Immunochemical techniques

Background

Immunoassay is a powerful qualitative and quantitative analytical technique used to detect and measure a wide range of clinically important analytes. The extreme sensitivity and specificity of immunoassays have allowed detection and quantitation of analytes present at very low concentrations not easily measured by other analytical techniques.

Content

This chapter describes the scope of immunologic assays, including the basics of antigen-antibody binding, antibody production, and nonantibody binding agents (e.g., aptamers, molecularly imprinted polymers, boronates). Qualitative methods described include the precipitin reaction, the agglutination reaction, passive gel diffusion, immunoelectrophoresis (IEP; crossed and counterimmunoelectrophoresis [CIE]), immunofixation, blotting (Western and dot blotting), and cell- and tissue-based immunochemical techniques. Quantitative methods include radial immunodiffusion (RID), electroimmunoassay, turbidimetric and nephelometric assays, surface plasmon resonance–based immunoassay, and labeled immunochemical assays. Important aspects of the latter assay category are considered, including methodologic principles (competitive versus noncompetitive and heterogeneous versus homogeneous immunochemical assays), and analytical and functional sensitivity. Important types of labeled immunoassays are outlined, including radioimmunoassay, enzyme immunoassay (e.g., enzyme-linked immunosorbent assay, enzyme multiplied immunoassay technique, cloned enzyme donor immunoassay, fluoroimmunoassay [e.g., fluorescence resonance energy transfer]), phosphor immunoassay, chemiluminescence and bioluminescence immunoassay, electrochemiluminescence immunoassay, magnetic particle immunoassay, immuno–polymerase chain reaction, bio-barcode immunoassay, and digital immunoassay. Multiplexed immunoassays based on distinguishable labels and location of a solid support (e.g., protein microarray) are illustrated, as well as simplified immunoassays designed for point-of-care applications.

Immunologic assays are prone to interferences, and the scope of these interferences is described and exemplified (e.g., hook effect, false-negative or false-positive results caused by anti–animal immunoglobulin antibodies).

Immunochemical reactions form the basis of a diverse range of sensitive and specific clinical assays. In a typical immunochemical analysis, an antibody is used as a reagent to detect an antigen of interest. The exquisite specificity and high affinity of antibodies for their antigens, coupled with the ability of antibodies to cross-link antigens, allow the identification and quantitation of specific substances by a variety of methods. The principles of the methods most commonly used in the clinical laboratory are discussed in this chapter. This introduction is intended to acquaint the reader with the structure and function of antibodies (immunoglobulins) in relation to their use as reagents in immunoanalysis.

Antibodies

Antibodies are immunoglobulins capable of binding specifically to a wide array of natural and synthetic antigens, including proteins, carbohydrates, nucleic acids, lipids, and other molecules. Immunoglobulins consist of five general classes designated as immunoglobulin (Ig)G, IgA, IgM, IgD, and IgE. IgG is used most commonly in immunochemical reagents. A schematic diagram of the IgG molecule is shown in Fig. 26.1 . IgG is a glycoprotein (molecular weight [MW], 158,000 Da) composed of two heavy (γ) and two light (κ or γ) chains joined by disulfide bonds. Each chain (H or L) is the product of three (L) or four (H) distinct gene segments. These are the constant (C), joining (J), diversity (D), and variable (V) genes that undergo combinatorial joining during B cell development. Several hundred germline V genes, 5 to 10 J genes, 15 D genes (H chain only), and a single C gene have been identified for each heavy or light chain class. During B cell development, the V, D, and J (H chain) or V and J (L chain) undergo random rearrangement and splicing, and this recombined product is then spliced to the constant region gene. This combinatorial diversity, along with somatic mutations that occur at the splicing sites, generates a tremendous diversity of antibody specificities. When a B-cell clone expressing a particular antibody specificity on its surface is selected by an antigen, it expands and differentiates into a plasma cell that secretes the specific antibody.

The variable amino acid sequence at the amino terminal end (∼105 amino acids) of each chain determines the antigenic specificity of the particular antibody. Each unique variable region is a product of a single plasma cell line or clone. A complex antigen is capable of eliciting a multiplicity of antibodies with different specificities that are derived from different cell lines. Antibodies derived in this manner are termed polyclonal and exhibit diverse specificities in their reactivity with the immunogen. Each unique region of the antigen molecule that will bind a complementary antibody is termed an epitope (antigenic determinant). See Chapter 31 for additional information.

Immunogens

An immunogen is a protein or a substance coupled to a carrier that, when introduced into a foreign host, is capable of inducing the formation of an antibody in the host. The antibody produced may be circulating (humoral) or tissue bound (cellular).

A hapten is a small, chemically defined determinant that, when conjugated to an immunogenic carrier, stimulates the synthesis of antibody specific for the hapten. It is capable of binding an antibody but cannot by itself stimulate an immune response.

Continued stimulation by an immunogen results in increased production of immunoglobulins of different types and of high-affinity binding characteristics for antigens. After the first exposure to an immunogen, a latent period (induction) occurs during which no antibody is present in serum; this period may last from 5 to 10 days.

The strength or energy of interaction between the antibody and the antigen is described by two terms. Affinity refers to the thermodynamic quantity defining the energy of interaction of a single antibody-combining site and its corresponding epitope on the antigen. The affinity can be influenced by thermodynamic factors such as pH and temperature. Avidity refers to the overall strength of binding of an antibody and its antigen and includes the sum of all the individual binding affinities of all the combining sites on the antibody. The avidity is also dependent on the valency and structural arrangement of the antibody and antigen. For example, IgG has two antigen-binding sites, whereas IgM has 10 antigen-binding sites per antibody molecule. For polyclonal antibodies, affinity and avidity are difficult to determine primarily because of the diversity of the antibody population.

Polyclonal antiserum is raised in an animal host in response to immunogen administration. In contrast, monoclonal antibodies are produced in a very different manner and represent the product of a single clone or plasma cell line, rather than a heterogeneous mixture of antibodies produced by many plasma cell clones in response to immunization. Monoclonal antibodies are currently widely used as reagents in immunoassay techniques. The usual method of production of monoclonal antibodies involves fusing antibody-producing plasma cells from the spleens or lymph nodes of immunized mice with a murine myeloma cell line from tissue culture.

Because of the unique ability of a monoclonal antibody to react with a single epitope on a multivalent antigen, the majority of monoclonal antibodies will not cross-link and precipitate macromolecular antigens. Consequently, monoclonal antibodies have not found broad applicability in traditional precipitin methods. A practical advantage of using monoclonal antibodies is that two different antibody specificities can be combined in a single incubation step. A solid-phase antibody specific for a unique epitope and another labeled antibody specific for a different epitope can react with an antigen in a single step. This eliminates the incubating and washing steps that usually would be required for polyclonal antibodies.

Phage display technology provides an in vitro approach for producing antibodies (single-chain Fv fragments, Fab fragments, and whole antibody molecules) that mimic the immune system but do not require B-cell immortalization. V genes coding for the heavy- and light-chain variable domains of immunoglobulin isolated from lymphocytes are amplified by the polymerase chain reaction (PCR) and ligated into a filamentous bacteriophage vector to form combinatorial libraries of V _H and V _L genes. Individual bacteriophages display copies of a specific antibody on their surface, and the phage library can be screened for the antibody of a defined specificity using immobilized antigen (“panning”). Large libraries displaying antibodies formed from more than 10 ¹² different V _H and V _L combinations can be constructed; this provides a rich source of antibodies with high affinity.

Binding forces

The strength of the binding of an antigen to an antibody depends on several forces acting cooperatively. These include van der Waals-London dipole-dipole interaction, hydrophobic interaction, and ionic coulombic bonding.

Reaction mechanism

The binding of antigen to antibody is not static but is an equilibrium reaction that proceeds in three phases. The initial reaction (phase 1) of a multivalent antigen (Ag _n ) and a bivalent antibody (Ab) occurs very rapidly in comparison with subsequent growth of the complexes (phase 2) and is depicted by the following equation:

where k ₁ >> k ₂ , n is the number of epitopes per molecule, and a and b are the numbers of antigen and antibody molecules per complex. Phase 3 of the reaction involves precipitation of the complex after a critical size is reached. The speed of these reactions depends on electrolyte concentration, pH, and temperature and on antigen structure and antibody class and the binding affinity of the antibody. The concentration of sodium chloride (NaCl) is important, and in most cases saline (NaCl, 0.15 mol/L) is used. Higher concentrations of NaCl can lead to smaller amounts of precipitate; this is due not to increased solubility of the antigen-antibody complex, but to an equilibrium shift causing a given amount of antigen to combine with smaller amounts of antibody. Decreasing the NaCl concentration can lead to increased precipitation of other proteins.

It is best to use dilute Ab and Ag solutions for determining the influence of such factors as (1) ionic species, (2) ionic strength, and (3) pH. Use of dilute solutions slows the growth of antigen-antibody complexes; this results in more stable and homogeneous complexes.

Factors influencing binding

Factors that influence the strength of binding between an antigen and an antibody include ion species, ionic strength, and polymers used in the solution.

Types of reactions

Types of antigen-antibody reactions that are of analytical importance include the precipitin reaction and those noted at a solid-liquid interface.

Precipitin reaction

If the number of antibody-combining sites is notably greater than the antigen-epitope sites ([Ab] >> [Ag]), then antigen-binding sites are quickly saturated by antibodies before cross-linking can occur, along with the formation of small insoluble antigen-antibody complexes ( Fig. 26.2A ). When an antibody is in moderate excess (i.e., [Ab] > [Ag]), the probability of cross-linking of Ag by Ab is more likely and hence large insoluble complex formation is favored (see Fig. 26.2B ). When [Ag] is in great excess, large complexes would be less probable (see Fig. 26.2C ). This model describes the results observed when antigens and antibodies are mixed in various concentration ratios. The curve shown in Fig. 26.3 is a schematic diagram of the classic precipitin curve. Although the concentration of total antibody is constant, the concentration of free antibody [Ab] _f (i.e., not bound to antigen) and free antigen [Ag] _f varies throughout the range for any given Ag/Ab ratio. A low Ag/Ab ratio exists in A of see Fig. 26.3 (zone of antibody excess). Under these conditions, [Ab] _f exists in solution but [Ag] _f does not. As total antigen increases, the size of the immune complexes increases up to equivalence (see Fig. 26.3B ), in which little or no [Ab] _f or [Ag] _f exists. This is the zone of maximum immune complex size. This equivalence zone does not represent a ratio of exact molar equivalence of reactants but is the optimal combining ratio for cross-linking in the particular system under evaluation. As Ag/Ab increases (see Fig. 26.3C ), the immune complex size will decrease and [Ag] _f will increase (zone of antigen excess). No [Ab] _f should exist in this area of the curve. However, for a given Ag/Ab ratio, the population of immune complexes formed at equilibrium will be heterogeneous with respect to size and composition.

Passive gel diffusion

Many qualitative and quantitative immunochemical methods are performed in a semisolid medium, such as agar or agarose. The primary advantage of using a gelatinous medium is that visualization of precipitin bands is allowed for qualitative and quantitative evaluation of the reaction. Antigen-to-antibody ratio, salt concentration, and polymer enhancement have the same influence on the antigen-antibody reaction in gels that they have on reactions in solution.

The initial concentration of antigen and antibody is critical. Each molecule in the system will achieve a unique concentration gradient with time. When the leading fronts of antigen and antibody diffusion overlap, the reaction will begin, but formation of a precipitin line will not occur until moderate antibody excess is achieved ( Fig. 26.3B ). A precipitin band may form and be dissolved many times by an incoming antigen before equilibrium is established and the position of the precipitin band becomes stable. Because heavier molecules diffuse more slowly, the position of the precipitin band is in part a function of the molecular masses of both antigen and antibody. The precipitin band acts as a specific barrier; neither specific antigen nor antibody can penetrate without being precipitated by the other, but unrelated molecules can cross the band of precipitation freely. Basic approaches to passive diffusion include simple diffusion and double diffusion. With simple diffusion, a concentration gradient is established for only a single reactant. Single immunodiffusion usually depends on diffusion of an antigen into agar impregnated with antibody. A quantitative technique based on this principle is RID, which is discussed later. The second approach is double diffusion, in which a concentration gradient is established for both reactants (antigen and antibody).

Double immunodiffusion in two dimensions is a historical immunotechnique known as the Ouchterlony method. It allows direct comparison of two or more test materials and provides a simple method for determining whether the antigens in the test specimens are identical, cross-reactive, or nonidentical.

The simplest method uses an agar dish or slide with holes cut as shown in Fig. 26.4 . When the same antigen is in adjacent wells, the lines of precipitation fuse and are continuous—this is a reaction of identity (see Fig. 26.4A ). When the precipitin bands cross each other, this is a reaction of nonidentity (see Fig. 26.4B ); if the two antigens are related but are not identical, a reaction of partial identity is observed (see Fig. 26.4C ). Here the cardinal point is that the precipitate serves as a barrier that does not block unrelated diffusing reactants. As shown in see Fig. 26.4D , when the two related antigens Ag and Ag ₁ are in separate wells and the respective antibodies, Ab and Ab ₁ , are in the third well, an AgAb precipitate forms on one side and blocks further diffusion of Ab from the antibody well. However, on the other side, the Ag ₁ Ab ₁ precipitate does not stop Ab from migrating further and forming an AgAb spur.

Note that a negative reaction does not necessarily imply absence of antibody or antigen. A negative reaction can result from using amounts of material too small for the detection limit of the method, or the antibody may be nonprecipitating.

Immunoelectrophoresis

If several antigens of interest exist in a solution (e.g., spinal fluid or serum), the various protein species can be separated and identified by IEP. This technique has been used extensively for the study of antigen mixtures and evaluation of the specificity of antiserum. ^,

The procedure is performed using an agarose gel medium poured onto a thin plastic sheet. The sample to be analyzed is placed in a reservoir in the gel, and an electrical field is applied across the gel surface. During electrophoresis, the proteins in the serum are separated according to their electrophoretic mobilities ( Fig. 26.5 ). After electrophoresis, an antiserum against the protein of interest is placed in a trough parallel and adjacent to the electrophoresed sample. Simultaneous diffusion of the antigen from the separated sample and the antibody from the trough leads to the formation of precipitin arcs, whose shape and position are characteristic of individual separated proteins within the specimen. By comparison with a known control separated on the same plate, individual proteins can be tentatively identified.