CHAPTER 4 Theoretical Considerations

Antigenic Considerations Inherent in the understanding of what is being measured in any ELISA are the definition of terms involving antigens and antibodies and an under- standing of the

CHAPTER 4

A level of technical knowledge and expertise is required before any immunoassay can be understood and performed Tests require basic skills, and it is assumed that workers have acquired some skills through use of laboratory equipment and performance of other test systems There are areas that are essential in the use of ELBA that will be dealt with before studying the predominantly system-oriented practical chap- ters in detail, This chapter will deal with a collection of “I hope” useful information An excellent manual of techniques relevant to ELISA is available (I), and should be bought by all scientists involved in experi- mental work involving antibodies See also refs 2 and 3, which contain

a large amount of relevant practical information

1 Setting Up and Use of ELISA The main aim in the development or use of established ELISAs is to measure some reactant It is the need to measure substances that is the major reason for the assay ELISAs can be used in the pure and applied fields of science The chief reason they are worth developing is their high sample handling capacity, relative sensitivity, and ease of performance One more factor is their possible ease of reading, so that excess time is not wasted where a test has “gone wrong.” The ELBA can be roughly assessed by eye before machine reading, so time is not wasted in reading, for example, 1000 sample points before this insight is obtained (as in radioimmunoassay) Care must be taken not to discard successful tests merely because ELISAs are in fashion The relationship between ELISA results and other test system results also must be established, because a large amount of comparative work using ELISA and one or more assays might be involved in setting up the ELISA as a standard assay

2 What Is Already Known

A body of knowledge is often available on any problem faced by a worker This is to be found in the scientific literature, so survey is necessary This

99

100 Theoretical Considerations

would mainly involve the detailing of work concerning the biological agent (antigen) being examined and any work involved with its relationship to defined hosts in experimental (laboratory animals) and field studies The knowledge can be divided into (1) the biochemical/molecular bio- logical aspects of the agent, and (2) the immunological aspects (serology and immunology per se)

The literature may deal with the exact agent that the worker wishes to study or a similar agent, and also reveal whether ELISAs have been per- formed Obviously, the two aspects of biochemistry and serology are related via the host The main task at the beginning of any study is to define the aims properly The scientists should also examine published work with care, since often assays have been poorly devised and/or have been validated with little data Scientists should also seek advice from others in related fields and examine whether reagents have already been produced that might be applicable to their own problems A particular area is the use of monoclonal antibodies (MAbs)

An excellent catalog of immunological reagents, “The Linscott Cata- logue,” (4) is available, which lists and updates reagents and suppliers of thousands of polyclonal and monoclonal reagents of direct relevance to ELISA A great deal of information is also available in commercial firms” catalogs, which often have detailed technical descriptions of the use of their products

3 Complexity of Problems This complexity is manifested throughout the interrelationship between the agent and host The concept of the relationship between antigen- icity:immunogenicity and protection should be examined in this light

3.1 Antigenicity This the ability of proteins/carbohydrates to elicit the formation of anti- bodies that, by definition, bind specifically to the antigens used for injection into animals Antibodies may be produced as a consequence of replication of

an agent or by injection of inactivated whole or parts of that agent This can further be refined so that defined peptides or polypeptides are used The antigens used to elicit antibodies can, in turn, be used as in the ELBA

3.2 Immunogenicity This is a measure of the effect of binding of antibodies elicited by any

of the preparations mentioned above More specifically, the effect is one

Antigenic Considerations 101

of producing some degree of immunity against the disease agent Gener- ally, such measurements are made in vitro or in animal systems other than that being examined in the field

3.3 Protection Production of antibodies and demonstration of immunogenic responses

do not necessarily mean that animals will be protected against challenge

of the disease agent The relationship of immunoassay results to protec- tion is never straightforward owing to the many other factors involved in immunological responses, e.g., cellular immunity

4 Antigenic Considerations Inherent in the understanding of what is being measured in any ELISA are the definition of terms involving antigens and antibodies and an under- standing of the implications of the size, number of possible antigenic sites (epitopes), distribution of epitopes (distance between them), variability of epitopes, the effect on variation in epitopes on different assay systems, and

so forth Figure 16 in Chapter 2 shows some possible antigenic configura- tions, elements of which are reproduced below As the size (molecular weight) of disease agents increases, there is an increase in complexity

4.1 Size Considerations Immunoassays must be developed with as much knowledge as pos- sible of all previous studies As already stated, the complexity of agents generally increases with their size, On theoretical grounds, we can dem- onstrate the relationship of size to possible complexity by examining spherical agents of different diameters, beginning at 30 nm (approximate size of a foot-and-mouth disease virus [FMDV] particle)

One can calculate that the area bound by an Fab molecule (single-arm combining site of antibody without Fc) is 15 nm2 This represents an antigenic site (epitope)

Thus, the number of possible sites on the virus is the surface area of the virion divided by the surface area of the combining site The surface area of a sphere is 4x9, therefore 4 x 3.14 x 152/15 = 186 If the diam- eter of the agent is increased by twofold (60 nm), we have, using the same calculation, 942

These are small agents If we do the calculations for agents increasing

in diameter by lo-fold steps, we have for Fab and whole IgG (binding bivalently; effective area 60 nm2) data as shown in Table 1

102 Theoretical Considerations

Table 1 Relationship of Diameter of Agents

to Number of Binding Sites

Sites

300 18,600 5000

3000 1,860,000 500,000 30,000 186,000,000 50,000,000

These figures illustrate that the surface area increases with the square

of the diameter Such a calculation is based on the fact that the whole surface is antigenic (rarely true) and that the molecules bind maximally However, experimentally, relative figures close to these are obtained This has implications in immunoassays, since one can calculate how much antibody is needed to saturate any agent or measure the level of antibody attachment as a function of available surface

Since we know the molecular weight of IgG (and Fab), we can calcu- late the weight of a number of molecules Thus, mol wt IgG = 150,000 Daltons

Using Avagadro’s number (approx 6 x 1023) we have:

1 g IgG contains 6.0 x 1023/1S x lo5 mol= 4 x 1018

1 mg IgG has 4 x 101*/103 = 4 x 1Ol5 mol

1 pg IgG has 4 x 1018/106 = 4 x 1012 mol

1 ng IgG has 4 x 10**/109 = 4 x lo9 mol

1 pg IgG has 4 x 1018/1012 = 4 x lo6 mol

Such a model calculation helps to understand at the molecular level what one is dealing with when faced with different antigens

4.2 Definitions There may be some confusion concerning the terminology used in immunological and serological circles This section provides some work- ing definitions that will aid understanding of the mechanisms involved

in ELISAs

4.2.1 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

Antigenic Considerations 103

can be simple, e.g., peptides of mol wt around 5oo0, to complex See Fig 16

in Chapter 2 for a summary, Antibodies specific for the antigen are pro- duced Definition can be extended to molecules that evoke any specific immune response, including cell-mediated immunity or tolerance

4.2.2 Antigenic Site This 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 Epitope This is the same for antigenic site, but where a greater specificity of reaction has been defined, e.g., using tests involving MAbs where a single population of antibodies identifies a single chemical structure on an antigen

4.2.4 Epitype This 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 It can be regarded as identifying slightly different specificities of antibodies reacting with the same antigenic site

4.2.5 Continuous Epitope This is an epitope 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 Discontinuous Epitope This is an epitope 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 and Section 4.2.7.)

4.2.7 Linear Epitope This is the same as for the continuous epitope, recognition of atoms in

a linear sequence See Fig 1

4.2.8 Conformational Epitope This is an epitope formed through the interrelationship of chemical elements combining so that the three-dimensional structure determines the specificity and affinity Such epitopes are usually affected by dena- turation See Fig 2

104 Theoretical Considerations

External surfa

Internal surface +

Fig 1 Representation of a linear or continuous site Black area shows paratope of antibody with specificity for site

4.2.9 Antibody-Combining Site This 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 Paratope This is the part of the antibody molecule that binds to the epitope It is most relevant to MAbs where a single specificity for a single epitope can

be defined

4.2.11 Affinity and Avidity These relate to the closeness of fit of paratope and epitope Considered

in thermodynamic terms, they are the strength of close-range noncova- lent forces Mathematically, they are expressed as an association con- stant (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 because of 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 aver-

Antigenic Considerations

A

External surface

Fig 2 Representation of two types of conformational epitopes Black area shows paratope of antibody molecule with specificity for the sites

age binding energy from the sum of all the individual affinities of a popu- lation of antibodies binding to different antigenic sites

4.2.12 Polyclonal Antibodies This is the serum product of an immunized animal containing many different antibodies against the various mixture of antigens injected The antiserum is the product of many responding clones of cells and is usu- ally heterogeneous at all levels These levels include the antibodies’ specificity, classes and subclasses, titers, and affinities The response to

106 Theoretical Considerations

a,b,c,d o yw?-

Specific antibodies nga’mst differtut sites are mixtures of different antibodies with different affh&ies, classes and isotypes Resulting mixture of all antibodies

is a polyclonal antiserum

a,b,c,d II This represents a range of slightly different antibody populations

recognising the same site for antigenie sites x, y and z

a-

X ” + Single population of antibody molecules

with single afkity against epitope in antigenic site X

J’ ’ + Single population of antibody molecuks

Or with single affMty against epitope in

2+

antigenic site Y

Za + Single population of antibody molecules

with single affinity against epitope in antigenic site 2

Antigenic site A

Antigenic site B

0 Antigenic site C

Fig 3 Comparison of polyclonal (A) and monoclonal (B) antibodies 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 Monoclonal Antibody These are antibodies derived from single antibody-producing cells immortalized by fusion to a B-lymphocyte tumor cell line to form hybri- doma clones The secreted antibody is monospecific in nature and, thus, has a single affinity for a defined epitope See Fig 3

5 Other Techniques The performance of good immunoassays also requires practical exper- tise in immunochemical techniques (or at least theoretical knowledge of when such techniques are to be used) Below is a list illustrating some

Units 107

techniques of use in the purification and characterization of antigens and antibodies As already stated, an excellent manual for a whole variety of immunochemical techniques is available Techniques of use include: Sucrose density gradient centrifugation;

Polyacrylamide gel electrophoresis (PAGE);

PAGE-followed by immunoblotting;

Isoelectric focusing;

Immunodiffusion m agar/agarose;

Gel-chromatography (DEAE, affinity, Sephadex);

Salt fractionation of IgG; and

Enzyme conjugation methods

The nature and preparation of antibody fractions and their relevance in disease and assay should also be examined, i.e., whole molecule, Fc, Fab, Fab’2, IgM, IgA

6 Units Successful assays depend on a good knowledge of units of volume and weight The concepts of accuracy in dilutions and the relevance of pipet- ing methods also fall within the necessary practice needed for assays

6.1 Volumes The pipets used in microtiter plate assays are graduated in microliters (PL) The relationship of volumes is shown below:

Cubic Volume Symbol centimeter Microliters

6.2 Weights Note the relationship of weights:

Gram

Milligram

Microgram

Nanogram

Picogram

Femtogram

Attogram

Relationship

to gram

1 10-s 10”

10-g

1 O-12

1 O-15 lo-‘*

108 Theoretical Considerations

7 Making Dilutions Difficulties are often encountered in the making of dilutions An exam- ple of a “difficulty” is: make up a l/1200 dilution of a sample already at

a l/50 dilution in a final volume of 5.5 rnL

Often workers attempt to round-up volumes so that larger than neces- sary volumes are made, which is wasteful of reagents, e.g., conjugates Other problems, such as making a l/20,000 dilution in a final small volume, such as 3 mL, arise These problems are eased if all volumes in the calculation are converted to microliters (PL) A few worked examples will illustrate this

1 Make a l/100 dilution of neat sample at a final volume of 10 mL Neat infers a sample is undiluted Thus, a final volume of 10 mL is required Convert this to microliters: = 10 x 1000 pL = 10,000 p.L The dilution required is l/100; divide this into the required final volume = 10,000/100 This is the volume to be added of neat sample in microliters, which is 100

pL This, of course, would be conveniently calculated using milliliters, thus, 10 mL/lOO = 0.1 mL Thus, one would add 0.1 mL of the neat sample

to 9.9 mL of diluent (final volume minus the volume of the added neat sample) The conversion of the 0.1 mL in to the units of the micropipet would then have to be done, i.e., 0.1 mL = 100 p,L

A slightly more complex calculation illustrates the benefit of initial conversion to microliters of all the volumes

2 Make a l/200 dilution of a neat sample in a final volume of 4 mL Convert final volume to microliters: We have 4 mL = 4000 pL Dilution factor = 200; therefore, 4000/200 = 20 pL of neat A check on such calculations should always be made Thus, dilution factor x vol of sample being diluted should equal the required volume Therefore: 200 x 20 pL = 4000 FL (4.0 mL) Notice here that using milliliters, we would have 4/200 = 0.02 mL This become a little bit more difficult to relate to the microliter setting of the micropipets

3 Where a sample is already diluted, include this in the calculation, Thus, we have an already diluted sample at l/50 We require a final dilution of l/1000

in a final volume of 20 mL This type of calculation causes the most prob- lems Convert fmal volume to microliters Final volume required is 20 mL = 20,000 pL Dilution factor = 1000 Now assuming the sample were not already diluted, we would add 20,000/1000 = 20 p.L of neat sample Now, since the sample is already diluted l/50, we need to add more, The factor is determined by the known dilution factor (50), so multiply the neat value