IAPTER 1 Basic Immunology

An individually acti- vated B-cell proliferates and differentiates to form plasma cells that begin to produce identical antibodies with a single antigen specificity at the rate of 3000-3

Basic Immunology

1 The Immune Response

A knowledge of immunology is essential in developing ELISAs Infor- mation about the specific system and similar systems already studied from the biochemical and immunological aspects is important to allow development of assays and to determine the significance of results It is not the intent of this chapter to provide an in-depth understanding of immunology; however, it is necessary for the ELISA operator to have a basic understanding of:

1, The immunology of infectious diseases

2 The properties of certain components of the immune system

3 Aspects of serology

Mammals possess a system of surveillance called the immune system that protects them from disease-causing (pathogenic) microorganisms, such as viruses, bacteria, and parasites The immune system specifically recognizes and eliminates pathogens The protection afforded by the immune system of the mammal is divided into two functional divisions, namely, the innate immune system and the adaptive immune system, both

of which respond specifically to these foreign substances

Innate immunity acts as the first line of defense against infectious agents, and most potential pathogens are checked before they can estab- lish infection If these defenses are overcome, then the adaptive immune system is activated The adaptive system produces a specific reaction against each infectious agent, and also remembers that particular agent and can prevent it causing disease in the future Most of the applications

of ELISA involve studies on the adaptive immune system, so this will be featured in more detail Excellent books on basic immunology have been written, in particular (I), which has extensive references for specific areas outlined below

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1.1 Innate Immunity

The factors involved in innate immunity include biochemical and physical barriers, e.g., the skin, acting as an impenetrable barrier to infec- tious agents, and the presence of lysozyme in tears, which destroys bac- teria, such as S aureus The key difference in this system from that of the adaptive immunity is that resistance is not improved by repeated infec- tion and that the system is aspecific in nature Thus, if organisms do penetrate an epithelial surface, they encounter phagocytic cells of the reticuloendothelial system (RE), where the cells are of many types derived from bone marrow cells Their function is to engulf, internalize, and destroy infectious agents For this purpose, they are placed strategi- cally where they might encounter particles, e.g., the Kupffer cells of the liver line the sinusoids along which blood flows The blood phagocytes include neutrophil polymorph and the blood monocytes, both of which can migrate into tissues as a result of an invasive stimulus Other cells, such as natural killer (NK) cells, are leukocytes capable of recognizing cell surface changes on virus-infected cells Such cells bind and kill cells under the influence of substances called interferons, which are produced

by the virus-infected cells or sometimes by lymphocytes

Other factors involved in innate immunity involve certain serum pro- teins These are referred to as acute-phase proteins The concentration of such proteins rises dramatically on and is maintained throughout infec- tion The various proteins have defined properties and produce protec- tive effects through complex interactions with other serum components, such as complement followed by lysis of disease agents

Visible signs of an early immune response are observed in inflamma- tion, which is the body’s reaction to an injury, such as invasion with an infectious agent Three major events occur, namely:

1, An increased blood supply to the infected area,

2 An increase m the permeability of the capillarres caused by retraction of endothelial cells, allowing larger molecules to cross the endothelium, e.g., soluble mediators

3 Migration of leukocytes ( neutrophils, polymorphs, and macrophages) from capillaries to surroundmg tissues,

These features, whereby phagocytes are attracted to sites of injury, are important in immunity and initiate all levels of potential innate immune mechanisms

1.2 Adaptive Immunity Innate immunity relies on stimulation of factors through aspecific rec- ognition of infectious agents Problems arise when the recognition pro- cess is not activated, e.g., when phagocytes are unable to recognize the infectious agent either because they lack a suitable receptor for the agent

or because the agent does not activate soluble factors What is needed in such a situation is a specific molecule that can attach at one end to the infectious agent and at the other end to the phagocytic cell Such mol- ecules, called antibodies, are produced in the mammalian systems Antibodies are produced by B lymphocytes of the adaptive immune system, which act as flexible adaptors between infectious agents and phagocytes Any particular antibody molecule can bind only to one type

of infectious agent, and the other end of the molecule binds to the phago- cyte by way of a receptor, the Fc receptor

A stylized structure of an antibody is shown in Fig 1A Figure 1B shows that IgG is a rather bulky structure when examined at the molecu- lar level Antibodies are effectively bifunctional molecules One part, which is extremely variable between different antibodies, binds to all the various infectious agents, whereas the second, constant portion binds to receptors of cells and also activates complement

2 Antigens The mammalian immune system has the ability and capacity to recog- nize surface features (topography) of foreign macromolecules or micro- organisms that are not normal constituents of that mammal (e.g., pathogenic microorganisms) This recognition of surface features is nor- mally specific, and the components of the mammal that carry out this specific recognition of surface features of macromolecules or microor- ganisms are protein molecules called antibodies Foreign substances have specific surface features, called antigens, that antibodies recognize The portion of the antigen to which the antibody binds is called the antigenic determinant or epitope Antibody specifically binds to epitopes

on the antigen by multiple noncovalent interactions similar to the inter- actions that confer specificity to enzyme-substrate reactions The part of the antibody that binds to the antigenic determinant is termed the anti- gen-combining site or paratope (which is complimentary to the epitope)

An antigen eliciting a response from the immune system is referred to

as an immunogen Microorganisms, macromolecules, such as foreign

Fig 1 (A) Representation of basic structure of an IgG molecule (B) Model

of IgG molecule based on X-ray crystallographic analysis

proteins, nucleic acids, carbohydrates, polysaccharides, and so forth, are usually effective immunogens Molecules with mol wt below 5000 usu- ally are not effective immunogens However, many of these small nonimmunogenic molecules, when covalently attached to a large mol- ecule, can stimulate an immune response These molecules, which are nonimmunogenic, are termed haptens, and the large molecules to which they can be covalently attached, generally proteins, are termed carriers Once the hapten is attached covalently to the carrier protein and intro- duced into an organism, a specific antibody response to the hapten and the carrier (if the latter is recognized) occurs This antibody response can

be specific to the hapten Thus, nonimmunogenic molecules (haptens) can

be recognized by an organism when covalently attached to a carrier protein

2.1 Antigen Presentation, Processing, and Recognition Lymphocytes account for between 20 and 80% nucleated cells in the blood, and over 99% nucleated cells in lymphatic fluid Lymphocytes contact and respond to antigens in specialized lymphoid organs Included

in these specialized organs are the spleen, thymus, lymphatic tree and the lymph nodes positioned along them, bone marrow, and Peyer’s patches (appendix, adenoids, tonsils [Bursa of Fabricius])

The lymphoid system has three principal functions, namely:

1 Concentration of antigens from all parts of the body into lymphoid organs

2 Circulation of lymphoid cells through these organs to ensure antigen expo- sure to antigen-specific lymphocytes in a short period of time

3 Transmission and dissemination of the products of the immune response, e.g., antigen-specific effector B + T-cells, humoral antibodies, throughout the body

Antigens are collected and processed by different lymphoid organs depending on their route of entry into the body, i.e., respiratory system, gastrointestinal tract, skin, vector transmission, venereal, and so on Anti- gen processing in all these lymphoid organs involves macrophages, a short period of time after infection (injection) The antigen(s) becomes incor- porated in special vesicles (phagolysosomes) within the macrophage The macrophage cell surface either retains or receives a small amount of immunogenic material for presentation to antigen-specific lymphocytes Binding of this macrophage surface antigen to a B-cell or T-cell (pre- sentation of antigen) induces a general activation of the cell This pro- cess, known as blast-transformation, causes the B-cell (or T-cell) with the appropriate receptor specificities to recognize the antigen-presenting macrophage to enlarge Such activated cells initiate DNA synthesis, divide, and give rise to effector cells and memory cells of the B-cell (or T-cell) lineage

Most memory B-cells re-enter the general circulation, whereas most effector B-cells are retained in the lymph node An individually acti- vated B-cell proliferates and differentiates to form plasma cells that begin

to produce identical antibodies with a single antigen specificity (at the rate of 3000-30,000 molecules/cell/s)

An organism’s total response even to a simple antigen is almost always heterogeneous with respect to antibody specificity because most anti- gens have multiple epitopes, which can trigger the activation of different

B-cells Consequently, the serum of the host reflects the heterogeneous collection of immunoglobulin molecules previously secreted

Although individual B-cells are committed to produce ,one or at most two antibody isotypes, the B-cell response to any antigen can produce antibody molecules of all five classes of immunoglobulins Since anti- body molecules of different classes differ in their heavy-chain-constant regions (Fc), they may exhibit identical antigen-binding specificities and, hence, identical variable regions Antibodies of the five classes mediate different physiological effector functions, and are present in serum at different concentrations and for different half-life periods The different classes are also produced in different relative amounts in primary and secondary immune responses

2.1.1 IgM This is the first antibody produced in response to an irnmunogen and is particularly effective against invading microorganisms It is a pentamer in serum, and although the affinity of each active site on the pentamer (10 in number) for an epitope may be low, the overall avidity of the pentamer for a complex antigen is high because of the repeating nature of epitopes on many cell membrane antigens Because it is present as a pentamer in serum, IgM is about 1000 times more effective (on a molar basis) at agglutinating cells by crosslinking them, than a monomeric antibody against the same epitope IgM coated onto the antigen of target cells stimulates target cell ingestion by macrophages and target cell destruction (lysis) by compliment fixation

2.1.2 IgG This monomeric antibody is normally produced later in the immune response than IgM This is the most prevalent antibody in blood and tis- sue spaces, and is capable of fixing complement It also activates mac- rophage ingestion of opsonized (coated) antigen particles IgG is the only class of antibody that can cross the placenta to provide passive immunity

to the developing fetus Normally, the affinity of IgG antibodies toward

a specific antigen increases with time after immunization, a process known as affinity maturation

2.1.3 IgA This is also produced later in the immune response than IgM It can exist as a monomer, dimer, or trimer of the basic Y-shaped structural unit IgA antibodies are important at numerous epithelial surfaces and

act as a potential protective barrier at several points of entry, e.g., gastro- intestinal tract, respiratory tract, genitourinary tract, eyes, and so forth Some epithelial cells produce a polypeptide, called the secretory compo- nent, which complexes to the Fc region of IgA and mediates their trans- port across the epithelial cell surface to the lumen IgA B-cell precursors are especially frequent in lymphoid organs draining the gastrointestinal tract and in the mammary glands IgA is the major immunoglobulin in colostrum and milk, and is also present in sweat, tears, and saliva

2.1.4 IgE This monomeric antibody is heat-labile It is present in blood in very low concentrations IgE antibodies are produced in response to infection mainly the helminth parasites and in allergic atopic conditions IgE anti- bodies can bind via their Fc regions to mast cells or blood basophils Further interaction of this bound IgE with a cognate (known and recog- nized by IgE) antigen can trigger cell degranulation and the liberation of vasoactive compounds, such as histamine and heparin

2.1.5 IgD This monomeric antibody is present only in minute concentrations in blood Its functions are unknown

3 Adaptive Immunity and Clonal Selection

The immune system as a whole can specifically recognize many thou- sands of antigens The specificity of the adaptive immune response is based on the specificity of the antibodies and lymphocytes, and since it has been shown that each lymphocyte is only capable of recognizing one particular antigen, this means that the lymphocytes recognizing any par- ticular antigen are a very small proportion of the total Thus, we have to explain how an adequate response to an infectious agent is mounted The answer is clonal selection, whereby antigen binds to a small number of cells that can recognize it and induces them to proliferate Thus, the anti- gen selects the specific clones of antigen-binding cells

This is illustrated in Fig 2 This process occurs in both B-lympho- cytes, where they mature into antibody-producing cells, and T-lympho- cytes, which are involved in the recognition and destruction of infected cells

A basic requirement for the production of an antibody response is that the immunogen possesses surface features that are recognized as foreign

in the animal into which it is introduced or in which it occurs

Antigen selection

Production of antibody 2

Fig 2 The antibody-producing cells (B-cells) are programmed to make a single antibody only The antibody is placed on an Fc receptor on the cell’s surface Each B-cell has a different receptor, and antigen binds to those cells with the appropriate receptor The cells become stimulated to multiply and mature onto antibody-producing cells and memory cells, which can live longer All the cells have the same antigen-binding capacity

4 Antibodies Antibodies are fundamental reagents in ELISA, and the determination

of their presence and/or concentration in the blood is vital in understand- ing disease processes and in diagnosis of disease A knowledge of the properties of antibodies is fundamental to the development of specific assays An understanding of the variation in antibody composition of different mammals is also important

ANTIGEN BINDING SITE (PARATOPE)

Fig 3 Structural elements of an IgG molecule

4.1 Antibody Structure and Function

Antibodies form a group of glycoproteins present in the serum and tissue fluids of all mammals The group is also termed immunoglobulins, indicating their role in adaptive immunity All antibodies are immuno- globulins, but not all immunoglobulins are antibodies, i.e., not all the immunoglobulin produced by a mammal has antibody activity

Five distinct classes of immunoglobulin molecule have been recog- nized in most higher mammals These are immunoglobulin (Ig) G (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 The basic polypeptide structure of the immunoglobulin molecule is shown

in Fig 3

The basic structure of all immunoglobulin molecules is a unit of two identical light (L) polypeptide chains and two identical heavy (H) polypeptide chains linked together by disulfide bonds

The class and subclass of an immunoglobulin molecule are determined

by its heavy-chain type Thus, in the human, there are four IgG subclasses, IgGl, IgG2, IgG3, and IgG4, which have heavy-chains called 1,2,3, and

4 The differences between the various subclasses within an individual immunoglobulin class are less than the differences between the different classes Thus, IgGl is more closely related to IgG2, and so on, than to IgA, IgM, IgD, or IgE The most common class of immunoglobulin is IgG IgG molecules are made up of two identical light chains of mol wt 23,000 Daltons and two identical heavy chains of mol wt 53,000 Daltons Each light chain is linked to a heavy-chain by noncovalent association, and also by one covalent disulfide bridge For IgG, each light-heavy- 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 immunoglobulin isotypes The structures of these other classes and sub- classes differ in the positions and number of disulfide bridges between the heavy chains, and in the number of light-heavy-chain pairs in the molecule IgG, IgE, and IgD are composed of one light-heavy-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 light-heavy-chain pair 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 In contrast, in the C-termi- nal 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 region (V) is approx

110 amino acid residues in length, and the constant region (C) of the light chain is similarly about 110 amino acids in length The variable region of the heavy chain (Vu) is also about 110 amino acid residues in length, but the constant region of the heavy-chain (C,) 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 immunoglobulin 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 In contrast, the constant regions

of the heavy chain make up the part of the molecule that carries out the effector functions, which are common to all antibodies of a given class From Fig 3, it can be seen that there must be two identical antigen- binding sites (more in the case of serum IgM and secretory IgA) Hence, the basic Y-shaped immunoglobulin 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 vari- able regions of the light- and heavy-chain pair The size and shape of this crevice can vary because of differences in the relationship of VL and VH regions, as well as differences in the amino acid sequence variation, Thus, the specificity of antibody will result from the molecular complement- arity between determinant groups (epitopes) on the antigen molecule and amino acid residues present in the active site

From this we can see that an antibody molecule has a unique three- dimensional structure However, a single antibody molecule has the abil- ity to combine with a range (spectrum) of different antigens This phenomenon is known as multispecificity Thus, the antibody can com- bine 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 opera- tor is assaying for the correct or desired antigen; therefore, careful plan- ning of negative and positive controls is essential

Figure 4 demonstrates the digestion of IgG using papain or pepsin proteolytic enzymes Mild proteolysis of native immunoglobulin 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 con- stant regions of the light chain and the variable and part of the constant (ChI domain) regions of the heavy-chain Therefore, each Fab carries

one antigen-binding site The third fragment, consisting of the remainder

of the constant regions of the heavy-chains, is readily crystallizable and

is called “fragment crystallizable” or Fc

Pepsin digestion cleaves the Fc from the molecule, but leaves the disul- fide bridge between the Fab regions This molecule contains both anti- gen-combining sites and is bivalent

The five immunological classes (isotypes) can be distinguished struc- turally by differences in their heavy-chain constant regions (i.e., mainly the Fc portion) These heavy-chain classes define the corresponding immunoglobulin classes IgA, IgG, IgD, IgE, and IgM Some classes can

be divided further into subclasses

In addition, two major typesS of light chains exist, based on the differ- ences in the constant region Cl and are known as kappa (K) and lambda (h) Immunoglobulins from various mammals appear to conform to the above format However, the subclass designation and variety may not be the same in all species examined, e.g., mice have IgGl, IgG2a, IgG2b, and IgG3; cows have IgGl and IgG2

4.2 Antibody Production

in Response to Antigenic Stimulus

The antibodies produced in a humoral response to antigenic stimulus am heterogeneous in specificity and may include all immunoglobulin classes This heterogeneous response is owing to the fact that most antigens have multiple antigenic determinants that trigger off the activation of different B-cells There- fore, the serum of any mammal(vertebrate) contains a heterogeneous mix- ture of immunoglobulin molecules The specificities of these immunoglobulin molecules will reflect the organism’s past antigenic exposure and history The first antibody produced in response to a primary exposure of an immunogen is IgM When the immunogen is persistent or the host (mam- mal) is re-exposed to the immunogen other classes of antibody may be produced as well as IgM Thebbody compartment in which the immuno- gen is presented can determine the predominant antibody isotype pro- duced (e.g., IgA in the gastrointestinal tract) In general, primary exposure

to an immunogen stimulates the production of IgM initially, followed by the appearance of IgG, as shown in Fig 5

If no further exposure occurs or the immunogen is removed by the mammal, a low level of IgM and IgG can be detected If re-exposure occurs, a similar peak of IgMiantibody is produced, which declines in a similar kinetic manner to the primary IgM response, but the IgG response

is not only more rapid (over time), but also reaches higher serum levels, which persist for a longer period of time This IgG response to re-expo- sure is known as the “anamnestic response.”

Where complex antigens occur, as in infectious diseases, the dosage (infection level), type of antigen (viral, bacterial, protozoan, and helmin- thic), route of infection (oral, respiratory, cutaneous), and species of mammal infected (cow, pig, camel, and human) will all affect the degree and speed by which IgG replaces IgM

These considerations are vital for the immunoassayist concerned with diagnosing infectious diseases of mammals, and great care and planning

0 5 10 15 20 25 30 35 40 45 50 55

Days after primary dose of antigen

Fig 5 Anamnestic response following second administration of antigen Pri- mary response following initial antigen dose has a lag phase, where no antibody

is detected (4-5 d) This is followed by a log phase, where antibody is produced

A plateau phase follows where antibody titers stabilize after which a decline in titer is observed On secondary stimulation, there is an almost immediate rise in titer and higher levels of antibodies are achieved that are mainly IgG

should be exercised before undertaking such immunoassays It should also be noted at this stage that different infectious disease agents can stimulate different antibody isotypes For example, certain viral patho- gens stimulate predominantly IgM agglutinating responses, bacterial polysaccharides stimulate IgM (and IgG2 in humans) antibodies, and helminthic infections stimulate IgE antibody synthesis In general, it can

be stated that during the development of immunity to infectious disease agents, the antibodies produced become capable of recognizing antigens better, as demonstrated by improved antigen-antibody interaction, three dimensional fit, and wider epitope recognition

The multispecificity of antibody molecules, i.e., the ability to combine with a variety of epitopes containing similar molecular structures, is depen-

1 Antigen

Water excluded

Fig 6 Attractive forces binding antigen to antibody A close approach of interacting groups IS needed for these forces to interact Hydrogen bonding results from formation of hydrogen bridges between atoms; electrostatic forces are the result of attraction of oppositely charged groups on two protein side chains Van der Waals forces are generated by interaction between electron clouds, and hydrophobic bonds rely on the association of nonpolar, hydropho- bic groups, so that contact with water molecules is minimized Half the total binding may be the result of the hydrophobic bonding

dent not only on the heterogeneity of the epitope in question, but also on the

molecular construction of the antigen-reactive sites (paratope) of the anti-

body molecules Since the binding of antibody to antigen is mediated by several types of noncovalent bonds (e.g., electrostatic, hydrogen bonds, Van der Waals forces, and hydrophobic forces) (see Fig 6), the strongest binding must occur when the paratope matches the epitope perfectly (best fit)

Good tit

High attraction Low repulsion High affinity

Fig 7 A good fit between antrgenic sites and antibody-combining sites cre- ates an environment for the intermolecular attractive forces to be created and limits the changes of repulsive forces The strength of the single antigen-anti- body bond is the affinity that reflects the summation of the attractive and repul- sive forces

4.2.1 Affinity and Avidity

The binding energy between an antibody molecule and antigen deter- minant is termed affinity Thus, it can be seen that antibodies with paratopes that recognize epitopes perfectly will have high affinity (good fit) for the antigen in question, whereas antibodies with paratopes that recognize epitopes imperfectly will have low affinity (poor fit) for the antigen in question Low-affinity antibodies, where the fit to antigen is less than perfect, will have fewer noncovalent bonds established between the complex, and the strength of binding will be less, as shown in Fig 7 With simple immunogens containing few epitopes, it is seen that as the antibody response develops (in response) to this immunogen, its rec- ognition by antibody will become better/closer, e.g., low-affinity anti- bodies will be replaced by high-affinity antibodies, which will cause the interaction between antigen and antibody to be more stable Antibodies produced later on during infection are generally of higher affinity than

those produced early on during infection Hence, the IgG antibodies pro- duced in response to re-exposure will be of higher affinity than those produced in response to initial exposure

In a serum sample, where there has been polyclonal stimulation of antibody production by antigen, there will be a variety of affinities pres- ent within the antibodies The match (fit) between antibodies to that anti- gen will be variable, and the antibodies present in that serum sample will bind to antigen differentially Thus, it can be seen that not only can an antigen stimulate different antibody isotypes, but also antibodies with different affinities for the antigenic determinant Avidity can be regarded

as the sum of all the different affinities between the heterogeneous anti- bodies contained in a serum and the various antigenic sites (epitopes)

We have already discussed the reasons for affinity variation from the points of view of the antibodies and antigens, each interaction between

an antibody population and a specific antigenic site has an individual affinity or equilibrium constant for the defined reaction Thus, the avid- ity can be regarded as the average of all the affinity constants for all interactions between the serum and antigen(s) It is important to realize that the avidity of a serum may change on dilution, since one may be diluting out particular populations of antibodies

As an example, one could have a serum containing a low quantity of antibodies showing high-affinity for a particular complex antigen and a high quantity of low-affinity antibody Under immunoassay conditions where that serum is not diluted greatly, one would have “competition” for antigenic sites between the high- and low-affinity antibodies, and the high-affinity antibodies would react preferentially On dilution, however, the concentration of the high-affinity antibodies would be reduced until one could only be left with low-affinity antibodies Such problems are important where one is using immunoassays to compare antigens by their differential activity with different antisera The dilution of any serum can affect its ability to discriminate between antigens owing to the dynamics

of the heterogeneous antibody population (relative concentrations and affinities of individual antibody molecules) Such problems of quality and quantity do not apply to monoclonal antibodies (MAb), since by defi- nition the immunoglobulin molecules in the population are identical They all have the same affinity and, therefore, the avidity equals affinity, Thus, the population reacts identically to any individual molecule in that population After diluting the monoclonal population, there is no alter-