a typical non-competitive immunoassay work. 3. Define the following: Polyclonal vs. monoclonal antibodies, competitive vs. immunometric immunoassay, homogenous vs. heterogeneous immunoassay, RIA, Log-logit plot, excess reagent vs. limited reagent immunoassay, Nephelometry and Turbidimetry, antigen, hapten, epitope, immunogen, antiserum, avidin, biotin, amplification, matrix effects, cross-reactivity, heterophilic antibodies, and cut-off. 4. Describe the use of conjugation in preparing reagents for immunoassays. 5. Which factors affect the sensitivity of immunoassays? 6. Describe common types of isotopic and non-isotopic labels used in immunoassay. 7. List two enzymes commonly used in enzyme immunoassays. 8. List common separation techniques used by heterogeneous and homogenous immunoassays. 9. Outline the principle behind the following immunoassay techniques: RIA, EMIT, ELISA, FPIA, CEDIA, and use of microparticles. 10. Describe immunoassay formats commonly used for therapeutic drug monitoring and screening for drugs of abuse testing. A. The Antigen-Antibody Complex 1. Antibody Antibodies are immunoglobulins produced by B-lymphocytes in response to exposure to an antigen (any substance that induces an immune response). These proteins are Y- shaped and fall into 5 classes: IgG, IgM, IgA, IgE, and IgD. About 90% of immunogloblins in serum are IgG. IgG is a glycoprotein (158,000 mwt) containing 4 polypeptide chains, 2 heavy chains and 2 light chains joined by disulfide bridges. IgG contains three key domains: two identical Fab regions at the top of the Y arms and on Fc region that forms the base of the Y. The Fab region contains a variable amino acid sequence that determines the antigenic specificity of the antibody. This segment is sufficient in itself for antigen binding. Each unique amino acid sequence forming a unique antibody is a product of a single plasma cell line. The Fc portion is important in immune regulation. Only IgG is used in immunoassays. Binding of antibodies to antigens is reversibly and very specific. The induction of an immune response depends on the size of the antigen. Small molecular weight compounds (<2000 Daltons) such as drugs are unable to induce antibody formation, and can only do so when bound to a carrier protein to form an immunogen (see section on conjugation). An immunogen must have a stable structure in order to induce this response. When an immunogen is injected into an animal it will induce an immune response. Some of the antibody produced will be specific for the small molecule (hapten). Immunoassays require the production of large amounts of antibodies. Dr. Edward Randell 3/29/2007
  • 2. Pharmacy 2203: Immunoassay Methods 2 a) Polyclonal Antibodies are derived from many cell lines. a Antigenic Conventional immunization Immunize with Ag Ag Determinants involves injecting antigen into an To produce Antibodies c b (a,b,c) animal. This stimulates the production of a heterogeneous population of antibodies that Remove Polyclonal differ in affinity and specificity Centrifuge Blood & purify antibodies for the antigen of interest. Each antibody differs from others in the parts of the antigen that it Antiserum recognizes (epitopes). An antigen can have many epitopes depending on its size. The figure outlines the steps involved in producing polyclonal antibodies. The production of polyclonal antiserum involves: 1. injection of an immunogen; 2. waiting for immune response; 3. collection of blood; and 4) separation of the antiserum. Host animals are typically rabbit, sheep, or goats. The animal should be large enough to produce large volumes of blood on a regular basis without causing significant harm to it. b) Monoclonal Antibodies are derived from a single cell line. Monoclonal antibodies are specific Remove antibody-forming Spleen cells toward a single epitope on an antigen. cells from spleen Hybridoma technique is used. During the production of monoclonal antiserum, Fuse antibody forming cells with tumor cells + lymphocytes from an immunized mouse are fused with mouse myeloma cells to Hybridoma Tumor cells grown In cell culture produce a hybridoma. This is used to produce antibodies in cell culture. The myeloma portion allows the hybridoma to propagate in a culture media. The Screen hybridomas for specific Antibody production lymphocyte portion produces antibodies. A hybridoma that produces the desired type of antibody is selected by cell- sorting techniques and then grown in Monoclonal Unsatisfactory Monoclonal culture. Each antibody in the selected Antibodies to b Production Antibodies to a culture medium has identical binding properties. The antiserum is very specific for a specific epitope on an antigen (unlike polyclonal anti-serum) and will have the same properties as long as the selected cell line is maintained. The advantages of monoclonal antibodies over polyclonal antibodies are: 1. High specificity (Due to a single epitope determinant on the antigen); 2. Large quantity (An almost unlimited supply can be produced); 3. Selectivity (one can select the antibodies for desired characteristics). c) Affinity vs. Avidity Immunoassays work because of specific recognition of the antigen by the antibody and the formation of an antigen-antibody complex. The antigen-antibody binding is described Dr. Edward Randell 3/29/2007
  • 3. Pharmacy 2203: Immunoassay Methods 3 in terms of affinity and avidity. Affinity is a property of the antigen and describes the strength of binding of its epitope to a single binding site on the antibody. Avidity is a property of the antibody and describes the overall binding of one antibody molecule to antigen. It includes the sum of all binding sites the antibody may have. For example: Fab fragment has one binding site, IgG has 2, and IgM has 10. d) Interaction between antigen and antibody The strength of binding is determined by the equilibrium binding constant (K) for the antigen-antibody complex formation. The binding follows the basic thermodynamic principles of a reversible reaction between two molecules. This relationship is described by the chemical reaction shown below where Ag is the antigen, Ab is the antibody, and Ag-Ab is the antigen-antibody complex. K is the equilibrium constant. [ AgAb ] Ag + Ab ⇔ AgAb K= (K is typically 107 to 109 mol-1) [ Ag ][ Ab] This means that higher affinity antibodies (larger K) bind larger amounts of antigen in a shorter time period. The complexes that form are also more stable. K is affected by temperature, pH and buffer composition. K is estimated graphically for monoclonal antibodies by creating a double reciprocal plot (1/f vs. 1/b) or a Scatchard plot (b vs. b/f) or from the slope of the dose response curve (below). “f” indicates free antigen and “b” indicates antibody bound antigen. Key aspects of the Antibody-Antigen interaction: 1) Reaction is reversible and favors complex formation under physiological conditions. 2) Binding depends on hydrogen bonds, van der Waals forces, ionic bonds, hydrophobic interactions. 3) Binding is very specific and requires the correct 3-D structure of an antigen. 4) The amount of complex formed depends on concentration of antibody and antigen. 5) Both antibody and antigen (if large enough) have multiple sites for binding to occur. Therefore, extensive cross-linking can occur when both are present in solution. When antibody and antigen reach equivalence large immune complexes form which can precipitate out of solution. B. Immunoassay Immunochemical methods exploit the properties of the antibody-antigen complex and use antibodies as reagents to detect and quantify antigens. A major advantage of immunoassay over many other quantitative techniques is that it requires minimal purification of the antigen. Methods are subdivided into two subtypes: 1) Particle methods: involve 1) the precipitation of large immune complexes (immunodiffusion and immunoelectrophoresis); 2) measurement of light scattering by large immune complexes in solution (nephelometry and turbidimetry). Dr. Edward Randell 3/29/2007
  • 4. Pharmacy 2203: Immunoassay Methods 4 2) Label methods involve use of a “label” on either the antibody or antigen to identify antigen-antibody complex formation. The first ‘label type’ immunoassay was developed in the 1950’s by Yalow and Berson and quantified human antibodies to bovine insulin in the blood of patients with diabetes who had acquired immune response to insulin. This first immunoassay used radioactively labeled insulin. The procedure involved mixing radioactively labeled insulin with the patient’s blood, separating the bound and non-bound insulin and then measuring the radiation associated with the labeled insulin bound to the patient’s immunoglobulins - radioimmunoassay (RIA). Today non-isotopic labels are more commonly used. A variety of compounds can be quantified by immunoassays, ranging in size from large proteins to small molecules. Immunoassays involve two different approaches: 1. Competitive immunoassay; 2. Non-competitive (Immunometric) assays. C. The Competitive Immunoassay 1. Example RIA The traditional RIA uses the Steps involved in Competitive competitive immunoassay Immunoassay approach. A competition is Add labeled drug set up between the unlabeled * * * * antigen and the labeled antigen (radioactive drug, for Add sample 1 (reference example) because of a solution containing drug and incubate) If low drug * * limited number of binding concentration sites on an antibody. The * * Decant figure below shows a tube * * with antibodies attached to If high drug * the bottom. In a typical concentration experiment a number of assay tubes are set up and a constant amount of radioactively-labeled drug is added to each. The antibodies are specific for the drug and can bind to it. Reference solutions containing known concentrations of drug are added to specific tubes. These serve as standard solutions to construct a standard curve. To other tubes, unknown samples are added. When there is high concentration of drug in the reference solution or sample, it will displace a large amount of labeled drug that was originally bound to the antibodies. Conversely, samples with low drug concentrations will displace only a small amount of labeled drug. After a suitable time period of incubation, the supernatant of each tube is removed and the radioactivity remaining in each tube - the bound fraction is measured. A calibration (standard) curve is then constructed, plotting some measure of the bound drug concentration against drug concentration in the reference solutions. The concentration of drug for each unknown sample is then determined by comparing the amount of measured Dr. Edward Randell 3/29/2007
  • 5. Pharmacy 2203: Immunoassay Methods 5 bound drug (radioactivity) on the ordinate (y-axis) of the standard plot with the corresponding concentration of drug added on the abscissa (x-axis). The math that describes the relationship follows: [ AgAb ] Ag + Ab ⇔ AgAb K= [ Ag ][ Ab] From these relationships the following is derived: b = ( AgAb) /[ Ag ] ( b is bound antigen / free antigen) f f ( Ag ) = ( Ag T ) − ( AgAb) ( Ab) = ( AbT ) − ( AgAb) If we substitute for [AgAb] and rearrange, we produce the following quadratic equation: ( b ) 2 + ( b )( KAg T − KAbT + 1) − KAbT = 0 f f This mathematical function described the shape of the dose response curve. 2. Dose Response Curve In an immunoassay the total amount of antigen for the standard solutions is known. Plotting b/f (or b/AgT or % bound) against total antigen produces the standard curve. The value for b/f is determined by b/f the radioactivity (or other label signal) remaining bound divided by the total amount of radioactivity (or other label signal) added in the beginning - the radioactivity (signal) is proportional to the concentration of the labeled antigen. Using this standard curve and the b/f for an unknown sample the antigen Concentration of antigen concentration can be calculated. The dose response curve shown is that typical of a RIA standard curve. 3. Transformation to linear plot a) Log-logit It is more convenient to work with 2 linear responses rather than curves. 1.5 Therefore, a number of approaches 1 are used to linearize data. One of Logit(B/Bo) 0.5 these is the log-logit 0 -0.5 ⎡ B ⎤ -1 B ⎢ ⎥ -1.5 Logit = In ⎢ Bo ⎥ Bo B -2 ⎢1 − ⎥ -2.5 ⎣ Bo ⎦ 3 4 5 6 7 8 Dr. Edward Randell 3/29/2007 Ln(Cyclosporin conc.)
  • 6. Pharmacy 2203: Immunoassay Methods 6 transformation which is shown. In this case, B is the bound-labeled antigen; Bo is the maximum binding of labeled antigen that occurs when no unlabeled antigen is present. Typically, the concentration of a drug is log transformed and plotted (on x-axis) against logit B/Bo (on y-axis). The plot is linear. A competitive immunoassay is based on the following assumptions: 1. Antigen is present as a single chemical species. 2. Antibody is present in a single form. 3. A single molecule of antigen can only react with a single molecule of antibody. 4. Both the labeled and unlabeled antigen have similar physical-chemical properties; 5. The antigen-antibody reaction is governed by the law of mass-action (a single rate constant describes the reaction). 6. The reaction must reach equilibrium. 7. Separation of the bound and free antigen does not disturb the equilibrium. 8. The ratio of bound/free antigen (or the ratio of bound/total antigen) is measured perfectly. D. Non-Competitive (Immunometric) Assay This technique is very sensitive but is not suited for measurement of small antigens like drugs. Immunometric assays utilizing the sandwich approach are common for large molecules. In order for + + the sandwich type assay to work, the antigen must Solid phase antibody Antigen Labelled antibody (excess) have at least two distinct (excess) binding sites that are recognized by two + different antibodies. These assays come in two types: simultaneous or Separate and count activity bound to solid phase sequential assays. Simultaneous assays involve incubating labeled and unlabeled antibodies with the unknown sample, all at the same time. Sequential assays are more time consuming and involve several incubation and wash steps that separate reagent addition steps. E. Classification 1. Competitive vs. non-competitive: In a competitive immunoassay there is a competition between labeled and unlabeled antigen for a limited number of binding sites on the antibody. All reactants are mixed together either simultaneously or sequentially (example RIA). In a typical non-competitive assay (immunometric assay) an excess amount of an antibody is used to capture the analyte from the sample. A labeled second antibody is added and binds to the first antibody-antigen complex to form a sandwich. The complex formed is then measured. Non-competitive techniques are more sensitive and reproducible than competitive ones. Dr. Edward Randell 3/29/2007
  • 7. Pharmacy 2203: Immunoassay Methods 7 2. Homogenous vs. Heterogenous: Heterogenous assays involve the binding of antigen to antibody followed by physical separation of the bound antigen from the unbound antigen prior to measurement. Heterogenous assays tend to be more versatile and sensitive. If the immunoassay does not require any physical separation step after mixing antigen and antibody and measurement of bound label occurs in the presence of unbound label, the assay is homogenous. 3. Isotopic vs. non-isotopic: Isotopic immunoassays use radioactive labels on the antigen or antibody. Non-isotopic methods use enzyme reactions, chromophores, fluorescent labels, or luminescent labels in order to detect the antigen-antibody complex. 4. Limited reagent vs. excess reagent: In limited reagent assays, the number of available binding sites on the antibody is limited compared to the concentration of labeled and unlabeled antigen (a competitive immunoassay). Non-competitive assays are reagent excess assays. II. Immunoassay Components The design of immunoassays involves careful selection of antibodies with appropriate binding strength and specificity, and paying close attention to the selection of an appropriate label and detection technique for measurement of antigen-antibody binding. Labeled antigen and antibodies are prepared by covalent attachment (conjugation) of the label. In some assays washing steps are necessary to physically separate bound antigen from free antigen. Washing can also remove potential interferences from subsequent steps in the binding and measurement reactions. Competitive assays require labeled antigen, antibody, and a method of separating the bound from the free antigen. Non- competitive assays require antibody, a labeled second antibody, and a separation technique. Separation techniques permit the separation of bound label antigen from unbound label for measurement. A. Labeling and Detection techniques. 1. Conjugation Conjugation is the process of linking either antigen or antibody of another molecule to enable 1) recognition or 2) detection. 1) Recognition: A hapten (a drug for example) is conjugated to a suitable carrier protein to produce an immunogen. Conjugating hapten to a large protein involves first derivatizing the hapten to add a functional group that can be used to allow conjugation. In general, the hapten is very small in comparison to the size of the carrier protein. The immunogen is designed such that several bond-lengths separate the hapten from the carrier protein. This makes the hapten more spatially unique causing the host animal’s immune system to produce higher concentrations of antibodies that specifically bind to it. Examples of carrier proteins include: bovine serum albumin, gamma-globulins, egg albumin, fibrinogen, thyroglobulin, and hemocyanin. Dr. Edward Randell 3/29/2007
  • 8. Pharmacy 2203: Immunoassay Methods 8 2) Detection: Conjugation is also used to conjugate an antigen to an enzyme or other label in a competitive enzyme immunoassay or to conjugate an antibody to an enzyme or other label in an immunometric assay. Antigens and antibodies can also be conjugated to Biotin, Avidin, or to polystyrene beads. 2. Antibody Monoclonal antibodies or combinations of monoclonal and polyclonal antibodies are most commonly used in immunoassay. 3. Labels Either antibody (non-competitive) or antigen (competitive) is labeled to provide a signal for quantification. Labeled antigens must have 2 characteristics: 1) must be immunologically similar to the compound being tested to successfully compete with the antibody; and 2) must be lent to sensitive detection free from interference from common matrices. There are two main types of labels: a) Isotopic labels. The commonly used isotopes in immunoassay are: 3H, 125I, and 14C. 3H and 14C, emit beta radiation only, and can be incorporated into the structure of the compound of interest. 125I is a gamma radiation emitter and has the advantage of high activity and ease of radioactive counting. b) Nonisotopic labels. Nonisotopic labels use a variety of detection techniques: (1) Detection of Light Scattering (A particle Method) Light is scattered by large molecules when their hydration diameter is similar to the wavelength of incident light. This is applied to measuring the formation of large immune complexes by Turbidimetry and Nephelometry. Turbidimetry measures light scattering as a reduction in the amount of incident light (↑absorbance). Nephelometry measures light scattered light directly. Rates of immune complex formation (rate nephelometry) can be applied to measure some drug concentrations for Therapeutic purposes. In these cases several molecules of drugs are conjugated to a larger molecule and permits immune complex formation. (2) Enzymatic labels The enzyme is conjugated to the antigen or the antibody and enzyme activity is measured to quantify the antigen. Enzymes are not always suitable for labeling small antigens as the large enzyme may block epitopes. Binding reactions are detected by enzyme activity monitored using photometric (chromogenic), fluorometric or luminometric techniques. Cloned-enzyme donor immunoassay (CEDIA) and enzyme multiplied immunoassay technique (EMIT) are competitive techniques using enzyme labels. The most commonly used enzymes used to assist photometric detection of immunoassays are horseradish peroxidase and alkaline phosphatase. In non-competitive assays horseradish peroxidase labeled antibody is measured by the production of peroxide and reaction with o- phenylenediamine to form a colored product that can be monitored at 290 nm. Conjugated alkaline phosphatase can be measured photometrically by hydrolysis of p- Dr. Edward Randell 3/29/2007
  • 9. Pharmacy 2203: Immunoassay Methods 9 nitrophenylphosphate to a colored product. Some ELISA (enzyme-linked immunosorbent assay) assays use this. (3) Photometric/Visual detection Antibody or antigen can be conjugated to colored microparticles which can be seen visually. Many over-the-counter home diagnostic kits make use of these labels. (4) Fluorometric detection Fluorescence involves excitation of a molecule by a photon of appropriate energy. On return of the molecule to its lower energy ground state it emits a photon of longer wavelength. Fluorimetry is more sensitive than photometric detection but is prone to more interference by substances commonly found in biological samples. Techniques to reduce interfering compounds include precipitation of proteins and other naturally occurring fluorescent compounds, or washing to separate interferences. The separation step which separates free and bound antigen, can also serve to remove interfering fluorescent compounds from the sample. The fluorescent signal is produced either by fluorescent labels or as enzyme substrates. Specific fluorescence techniques include: 1. Time-resolved fluoroimmunoassay takes advantage of the differences in decay time between the fluorescent probe and interfering substances. Most biological materials have short lived fluorescence in the order of 1-20 ns. Rare earth metals like lanthanides (europium, terbium) are long life fluorophors in the neighborhood of 10- 1000 nanoseconds and can be used as fluorescent probes. By using a time-resolved fluorimeter one can measure the long-lived fluorophors while rejecting the short-lived fluorescence of the interfering substances. 2. One of the most widely used fluorescence immunoassay techniques is fluorescence polarization assay (FPIA). When fluorescent molecules are excited with polarized light, the emitted light depends on the rotational property of the molecule. A small molecule with a fluorescent label rotates very quickly in solution. A large molecule will rotate very slowly; therefore a small fluorescently labeled molecule bound to an antibody would rotate very slowly compared to the free antigen in solution. When polarized light excites the quickly rotating small molecule, the polarization signals decrease more than that of the large molecule bound fluorescent signal. 3. Enzymatic fluorescent immunoassay work by converting a non-fluorescent substrate into a fluorescent product. Alkaline phosphatase converts 4-methyl-umbelliferyl phosphate to product 4-methyl-umbelliferone. This fluorescent product is monitored at its excitation wavelength of 335nm and at an emission wavelength of 448 nms. The most common fluorescent labels are fluoresceins, rhodamines, and umbelliferones. Fluorescent labels are well suited for small antigens. (5) Luminometric detection Luminescence requires excitation of the molecule to a state upon which the return to ground state allows the emission of light which is measured. Luminescence reactions involve the production of light either by chemical reaction (chemiluminescence) or by biochemical reaction (bioluminescence). Luminescence immunoassays have the potential to produce the greatest sensitivity. Bioluminescence is the process by which fireflies Dr. Edward Randell 3/29/2007
  • 10. Pharmacy 2203: Immunoassay Methods 10 produce light. They have an enzyme called luciferase and produce a light generating molecule called luciferin. Chemiluminescence is more commonly used. Reagents used to emit light in chemiluminescence assays include luminol, Dioxitane, and acridium ester. Alkaline phosphatase and peroxidase are also used as reagents to assist the process. c) Amplification Enzyme labels produce amplification (each enzyme label produces many detectable product molecules). Immunoassay label detection limits Combining amplification with ultrasensitive Label Type Detection limit (M) detection reactions Fluorescence 1 · 10-10 produce the most sensitive immunoassays. Chemiluminescence 1 · 10-13 Amplification can also be Fluorescence polarization 1 · 10-14 assisted using the biotin- Radioactive 1 · 10-15 avidin system. Biotin and avidin (a biotin binding Time-resolved fluorescence 1 · 10-17 protein) bind to each Electrochemiluminescence 2 · 10-20 other with high affinity. Several biotin molecules Enzyme-based: Photometric 1 · 10-16 can also be conjugated to Fluorescence 1 ·10-19 antibody and avidin to an Coupled enzyme reaction 1 ·10-20 enzyme label. Following Chemiluminescence 1 ·10-21 binding of the antigen to a single antibody molecule, the biotin molecules can each attract an avidin bound enzyme molecule. Because the amount of signal produced is proportional to the amount of enzyme, much more signal can result from a single antigen-antibody binding interaction. The table compares the detection limit of different labels. B. Separation of free and bound antigen Immunoassays are divided into two groups according to separation technique: 1 Heterogeneous assays rely on separating the free and bound antigen based on chemical or immunologic difference between the free antigen and antigen-antibody complex. Separation techniques include: i Double antibody method. An antibody against the primary antibody is used to precipitate the primary antigen/antibody complex. Complexes are then centrifuged out. ii Coated tube or plate. The reaction tube or microtitration plate is coated with primary antibody that binds antigen and holds it during washing steps. iii Beads and Particles – Cellulose, polystyrene or sephadex beads, or magnetic particles are coated with the antibody that binds to holds the antigen during washing. iv Adsorption – Charcoal adsorbs free antigen while leaving the bound antigen in solution. The charcoal bound free-antigen is removed by centrifugation or filtration. v Solvent or salt are used to precipitate the antibody with any bound antigen. The precipitate can be removed by centrifugation. Dr. Edward Randell 3/29/2007
  • 11. Pharmacy 2203: Immunoassay Methods 11 vi Column separation – ion exchange or gel filtration is used to separate bound antigen from free antigen. 2 Homogenous assays do not involve physical separation of bound antigen from unbound antigen. Most homogenous assays take advantage of the great difference in size between the antigen molecule and the bound antigen-antibody complex. Most homogenous assays measure antigen molecules that are very small in size). Homogenous assays that use enzymes rely on either: i Conformation change in an enzyme; ii Inhibition of enzyme. C. Optimizing Analytical Sensitivity Immunoassay analytical sensitivity (how little antigen can be measured) depends on two main factors: 1) how strongly the antibody binds to the antigen and 2) the sensitivity of the label detection method (see table above). 1. Factors affecting sensitivity in Competitive Assays For competitive immunoassays analytical sensitivity is mainly determined by the binding affinity of the antigen for antibody. A number of factors affect binding strength: 1) Temperature: As temperature increases, the rate of binding reaction increases but the binding affinity is reduced. Hence, higher sensitivity can be achieved at lower temperature. Higher temperature can also increase protein and reagent denaturation which could adversely affect the assay. 2) The pH: the binding affinity of antibody to antigen is pH dependent. 3) Time: Incubation of antigen and antibody to equilibrium requires a longer time unless the concentration of antigen are relatively high. In Sequential assays the addition of unlabeled antigen before the labeled antigen can increase the sensitivity and reduce the incubation time. This is also useful for assays where the limited antigen is sensitive to degradation. 4) Electrolyte concentrations in buffers. Salts can inhibit antibody binding. 2. Factors affecting sensitivity in Immunometric Assays Sandwich techniques are generally very sensitive but depend on the antigen having 2 well-spaced epitopes. The sensitivity of immunometric assay is essentially determined by the least detectable amount of labeled antibody. Maximum sensitivity therefore requires: (1) high activity of the labeled antibody, (2) low non-specific binding of the antibody, (3) high affinity of the labeled antibody for antigen, (4) small experimental error in measuring the bound labeled antibody, (5) high labeled antibody concentrations. D. Assay Format Assay format describes how all the various steps come together to produce a functioning assay. The variables include: 1) # of Steps in the assay procedure; 2) Placement of Label (Ab or Ag); 3) Order of reagent/sample addition; 4) Volume/Concentration of reagents/sample; 5) Incubation times; 6) Incubation temperature; 7) Separation steps. Dr. Edward Randell 3/29/2007
  • 12. Pharmacy 2203: Immunoassay Methods 12 1. Formats Commonly used for Drugs: Heterogenous Assays: RIA: Often uses 125I as a label in a competitive format. Separation is commonly based on double-antibody or coated tube technique. With double antibody technique polyethylene glycol is often added to aid in precipitation of the immune complexes prior to separation by centrifugation. When coated tubes are used the supernatant containing the unbound free label can be poured off without the need for centrifugation. In the case of 125I, a gamma counter is used to determine bound cpms (radioactivity). ELISA: Uses antibody bound to the bottom of a microtitre plate. In one format the drug is conjugated to horseradish peroxidase. When the drug-enzyme conjugate is free in solution the enzyme converts a substrate to a colored product, but when the drug-enzyme conjugate is bound to antibody the enzyme activity is inhibited. The addition of drug in a patient’s sample results in displacement of more of the bound drug-enzyme conjugate from the bottom of the plate as the free active form. Microparticle: Some drug of abuse test kits rely on microparticle bound drugs that compete with antibodies bound to solid phase supports where separation occurs by a wick flow type format. Similar technique is applied in other over-the-counter diagnostic kits. Homogenous Assays: Non-enzymatic: FPIA: In a competitive FPIA unlabeled antigen competes with the fluorescein-labeled antigen for the antibody binding site. With increasing concentration of unlabeled antigen, more fluorescent label becomes unbound. Therefore, the fluorescence polarization signal decreases. Fluorescein absorbs light at 485 nm and emits at 525 to 550 nm. This is commonly used for therapeutic drug monitoring (TDM). Rate Nephelometry: The basis of this technique is inhibition of immunoprecipitation by drugs in a sample. The assay depends on a conjugate prepared by linking several drug molecules to a larger carrier molecule. The conjugate competes with the free drug for binding to available sites on the antibody. Increased drug concentration in the sample results in decreased rate of formation of immune complexes. Enzymatic: EMIT: The majority of EMIT assays are for TDM and drugs of abuse testing, and measurements are made using a spectrophotometer or automated chemistry analyzer by photometric technique. EMIT assays use glucose-6-phosphate-dehydrogenase, malate dehydrogenase, or lysosyme as labels. The enzyme label is conjugated to a drug derivative. Only the unbound form of the enzyme-labeled drug derivative is active in solution forming a product. Binding of the enzyme labeled drug to the antibody, results in a decrease in the activity of the enzyme. The enzyme activity is measured by the change in NADH concentration at 340 nms. CEDIA: uses genetically engineered fragments of ß-galactosidase conjugated to the drug of interest. A second fragment (acceptor) required for enzyme activity is included in the Dr. Edward Randell 3/29/2007
  • 13. Pharmacy 2203: Immunoassay Methods 13 reaction mixture. The intact functional enzyme converts chlorophenol red-ß-galactose (colorless) to chlorophenol red (absorbs at 570 nm) and galactose, and requires binding of the drug-enzyme fragment to the acceptor fragment. Binding of antibody to the drug- enzyme fragment prevents binding to the acceptor fragment, and enzyme activity is reduced. Competition with drug in the patient sample results in increased enzyme activity. E. Factors affecting result validity. 1. Immunometric Methods A major problem is interference by heterophilic antibodies and rheumatoid factor. Heterophilic antibodies bind to reagent antibodies and interfere with the assay. Another disadvantage is the high dose hook effect where antigen concentrations are so high that it prevents the formation of the “sandwich”. Both the solid-phase antibody and the detection antibody become saturated with antigen. This results in a gross underestimation of the amount of antigen present. 2. Matrix effects Matrix effects are due to interferences from the sample matrix (endogenous compounds) and reagents. For example, substances with structural similarities to the drug of interest, or substances with colorimetric or absorptive properties may interfere with detection of the label. The impact of matrix effects can be limited through the assay design. Knowledge of the limitations of a specific assay and the effect of well-known matrices is required. To avoid matrix effects assay, conditions are often changed when the sample type is changed (For example: an assay designed to detect a drug in blood plasma, may not give an accurate result if a urine sample is used) 3. Cut-offs “Cut-off” defines the limit above which the assay test result is positive for a substance of interest. Likewise, a concentration of drug (antigen) below the cutoff is considered to be negative for the drug although trace amounts of the drug may be present. The cutoff can be set at the limit of detection of the assay (the lowest concentration that can be measured by the assay) or arbitrarily set at a higher concentration that is of clinical, administrative, or legal significance. Cutoffs are commonly applied to clinical toxicology, in which case, agencies mandate administrative cutoffs well above the limit of detection of the method to ensure that most laboratories achieve accurate results at this concentration. A result below a cutoff value does not indicate that the drug is not present. As well, a result above a cutoff value does not indicate that the drug is present, as there are many causes of false- positives and interferences. 4. Cross-reactivity A major challenge in designing immunoassay kits is to produce a product that will test specific drugs within a class of structurally related drugs, and exclude others. An excellent example is in the development of the method to screen for amphetamines. A common problem is that many over-the-counter cold medications contain substances (e.g. pseudoephedrine) that resemble amphetamine and methamphetamine in chemical Dr. Edward Randell 3/29/2007
  • 14. Pharmacy 2203: Immunoassay Methods 14 structure, and at high enough levels will cross-react with the antibody giving a positive result. Most manufacturers examine cross-reacting substances, and list these in package inserts. Inactive drug metabolites may also cross react. F. Applications 1. Automation Clinical laboratories use highly automated equipment to perform immunoassays. These technologies make use of non-isotopic labels that enable high throughput automation of both homogenous and heterogeneous assays. 2. TDM A special application of immunoassay is in the analysis of drugs for therapeutic purposes. The most commonly used techniques are FPIA, EMIT and CEDIA. All of these technologies lend themselves to measurement of very small molecules with the homogenous format preferred. It is unusual to find RIA ELISA, or Nephelometric/turbimetric based methods for TDM. 3. Point of Care tests and Urine Drug Screens In addition to automated immunoassays, some simplified immunoassays are applied to toxicology (Urine drug screens), and other forms of point of care testing (POCT) like screens for pregnancy and ovulation detection (Later Lecture). Different formats have been applied to urine drug testing. Most POCT devices use a negative-indicating reaction in which a band in a test window is absent if drug is present. These tests vary considerably in complexity and number of steps in the process. The more complex versions require incubation of the sample with antibody followed by transfer of the mixture to the test device. A wash step is also involved. Other devices require neither fixed sample size nor sample mixing. The simplest version has the testing device incorporated into the urine collection cup itself. The major issues affecting the use of these devices are related to sensitivity and specificity for specific drugs. Sample adulteration is also a major concern. Many of these are available to the general public for purchase. Reference: Hand and Baldwing. Chapter 20 Immunoassays. In Clarks analysis of drugs and poisons (Moffat, Osselton, Widdop eds) Pharmaceutical Press 2004 IL USA p301-312. (Copy on reserve at Library) Handbook of Pharmaceutical Analysis (Ohannesian and Streeter, eds) Marcel Dekker, Inc, New York, Basel p.225-312. (On Reserve at HSC Library) Dr. Edward Randell 3/29/2007