The Elisa guidebook

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Table 1

Relationship of Diameter of Agents to Number of Binding Sites

Diameter	Fab sites	IgG sites
25	98	32
250	9800	3200
2500	980,000	320,000
25,000	98,000,000	32,000,000

Since we know the molecular weight of IgG (and Fab), we can calculate the weight of a number of molecules. Thus, the mol wt of IgG = 150,000. Using Avogadro's number (approx 6 ¡Á 1023) we obtain the following results:

1.1 g of IgG contains approx 6 ¡Á 1023/1.5 ¡Á 105 = 4 ¡Á 1018 molecules.

2.1 mg of IgG contains approx 4 ¡Á 1018/103 = 4 ¡Á 1015 molecules.

3.1 µg of IgG contains approx 4 ¡Á 1018/106 = 4 ¡Á 1012 molecules.

4.1 ng of IgG contains approx 4 ¡Á 1018/109 = 4 ¡Á 109 molecules.

5.1 pg of IgG contains approx 4 ¡Á 1018/1012 = 4 ¡Á 106 molecules.

Such a model calculation helps us to understand at the molecular level what we are dealing with when faced with different antigens.

4.2¡ª Definitions

There may be some confusion concerning the terminology used in immunological and serological circles. This section provides some working definitions that will aid the understanding of the mechanisms involved in ELISAs.

4.2.1¡ª Antigen

An antigen is a substance that elicits an antibody response as a result of being injected into an animal or as a result of an infectious process. Antigens can be simple (e.g., peptides of mol wt about 5000) to complex. Antibodies specific for the antigen are produced. The definition can be extended to molecules that evoke any specific immune response including cell-mediated immunity or tolerance.

4.2.2¡ª Antigenic Site

An antigenic site is a distinct structurally defined region on an antigen as identified by a specific set of antibodies usually using a polyclonal serum.

4.2.3¡ª Epitope

An epitope is the same as an antigenic site, but in which a greater specificity of reaction has been defined, e.g., using tests involving mAbs in which a single population of antibodies identifies a single chemical structure on an antigen.

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Fig. 1.

Representation of a linear of continuous site. Black area shows paratope of antibody with specificity for site.

4.2.4¡ª Epitype

An epitype is an area on an antigen that is identified by a closely related set of antibodies identifying very similar chemical structures (e.g., mAbs, which define overlapping or interrelated epitopes). An epitype can be regarded as an area identifying slightly different specificities of antibodies reacting with the same antigenic site.

4.2.5¡ª

Continuous Epitope

A continuous epitope is produced by consecutive atoms contained within the same molecule. Such epitopes are also referred to as linear epitopes and are not usually affected by denaturation (see Fig. 1).

4.2.6¡ª

Discontinuous Epitope

A discontinuous epitope is produced from the interrelationship of atoms from nonsequential areas on the same molecule or from atoms on separate molecules. Such sites are also usually conformational in nature (see Fig. 2).

4.2.7¡ª

Linear Epitope

A linear epitope is the same as a continuous epitope, with recognition of atoms in a linear sequence (see

Fig. 1).

4.2.8¡ª

Conformational Epitope

A conformational epitope is formed through the interrelationship of chemical elements combining so that the 3D structure determines the specificity and affinity. Such epitopes are usually affected by denaturation (see Fig. 2).

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Fig. 2.

Representations of two types of conformational epitopes. Black area shows papatope of antibody molecule with specificity for the sites.

(A) Recognition of three-dimensional (3D) relationship of atoms from nonconsecutive atoms on same protein molecule; (B) recognition of 3D relationship of atoms on two different protein molecules.

4.2.9¡ª Antibody-Combining Site

An antibody-combining site is the part of the antibody molecule that combines specifically with an antigenic site formed by the exact chemical nature of the H and L chains in the antibody molecule.

4.2.10¡ª Paratope

A paratope is the part of the antibody molecule that binds to the epitope. It is most relevant to mAbs antibodies in which a single specificity for a single epitope can be defined.

4.2.11¡ª

Affinity and Avidity

Affinity and avidity relate to the closeness of fit of a paratope and epitope. Considered in thermodynamic terms, it is the strength of close-range non-

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covalent forces. Mathematically it is expressed as an association constant (K, L/mol) calculated under equilibrium conditions. Affinity refers to the energy between a single epitope and paratope. Antisera usually contain populations of antibodies directed against the same antigenic site that have different affinities owing to their differences in exactness of fit. Antisera of multiple specificity (i.e., specific to many determinants on an antigen) cannot be assessed for affinity, however, they can be assessed for overall binding energy with an antigen in any chosen assay. This is termed the avidity of the serum. The avidity represents an average binding energy from the sum of all the individual affinities of a population of antibodies binding to different antigenic sites.

4.2.12¡ª

Polyclonal Antibodies

Polyclonal antibodies are the serum product of an immunized animal containing many different antibodies against the various mixtures of antigens injected. The antiserum is the product of many responding clones of cells and is usually heterogeneous at all levels. These levels include the specificity of the antibodies, classes and subclasses, titer, and affinity. The response to individual epitopes may be clonally diverse, and antibodies of different affinities may compete for the same epitope. This variation means that polyclonal antisera cannot be reproduced (see Fig. 3).

4.2.13¡ª

Monoclonal Antibodies

mAbs are antibodies derived from single antibody-producing cells immortalized by fusion to a B- lymphocyte tumor cell line to form hybridoma clones. The secreted antibody is monospecific in nature and thus has a single affinity for a defined epitope (see Fig. 4).

5¡ª Antibodies

Antibodies form a group of glycoproteins present in the serum and tissue fluids of all mammals. The group is also termed immunoglobulins (Igs) indicating their role in adaptive immunity. All antibodies are Igs, but not all Igs are antibodies, that is, not all the Ig produced by a mammal has antibody activity. Five distinct classes of Ig molecules have been recognized in most higher mammals. These are Ig, IgG, IgA, IgM, IgD, and IgE. These classes differ from each other in size, charge, amino acid composition, and carbohydrate content. There are also significant differences (heterogeneity) within each class. Figure 5 shows the basic polypeptide structure of the Ig molecule.

5.1¡ª

Antibody Structure

The basic structure of all Ig molecules is a unit of two identical light (L) polypeptide chains and two identical heavy (H) polypeptide chains linked

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Fig. 3.

Specific antibodies are produced against different sites and can vary in affinity against the same site. This, a,b,c,d-----n represents a range of slightly different antibody populations recognizing antigenic sites x, y, and z. The antibodies have different affinities, classes, and isotypes.

The resulting mixture is a polyclonal antiserum.

Fig. 4.

mAbs are monospecific in terms of antigenic target and affinity.

together by disulfide bonds. The class and subclass of an Ig molecule is determined by its heavy chain type. Thus, in the human, there are four IgG subclasses¡ªIgG1, IgG2, IgG3, IgG4¡ªthat have heavy chains called 1, 2, 3, and 4. The differences between the various subclasses within an individual Ig class are less than the differences between the different classes. Therefore IgG1 is more closely related to IgG2, and so on than to IgA, IgM, IgD, or IgE. The most common class of Ig is IgG.

IgG molecules are made up of two identical light chains of mol wt 23,000 and two identical heavy chains of mol wt 53,000. Each light chain is linked to

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Fig. 5.

Representation of basic structure of an IgG molecule.

a heavy chain by noncovalent association, and also by one covalent disulfide bridge. For IgG, each lightheavy chain pair is linked to the other by disulfide bridges between the heavy chains. This molecule is represented schematically in the form of a Y, with the amino (N-) termini of the chains at the top of the Y and the carboxyl (C-) termini of the two heavy chains at the bottom of the Y shape. A dimer of these light-heavy chain pairs is the basic subunit of the other Ig isotypes. The structures of these other classes and subclasses differ in the positions and number of disulfide bridges between the heavy chains, and in the number of L-H chain pairs in the molecule. IgG, IgE, and IgD are composed of one L-H chain pair. IgA may have one, two, or three light-heavy chain pairs. IgM (serum) has five light-heavy chain pairs, whereas membrane-bound IgM has one. In the polymeric forms of IgA and IgM, the light-heavy chain pairs are held together by disulfide bridges through a polypeptide known as the J chain.

In both heavy and light chains, at the N-terminal portion the sequences vary greatly from polypeptide to polypeptide. By contrast, in the C-terminal portion of both heavy and light chains, the sequences are identical. Hence, these two segments of the molecule are designated variable and constant regions. For the light chain, the variable (V) region is about 110 amino acid residues in length

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and the constant (C) region of the light chain is similarly about 110 amino acids in length.

The variable region of the heavy chain (VH) is also about 110 amino acid residues in length, but the constant region of the heavy chain (CH) is about 330 amino acid residues in length. The N-terminal portions of both heavy and light chain pairs comprise the antigen-combining (binding) sites in an Ig molecule. The heterogeneity in the amino acid sequences present within the variable regions of both heavy and light chains accounts for the great diversity of antigen specificities among antibody molecules. By contrast, the constant regions of the heavy chain make up the part of the molecule that carries out the effector functions that are common to all antibodies of a given class.

Figure 5 shows that there must be two identical antigen-binding sites (more in the case of serum IgM and secretory IgA); hence, the basic Y-shaped Ig molecule is bivalent. This bivalency permits antibodies to crosslink antigens with two or more of the same epitope. Antigenic determinants that are separated by a distance can be bound by an antibody molecule.

The antigen-combining site (active site) is a crevice between the variable regions of the light and heavy chain pair. The size and shape of this crevice can vary owing to differences in the relationship of VL and VH regions as well as to differences in variation in the amino acid-sequence. Thus, the specificity of antibody will result from the molecular complementarity between determinant groups (epitopes) on the antigen molecule and amino acid residues present in the active site.

An antibody molecule has a unique 3D structure. However, a single antibody molecule has the ability to combine with a range (spectrum) of different antigens. This phenomenon is known as multispecificity. Thus, the antibody can combine with the inducing antigenic determinant or a separate determinant with similar structures (crossreacting antigen). Stable antigen-antibody complexes can result when there is a sufficient number of short-range interactions between both, regardless of the total fit. This is a problem for the immunoassayist, and care must be taken to ensure that the operator is assaying for the correct or desired antigen; therefore, careful planning of negative and positive controls is essential.

5.1.1¡ª

Antibody Digestion

Figure 6 demonstrates the digestion of IgG using papain or pepsin proteolytic enzymes. Mild proteolysis of native Ig at the hinge regions of the heavy chain by papain will cleave IgG into three fragments. Two of these fragments are identical and are called fragment antigen binding or Fab. Each Fab consists of the variable and constant regions of the light chain and the variable part of

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Fig. 6.

Enzymatic cleavage of human IgG. Pepsin cleaves the heavy chain to give F(Ab') and o/fc' fragments. Further action results in greater fragmentation of central protein to peptides. Papain splits the molecule in the hinge region to give two Fab2 fragments

and the Fc fragment. Further action on the Fc can produce Fc'.