CHAPTER 2 Basic Principles of ELISA

CHAPTER2

Basic Principles

of ELISA

1. Reaction
Schemes
This chapter introduces the basic test formats for the performance of
solid-phase heterogeneous ELISA. This form of ELISA has been so useful because:
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 commercially 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 classified 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, antiAb 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

2. Direct

ELISA

2.1. Direct-Labeled

I-Ag

+

Ab*E

W

+

Antibody

S + Read

W

Stage (i): Passive adsorption of antigen to plate by incubation in defined buffer.
Stage (ii): Wash.
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 enzymelabeled antiserum. This has the disadvantage that sera raised against different 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.
2.2. Direct-Labeled
I-Ab

+
W

Ag*E

+

Antigen

S + Read

W

Stage (i): Passive adsorption of antibody to plate.
Stage (ii): Wash.

Stage(iii): Addition and incubation of enzyme-labeledantigen.


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 fractionation 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 progesterone, 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 antigen 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 analogous to many radioimmunoassay methods.
3. Indirect
I-Ag

ELISA

+
Ab
+ Anti-Ab*E + S + Read
W
W
W

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 developmentsystem.
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 antibody 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 assaysoffer immediate advantagesover 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
I-Ab

+

W

Ag

W

+

AB*E

+

S + Read

W

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 speciesreact with antigen attached to the solid phase. Any bound antibodies are detected by the addition 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 “captured” 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 speciesto 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 antigen takes place in the sandwich conditions. Such assayshave been described

as capture or trapping assays,referring to the property of the bound antibody to bind the antigen to the plastic surface.
Note also that antigens must contain at least two antigenic sites, capable 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 surface. Some difficulties can be encountered using the same MAb in sandwich 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 disadvantage that all the detecting antisera have to be conjugated. Figure 4 shows

the scheme diagrammatically.
4.2. Indirect
I-Ab

+
w

Ag

+
w

AB

Sandwich
+

w

Anti- AB *E +
W

S + Read

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 systemexploits the antibodies attachedto
the solid phase to capture antigen. This is then detectedusing 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 adifferent
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

Competition

A
Incubate

Inhibition/Blocking

B

Wash

+

Incubate

Incubate

C
Incubate

Incubate

No
wash

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 assaysare called blocking or inhibition assays.Competition implies simultaneous addition of reagents.

Inhibition or blocking assays are similar, except that one of the reagents being examined for “competing” ability of a system is added and
incubated before the second “competitor” is added. This can be illustrated simply as shown in Fig. 6.
5.1. Direct
I-Ag

+ Ab*E

+
W

W
+AB

Antibody
S + Read

Competition


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.

assays are defined as assays where the detecting (pretitrated labeled) antibodies are added with the sample simultaneously.
Competition

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 antibody (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 containing no competing serum). Such assays are of increasing importance particularly where MAbs are used. Note that any serum from any species
can be used as competitor. The scheme is illustrated in Fig. 7.
5.2. Direct
I-Ag

Antigen

+ Ab*E
W

+ S + Read
W
+ Ag as dilution range

Stage (i): Passive adsorption of antigen,
Stage (ii): Wash.

Competition


46

Basic Principles
Pre-titration

I

of antigen

and labelled

of ELISA

antibodies

Competition-addition
of serum containing
common to pretitrated
conjugate

antibodies

Addition of samples with
antibodies in common with
pre-titrated labelled serum
will bind to same antigenic
sites
E

>
c
>

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

E 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

47

ELISA
Pre-titration

Competition

of labelled

antibodies

with sample possibly

Addition
of antigen with same
antigenic sites as antigen on
solid phase

and antigen

containing

same antigen(s)

Addition
of antigen with no common
antigen sites with solid phase antigen

No colour
4
100°AOCompetition
i-00
“2 ’o@00000@

0%

Competition
0

0

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 substance 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 reduction 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.


Basic Principles

48
5.3. Indirect
I-Ag

+

Ab

+

Antibody

Competition

Anti-Ab*E

w
+AB

w

of ELISA

+ S + Read
w

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 antispectesconjugate 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 competing antibody is added to the solid-phase antigen either before or simultaneously 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 conjugate 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.
5.4. Indirect
I-Ag

+
W

Ab

+
W

Antigen

Competition

Anti-Ab*E

+ S + Read
W

+Ai3
Stage (i): Passive adsorption of antigen.
Stage (ii): Wash.
Stage (iii): Simultaneous incubation of free antigen (test sample): with antibody 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.


Competition

ELBA

49
Pre-titration

Competition-addition

of indirect system

of samples containing antibodies?

Serum contains antibodies
which bind to antigen &,

Serum contains NO antibodies
which bind to antigen

These block pre-titrated
antibodies binding

4
On addition of anti-species enzyme labelled conjugate
A

Conjugate does not bind

No color
COMPETITION

Conjugate binds

Color
NO COMPETITION

Fig. 9. Competition ELISA-indirect antibody. A pretitrated system for antibody binding to antigen is challenged by the addition of another serum (test)
sample. If antibodies bind to the sites in common with the pretitrated antibodies, they block (if added before pretitrated antibody) or compete with (if added
simultaneously) this reaction. Since an antispecies conjugate is used, the competing sample serum cannot be from the same species.
This is an indirect ELISA where antibody is pretitrated against the solidphase bound antigen by the use of antispecies conjugate, which is challenged by
the addition of dilution ranges of antigen in the liquid phase. Again, the amount


Basic Principles

50
Petitration

Addition of same or similar
to that on solid phase

of indirect

antigen

of ELISA

system

Addition of antigen
on solid phase

different

to that

Wash and add conjugate

Add substrate

No color-100%

competition

Fig. 10. Competition ELBA-indirect antigen. The pretitrated indirect ELISA
is competed for by antigen. If the antigen shares antigenic determinants with
that of the solid-phase antigen, it binds to the pretitrated antibodies preventing
them from reacting with the solid-phase antigen. If there is no similarity, the
antibodies are not bound and can react with the solid-phase antigen. Addition
of the antispecies enzyme conjugate quantifies the bound antibodies.
of pretitrated antibody should be about 70% of the maximal reaction (solidphase antigen excess). Competition is reflected by a decrease in the expected
color obtained without competitor. The scheme is illustrated in Fig. 10.
6. Choice of Assays
The most difficult question to answer when initiating the use of
ELISAs is which system is most appropriate. This section will attempt to


51

Choice of Assays

C

D

Fig. 11. Basic ELISA methods. (A) Direct, (B) Indirect, (C) Sandwich (direct),
(D) Sandwich (indirect)
investigate the relationships between the various systems to aid in assessing their suitability. Questions that must be addressed are:
1. What is the purpose of the assay?
2. What reagents do I have?
3. What do I know about the reagents?
4. Is the test to be developed for a research purpose to be used by me only, or
for applied use by other workers?
5. Is the test to be used in other laboratories?
6. Is a kit required?
These questions have a direct affect on the three phases that might be
put forward as a general rule for the development of any assay, i.e.:
1. Feasibility-proof that a test system(s) can work.
2. Validation-showing that test(s) is “stable” and that it is evaluated over
time and under different conditions.
3. Standardization-quality control, establishment that the test is precise and
can be used by different workers in different laboratories.
Figures 11-15 summarize the exploitation of ELISA methods, highlighting the relationship of assays to the relative purity and concentration
of reactants, and indicating the use of direct and indirect methods for
competition assays. These will be relevant when examining the possibilities outlined in Section 6.1.


Basic Principles

52

of ELISA

*ENZ
t

A

+ENZ
t
s-

B

Fig. 12. Competition for direct ELISA.
6.1. Phase

1 in Developing

an ELISA

Feasibility

Phase 1 involves the trial of various systems of ELISA with existing
and newly prepared reagents to be able to obtain the desired aim. This
phase includes identification of needs based on preliminary experiments
and a good knowledge of the biology of the system. The latter point may
become more important when attempts at using ELISA fail because of
lack of knowledge. Thus, as an example, we may wish to estimate the
antibody titer in cattle sera against a particular antigen. The possibility of performing all the ELBA systems and obtaining the most appropriate system will depend on the availability of various reagents and their
specificities.

Choice of Assays

53

INDIRECT

A

Fig. 13. Competition for indirect ELBA.
6.1.1. Assessing What Is Available
As examples: We may have only the relevant antigen. Figure 16 shows
different types of antigens of ,varying complexity. Thus, we may know a
great deal about the antigen or very little. We may have a high concentration of a defined protein/polypeptide/peptide of known amino acid sequence
or have a thick soup of mixed proteins containing the antigen at a low
concentration contaminated with “host cell” proteins. We may have an
antiserum against the antigen. This could be against purified antigen or
against the crude soup. The antibody may have been raised in a given
species, e.g., rabbit. We may have an IgG fraction of the antiserum (or


54

Basic Principles

of ELISA

AG COMPETITION

AB COMPETITION

+

0

+#

+
YI
I, 2 d 3 ALTRRNATNE

Yf

Y
llbii?S

+o

FOR ADDI’KION

Fig. 14. Competition for sandwich ELISA-same
detection.

OF COMPETITORS

antibody for capture and

could easily make one). We may have field sera against the antigen (bovine
sera). We may have an MAb. We may have antisera from different species, e.g., rabbit and guinea pig sera. ELISAs for similar systems may

have been developed and can be found in the literature.
We will require an enzymic reaction in the assay. Thus we will need
an antispecies conjugate (commercial, most probably) or will have to
label an antigen-specific serum with enzyme (facilities to do this?). We
have to decide which commercial conjugate to buy. This will depend on
the desired specificity of the conjugate (antiwhole molecule IgG, anti-Hchain IgG, anti-H chain IgM, and so forth). The choice is somewhat determined by the aims of the assay and its design. Thus, we may wish to
determine the IgM response of cattle to our antigen, which will require
an anti-IgM (specific) somewhere in the ELISA protocol. Obviously the
basic needs for performing the ELISA must be addressed in terms of
plates, pipets, buffers, reader, and so on.


55

Choice of Assays
I
&3ENZ
+s-+

*ENZ

AG COMPETITION

I,2 & 3 ALTERNATIVE

TIIUES FORADDITION

OF COMPET1mR.S

J

AB COMPETITION

Fig. 15. Competition for sandwich ELBA-different
and detection.

antibody for capture

6.1.2. Examination
of Possible Assays with Available Materials
Obviously, the reagents available must be examined first as previously
stated. This section will deal with some extremes in order to illustrate the
relationship of the assays available and their particular advantages. As
for Section 6.1.1.) perhaps we have to examine the level of antibodies in
bovine serum. Some scenarios are described with different available
reagents. These will probably cover most of those that are met in practice. This assumes that there are sera to test from infected and noninfected
animals. Further subtleties can be examined by defining the specificities
of the conjugates (anti-IgG, IgM, or whether they are H-chain-specific).
The increase in choice of reagents and the possibilities for performing
different ELBA configurations follow:
1. a. Crude antigen (multiple antigenic sites)
b. Antibody raised against crude antigen in rabbits
c. Anticow conjugate


Basic Principles

56

Large multivalent antigen,

sequential and conformational

Smaller multivalent

of ELISA

epitopes

antigen

Smaller antigen, univalent.
Polypeptide, linear sequential epitopes.
Polypeptide, sequential and conformational

epitope.

Peptide.
Peptide, linked to carrier protein with
conformational epitope.

Fig. 16. Different forms of antigens for use in ELBA and for antiserum production. These antigens could be contaminated with “host cell” proteins.
d. Postinfected and d 0 (uninfected) cow sera
2. a. Purified antigen (small amount, e.g., 100 pg)
b. Crude antigen (large amount)
c. Antibody raised in rabbits against pure antigen
d. Antirabbit conjugate
e. Anticow conjugate
f. Postinfected and d 0 (uninfected) cow sera
3. a. Crude antigen (as in 1.)
b. Antibody against pure antigen (rabbit)

c. Antibody against pure antigen (guinea pig)
d. Antiguinea pig conjugate
e. Postinfected and d 0 (uninfected) cow sera
f. Anticow conjugate
g. Antirabbit conjugate.


Choice of Assays

57

6.1.2.1. SITUATION 1
Here the use of crude antigen directly on an ELISA may well be unsuccessful, since it may be at a low concentration compared to other proteins and thus only attach at a low concentration. This does not allow the
ELISA approaches as shown in Fig. 1 lA,B and, thus, competitive methods based on these, as in Figs. 12B and 13B.
Since a rabbit serum against the antigen is available, this may be used
as a capture serum (or a capture IgG preparation) coated on wells to capture the crude antigen to give a higher concentration of antigen to allow the
binding of antibody as in Fig. 1lC,D. This also allows competitive techniques as shown in Fig. 15B.
The bound antibody would be from cows and would be detected using
the antibovine conjugate. There may be problems, since the crude antigen was used to raise the rabbit serum. Thus, antibodies against the contaminating proteins may be produced in the rabbit. The cow sera being
tested may react with such captured contaminants. However, where the
antigen is an infectious agent, antibodies against the contaminating proteins may not be produced, thus eliminating the problem. Where the antigen is used as a vaccine, whereby relatively crude preparations similar to
the crude antigen are used to formulate the vaccine, then this problem
will be present. Attempts can be made to make the rabbit serum specific
for the desired antigenic target. Solid-phase immunosorbents involving the
contaminating crude elements (minus the desired antigen) can be used to
remove the anticrude antibodies from the rabbit serum, which could then be
titrated as a capture serum. An example can be taken from the titration of
foot-and-mouth disease virus (FMDV) antibodies. The virus is grown in
tissue culture containing bovine serum. Even when virus is purified from
such a preparation, minute amounts of bovine serum contaminate the

virus. When this “purified” virus is injected into laboratory animals as an
inactivated preparation, there is a large amount of antibovine antibodies,
as well as antivirus antibodiesproduced.This serum cannot be used in a capture system for specifically detecting virus grown as a tissue-culture sample
(containing bovine serum), since it also capturesbovine serum. The capture
serum is also unsuitable for capturing relatively pure virus for the titration of bovine antibodies from bovine serum samples, since the capture
antibodies react strongly with the detecting cow serum. Thus, the capture serum has to be adsorbed with solid-phase immunosorbents, e.g.,
those produced through the attachment of bovine serum to agarosebeads,


Basic Principles

58

of ELISA

Solid Phase

Coating with Guinea pig IgG

1

Wash

Addition

1

of anti-guinea

pig IgG conjugate

1
Wash

Addition

1

of substrate/chromogen

Scheme 1.

Once the specificity of the capture serum is established, the optimization of the crude antigen concentration can be made using a known or
several known positive cow sera using full dilution ranges. Inclusion of
dilution ranges of negative sera allows an assessment of the difference
between negative and positive sera at different dilutions of serum. The
diagram below illustrates the use of the reagents to set up a sandwich
ELISA. The assay is made possible through the specific capture of
enough antigen by the solid-phase rabbit serum as in Fig. 11D.
I-Ab + Ag + AB + Anti-AB*Enz
Rabbit Crude Cow Anticow

+ Substrate + Read

6.1.2.2. SITUATION 2
This situation is not very different from the first. However, we have
more reagents! We have the antigen purified and used to raise antibodies
in rabbits (see Scheme 1). Thus, with due reference to the reservations

Choice of Assays

59

already described in Section 6.1.2. l., we have the basis of setting up a
capture ELISA, since the rabbit antibodies may capture the antigen at a
high concentration from the crude antigen, which we have in a large
amount. The development of the capture ELISA as shown in Fig. 1 lC,D
is as described above.
The availability of the antirabbit conjugate may allow development of
competitive assays if enough specific antigen binds to plates, although
this is unlikely, as indicated above. The antigen and rabbit serum could
be titrated in an indirect ELISA (Fig. 11B) in a chessboard fashion, enabling the optimization of the antigen and serum. These optimal dilutions
could be used to set up competitive ELISAs, whereby cow sera would be
competed for the pretitrated antigen/rabbit/antirabbit conjugate system,
as in Fig. 13B. Again it must be emphasized that this is unlikely since the
antigen is crude and some form of capture system will be needed to allow
enough antigen to be presented on the wells.
Since this situation has some purified antigen, this could be used in the
development of a similar competitive assay. This will depend on the
availability of this antigen, which can be determined after the initial
chessboard titrations where the optimal dilution of antigen is calculated,
The chief benefit of obtaining purified antigen was to obtain a specific
serum in rabbits, allowing specific capture of antigen from the crude
sample. In many cases, there is enough antigen of sufficient purity to be
used in such assays. Another alternative, as shown in Fig. 14B, is available if the rabbit serum can be conjugated with enzyme.
6.1.2.3. SITUATION 3
Here we have all the possibilities of the first two situations plus the
production of a second species (guinea pig) serum against the purified
antigen (seeScheme2). This allows the development of competitive assays

as in Fig. 15B using either the rabbit or guinea pig as capture serum or
detector with the relevant antispecies conjugate. Different species may
have better properties in acting as capture reagents and also show varying specificities. This can be assessed in chessboard titrations. This is
relevant since we require results on the detection and titration of cattle
sera so that the competitive phase relies on the interruption of a pretitrated
antibody as close to the reaction of cattle serum with antigen as possible.
The rabbit or guinea pig serum may differ in their specificities as compared to cattle sera.