136
Experimental Section
A Review of Enzyme-Immunoassay and a Description
of a Competitive Enzyme-Linked Immunosorbent
Assay for the Detection of Immunoglobulin Concen-
trations
RICHARD O'KENNEDY, MONICA BYRNE, C1ARAN
O'FAGAIN and GERARD BERNS
School o f Biological Sciences
Dublin City University
Glasnevin, Dublin 9, Ireland
Introduction
In the 1960s the development of the principle of enzyme
immunoassay and methods for the labelling of antibodies, or
antigens, with enzymes provided a whole new surge of research
in immunoassay procedures.
The principle of the use of enzyme labels is shown in Figure 1.
Basically, the binding of the antibody to the antigen is detected
using an enzyme label. The enzyme acts on the colourless
substrate to give a coloured product which is readily detectable.
substrote
tlllllll /llllllll l/l/l/l~/~
Antigen Antibody( ~ - ) Lal0eLLed Enzyme
(L~ } with enzyme ((~)) Antibody surrounded
bound to by cotoured
antigen product
Figure 1 The presence of the antigen is detected using the enzyme-
labelled antibody. The amount of coloured product is propor-
tional to the concentration of the enzyme and thus to the
concentration of the antigen
BACKGROUND
Enzyme Immunoassays
There are various ways in which enzyme immunoassay systems
may be classified. One simple way is to divide enzyme
immunoassays into two categories, heterogeneous and homo-
geneous.
In the heterogeneous assay, antigen-antibody complexes are
physically separated from free antigen and antibody using some
type of solid phase system. In the homogeneous enzyme-
immunoassay no such separation is necessary and the elimi-
nation of this step is.an advantage. Here the binding reaction of
antigen and antibody modifies the activity of the enzyme label.
The extent of this modification is proportional to the amount of
binding and hence the concentration of material being
measured.
Enzyme linked immunosorbent assay (ELISA) is a good
example of a heterogeneous assay. Enzyme multiplied immuno-
assay technique (EMIT) is an example of a homogeneous assay
system. Both types of assay have their advantages and disadvan-
tages, as will be discussed later.
Enzyme immunoassays may be further subdivided into two
main divisions, competitive and non-competitive assays.
The Competitive ELISA Method
There are several types of competitive ELISA assays.
Competitive-ELISA for measurement of antigen
system an enzyme-labelled antigen is used which competes with
the unlabelled antigen in the sample for binding to the
immobilised antibody. The greater the concentration of the
BIOCHEMICAL EDUCATION 18(3) 1990
antigen in the sample the lower the amount of labelled antigen
binding to the antibody. The assay is set up as follows (see Fig 2).
(1) Attach antibody (Y) to solid phase
(2) Zncubate with enzyme-LabeLLedantigen
( < ~ ) with or without unknown sampLe
antigen (<])
~) ~" ~ (3l Antigen binds and unbound antigen is
/z/z/z/////z/////////z removed by washing
/MI~'//////)t///// /f// 141 Incubatewith enzyme substrote and
measure enzyme product formation
Figure 2 Competitive ELISA for measuring antigen concentration
(i) Antibody to the antigen of interest is immobilised on to the
solid phase. This may be a plastic tube, microtitre plate, plastic
strip, etc.
(ii) Washing is carried out to remove unbound antibody.
(iii) A mixture of labelled antigen and sample solution,
containing the antigen of interest, is added. Both labelled and
unlabelled antigen compete for binding to the antibody. Con-
trols containing labelled antigen only and a set of standards
containing known amounts of unlabelled antigen are also used.
Suitable blanks are also included.
(iv) The plate is washed to remove all unbound antigen.
(v) Enzyme substrate is added and after a suitable incubation
period the levels of enzyme activity are measured.
(vi) A graph of enzyme activity versus antigen concentration is
drawn and the levels of antigen in the sample determined. The
amount of enzyme activity is inversely proportional to the
concentration of unlabelled antigen in the sample (Fig 3).
100
80 ~ , X=concentration
Unknowanntg i en
y_ 60
o
40
20
Ixl
0 x
Antigen concentration
Figure 3 Determination of antigen concentration using a com-
petitive binding antigen-enzyme-labelled assay
Competitive ELISA using enzyme-labelled antibody to measure
antibody and antigen levels This type of assay involves the
immobilization of the antigen on to a solid phase. Enzyme-
labelled antibody and antibody in the sample then compete for
binding to the antigen. This assay measures the level of specific
antibody present in the sample. A good example of its use is the
detection of antibodies to disease-causing organisms, eg HIV
In this assay virus in AIDS. As in competitive enzyme-labelled antigen
ELISA tests the amount of product formed is inversely propor-
tional to the concentrations of standard or sample antibody (Fig
4).

('1) TmmobiLise antigen on solid phase and wash
(2) Incubate with enzyme-LabeLLed and unLabeLLed
antibody
( 3 ) Antibodies bind to antigen and unbound antibodies
are removed by washing
( 4 ) FoLLowing addition of substrate the amount of
coLoured product formed is inversely proportional
to standard and test sample antibody LeveLs
Figure 4 Competitive ELISA using immobilised antigen and
enzyme-labelled antibody
A variation that is also extensively used involves firstly the
immobilization of the antigen on to a solid phase. A solution of
enzyme-labelled antibody and samples containing standard or
unknown antigen concentrations are then added. The greater the
level of the antigen in the added sample the smaller the number
of enzyme-labelled antibodies available for binding to the
immobilised antigen. The amount of enzyme product formed is
inversely proportional to the concentration of antigen in the
standards or test solutions.
Many of the above systems employ a two-step assay using a
second enzyme-labelled anti-immunoglobulin antibody. This is
shown in the accompanying diagram (Fig 5). The second
antibody is raised in a different species from the first antibody.
Enzyme- LabeLLed -~E~-
anti- immunogLobuLin antibody
Antibody to antigen
TmmobiLised antigen
iiiiiii/i/1111/11/11111/
Figure 5 Use of enzyme-labelled anti-immunoglobulin antibody
The advantages of this are firstly that more than one labelled
second antibody will bind to the primary antigen-specific
antibody. This will amplify the signal produced. The second
main advantage is that the use of the second antibody removes
any problems associated with the production of a range of
labelled antigen-specific antibodies. However, it does introduce
an extra step in the assay. The extra sensitivity achieved more
than compensates for this. These systems are shown in Figs 6 and
7.
Non-Competitive Assays
These assays involve the use of either immobilised antibody or
antigen.
{1) Antibody is immobiLised on solid phase
(2) Antigen (<~)-containing solutions are then added
A second enzyme-LabeLLed antibody to the antigen is added.
(3)
This antibody reacts with a different epitope to the first
antibody
(4) Enzyme activity Is measured foLLowing addition of
substrate
Figure 6 Non-competitive enzyme assay using immobilised
antibody
BIOCHEMICAL EDUCATION 18(3) 1990
137
A A A
H,IIrIHIIHrlttH,,,H Enzyme-LabeLLed antibody against second
antibody
Second antigen-specific antibody
Antigen rrr~~,~
HZHrtH/HHHtHHI Antibody specific to antigen immobitised on solid HH ,Hr
phase
Illllllllllllllllllll
Figure 7 Use of second enzyme-labelled antibody in non-
competitive assays
Use of Immobilised Antibody In these assays the antibody is
immobilised on to the solid phase. Following washing, the
antigen solutions (standards or test samples) are added. Labelled
antibody is then added. After the substrate is added the level of
coloured product formation is measured and this is proportional
to the amount of antigen present in the standard and test
samples. These assays are often called sandwich assays due to
the use of immobilised antibody, antigen and labelled antibody.
An amplification system, involving the use of enzyme-labelled
antibody may be used.
Use of Immobilised Antigen It is also possible to have the
antigen immobilised on the solid phase. Any antibody, specific
for that antigen, in the standards or test solutions, will bind to it.
A second enzyme-labelled antibody specific to this antibody is
then added. The amount of enzyme activity is again directly
proportional to the antibody levels in the samples. This type of
assay is of great importance in relation to the screening of
hybridomas for antibody production to specific antigens.
Advantages of Enzyme Immunoassays
There are many advantages in enzyme immunoassays including:
(i) High sensitivity, (ii) High specificity, (iii) Assays are
relatively cheap and require small amounts of reagents, (iv)
Assays are rapid and can give both qualitative and quantitative
results, (v) Detection is easy: results can be detected visually or
using special readers, (vi) Results are reproducible, (vii)
Automated, high throughput and manual methods are available,
(viii) Very versatile, (ix) No problems with radiation or disposal
of waste, (x) Simple portable field systems are available, (xi)
Can use monoclonal or polyclonal antibodies. Thus it can be
seen that these assay systems have ideal qualities for analytical
assays.
Problems Associated with Enzyme Immunoassays
Some problems may be encountered with enzyme immuno-
assays. These include: (i) Specificity of antibody for antigen, (ii)
Non-specific binding of antibodies to solid phase, cells etc, (iii)
Insufficient washing, (iv) Quality and purity of enzyme-labelled
antibodies, (v) Feasibility of labelling: this is of particular
relevance to the antigen, (vi) Effect of labelling on antibody
binding characteristics, (vii) Sensitivity of assay, (viii) Leaching
IHrYt',H
of immobilised antibody or antigen from solid phase, (ix)
Stability of antibody, (x) Stability of enzyme.
With polyclonal antibodies there may be a problem with the
specificity of different batches. These problems may be over-
come by purification and adequate quality control before the use
of any antibody for analytical purposes. The advent of mono-
clonal antibodies has helped to overcome such problems.
IIHHHHrt Non-specific binding of antibodies to solid phases can be
overcome by using adequate blocking measures. These may vary
depending on the assay used. Monoclonal antibody technology
frequently produces so called 'sticky' antibodies that bind to
plastics, glass, etc. Non-specific binding of antibodies to cells can
often be overcome by the use of Fab or F(ab')2 fragments
(constant portion removed by enzyme treatment). The constant
portion has a region that binds to cell surfaces thus giving rise to
138
non-specific effects. The use of enzyme-labelled F(ab')2 frag-
ment antibodies is of particular importance in screening hy-
bridoma supernatants for the production of antibodies where the
material used for screening is immobilised cells.
Insufficient washing, at any stage in a heterogeneous enzyme
immunoassay can result in spurious effects eg where not all
unbound enzyme-labelled antibody is removed prior to addition
of substrate.
The quality and purity of enzyme-labelled antibodies is
important. Firstly, the number of enzyme molecules attached to
each antibody molecule should be uniform in each batch and
there should be little batch to batch variation. Secondly,
enzyme-labelled preparations should be purified so that no free
enzyme or antibody is present. The same should be the case
where the antigen is labelled.
Enzyme labelling of antibodies is now well established and
most antibodies can be enzyme-labelled with little effect on
binding. However, there are exceptions and any preparation
should be tested to ensure that no major alterations have
occurred in binding characteristics.
In general enzyme immunoassays are very sensitive. The
development of amplification systems, eg second or third
antibodies, biotin- avidin, etc, have ensured that a very high
level of sensitivity can now be achieved.
Leaching (slow desorption) of antibody or antigen from the
solid phase can occur during longterm storage, incubations or
washing. Ideally, little or no leaching should occur as this may
lead to variability between assays. This problem can be
overcome by the use of agents that stabilise the attachment of
antibodies or antigens to the solid phase (eg poly-IMysine or
glutaraldehyde) by covalent bonding or by using specially
treated plates or solid phases.
In general, polyclonal antibodies are more stable than
monoclonals. This is due to the heterogeneity of the antibodies
in a polyclonal preparation, with a number of antibodies having
different stability characteristics. Stability is an important factor
in relation to storage and working-lifetime for a kit.
The importance of enzyme stability is obvious and this may
also limit the working life of the assay. Nowadays, efforts are in
progress to develop chemical methods to increase enzyme
stability.
In designing or using any enzyme assay all the above points
need to be taken into consideration in order to optimise the
system. Nevertheless, it is true to say that all the problems
described can be overcome and reliable assays can be produced.
Homogeneous Enzyme Immunoassays
The great advantage of homogeneous enzyme-immunoassays is
that the separation of free and bound label is unnecessary. The
use of a solid phase is generally not required in homogeneous
assays.
Competitive homogeneous enzyme immunoassays This system
is widely known as E M I T (Enzyme Multiplied Immunoassay
Technique) which is a trade name of the Syva Company. Here,
the binding of antibody and antigen modulates the activity of the
enzyme label either by steric hindrance or by changes in the
configuration of the enzyme (Fig 8). Enzymes commonly used
include isocitrate dehydrogenase and glucose-6-phosphate de-
hydrogenase. The activity of these enzymes is measured by
following the change in absorbance at 340 nm due to the
conversion of N A D + to N A D H .
Systems have been designed which either activate or inhibit
the enzyme. Some assays have utilised antigens conjugated to
the enzyme substrate. Here the binding of antibody inhibits
access of the enzyme to the substrate.
A number of non-competitive, homogeneous assay systems
are now available. These assays employ two different antibodies
that react with different epitopes on the antigen. Each antibody
BIOCHEMICAL EDUCATION 18(3) 1990
Enzyme
1 [ substmte
(1) /~ + ~ L'> "
Antibody Enzyme ( E ) - activity
LabeLLed
antigen (~>) Enzyme
UnkabeLLed
X if"
Antibody Unbound
__~,~ Activity
antigen -antigen enzyme-
from comptex LabeLLed
sample antigen
Figure 8 Enzyme Multiplied lmmunoassay Technique (EMIT)
(1) The binding of antibody to enzyme-labelled antigen sterically
hinders the active site of the enzyme thus preventing enzyme
activity. (2) When unlabelled antigen is"added it competes with the
labelled antigen for binding to the antibody. The greater the level
of unlabelled antigen added the greater the level of unbound
enzyme-labelled antigen and hence enzyme activity. No separ-
ation step is required
is labelled with a different enzyme eg glucose oxidase and
peroxidase. One enzyme produces the substrate for the other
and significant enzyme activity is only detected when both
enzymes are in close approximation. This occurs when their
respective antibodies are bound to closely associated epitopes on
the antigen.
Selection and use of Enzymes in Enzyme-Imunoassays
Enzymes used in enzyme immunoassays should have the
following properties: (i) High turnover number, (ii) High
stability, (iii) Low cost, (iv) Easy detection, (v) High purity, (vi)
Ease of preparation, (vii) No interfering compounds/conditions
in samples (eg enzyme inhibitors, extremes of pH etc.), (viii) No
endogenous enzyme activity in samples. This last would give
high background effects. In some cases samples can be treated to
remove such activity.
The enzymes and substrates most commonly used in ELISA
are shown in Table 1 but this is by no means an exhaustive list.
Table 1 Enzymes and Substrates used in EL1SA
Enzyme Substrate
Horseradish peroxidase o-phenylenediamine (OPD)
Alkaline phosphatase p-nitrophenylphosphate
13-galactosidase o-nitrophenyl-[3-D-galactopyranoside
Glucose oxidase 6-D-glucose
Urease urea
Use of Microtitre Plates and Strips
Microtitre plates, strips and clusters are used extensively in
ELISA. Microtitre plates are made of polystyrene or PVC and
have 96 wells. Strips and clusters can be made up containing any
number of wells. The simplest assay systems involve immobilis-
ation of the antibody onto the well. A solution of labelled
antigen and unlabelled sample antigen is then added followed by
washing to remove non-bound material. The amount of enzyme
activity detected is inversely proportional to the concentration of
sample antigen.
Microtitre plates are also used very extensively in screening
hybridomas for monoclonal antibody production. In one such
screening system the antigen is immobilised on the well. The well
is then blocked to prevent non-specific binding to the plastic
surface of the well. This is carried out by incubating the antigen
coated well in a solution containing a low concentration (0. l'Tr)
of a protein such as albumin. After washing, the hybridoma
supernatant is added. If antibody specific to the antigen is
present, it will bind to it. After a further washing step enzyme-

labelled second antibody is added. This antibody is raised in a
species different to that of the first antibody (eg goat anti-mouse
IgG).
Binding of the enzyme-labelled second antibody takes place
where mouse antibody has already bound to the antigen. After
further washing enzyme substrate solution is added. Positive
wells show colour due to enzyme activity. The presence of
specific antibody can thus be detected by visually examining the
plate or quantitatively measuring the absorbances. Blanks and
positive and negative controls should always be included. Only
plates with good binding and optical qualities should be used.
This methodology allows rapid screening of a very large
number of different clones for antibody production. This is very
important as useless clones can be quickly discarded and positive
clones detected early. The latter is important to prevent
overgrowth by non-producing clones.
Preparation of Enzyme-labelled Conjugates
Most enzyme assays involve labelling either the antibody or the
antigen. There are a number of well established methods for
chemically linking proteins, eg enzyme, to antibody. In the case
of antigens, the types of reactions used will depend on the nature
of the antigen. If the antigen is protein in nature, then the
coupling procedures are similar to those for the antibody.
However, because of the diverse nature of antigens, the coupling
may prove easy or difficult depending on the reactive groups
available on the antigen.
Enzyme-labelled reagents should always be carefully checked
for purity. In many cases free enzyme, antibody or antigen may
be present and they may cause interference. The extent of
conjugation should be controlled and should be reproducible in
terms of the amount of enzyme conjugated. All enzyme labelled
preparations should be purified. Affinity chromatography is a
good method to use. The purity of samples may be examined by
gel filtration or HPLC.
A wide variety of different chemicals have been used for
linking enzymes to antibodies or antigens. These include
glutaraldehyde, periodate oxidation and a range of homo and
heterobifunctional reagents.
Developing areas in relation to enzyme immunoassays
Enzyme immunoassay is a very fast developing area. Among the
most notable trends are the following:
(1) Use of a variety of methods to separate bound and unbound
reagents eg magnetic methods.
(2) Use of enzyme substrates that give fluorescent and lumi-
nescent products. This may greatly increase the sensitivity of the
assays.
(3) Development of amplification systems that increase sensiti-
vity eg use of enzyme labelled biotin-streptavidin.
(4) Use of simple strip assays eg Checkmate assays for the
determination of progesterone levels in cow's milk. These can be
used by farmers at the 'cow side'.
(5) Application of assays to measure levels of hormones, etc, in
saliva.
(6) Development of highly automated readers, plate washers
and associated computer software.
(7) Development of hand held mobile detectors.
(8) Use of a range of new solid phase materials.
(9) Development of a new range of homogeneous assays using
different enzymes and different methods for enzyme activation
or inhibition.
A range of applications of enzyme immunoassay is listed in
Table 2. This is by no means an exhaustive list but will give a
good idea of the wide use of these systems for analysis.
EXPERIMENT
Introduction
A simple ELISA system that can detect mouse immunoglobulin
BIOCHEMICAL EDUCATION 18(3) 1990
139
Table 2 Applications of Enzyme Immunoassay
Assay system used Examples
Homogeneous Detection and monitoring of drug levels eg
(eg EMIT) diazepam, theophylline, digoxin and
gentamicin and coumarins
Heterogeneous Measurement of protein levels associated
(mainly ELISA) with disease eg alphafoetoprotein in cancer;
SGOT in liver disease; Factor VIII in
haemophilia
Detection of antibodies to infectious agents
eg anti-HIV antibody in AIDS, Rubella,
Herpes I and II, Cytomegalovirus,
Mycoplasma pneumoneae, Toxoplasma
gondii, Brucella abortus, etc
Detection of hormone levels eg 13HCG (in
pregnancy), TSH, progesterone, oestrone
sulphate, insulin, etc
Detection of parasitic diseases, eg liver
fluke, malaria, schistosomiasis
Detection of food additives and
contaminants eg anabolic steroids,
antibiotics and staphlococcal enterotoxin
Detection of antibody levels, antibody class
and types eg IgG, IgM, IgE, IgGl, IgG2,
IgG4 and IgG3
(mouse IgG) is described. The system used is also a competitive
assay. However, in this case the antigen (mouse IgG) is
immobilised. Samples containing mouse IgG (non-immobilised)
and enzyme-labelled antibody are then added to wells of ELISA
plates, already coated with mouse IgG. Competition will occur
between binding of immobilised and non-immobilised IgG with
the enzyme-labelled antibody. The greater the amount of free
mouse IgG in the samples, the greater the degree it will bind to
the labelled antibody and, therefore, less labelled antibody will
be available for binding to the immobilised mouse IgG. After
washing the plates only enzyme-labelled antibody binding to
mouse IgG-coated on the wells will be available for detection.
Therefore, high concentrations of mouse IgG in added samples
or standards will result in low absorbance levels in the wells (see
Fig 9).
Materials and Equipment
Antigen (eg mouse IgG) may be obtained from Sigma. Phos-
phate-buffered saline (PBS), Dulbecco A, pH 7.3; 0.05 M
carbonate-bicarbonate buffer, pH 9.6; PBS-Tween, ie PBS
containing 0.5% (v/v) Tween 20; Blocking solution, 3% (v/v)
bovine serum albumin; 13-galactosidase-linked sheep anti-mouse
antibody; 1 M Na2CO3, used as stopping solution.
Diluent for conjugate: 100 ml PBS, 7 I~1 2-mercaptoethanol,
0.1 g MgCI2 and 0.1 g NAN3; Substrate: 100 ml PBS, 705 i~1 2-
mercaptoethanQl, 90.37 mg o-nitrophenyl-13-D-galactopyrano-
side (ONPG) and 0.1 g MgCI2. 96-Well ELISA plates (Nunc or
Titertek); autopipettes (50, 100, 200 p,1 etc); incubator at 37°C;
Microplate reader eg Multiskan from Flow Laboratories.
Experimental
The antigen, in this case mouse IgG, is dissolved in carbonate-
bicarbonate buffer (7.5 txg/ml works well): 100 Ixl of this
140

No coLour
Table 3 Results of typical ELISA Jrom student practical
Enzyme-LabelLed (*J / H / H H H H / / / /
antibody to Concentration of" competing antigen
mouse IgG ~f (p,g/ml) A4(~5
Free mouse \/ raised in a /
TmmobiLised mouse
IgG (added) - - goat /
TgG 0.0 1.477 _+ 0.132 (16)
1.0 0.954 _+ 0.141 (6)
2.5 0.678 _+ 0.063 (16)
HH/,'/,'H,'H//!
I IIIIIIIIIIIIIll E×ce~
ss 5.0 0.427 _+ 0.025 (15)
7.5 0.314 + 0.033 (14)
'~ /~CoLour 10.0 0.262 _+ 0.036
A405 is the absorbance at 405 nm. The results are shown as the mean -±
(14)

/~l/l/l/Ill//Ill
95% confidence limits with the number of determinations in parenthesis

1.6
Figure 9 Illustration of competition between immobilised mouse
lgG and free mouse IgG for binding with enzyme-labelled anti-
mouse IgG antibody. Binding of the latter to the immobilised
~ 1.2
mouse IgG can be detected by use of the conjugated enzyme
O

solution is added to each well or to a set number of wells of a "~ 0.8

microtitre plate and incubated in a humid chamber at 37°C for g
2 h, or 4°C overnight, to coat the plate with mouse IgG. The
wells are emptied by inverting and shaking and are then filled ~ 0.4
..Q
with 200 ill of PBS-Tween and incubated at room temperature <

for 3 min then emptied by inverting and shaking. The washing

procedure is repeated 2 - 4 times. Now 200 ill of blocking f I I i I i I J i f I
solution is added to each well and incubated at 37°C for 1.5 h. 20 40 60 80 1 O0

This blocks any remaining protein-binding sites on the plate and
thus prevents non-specific binding. The above PBS-Tween
washing procedure is repeated.
Dilutions of the competitive antigen (mouse IgG) are made up
in PBS: 10 lag/ml, 7.5 p.g/ml, 5.0 p.g/ml, 2.5 ~zg/ml and 1.0 ~g/
ml. These solutions are used as standards to test the system. The
enzyme-labelled conjugate (13-galactosidase-linked goat anti-
mouse IgG) is diluted 1/500 in diluent solution. 50 p.l of the
enzyme-labelled conjugate and 50 ~1 standard solutions of
mouse IgG are simultaneously added to the same wells coated
with immobilised mouse IgG. This allows the competition
reaction to occur. Duplicates of blanks, standards, controls and
samples are used. The plate is shaken for 10 min to ensure
efficient mixing and then incubated at 27°C for 30 min and at 4°C
for 1 h, after which the washing procedure is repeated.
Finally, 100 p.l of freshly-prepared substrate is added to each
well and incubated at 37°C for 30 min in a humid chamber,
followed by 50 p.l of stopping solution and the plate shaken to
ensure thorough mixing of the solutions in the wells. The
absorbance of the solutions in the wells at 405 nm is determined.
Comments
The plate-reader is blanked using wells containing all solutions
except the conjugate and competing antigen. PBS is used
instead. The results from a typical experiment, performed by
students for the first time, are shown in Table 3. The standard
graph is plotted as shown in Fig 10 and the concentration of
mouse IgG in analytical samples can be calculated. A skilled
operator will obtain better results.
The system is rapid, easy to set up and gives reproducible
results. It can be used to measure mouse IgG levels and also
provides a useful procedure to demonstrate ELISA. We have
used this system with a range of students having only a very basic
knowledge of ELISA. They have had no problem understanding
the concepts involved. Indeed, it sharpens their appreciation of
the concept of competitive assays. The assay method is much
more economical than commercially available assay kits.
Students have produced good results based on their first attempt
with such an assay.
Concentration of competing antigen (fig/mr.)
Figure 10 Standard curve for ELISA assay. Absorbance (405 nm)
is plotted against concentration of competing antigen
Acknowledgements
We wish to thank the Childrens Leukaemia Research Project,
Health Research Board, E O L A S and Baxter-Dade (Switzer-
land) for financial support and Ms Barbara Drew for typing the
manuscript
Reading List
Edwards, R (1985) Immunoassay, An Introduction, William Heinemann
Medical Books, London
Engvall, E (1980) Enzyme lmmunoassay EL1SA and EMIT, in Methods
in Enzymology 70,419-439
Maggio, E T (Editor) (1980) Enzyme hnmunoassay, CRC Press, USA
Tijssen, P (1985) Practice and Theory of lmmunoassays, in the series
'Laboratory Techniques in Biochemistry and Molecular Biology',
(Editors Burdon, R H and van Knippenberg, P H) Elsevier, Amsterdam
Voller, A, Bidwell, D E and Bartlett, A (1979) The Enzyme-Linked
Immunosorbent Assay (ELISA), Booklet sponsored by Dynatech
Europe, Borough House Rue du PrE, Guernsey, UK
O'Kennedy, R (1989) 'Enzymeimmunoassay - - A review of its
development, uses and recent trends', Clinical Chemist0' and Enzymol-
ogy Communications 1,313-328
Shattock, A G, Morris, M, Kinane, K and Fagan, C (1989) 'The
Serology of Delta Hepatitis and the Detection of IgM anti-HD by EIA
using Serum-Derived Delta Antigen', J Virol Meth 23, 223-240
Fagain, C, Sheehan, H, O'Kennedy, R and Kilty, C (1988) 'Maintenance
of enzyme structure-possible methods for enhancing stability', Process
Biochem 23, 166-171

BIOCHEMICAL EDUCATION 18(3) 1990