CHAPTER 2 Basic Principles of ELISA

Basic Principles of ELISA I = Solid-phase to which reagent is attached passively S = Substrate addition and color development + = Addition of reagents and incubation Read = Measurement o

1 Substances, e.g., antibodies or antigens, may be passively adsorbed to solid surfaces, such as plastics Microtiter plates in a 96-well format are com- mercially available for use in ELISA, along with suitable equipment for easy manipulation and dispensing of reagents This allows use of small volumes and gives the ELISA the potential of handling high numbers of samples rapidly

2 Smce one of the reactants in the ELISA is attached to a solid-phase, the separation of bound and free reagents is easily made by simple washing procedures

3 The result of an ELISA is a color reaction that can be observed by eye and read rapidly using specially designed multichannel spectrophotometers This allows data to be stored and analyzed statistically

The use of passive adsorption also allows a great deal of flexibility in assay design This chapter describes some basic test schemes These assays are dealt with in later chapters at the practical level Diagrams of the schemes are also included to reinforce the principles ELISAs may be clas- sified under four headings: direct, indirect, sandwich, and competition

In the following, the various ELISAs are represented using letters, as well as diagrams

Ag = Antigen

Ab = Antibody directed against antigen

AB = Antibody from another animal species as compared to Ab

Anti-Ab = Species-specific antiserum, e.g., if Ab was raised in a mouse, anti-

Ab is anttmouse serum

*E = Enzyme attached to a particular antibody (Anti-Ab*E = antispecies antibody linked to enzyme, e.g., antimouse)

35

Basic Principles of ELISA

I = Solid-phase to which reagent is attached passively

S = Substrate addition and color development

+ = Addition of reagents and incubation

Read = Measurement of color using spectrophotometer

W = Separation of bound and free reagents by washing

Stage (iii): Addition and incubation of enzyme-labeled antibody

Stage (iv): Wash

Stage (v): Addition and incubation of color development system

Stage (vi): Read

Antigen attached to the solid phase is reacted directly with an enzyme- labeled antiserum This has the disadvantage that sera raised against dif- ferent antigens all have to be labeled Thus, this is a poor assay if used to detect antigen from “crude” samples (containing a high concentration of contaminating substances), since low levels of antigen attach to wells owing to competition for plastic sites by such contaminants This is a typical assay for use in the estimation of the titer of enzyme-labeled antispecies conjugates This is illustrated in Fig 1 Thus, the Ag in this case might be the IgG from an animal of the appropriate species against which the Ab*E is made

The interaction in this assay is made use of mainly in assays involving monoclonal antibodies (MAbs), which are directly labeled with enzyme

In this case, a very important epitope might be detected by the MAb, so that the labeling of a single antibody might be justified and form the basis of other assays as described below

Stage (i): Passive adsorption of antibody to plate

Stage (ii): Wash

Stage (iii): Addition and incubation of enzyme-labeled antigen

Direct ELISA 37

Fig 1 Direct ELISA Antigen is attached to the solid phase After washing, enzyme-labeled antibodies are added After an incubation period and washing, the substrate system is added and the color allowed to develop

Stage (iv): Wash

Stage (v): Addition and incubation of color development system

Stage (vi): Read

Antibodies are adsorbed to the solid-phase usually after crude frac- tionation to obtain the immunoglobulin fraction (IgG) Enzyme-labeled antigens are then added and react with antibodies of suitable specificity This has a poor applicability to diagnostic problems Antigens are rarely labeled However, a modification of this assay is used to measure proges- terone, using competitive conditions The scheme is illustrated in Fig 2

38 Basic Principles of ELISA

Fig 2 Direct-labeled antigen Antigen is labeled with enzyme and can be captured with antibodies attached to the solid phase This system can form the basis of other assays, e.g., competitive techniques where either antibody or anti- gen can be incubated with the labeled antigen and the degree of inhibition of binding of the labeled antigen with the solid phase measured This form of assay has been used in the estimation of hormone concentrations and is analo- gous to many radioimmunoassay methods

Stage (i): Passive adsorption of antigen

Stage (ii): Wash

Stage (iii): Addition of antibody directed against Ag

Stage (iv): Wash

Stage (v): Addition of enzyme-labeled antispecies antibody

Stage (vi): Wash

Sandwich ELISA 39

Stage (vii): Addition of color development system

Stage (viii): Read

This is extensively used for the detection and/or titration of specific antibodies from serum samples The specificity of the assay is directed

by the antigen on the solid-phase, which, may be highly purified and characterized or relatively crude and noncharacterized After addition and incubation of the antigen, the wells are washed to get rid of unbound antigen Serum containing antibodies against this antigen can then be added and diluted in a buffer that prevents the nonspecific adsorption of protein for any free sites on the solid-phase not occupied by the antigen (blocking buffer)

Sera, may be added as a single dilution (common in epidemiological testing of large number of sera) or as a dilution range After incubation, the wells are washed to get rid of unbound antibodies Bound antibody is then detected after incubation, with a single dilution of antispecies anti- body conjugated to an enzyme This is diluted in blocking buffer The amount of specific antibody binding to the antigen is quantified after addition of color development reagents (enzyme substrate or substrate/ dye combination) Such assays offer immediate advantages over the direct tests since only a single antispecies enzyme conjugate is needed to titrate antisera from many animals of a single species The scheme is shown diagrammatically in Fig 3

4 Sandwich ELISA 4.1 Direct Sandwich

Stage (i): Passive adsorption of antibody

Stage (ii): Wash

Stage (iii): Addition of antigen

Stage (iv): Wash

Stage (v): Addition of enzyme-labeled antIbody* against antigen

Stage (vi): Wash

Stage (vii): Addition of color development system

Stage (viii): Read

Enzyme-labeled antibody can be produced in the same animal (can be the same serum) that produced the passively adsorbed antibody, or from

a different species immunized with the same antigen that is captured

40 Basic Principles of ELISA

Fig 3 Indirect ELISA Antibodies from a particular species react with anti- gen attached to the solid phase Any bound antibodies are detected by the addi- tion of an antispecies antiserum labeled with enzyme This is a widely used system in diagnosis

This is similar to Section 2.2., except that antibody is attached to the solid-phase, usually as an IgG fraction of the whole serum A constant dilution of antibody is attached to the solid phase, and after incubation, unadsorped antibody is washed away Antigen at a single dilution or as a dilution range is then added, in a buffer that prevents nonspecific bind-

Sandwich ELBA

ing of the serum proteins to any available plastic sites After incubation, unbound antigen is washed away Bound antigen is then detected by the addition of enzyme-labeled antibody specific for the “trapped” or “cap- tured” antigen

This antibody can be:

1 The same as that used on the solid phase

2 Produced in the same species as the trapping serum

3 Produced in a different species to that in which the trapping antibody was made After incubation and washing away of unreacted conjugate, the color detection system is added and color is measured Note that certain anti- gen preparations cannot be directly attached to microplates, since they are at low concentration and/or they are contained in high concentrations

of contaminating protein The antibody attached to the plastic should bind antigen specifically, so that selection and concentration of the anti- gen takes place in the sandwich conditions Such assays have been described

as capture or trapping assays, referring to the property of the bound anti- body to bind the antigen to the plastic surface

Note also that antigens must contain at least two antigenic sites, capa- ble of binding to antibody, since at least two antibodies act in the sandwich This is important where lowerTmo1 wt antigens are being used of limited antigenic potential, or where antigenic sites are concentrated on one sur- face Some difficulties can be encountered using the same MAb in sand- wich assays, since the capture step may bind to the only epitope expressed

on a “small” antigen As in the direct ELISA, this test has the disadvan- tage that all the detecting antisera have to be conjugated Figure 4 shows the scheme diagrammatically

4.2 Indirect Sandwich

Stage (i): Passive adsorption of Ab

Stage (ii): Wash

Stage (iii): Addition of antigen

Stage (iv): Wash

Stage (v) Addition of antibody from different species vs antigen

Stage (vi): Wash

Stage (vii): Addition of enzyme-labeled antispecies (directed against AB)

42 Basic Principles of ELISA

Fig 4 Sandwich ELISA-direct This system exploits the antibodies attached to the solid phase to capture antigen This is then detected using an enzyme-labeled serum specific for the antigen The detecting antibody is labeled with enzyme The capture antibody and the detecting antibody can be the same serum or from different sources The antigen must have at least two different antigenic sites

Stage (viii): Wash

Stage (ix): Addition of color development system

Stage (x): Read

Figure 5 shows the scheme diagrammatically

This is the same as Section 4 l., except that the second antibody is produced in a different species from the trapping antibody Thus, the second antibody can be detected using a species-specific antiserum con-

Competition ELISA 43

Fig 5 Sandwich ELBA-indirect The detecting-antibody is from a different species than the capture antibody The antispecies enzyme-labeled antibody binds to the detecting antibody specifically and not to the capture antibody

jugate that does not react with the antibody on the plastic The advantage

is that many second antibodies, may be titrated with a single conjugate

5 Competition ELISA Competition assays imply that two reactants are trying to bind to a third Proper competition assays involve the simultaneous addition of the two competitors

44 Basic Principles of ELISA

Fig 6 Comparison of competition and inhibition ELISA The test scheme involves the reaction of two antibodies with an antigen attached to the solid phase Where one of the antibodies is incubated first, the assays are called block- ing or inhibition assays Competition implies simultaneous addition of reagents

Inhibition or blocking assays are similar, except that one of the reag- ents being examined for “competing” ability of a system is added and incubated before the second “competitor” is added This can be illus- trated simply as shown in Fig 6

5.1 Direct Antibody Competition

+AB

Competition ELISA 45

Stage (i): Passive adsorption of antigen

Stage (ii): Wash

Stage (iii): Addition of competing antibody at various dilutions

Stage (iv): (Optional washing step after incubation with AB alone)

Stage (v): Addition of enzyme-labeled antibody (pretitrated to give optimal color development)

Stage (vi): Wash

Stage (vii): Addition of color development system

Stage (viii): Read

Competition assays are defined as assays where the detecting (pre- titrated labeled) antibodies are added with the sample simultaneously When the sample is added for a period of incubation before the pretitrated antibodies, a blocking assay or inhibition assay results

The blocking assay can involve incubation of the test sample followed

by washing and then addition of the labeled antibodies or their addition without washing away nonbound antibodies from the sample

Direct antibody competition involves the adsorption of antigen to the solid phase After washing away unadsorbed antigen, a specific antibody labeled with enzyme is added This is pretitrated so that the antigen is saturated, and no free antigenic sites are available for further antibody combination

This interaction of antigen and pretitrated antibody (identical to the direct ELBA) is upset if the labeled antibody is mixed with another anti- body (competing antibody) that is able to react with the solid-phase bound antigen The competing antibody is added as a dilution range Thus, this replaces all or some of the pretitrated conjugated antibody

After washing and development of the assay, the replacement is observed

as a decrease in the color expeated (that found in control wells contain- ing no competing serum) Such assays are of increasing importance par- ticularly where MAbs are used Note that any serum from any species can be used as competitor The scheme is illustrated in Fig 7

+ Ag as dilution range Stage (i): Passive adsorption of antigen,

Stage (ii): Wash

46 Basic Principles of ELISA

Pre-titration of antigen and labelled antibodies

Competition-addition of serum containing antibodies

I common to pretitrated conjugate

Pre-titrated enzyme-labelled antibodies are blocked by the test antibodies reacting with common antigenic sites

Where all sites are blocked there

is no colour development since

no enzyme-labelled antibodies are attached to antigen

Fig 7 Competition ELISA-direct antibody The degree of inhibition by binding of antibodies contained in a serum for a pretitrated enzyme-labeled antiserum reaction is determined

Stage (iii): Simultaneous incubation of free antigen with enzyme-labeled antibody (pretitrated): directed against antigen on plastic

Stage (iv): Wash

Stage (v): Addition of color development system

Stage (vi): Read

Competition ELISA 47

Pre-titration of labelled antibodies and antigen

Competition with sample possibly containing same antigen(s)

Addition of antigen with same

antigenic sites as antigen on

Fig 8 Competition ELBA-direct antigen Reaction of antigen contained in samples with the enzyme-labeled antibody directed against the antigen on the solid phase blocks its binding to the solid phase If the antigen has no crossreactivity with the solid-phase antigen, then the labeled antibody binds, and a color reaction is observed

This is as described in Section 5 l., except that the competing sub- stance for the pretitrated conjugated antibody is antigen If the labeled antibody reacts with the dilution range of added antigen (competitor) in the liquid phase, it is washed away after the incubation step Thus, labeled antibody is unavailable to react with the solid-phase antigen, and a reduc- tion in expected color is observed Such assays can be used to quantify antigens or to compare the relative affinity of binding of two antigens for the same serum The scheme is illustrated in Fig 8

48 Basic Principles of ELISA

+AB Stage (i): Passive adsorption of antigen

Stage (ii): Wash

Stage (iii): Addition of test antibody AB at various dilutions

Stage (iv)*: (Optional washing step after incubation with AB alone)

Stage (v): Addition of antibody (pretitrated): standard serum

Stage (vi): Wash

Stage (vii): Addition of antispectes conjugate against standard antiserum (Ab) Stage (viii): Wash

Stage (ix): Addition of color development system

Stage (x): Read

This is essentially the same as the indirect ELISA, except that a com- peting antibody is added to the solid-phase antigen either before or simul- taneously with pretitrated specific antibody

The level of antibody used is usually about 70% maximal reactivity (solid-phase antigen excess) The competing antibody must be from a different species from the pretitrated antibody, since the antispecies con- jugate must not react with both If the competing antibody is able to bind

to the antigen, then it prevents the pretitrated antibody reacting, and this

is observed as a decrease in the expected color as compared to controls without competitor The scheme is illustrated in Fig 9

+Ai3 Stage (i): Passive adsorption of antigen

Stage (ii): Wash

Stage (iii): Simultaneous incubation of free antigen (test sample): with anti- body directed against antigen on plastic at pretitrated dilution

Stage (iv): Wash

Stage (v): Addition of enzyme-labeled antibody against Ab

Stage (vi): Wash

Stage (vii): Addition of substrate

Stage (viii): Read