PART 11

Practical Exercises
The aim of Chapters 5-8 will be to illustrate the principles of ELISA
fully by:
1. Showing worked examplesof eachassay,including diagramsof platesand
representationaldata from assays;
2. Analyzing such data in terms of important rules that are learnedat each
stage;and
3. Providing full working instructronsfor workers to be ableto perform each
assayso that they obtain their own data to be analyzedas describedin (1)
and (2). This includes full instructionson the preparationand standardiza-
tion of reagents.
The chapters can therefore be used in several ways. Workers without
accessto reagentswill obtain a working knowledge of ELISA through the
examples. The chapterscan also be used in training courses where reagents
may be provided (as indicated in the text). The information will also be
useful to workers who have already had some experience of the tech-
nique and who may have had difficulties in obtaining and analyzing data.
Remember that it is the application of the ELISA to specific problems,
and not the methodology for its own sake, that is the most important
reason the techniques should be mastered.
1. Test Schemes
You may be already familiar with the concepts in ELISA, whereby an
antigen binds to an antibody that can be labeled with an enzyme, or be in
turn detected with a species-specific antibody (enzyme labeled). All the
ELISAs are variations on this theme. Inherent in the methods of ELISA
described in these chapters is the fact that one of the reagents is attached
to a solid-phase, making the separation of bound (reacted) and unbound
(nonreacted) reagents simple by washing. Before performing ELISA on
disease agents, it is useful to train using reagents of defined reactivity,
which are easily available and which provide security problems. An ideal
115
116 Practical Exercises

system is to use an imrnunoglobulin (Ig) and more particularly an immu-

noglobulin G (IgG) as an antigen. Do not get confused here, since you
have learned that the antibody population contains high levels of IgG
acting as antibody. In the context of learning the principles, we are using
IgG as an antigenic protein, since:
1. IgG from one animal species can be injected into another animal species so
that a specific antiserum to that IgG is prepared.
2. Such antibodies can be labeled with enzyme, or detected with a second
species-specific antibody labeled with enzyme.
Such reagents are defined, easy to standardize, stable, and available
commercially. The particular IgG system chosen in most of the chapters
involves guinea pig, but similar tests can be performed with other spe-
cies IgG using the appropriate antispecies reagents. The practical ele-
ments of all the assays are very similar, i.e., reagents and equipment
needed. The systems described are analogous to the ones most commonly
used to examine problems associated with diagnosis.
The schemes will be described using symbols where:
I- = solid phase microtiter plate well
Ag = antigen
Agl, Ag2, etc. = particular antigens highlighted in assay
I-Ag = antigen passively adsorbed onto wells
I-Ab, I-AB = particular antibodies passively coated onto wells
Ab = antibody
AB = antibody from a different species to Ab
Abx, Aby = different antibodies identified by subscript letters
Anti-Ab = antispecies specific antibody (against species in which Ab was
produced)
Anti-Ab*E = antispecies specific antibody labeled with enzyme
W = washing step, involving separation of bound and free reagents
+ = addition of reagents and incubation step
S = substratekhromophore addition
Read = read test in spectrophotometer at 492 nm
Throughout Chapters 5-8, many of the practical stages are the same.
The conjugates described are all made with horseradish peroxidase and
the substratekhromophore is hydrogen peroxide/orthophenylamine diamine
(OPD). The preparation and use of this is described in detail below.
1. Substratekhromophore: This is easiest made up from commercial tablets
of OPD that are preweighed. Commercial sources also supply citrate/phos-
Test Schemes 117

phate buffer tablets (pH 5.0). Thus, the volume of OPD can be made as
required by following the recommendations by the supplier. As an example,
30 mg tablets are available that make 75 mL of chromophore solution in
buffer. Unused OPD solution (without added hydrogen peroxide) can be
frozen at -20°C. This can then be thawed and used later. Close inspection
should be made to ensure that the OPD is not drscolored. Use complete
chromophore/substrate as soon as possible.
Larger volumes of OPD in citrate/phosphate buffer can be made and
frozen in a tightly stoppered brown bottle in small volumes. The OPD
solution should be made and frozen as quickly as possible. Do not use
solutions that show discoloration after freezing.
Hydrogen peroxide (HzO,) is the substrate for horseradish peroxidase
enzyme. This is purchased usually as 30 or 6% w/v and should be stored as
recommended by the supplier. The hydrogen peroxide should be kept
refrigerated and not subjected to heating. The addition of the hydrogen
peroxide should be made immediately before the use of the OPD in the
test. Add 5 PL of hydrogen peroxide (30% w/v) to every 10 mL of OPD
solution (pH 5.0), or 25 pL of 6% hydrogen peroxide to every 10 mL of
OPD solution. Use the substrate/chromophore immediately. OPD is a
mutagen, so care is needed in its handling and disposal.
2. Washing solution used in washing steps: This is PBS without the addition
of Tween 20. Washing requires the flooding and emptying of wells 4 times
with PBS.
3. Blocking buffer: This is PBS containing a final concentration of 1% bovine
serum albumin (BSA) and 0.05% Tween 20. This should be made in small
volumes as required, but can be stored at 4°C. Care should be taken to
avoid contaminated buffer.
4. Stopping solution: This 1M sulfuric acid in water. Care should be taken in
its preparation and handling.
5. Read: This implies reading plates using a multichannel spectrophotometer
at the appropriate wavelength for the color developing in the ELISA. In all
cases for Chapters 5-8, this is 492 nm for OPD. Plates should also be
assessedby eye to ascertain whether the test results are as expected.
 CHAPTER8

Competitive ELISA
1. General Information
The direct, indirect, and capture ELISAs have now been examined.
You should be able to optimize the conditions of the tests and be able to
use them to measure antigen or antibody in a variety of formats. Com-
petitive ELISAs involve the principles of all these types of assay.
Basically they involve methods that measure the inhibition of a reac-
tant for a pretitrated system. The degree of inhibition reflects the activity
of the unknown. We can, therefore, measure antibody or antigen, and
even begin to subtly compare small differences in the binding of anti-
gens or antibodies so that antigenic subtyping may be performed by com-
paring the relative avidity of one antiserum for two antigens in the same
system. As a reminder, let us consider the competitive assays based on
the indirect and the capture ELISAs for the detection of antigens or anti-
bodies in a diagrammatic way. The symbols used are:
I = Solid phase microtlter plate.
I-Ag = Antigen attached to solid phase by passive adsorption.
Ab = Antibody against Ag.
AB = Antibody produced m a different species to Ab.
Anti-Ab or anti-AB = Antispecies serum against particular Ab or AB,
Anti-Ab*E or Anti-AB*E = Antispecies Ab or AB conjugated serum.
W = Washing step.
S = Substrate/chromophore.
+ = Addition of reagents and incubation.
1.1. Indirect Assay-Antigen Detection by Competition
I-Agl +AB + Anti-AB*E + S +Read
+ Ag2
W W W
Here a pretitrated indirect assay with optimal Agl, AB, and conju-
gated anti-AB is competed for by Ag2, as a dilution range, in the liquid

177
 Competitive EiX3A

phase. If Ag2 can bind AB, then this prevents Al3 binding, which would
normally react with Agl on the plate. The maximum expected OD for
the pretitrated system without competitor is therefore reduced in the pres-
ence of the competitor Ag2. The degree of inhibition of the pretitrated
reaction is proportional to the relative amount of the competitor.
1.2. Indirect Assay-Antibody Detection by Competition
I-Ag +AB + AntI-AB*E + S + Read
+Ab
W W W
Here a pretitrated system is challenged by a dilution range of Ab. The
competing antibody has to be from a species that is not the same as that
of the AB in the optimized system. The degree of inhibition of the
pretitrated system depends on the concentration and interaction of the
Ab competitor with the Ag, this time on the solid-phase.
The direct ELISA could also be used for both systems 1.1 and 1.2. Note
that in the direct assayany speciesof competing antibody can be used since
the AB is labeled with conjugate. Such assaysare becoming increasingly
relevant where monoclonal antibodies (MAbs) are being used.
1.3. Capture Assay-Antigen Detection by Competition
I-AB-Agl +Ab + Anti-Ab*E + S + Read
+ Ag2
W W W
Here the capture assay is optimized to detect the Agl trapped on the
plates using Ab. The competition is achieved where Ag2 is mixed with
the Ab in the liquid phase. If this reacts, the amount of Ab available for
reaction with the trapped Agl is reduced.
1.4. Capture Assay-Antibody Detection by Competition
I-AB +Ag + Abx + Anti-Abx*E + S + Read
+ Aby
W W W W
Here the capture antibody is optimized to bind Ag, which is detected
using a constant amount of Abx (from animal speciesX). The competition
involves the reaction of the Ag with antiserafrom species Y, which should
not interact with the conjugate Anti-Abx in the liquid-phase. Remaining
Ag after the competition phase is then captured and titrated by the Abx
Direct Competitive ELISA

and the conjugate. Reduction in the expected color for the system with-
out any AbY represents competition.
Three assays will be dealt with practically.
1. Direct assay-antigen detection;
2. Indirect assay-antigen detection; and
3. Indirect assay-antibody detection: (a) full titration curves and (b) spot
test assessmentof sera.
2. Direct Competitive ELISA
for Antigen Detection and Quantification
This has assumed an increased importance with the development of
MAbs. A single MAb can be the one reagent that dominates a diagnostic
assay and therefore is worth labeling for use in an assay. The specificity
of the assay is ensured and relatively crude antigenic preparations can be
coated for use in a direct test format (providing enough antigen attaches).
This is also relevant to polyclonal antibodies. The demonstrated assays
involve IgG/anti-IgG systems.
2.1. Learning Principles
1. Optimization of homologous system.
2. Competition curves.
2.2. Reaction Scheme
I-Agl + Ab*E + S + Read
+ Ag2
w w
I-Agl = Microplate with optimum concentration of antigen attached.
Ag2 = Competing antigen as a dilution range.
Ab*E = Optimum dilution of conjugated Ab specific for the Agl.
S = Substrate/color detection system.
+ = Addition and incubation steps.
W = Wash.
Read = Spectrophotometric reading at 492 nm.
This exercise will most simply demonstrate the principles involved
with competitive assays.
2.3. Materials and Reagents
1. Agl = guinea pig IgG at 1 mg/mL for attachment to solid phase.
2. Ag2 = two samples: (a) guinea pig IgG (known concentration) and (b)
rabbit IgG at 1 mg/mL.
 180 Competitive ELISA

Table 1
Data From Chessboard Titration of Guinea Pig IgG
and Anti-Guinea Pig Enzyme Conjugate in Exercise 2.4.
1 2 3 4 5 6 7 8 9 10 11 12
A 1.89 1.88 1.67 1.34 1.10 0.97 0.86 0.57 0.44 0.32 0.31 0.31
B 1.87 1.86 1.63 1.29 1.04 0.93 0.84 0.53 0.34 0.24 0.23 0.21
C 1.68 1.45 1.32 1.14 0.96 0.86 0.64 0.45 0.29 0.19 0.17 0.16
D 1.14 1.03 0.94 0.83 0.57 0.45 0.38 0.29 0.19 0.18 0.15 0.16
E 0.99 0.91 0.74 0.54 0.46 0.36 0.29 0.19 0.18 0.15 0.13 0.14
F 0.66 0.44 0.39 0.33 0.24 0.21 0.19 0.15 0.18 0.16 0.14 0.12
G 0.34 0.20 0.16 0.18 0.16 0.18 0.15 0.16 0.14 0.12 0.14 0 13
H 0.30 0.19 0.15 0.16 0.15 0.17 0.13 0.12 0.13 0.13 0.15 0.16

3. Ab*E = rabbit antiguinea pig IgG conjugated to horseradish peroxidase.

4. Microplates.
5. Multichannel and single channel 10 mL and 1 rnL pipets.
6. 0.05M carbonate/bicarbonate, pH 9.6.
7. PBS containing 1% BSA, 0.05% Tween 20,
8. Solution of OPD in citrate buffer.
9. Hydrogen peroxide.
10. Washing solution.
11. Paper towels.
12. Small-volume bottles.
13. 1M sulfuric acid in water.
14. Multichannel spectrophotometer.
15. Clock.
16. Graph paper.
17. Calculator.
2.4. Practical
Repeat exercise 5 in Chapter 5 involving the chessboard titration of anti-
gen and enzyme-linked antibody. You should obtain a similar picture. Com-
pare the results. The labeled conjugate dilutions are made from A-H,
IgG is diluted l-l 1, and 12 has no antigen. Plot the chessboard titrations
of guinea pig IgG against the conjugate as shown in Table 1 and Fig. 1.
2.4.1. Assessment of Data, Choice of Conditions for Competition
We are trying to compete the antigen (guinea pig IgG) and a different
antigen (IgG from the rabbit) for a pretitrated homologous solid phase
reaction. The ultimate sensitivity of the assay depends on the exact rela-
Direct Competitive ELJSA 181

0 12 3 4 5 6 7 8 9 10 1112

Dilution of antigen added to wells.

+1 +2 *3 *4 *5 *6 *7 *S

Fig. 1. Data from Table 1 relating antigen titrations at different concentra-

tions of conjugate.

tionship of the antibody and antigen attached to the solid-phase. If we

use too much antibody, so that it is in excess of that required to saturate
the Ag, we will have a quantity of free antibody that may bind to the com-
petitor and there will still be an amount left to react with the solid-phase
IgG. Thus, competition will only occur where extremely high concentra-
tions of competitor are used. This can be illustrated by examination of
the titration curves sketched in Fig. 2.
Note that the plateau regions represent antibody excess for any given
antigen concentration. The extent of these plateau regions varies accord-
ing to the exact amount of antigen attached to the solid-phase. As we
reduce the antigen, the plateau height values decrease. At the highest
concentrations of Ag the titration curves are similar for different anti-
body concentrations, indicating that the antigen and antibody are behav-
ing at maximum saturating levels. On dilution of the antigen we see
(curve 4) that the plateau height is reduced, even where we know that the
182 Competitive ELISA

1.5

8
z
* l
cl
0

0.5

0 - I I I I I I I

0 1 2 3 4 5 6 7 8

Conjugate dilution

Fig. 2. Illustration of regions of conjugate excessand nonexcess when titrat-

ing conjugate against concentration of antigen.

antibody is available for higher OD values (curves 1 and 2). Here the antigen
is the limiting factor in color development. In the competition assay a
maximum plateau height, dependent on the amount of antigen attached,
of around 1.0-1.5 OD should be selected. That is to say, find out which
dilution of antigen produces serum titration curves giving a maximum
plateau of these values, e.g., curves 3 and 4. From this titration curve we
need to estimate the dilution of antibody yielding about 70% of the maxi-
mum plateau OD. Thus, using curve 4, we can illustrate this below.
The conditions are now set for competition. We have:
1. Antigen dilution as for a curve.
2. Antibody dilution as shown in Fig. 3.
Direct Competitive ELISA 183

8
m
1.5 770%
t
Maximum
%
1
8

OS

. . . . . . . . . . . . .

I I I
0
0 1 2 3 4 t 5 6 7 8

Dilution for test

Conjugate dilution -

Fig. 3. Estimation of conjugate dilution for use in competition stage.

2.5. Competition Assay Proper

1. Prepare the optimum antigen plates coated with guinea pig IgG as deter-
mined above.
2. Wash the plates.
3. Take the guinea pig IgG (homologous competitor) and the rabbit IgG
(heterologous competitor). Dilute each to 40 l.tg/mL in blocking buffer.
4. Add 50 l,tL of blocking buffer to each well of the IgG-coated plate.
5. See plate design in Fig. 4. Make a twofold dilution range of the guinea pig
and rabbit IgG by addmg 50 l.tL of the IgGs to row A. Do this in triplicate
(3 rows for the guinea pig Ig, lA, B, C, and 3 rows for the rabbit IgG,
column lD, E, F).
6. Double dilute the IgGs across the plate (l-l 1).
184 Competitive ELISA

Dilution range of competitor ->

Guinea pig IgG

000000000000
000000000000

000000000000

000000000000
Rabbit IgG
000000000000

000000000000
No competitor 000000000000

100% Competition 000000000000

Constant d&.&ionof conjugate

Fig, 4. Plate design for performance of competition assay.

7. Dilute the antiguinea pig conjugate (pretitrated level found above) in block-
ing buffer. Make up 6 mL. Do not allow tips to touch liquid in the wells.
8. Add 50 pL of the diluted conjugate to rows A, B, C, D, E, F, and G. Do not
add to row H. Avoid touching liquid in wells with tips when adding conju-
gate. Mix the contents of the plates by gentle tapping. Add 50 p.L of block-
ing buffer to row H.
9. Incubate for 1 h at room temperature (or under conditions you used to
titrate the conjugate). Rotate the plate to mix reagents every 10 min.
10. Wash the plates.
11. Add 50 pL/well of the OPD/H202 solution.
12. Stop the reaction after 10 min by addition of 50 pL/well of 1M H,S04.
13. Read OD in spectrophotometer at 492 nm.
2.5.1. Data-Typical Results
Table 2 shows the results from the spectrophotometric reading of plates at
the conclusion of the exercise. Figure 5 relates the OD values to the various
concentrations of competitors added. The results are processed by taking
the mean value for each of the triplicate dilutions (e.g., mean OD of col-
umn 1, wells A, B, C or column 5, wells D, E, F), as shown in Table 3.
2.5.2. Further Processing of Data
1. Take the mean OD reading of row G. This represents the OD resulting from
the reaction of the conjugate with the solid-phase IgG and the conjugate
 Direct Competitive ELISA 185

Table 2
Plate Data From the Competition of Samplesof GuineaPig
and Rabbit IgG for a Direct ELISA in Exercise2.5
1 2 3 4 5 6 7 8 9 10 11 12
A 0.04 0.05 0.07 0.10 0.23 0.35 0.56 0.78 0.98 1.12 1.34 1.34
B 0.06 0.06 0.08 0.12 0.25 0.41 0.61 0.79 1.01 1.14 1.35 1.38
C 0.07 0.05 0.09 0.13 0.21 0.43 0.58 0.81 1.05 1.17 1.36 1.34
D 1.12 1.23 1.34 1.35 1.34 1.36 1.29 1.37 1.36 1.41 1.32 1.34
E 1.14 1.24 1.35 1.35 1.36 1.39 1.34 1.36 1.33 1.34 1.32 1.38
F 1.13 1.25 1.34 1 38 1.38 1.41 1.42 1.35 1.33 1.38 1.34 1.32
G 1.35 1.34 141 1.35 1.36 1.32 1.29 1.34 137 139 1.32 1.45
H 0.05 0.06 0.05 0.07 0.08 0.04 0.07 0.05 0.04 0.07 0.08 0.04

only. This value should be similar to that obtained when you titrated the
conjugate. This representsthe 0% competition OD, where we get most color.
2. Take the mean of the OD from row H. This represents the 100% competi-
tion level, i.e., where there is a total inhibition of the binding of antibody
(not strictly true 100% control, since the conjugate was excluded from the
test, but approximates very well). Thus, we have the 100% competition
(degree of inhibition) and the 0% competition OD values.
3. Convert the OD values obtained for the wells that contained the two com-
petitors mto percent competition using the two values calculated above.
2.5.3. Example from the Above Data
Mean of row G = 1.35 (equivalent to 0% competition [a lot of color])
Mean of row H = 0.07 (equivalent to 100% competition [little color])
Subtract mean of row H from all values obtained. If value is minus then call
this 0.
This determines the 0% competition level, (i.e., the range is from O-1.29
OD). Using a simple formula, we can calculate the percent competition
of the samples.
% competition = 100 - (Test OD -background x lOO)/Range
As examples, we have for the guinea pig Ig competition: Range = 1.29.
Taking row 5 we have:
100 - [(0.16/1.29) x 1001 = 87.7%
Taking row 6 we have:
100 - [(0.33/1.29) x 1001 = 75%
186 Competitive ELISA

1.6

Rabbit IaG
1.4

1.2

0.8

0.6

Guine:
0.4

0.2

III III I Ill I

0
0 12 3 4 5 6 7 8 9 10 11 12

Dllutlon competmgIgG +

Fig. 5. Competition of guinea pig IgG and rabbit IgG for guinea pig system.

Taking row 7 we have:

100 - [(0.51/1.29) x 1001 = 60%
Repeat this exercise for your data! Plot the data relating the percent
competition against the concentration or dilution of the IgGs as shown
in Fig. 6.
2.5.4. Analysis of Data
1. Notice that as you dilute out the homologous competitor (the guinea pig
IgG), the competition reduces.
2. The plateau of 100% competition is where the competing IgG is in large
excess over that on the plate.
3. Suggest what is happening at the 50% competition point.
4. Notice that the competition curve IS sigmoidal.
Indirect Assay-Antigen Competition 187

Table 3
Mean Values of Data in Table 2 for Various Dilutions
of Competing Antigens in Exercise 2.5.
Mean ABC Mean DEF
minus row H minus row H
guinea pig IgG rabbit IgG
1 0 1.17
2 0 1.18
3 0 1.29
4 0.01 1.35
5 0.16 1.28
6 0.33 1.27
7 0.51 1.27
8 0.73 1.29
9 093 1.28
10 1.10 129
11 1.28 126
12 1.29 1.27
Mean row G - Mean row H = 1 29 = the range

5. Notice that the rabbit IgG hardly competes, and ask why.
6. How might the sensitivity of the assay be altered? Clue: What happens if
we reduce (1) the amount of antigen on the solid phase or (2) the amount of
conjugate in the test?

3. Indirect Assay-Antigen Competition

I-Ag 1 + Ab + Anti-AB*E + S + Read
+ Ag2
W W
This exercise is similar to that for the direct competition assayfor antigen
in Section 2. except that the antigen is detected by an unlabeled antibody
(rabbit antiguinea pig IgG), which in turn is detected using an antispecies
conjugate (antirabbit IgG linked to enzyme). The indirect assay condi-
tions are optimized as in Chapter 6, by chessboard titration of antigen
and antiserum using a constant dilution of antispecies conjugate. You
can use the results of the chessboard titration shown in Table 1 to assess:
(a) the best guinea pig concentration/dilution to adsorb to wells, and (b)
the optimum amount of antibody to give about 70% binding to the opti-
mum amount of antigen found above.
188 Competitive ELISA

120

100

g 80
.rl
5
gE 60
s
s 40

20

0
0 1 2 3 4 5 6 7 8 9 LO 1112
. .
Log ,,,dh.ztmn competitor v

Fig. 6. Percentage competition plots of guinea pig and rabbit IgG competing
for guinea pig system.

3.1. Materials and Reagents

1. Ag = guinea pig IgG at 1 mg/mL for attachment to solid-phase.
2. Ab = rabbit antiguinea pig IgG.
3. Ab*E = swine antirabbit IgG conjugated to horseradish peroxidase.
4. Microplates.
5. Multichannel and single channel 10 mL and 1 mL pipets.
6. 0.05M Carbonate/bicarbonate, pH 9.6.
7. 0.05% PBS containing 1% BSA, Tween 20.
8. Solution of OPD in citrate buffer.
9. Hydrogen peroxide.
10. Washing solution.
11. Paper towels.
Indirect Assay-Antigen Competition 189

0000000000
I 00 0000 000000
00 0000 000000
I2 00 0000 000000
*fi 00 0000000000
g 0000000000
.i 00 0000000000
2 00 0000000000
cl
1 2 3 4 5 0% 100%

Competitors

Fig. 7. Plate design for addition of competitors.

12. Small volume bottles.

13. 1M sulfuric acid in water.
14. Multichannel spectrophotometer.
15. Clock.
16. Graph paper.
17. Calculator.
18. Standard guinea pig IgG (known concentration).
19. Three samples containing unknown concentrations of guinea pig IgG.
20. Bovine IgG at 1 mg/mL.
3.2. Method
1. Coat wells of microplate with 50 pL of guinea pig IgG at optimum concen-
tration found from chessboard titrations.
2. Wash the wells.
3. Take the homologous guinea pig IgG competitor of known concentration
and the bovine IgG sample and dilute to 40 pg/mL in blocking buffer.
4. Take the 3 samples of unknown levels of guinea pig IgG and dilute l/10 in
blocking buffer.
5. Add 50 pL of blocking buffer to all the wells of the microplate that is
coated with the optimum guinea pig IgG.
6. Make twofold dilution range of all the diluted samples. Thus, add 50 PL of
the initial dilution as shown in the plate plan in Fig. 7. Prepare duplicate
columns of each by 8 wells.
190 Competitive ELISA

Fig. 8. Representation of plate of competition assay; data in Table 5.

7. Add 50 FL of pretitrated rabbit antiguinea pig IgG to columns l-l 1, Do

not allow tips to touch fluid in wells. Add 50 pL of blocking buffer to
column 12.
8. Incubate under the conditions in which initial chessboard titrations were
performed. Mix contents every 10 min if not rotating plates (unless over-
night incubation is being used).
9. Wash the wells and blot.
10. Add 50 pL/well of the swine antirabbit conjugate at optimal dilution.
11. Incubate plates at 37°C for 1 h. Wash plates.
12. Add the substrate/chromophore (50 ltL/well, OPD/hydrogen peroxide
solution), stop reaction after 10 min by addition of 50 PL 1M H2S04/well.
13. Read OD using a spectrophotometer at 492 nm.
3.3. Data-Typical Results
Figure 8 shows a diagrammatic representation of plates at the conclusion
of the assay. Table 4 shows the results read from the spectrophotometer.
3.3.1. Treatment of Data
1. Take the mean value of the OD from column 12 (0.08 in the example).
2. Subtract this from all the ODs of the rest of plate.
3. For each of the duplicate wells, find the mean OD for each competitor
dilution. Thus, for example, see Table 5.
 Indirect Assay-Antigen Competition 191

Table 4
Plate Data for Indirect Competition ELISA to Measure Antigen in Exercise 3
1 2 3 4 5 6 7 8 9 10 11 12
A 1.31 130 1.31 133 1.32 1.32 1.26 1.34 1.12 1 10 134 006
B 1.12 1.14 1.32 1.34 1.28 1.29 1.21 1.18 0.88 0.83 1.32 0.08
C 0.79 0.77 1.29 1.28 1.09 1.10 1.00 0.98 0.68 0.66 1.34 0.09
D 0.57 0.54 1.27 1.19 0.85 0.79 0.76 0.74 0.44 0.43 1.32 0.06
E 0.33 0.36 1.25 1.24 0.67 0.69 0.47 0.48 0.22 0.23 1.29 0.08
F 0.18 0.15 125 1.26 0.41 0.45 0.26 0.27 0.09 0.09 1.34 0.09
G 0.09 0.10 1.21 1.22 0.30 0.29 0.16 0.17 0.08 0.09 1.33 0.08
H 0.08 0.07 1.15 1.18 0.13 0.15 0.09 0.07 0.07 0.06 1.35 0.06
Guinea Bovine Guinea Gumea Guinea 0 100
pig test pig 1 Pii3 2 Pig 3 % %
control

Table 5
Mean Values of Plate Data Shown in Table 4
12 34 56 78 9 10 11
A 1.22 1.24 1.24 1.22 105 1.26
B 1.05 1.25 1.21 1.12 0.81 1.24
C 0.70 1.21 1.01 0.91 0.59 1.26
D 0.47 1.15 0.74 0.65 0.36 1.24
E 0.27 1.15 0.59 0.38 0.14 1.21
F 0.08 1.14 0.34 0.19 0.00 1.26
G 0.00 1.13 0.21 0.09 0.00 1.25
H 0.00 1.07 0.06 0.00 0.00 1.27

4. Take the mean result of column 1 1 = 1.26. This is the 0% competition value.
Use the formula to work out the percent competition of each IgG dilution.
% competition = 100 - [(OD test/range) x 1001
The worked examples are shown in Table 6.
Plot the competition curves relating competition to log,, dilution or
concentration as shown in Fig. 9.
3.3.2. Examination of Data
1. Bovine IgG competition is very low, and the slope of the curve is very
different from thoseof homologouscontrol guineapig IgG.
192 Competitive ELISA

Table 6
Competition Percent Values Calculated From Data Shown in Table 5
G. pig G. pig G. pig G. pig
control Bovine W Iii@ I@
W IgG A B C
% Competition
12 34 56 78 9 10
A 6 5 5 7 20
B 20 5 7 12 36
C 35 9 20 28 53
D 67 10 42 49 71
E 79 10 53 70 100
F 93 11 75 87 100
G 100 15 87 100 100
H 100 20 95 100 100

1 2 3 4 5 6 7 8
Log,, dilution competitor (2 fold)+

- G pig control + Bovine IgG

x G pig A * G pig B * G pig C

Fig. 9. Competition curves for various competitors; data shown in Table 6.
Indirect Assay-Antigen Competition

1 2 3 4 5 6 7 8
Log 1. dilution competitor (2 fold) ->

+ G pig control + Bovine IgG

* G pig A * G pig B * G pig C

Fig. 10. Competition curves for various competitors; data shown in Table 6.

2. The curves for all guinea pig competitors are of similar shape.
3. The curves for guinea pig IgG samples A, B, and C are displaced as com-
pared to the control IgG curve.
A standard curve relating the concentration of guinea pig IgG com-
petitor in the liquid-phase to the competition achieved is shown by the
control IgG. The concentration of IgG in the other samples can be deter-
mined with reference to this standard curve. Since the general slope of
the curves is similar, we can compare the relative concentration at any
point on the standard curve. Ideally the best comparison point is at 50%
competition. Thus, draw a line across the 50% competition point on your
graphs, as shown for the data in Fig. 10. Read the dilution of the test IgGs
that give 50% competition, and then relate this to the known IgG concen-
tration necessary to give 50% competition as determined from the stan-
dard curve at this point.
 Competitive ELISA

Thus, assuming starting concentration of guinea pig IgG at 2 pg/niL,

We have for standard IgG 50% competition = l/64
Dilution for IgG A = l/20
Dilution for IgG B = 1140
Dilution for IgG C = l/140
Multiply the dilution factor by the 2 p&L to get concentration/ml
for the test IgG.
IgG C = 140/64 x 2 pg/l.tL = 4.4 pg/mL
IgG B = 40/64 x 2 ug/pL = 1.25 l@mL
IgG A = 20/64 x 2 pg/pL = 0.63 pg/mL
Remember that the dilution range is in logic steps, so the antilog of the
value has to be taken to obtain the dilution factor at 50%.
3.4. Conclusions
1. We have used a standard curve relating a known concentration of homolo-
gous competitor to its competing ability to measureunknown concentrations
of the same IgG in samples. This has analogies to the radioimmunoassay
approaches used in the quantification of hormones.
2. Note that if it is known that the substance for detection and quantification
is the same immunologically (homologous) as the standard substance used
to compute the standard curve, a single dilution of test could be used, and
their competing ability read from a standard curve.
3. Such competition assayscan be used to determine the similarity of anti-
gens in the same system competing for a single antiserum. The slopes of
the competition lines can be compared to obtain a measure of antigenic
relatedness.
4. Indirect Competition Assay for Antibody Detection
4.1. Reaction Scheme
I-Ag +Ab + Anti-Ab*E + S + Read
+AB
W W W
I- = Microplate.
Ag = Antigen.
Ab = Retitrated antibodies against Ag.
AB = Competing antibody (from a different species to Ab).
Anti-Ab*E = Conjugated antispecies in which Ab was produced.
S = Substrate and chromophore.
Indirect Competition Assay for Antibody Detection 195

W = Wash.
+ = Addmon and incubation of reagents.
Read= Read in spectrophotometer.
In this exercise, the indirect assay is used to pretitrate the homologous
antibody, as for Section 3. The optimized system is then competed with a
dilution range of antibodies from another species (the conjugate must
not react with the competing antibodies). In this assay, the pretitration of
the homologous serum is slightly different than the antigen competition
indirect ELISA in that we need to add the amount of homologous anti-
bodies that just saturate the antigen coated on the plate, since we do not
wish to leave excess free antigenic sites that could react with the compet-
ing antibody and have little influence on the binding of the homologous
antiserum. Note that this kind of assay can be made using the direct
ELISA using a conjugated homologous serum, as for the direct antigen
competition ELISA. Such assays are becoming more common with the
advent of the use of MAb reagents.
4.2. Materials and Reagents
1. Ag = Guineapig IgG at 1 mg/mL for attachmentto solid phase.
2. Ab = Pig antiguineapig IgG.
3. Ab*E = Goat antipig IgG conjugatedto horseradishperoxidase.
4. AB = 1X rabbit antigumeapig IgG standardserum.
2 rabbit serafrom animals injected with guinea pig IgG (unknown
titer).
2 rabbit serafrom antibody-negativeanimals.
5. Multichannel and single channel 10 mL and 1 mL pipets.
6. 0.05M Carbonate/bicarbonate,pH 9.6.
7. PBS containing 1% BSA, 0.05% Tween 20.
8. Solution of OPD in citrate buffer.
9. Hydrogen peroxide.
10. Washing solution.
11. Papertowels.
12. Small-volume bottles.
13. 1M sulfuric acid in water.
14. Multichannel spectrophotometer.
15. Clock.
16. Graphpaper.
17, Calculator.
18. Microtiter plates.
196 Competitive ELISA

lr+-x 1 to 8 dilutions of serum

IgG dilutions

Fig. 11. Titration curves relating IgG dilutions on wells against different
serum dilutions.

4.3. Data
Figure 11 shows a graph relating pig antiguinea pig antibody titration
curves to the IgG concentrations on the wells. This was obtained by
chessboard titration of captured guinea pig IgG against dilutions of the
pig antiserum, with a constant optimal dilution of antipig conjugate. The
conditions for the indirect chessboardtitration were as for those described
for the titration of the rabbit antiguinea pig serum in Chapter 6. From
these data we can:
1. Assessthat the best antigen concentration for use is the competition assay.
Select the IgG concentration that gives a plateau maximum (in antibody
excess) of around l-l.5 OD. (curves 4 and 5 in Fig. 11).
2. Select the dilution of serum that just saturates this level of IgG (approx
l/100).
Indirect Competition Assay for Antibody Detection 197

4.3.1. Increasing the Confidence of the Titration Curve Results

Since in the chessboard titration we are only using a single dilution
range of antibody against the antigen, it is essential to titrate the antiserum
in multiple rows against the antigen level found to be optimal, i.e., we
adsorb IgG at a level equivalent to the fourth or fifth dilution used in the
above test, then titrate in quadruplicate a dilution series of serum against
it. In this way, we can observe the variation in results and assess the
confidence in the titer of antibody that just saturates the antigen used in
the competition assayproper. This may be necessary where, for example,
one obtains poor competition in the test proper with low sensitivity, indi-
cating that too high or very much too low a concentration of antiserum
was used.
4.4. Competition Assay Proper
1. Add 50 pL of guineapig IgG to plates at the optimal concentrationfound
in Section 4.3.1. Incubate plates under conditions used for coating in Sec-
tion 4.3.1.) and wash plates.
2. Take the rabbit antiguinea pig sera, label the standardantiserum 1, and
label the two unknown titers sera 2 and 3. Take the two sero-negative rab-
bit sera and label them 4 and 5.
3. Dilute the rabbit sera to l/50 in blocking buffer (make up 0.5 mL of each,
i.e., add 10 p.L serum to 0.5 mL of buffer).
4. Add 50 pL of blocking buffer to all the antigen-coated plate wells.
5. Add 50 p.L rabbit serum 1 to wells Hl and H2. Add duplicate rows of other
serainrowH(serum2inH3,4;serum3inH5,6;serum4inH7,8;serum
5 in H9, 10). Dilute the sera using a multichannel pipet, transferring and
mixing 50 pL in each step. We thus have a dilution range from l/100 (row
H) to l/12,800 (row A) for each of the sera.
6. Incubate for 30 min at 37°C.
7. Do not wash the plate.
8. Add 50 pL of the swine antiguinea pig serum at the optimal dilution to
each well from columns l-l 1. Do not touch pipet tips in liquid of wells
when adding reagent. Add 50 pL blocking buffer to column 12.
9. Incubate for 1 h at 37OC.
10. Wash the wells.
11. Add 50 pL of the optimal dilution antiswine conjugate to each well.
12. Incubate at 37°C for 1 h.
13. Add 50 pL/well of substrate and OPD; incubate for 10 min.
14. Stop the reaction by addition of 50 pL 1M HzS04 to each well.
 198 Competitive ELBA

Fig. 12. Representation of plate showing competition assay; data in Table 7.

Table 7
Plate Data From Exercise 4.3.
Showing Competition of Indirect Assay by Antibodies
1 2 3 4 5 6 7 8 9 10 11 12
A 1.12 1.16 1.21 1.20 0.78 0.84 1.14 1.13 1.14 1.15 1.11 0.07
B 1.07 1.09 1.21 1.19 0.56 0.58 1.15 1.12 1.16 1.14 1.15 0.09
C 0.89 0.91 1.10 1.09 0.34 0.32 1.13 1.09 1.15 1.12 1.17 0.07
D 0.63 0.61 0.87 0.89 0.21 0.19 1.10 1.09 1.13 1.15 1.16 0.06
E 0.42 0.41 0.63 0.65 0.09 0.08 1.16 1.09 1.14 1.13 1.15 0.08
F 0.23 0.26 0.43 0.45 0.08 0.07 1.13 1.14 1.14 1.16 1.15 0.07
G 0.13 0.12 0.23 0.25 0.07 0.08 1.15 1.12 1.16 1.15 1.17 0.06
H 0.08 0.09 0.12 0.10 0.08 0.07 1.14 1.16 1.14 1.15 1.15 0.07
Standard 1 2 3 4 0% 100%
Serum

4.4.1. Typical Data

Figure 12 shows a representation of the ELISA plate after stopping.
Table 7 shows the data.
4.4.2. Processing Data
This is similar to the other competition assays performed.
1. Column 12 = 100% competition value; take the mean OD = 0.08.
Indirect Competition Assay for Antibody Detection 199

Table 8
Mean Values of Data in Table 7
12 34 56 78 9 10 11
A 1.05 1.12 0.71 1.06 1.07 1.08
B 1.oo 1.12 0.49 1.06 1.07 107
C 0.90 1.01 0.25 1.03 1.06 1.09
D 0.52 0.80 0.12 1.01 1.06 108
E 0.34 0.56 0.00 106 106 107
F 0.16 0.36 0.00 1.06 1.07 1.07
G 005 0 16 0.00 1.06 1.07 1 09
H 0.00 0.03 0.00 1.07 1.06 1.07

2. Subtract this from OD values of all wells.

3. Take the mean OD of the duplicates for the competitors.
This is shown in Table 8.
Plot the data. Relate the loglo dilution of each of the antiserum to the
percent competition as illustrated in Fig. 13.
4.4.3. Analysis of Data
1. Note that the curves for the rabbit antisera are similar. All the samples com-
pete. Sample 3 is a strong competitor since it gives high competition at higher
dilutions than the standard.Sample 2 is aweaker competttor than the standard.
2. The negative sera give little or no competition even at low dilutions.
3. The activity of each of the two sera can be compared to the standard com-
peting antiserum. Arbitary units can be ascribed to the standard serum so
that serum titers could be expressed against this. As an example, let the
titer of the standard serum at 50% competition be 1000. The relative titers
of the other two test sera can then be related to this. Since the same dilution
range was used for the samples we have at 50% competition for serum 2, it
is 2x stronger than the standard, so we need twice as much antiserum to
compete to the same level as the standard. Therefore the relative titer
of the serum 1sl/500. For serum 3 at 50% we require 5x less antiserum to
give the same result as the standard, so the titer IS 5000. The difference in
the dilution factors necessary to give 50% competition is easily assessed
from the graphs shown above.
4. Note that this processing only holds true if the competition curves show
similar characteristics (shape). Considerable variation m slopes indicates
that there is a different population of antibodies in the competing serum.
As m all assays,the general picture of titration curves is best examined by
the assay of as many sera as possible.
200 Competitive ELISA

3 4
l + + + *
0
0 1 2 3 4 5 6 7 8

Log 1o dilution serum

Fig. 13. Competition curves for various competing sera;data in Table 8.

6. Indirect Assay Competition for Antibody-Detection

Using a Single Dilution of Test Serum
5.1. Reaction Scheme
The optimization of the antigen, homologous serum, and detecting
serum is as described in the last exercise. In this assay we use the standard
rabbit antiguinea pig serum as a full titration range in 3 rows of the plate.
The rest of the plate contains a number of rabbit sera of high, medium,
and low titer against guinea pig IgG as used in the ‘spot-test’ in Chapter
6. Not all the sera can be examined in this exercise since only a single
plate is being used. The assay is identical to that in Section 4.4., except
that duplicate samples of sera are assessedat a single dilution for their
competing ability. The titer of the serum is then read from the standard
curve obtained on full dilution of the standard serum.
The test therefore has two stages: (1) The titration of the homologous
antiserum and solid-phase antigen in a chessboard indirect ELBA, fol-
lowed by accurate titration of the serum using a constant amount of cap-
Detection Using a Single Dilution of Test Serum 201

tured IgG and replicate dilutions of the antiserum and (2) the competi-
tion assay proper.
5.2. Materials and Reagents
1. Ag = guinea pig IgG at 1 mg/mL for attachment to solid-phase.
2. Ab = pig antiguinea pig IgG.
3. Ab*E = goat antipig IgG conjugated to horseradish peroxidase.
4. AB = 1X rabbit antiguinea pig IgG standard serum.
32 rabbit sera, including high, medium and low titer sera against
guinea pig IgG and seronegative sera.
5. Multichannel and single channel 10 mL and 1 mL pipets.
6. 0.05M Carbonate/bicarbonate, pH 9.6.
7. PBS containing 1% BSA, 0.05% Tween 20.
8. Solution of OPD in citrate buffer.
9. Hydrogen peroxide.
10. Washing solution.
11. Paper towels.
12. Small-volume bottles.
13. lit! sulfuric acid in water.
14. Multichannel spectrophotometer.
15. Clock.
16. Graph paper.
17. Calculator.
18. Microtiter plates.
5.3. Titration-Stage
Repeat the chessboard titration and accurate antibody titration of pig
antiguinea pig system, as in Section 4.4., or use these results for condi-
tions. From the data the best antigen concentration, and the dilution of
swine antibody that just saturates the IgG, can be determined.
5.4. Competition Assay Proper
1. Add 50 pL of guinea pig IgG to plates at optimal dilution. Incubate using
the same regimen as in Section 5.3. Wash plates.
2. Take the rabbit test sera and dilute them to l/50 in blocking buffer. Use the
micronics racks for dilutions so that the samples can be added using the
multichannel pipet. Take the standard rabbit antiserum and dilute to l/50.
3. Add 50 pL blocking buffer to columns 1,2 and 11 and 12.
4. Add 50 p.L of the diluted standard rabbit serum to Hl and 2. Make a two-
fold dilution range of the serum to Al and 2.
5. Add the test samples to the wells as duplicates, as indicated in Fig. 14.
202 Competitive ELISA

Test sample duplicates

0% 100%
T
Standard
Controls

antibody
dilution
range

Fig. 14. Plate layout for “spot testing” in competition assay.

6. Incubate the plates for 30 min at 37°C.

7. Add 50 PL of the optrmum dilution of swine antiguinea pig serum in block-
ing buffer, incubate for 1 h at 37OC,and mix contents of plates every 10
min. Do not touch tips in liquid of the wells when adding this serum. Do
not add this serum to column 12. Instead add 50 pL blocking buffer.
8. Wash the plates.
9. Add 50 pL/well of the antiswine conjugate diluted in blocking buffer (opti-
mum dilution). Incubate 1 h at 37°C.
10. Wash the plate.
11. Add 50 pL/well of the substrate/OPD solution; incubate for 10 min at room
temperature.
12. Stop the reaction with 50 pL/well of 1M HzS04.
13. Read the plate using a spectrophotometer.
 Detection Using a Single Dilution of Test Serum 203

Table 9
Plate Data From Exercise 5.4. ‘Spot-Test’
1 2 3 4 5 6 7 8 9 10 11 12
A 1.21 1.24 0.34 0.32 1.23 1.21 1.12 1.12 0.09 0.09 1.23 0.07
B 1.03 1.05 0.19 0.18 0.43 0.45 0.56 0.58 0.78 0.78 1.21 0.09
C 0.91 0.90 1.13 1.15 067 0.69 1.11 1.13 0.12 0.14 1.24 0.08
D 0.76 0.73 0.98 0.96 0.13 0.12 0.16 0.13 0.78 0 80 1.24 0.09
E 0.53 0.54 0.06 004 0.34 0.36 0.16 0 18 1.23 1.21 123 0.08
F 0.31 0.34 0.34 0.36 0.14 0.16 1.17 1.19 0.08 0.10 1.21 0.07
G 0.12 0.13 1.21 1.23 1.14 1.11 0.09 0.07 0.67 0.69 1.26 0.05
H 0.06 0.08 0.06 0.09 0.15 0.12 0.23 0.27 0.10 0.12 1.23 0.06

Fig. 15. Representation of “spot testing” competition assay; data in Table 9.

5.4.1. Typical Data

Table 9 shows data obtained from the spectrophotometer. Figure 15
shows a diagrammatic representation of the stopped plate.
5.4.2. Treatment of Data
As for the other competition results:
1. Take the mean of column 12, and subtract this from all results from other wells.
2. Take the mean from column 11 (after subtraction above).
204 Competitive ELISA

Table 10
Mean Values of Test Sera From Table 9
Processed as Percent Competition Values
% competition results
12 34 56 78 9 10
A 0 73 2* 16 99
B 16 90 68 57 39
C 28 7* 48 9 95
D 41 23 96 93 38
E 59 100 76 91 0
F 78 76 93 4” 98
G 100 100 95 84 97

3. Express the other OD values as a percentage of the range 0 to the mean of

column 11, i.e., from O-l. 16 in the example above.
4. Take mean OD of the duplicate wells. Formula is percent competition in
each well.
100 - [(Test OD/1.16) x 1001
Plot the standard serum competition activity relating competition to
log,, of the dilution.
5. Read the relative titers of the other competition results from the curve.
6. Another approach to evaluation of spot-testing is that whereby accepted
negative sera are assessedascontrols. Several sera can be included in a test
so that their mean competition value and its variation can be assessed.
Thus, sera giving higher values of competition under the same conditions
(with prescribed confidence limits), can be assessedfor positivity. Studies
on a large number of negative sera give better population data as described
for the other assays,so that chosen negative controls may be added from
the defined population (see Table 10). In the above example, the sera with
asterisks could be the negative controls in order to test whether the system
was ideal. The percent value of their mean plus a defined interval as a
percent of this mean (as directed from large population studies) could be
given. Here we have mean = 3%. Assume that twice this mean is the
acceptable upper limit for negative competition values. Therefore, sera
could be ascribed as positive with competition values 26%. Actual titers
could be read as in Section 5. above.
5.5. Notes
We have used a dilution of l/100 for the test serain the example. This is
based on preliminary studies establishing the dilution as being optimal for
Overall Conclusions on Competition Assays 205

distinguishing positive and negative values. This must be attempted in

your laboratories for specific disease studies. The approach to examina-
tion of negative populations has already been discussed. In the case of
competition assays, a lower dilution of test serum might be used (effec-
tively increasing the sensitivity of the assay), since nonspecific factors
detected in the indirect assay do not seem to affect competition assay
results. Construction of full-scale serum titration competition curves of
many negative and positive sera will nominate the best dilution (with
definable confidence limits) of serum to be used. The sources of such
sera have already been discussed.
Thus, for any particular dilution used in the competition assay,an upper
limit of negativity should be definable (as a competition value) above
which positivity of antibody will be detected. Once competition assays
have been characterized in central laboratories it is usually simple to read
the assays by eye, with good levels of precision and sensitivity. In these
cases the selection of appropriate negative controls that define upper lim-
its of negativity as determined by eye, is important.
6. Overall Conclusions on Competition Assays
1. They provide a relatively simple method once the homologous systems
have been titrated.
2. These assayscan be read by eye, with some loss of sensitivity and reduc-
tion in confidence limits.
3. In all the examples above we have used 50 pL of competitor and 50 l.tL of
homologous serum as a mixture to compete for only 50 pL of antigen on
the solid-phase. You can alter the volumes to suit, for example:
a. 100 pL antigen solid phase vs 50 p,L homologous serum and 50 pL com-
peting antigen (or antibody). In this case,the pretitration would be with
100 l.tL solid-phase Ag vs 50 l.tL serum dilutions + 50 pL blocking buffer.
b. 50 pL solid-phase antigen vs 25 p.L homologous serum + 25 l,tL compet-
ing antigen (or antibody). In this case,the pretitration would be between
50 FL solid-phase antigen and 25 pL antibody dilutions + 25 ltL buffer.
c. The competitor and homologous serum can be mixed together in
another plate before addition to the solid-phase antigen plate. These
types of assayscan be termed Inhibition Assays since the reagents are
not directly competing in the same system,
Differences in results can be observed by alteration of the sequence of
reagents i.e., where true competition and inhibition methods are used, In
practice, the mixing of reagents in a true competition assay gives the most
sensitive assaysand best reflects avidity differences between reagents.