INTRODUCTION:

Assay systems involving use of antigens, haptens, or antibodies

labelled with an enzyme have recently been applied to the
measurement of substances in biological fluids.

These assay systems have been given various names: enzyme-,

enzymic-, enzymatic-, and enzymo- immunoassay (EIA),’ enzyme-
linked immunoassay, enzyme-labelled immunoassay, enzyme-
coupled imunoassay, immune enzymatic assay, and enzyme- linked
immunosorbent assay (ELISA).

These names are synonymous except for the tendency to use the last
name to describe the assay for specific antibodie.

The accuracy of the EIA is affected by all parameters which influence

the action of the enzyme, and a meaningful test cannot be developed
without a real understanding of the effects the test design may have
on the enzyme activity.

The primary interest in EIA is not in the mechanism of enzyme

action.
It is, nevertheless, essential to understand the elementary nature of
enzyme reaction and of the effect of external factors, such as pH,
temperature, ionic strength, other molecules and the solid-phase, on
enzymic activity. For an optimal assay it is necessary to know: (i) the
stoichiometric details of the reaction; (ii) the molecule(s) which
should be present or avoided; (iii) the kinetic dependence of the
reaction on these molecules; (iv) the optimization of experimental
conditions; and, (v) the accurate monitoring of the enzyme activity.
Knowledge of the kinetic behaviour makes it possible to estimate the
quantity of the immune reactant present and to compare EIA to other
assays.
Properties of enzymes used in EIA.

No enzyme fulfils all the criteria for an ideal label in EIA and a
compromise has to be made. In solid-phase EIA, the influence the
solid phase has on the enzyme should be minimal. Conjugation should
be easy and the conjugates should be active and stable. These are
undoubtedly major factors for the frequency of the selection of
horseradish peroxidase (POase) or β-galactosidase (BGase).

Alkaline phosphatase (APase), which is difficult to conjugate in

defined form (extensive polymerization), is, nevertheless, widely used
due to the absence of endogenous enzyme in plant tissues, high
stability, and to the technically undemanding preparation of very
stable conjugates.

Homogeneous, AM assays pose different requirements. Here, the

enzyme should be easily conjugated near the active site without
altering its activity. The reaction of hapten-or antigen-labeled enzyme
with antibody should affect strongly the enzyme activity, e.g., through
steric inhibition of the substrate at the catalytic site. The requirements
for optimal ionic conditions and temperature for both enzyme activity
and antigen-antibody interaction should be compatible.

EIH poses another set of requirements: (i) the enzyme should be small
to avoid problems of penetration into the fixed cell; (ii) the substrate
should be soluble and the product insoluble, so that the product is
deposited near the enzyme.

Enzymes used in activity amplification assays

EIA based on visual titration require clear-cut endpoints. Both

horseradish POase and urease produce easily detectable products,
whereas the products of APase or BGase are less strongly coloured.
The relative costs of the enzymes for EIH or EIA should not be
ignored. For example, BGase is about 26 times more expensive per
mg than POase and at least 150 times more expensive than POase.

Moreover, for 1:1 conjugates about 10 times more (in mg) BGase is
needed than POase due to the molecular weight, increasing the
relative cost of BGase by about 10 times. However, BGase is more
efficiently conjugated than POase which lowers its relative cost to
about 200 and is, under certain conditions, capable of detecting
smaller amounts of antigen than POase and shows fewer problems
with background staining by endogenous enzyme. If background
staining poses problems, APase, the usual alternative of POase, is
only slightly less costly than BGase, but conjugation methods for
APase and its detectability are far inferior.

It is evident that the frequently used APase is quite expensive indeed,

though its use on a small scale is not prohibitive. In general,
commercial conjugates are several-fold more expensive than those
prepared in the laboratory.

Horseradish peroxidase ( POase)

1.Physico-chemical properties of peroxidase

POase (hydrogen-peroxide oxido reductase, is the most widely used

enzyme in EIA and EIH.

Typical POases are hemoproteins and transfer hydrogen from

hydrogen donors (H-doPOases often occur as multiple isozymes and
are widely distributed, particularly in plants.

They appear to catalyze the same reaction, but differ markedly in

physicochemical and kinetic properties. It is very likely that the
different iso-POases serve specialized, albeit unknown biological
functions.
Three main types of POases have been identified: (i) the acidic
(probably cell-wall-associated) POases with a very high carbohydrate
content (ii) POases with a p l around neutrality (or slightly basic)
with a somewhat lower sugar content; and, (iii) very basicnors) (DH)
to H202

POases often occur as multiple isozymes and are widely distributed,

particularly in plants.They appear to catalyze the same reaction, but
differ markedly in physicochemical and kinetic properties. It is very
likely that the different iso-POases serve specialized, albeit unknown
biological functions. Three main types of POases have been
identified: (i) the acidic (probably cell-wall-associated) POases with a
very high carbohydrate content (ii) POases with a PI around neutrality
(or slightly basic) with a somewhat lower sugar content; and, (iii)
very basic POases (PI>11) of low sugar content.

Purification of horseradish peroxidase

POase is much more sensitive than APase to contaminants. Water

deionized with polystyrene resins is often toxic to POase; in fact,
POase is inactivated by polystyrene plates (or cuvettes) in solid-phase
EIA if Tween 20 is omitted. POase is also very sensitive to the
presence of bacteria or bacteriostatic agents (NaNJ and is inactivated
by oxygen (degassing recommended), hypochlorous acid and
aromatic chlorocarbons often found in laboratory water.

The rather expensive POase is usually purchased rather than purified.

The method presented here is exceedingly simple, fast, may decrease

the cost sometimes more than 10 times, and yields a preparation of
very high activity since isozymes with low activities are eliminated.

This method starts with a low-priced crude extract which is dissolved

in 2.5 mM sodium phosphate buffer, pH 8.0.

A DEAE-Sepharose column is equilibrated with the same buffer and

the POase solution is applied (up to 5 mg protein per ml gel).
Impurities and less active isozymes are retained whereas pure POase
passes directly and is collected by monitoring the eluate (at 401 nm or
visually). This POase preparation has higher RZ values (3.20-3.30)
and higher activities than the commercial ‘pure’ preparations; though
RZ values will be slightly higher if ‘pure’ commercial POase is
purified by this method.

POase can also be purified by affinity chromatography on a ConA-

Sepharose column followed by gel filtration. This method, however,
is not recommended, since it is time consuming, expensive, applicable
to small amounts (which become diluted) and does not remove the
less active isozymes.

Catalytic properties of horseradish peroxidase

The reaction supported by many studies dictates the correct

experimental conditions for POase assays: POase is divalently
oxidized by peroxide to Compound -I, which is, in turn, reduced to
the initial state by 2 successive univalent interactions with H-donors.
Compound-II is the one-electron oxidized, intermediate form. Some
H-donors, e.g. o-dianisidine, produce a direct, two-electron transfer
reduction.

Nitrogenous compounds (nucleophilic catalysts: pyridine, imidazole),

may stimulate this transfer.

An excess of substrate inactivates the enzyme by forming compound-

III(red) or IV(emerald green). This substrate inhibition is very
substantial in some of the current EIA procedures using POase.

Specific formation of Compound -I can be obtained only with certain

peroxides but many substances can serve as H-donors. Whether Km
can be obtained is a matter of controversy.

POases from different origins or their isozymes may have quite

different activities toward a certain donor. The k4 rate constants vary
largely.
In general, k1, is about 50-100 times greater than k4, thus k4 represents
the donor limiting step and k1 , the substrate limiting step. The rate of
substrate utilization is expressed as.

Formula 1

C isozyme reacts faster with the aromatic amines, whereas the acidic
isozymes react much faster with acidic donors.

Cyanide or sulphide reversibly inhibits POase at concentrations of

10-5 to 10-6 M. Cyanide inhibits all POases and has been used to
identify ‘true’ POase activity. Fluoride, azide, or hydroxylamine
inhibits only at concentrations higher than 10-3M. Dithionite reduces
ferri-POase to ferro-POase which then combains with carbon
monoxide. The enzyme is irreversibly inhibited by hydroxymethyl
hydrogenperoxide.

Galactosidase (BGase)

β-D-Galactoside galactohydrolase or β -Galactosidase (BGase), has

been detected in numerous microorganisms, animals and plants.

The most extensively investigated enzyme, from Escherichia coli (E.

coli), is most popular for EIA, and will be discussed here. Its large
size makes it less suitable for EIH.
Physicochemical properties of β -galactosidase

Native BGase, a tetramer, has a molecular weight of 465000 (from its

complete amino acid sequence) and a pl of 4.6.

The tetramer dissociates into inactive monomers at pH < 3.5 or > 11.5
or with mercurial.

Aggregated forms of BGase, i.e. multiples of the tetramers (16S) with

S020w values of 23, 27, 32, 36, and 41-45 S, are readily formed in
purified preparations in particular in the absence of SH compounds .

With 100 mM 2-mercaptoethanol (2-ME) and 10 mM MgC12 (2-ME

alone inactivates the enzyme) the enzyme is also much more heat
stable.
The enzyme is stable for at least 30 min at 40°C within pH 6.8, but its
stability at 40°C falls sharply below pH 6.0 and slowly above pH 8.0.
Heat stability is considerably lowered by various intermediates in the
glucose metabolic cycle.

It is of interest for EIA that BGase is an excellent immunogen, that

anti-BGase antibodies do not inhibit enzymic activity and that some
inactive BGase mutants can be activated through the reaction with
specific antibodies.

Purification of a- β galactosidase

In some E. coli strains about 5% of the total protein content is BGase

if lactose is the sole source of carbon. The large size and stability of
BGase render its isolation easy. The classical method of simple
affinity chromatography procedure, are described here.

E.coli K 12 cells (1 kg wet weight), grown in lactose medium, are

suspended in 21 standard buffer (10 mM Tris-acetate, pH 7.5,
containing 10 mM MgC12, 10 mM 2-ME, and 10 mM NaCl) and
sonicated.

The disrupted cell suspension is clarified by centrifugation at 2500x

rpm overnight. (NH4)2SO4 is added to 650 mM and the precipitate is
removed by centrifugation.

The (NH4)2S04co ncentration in the supernatant is then raised to 1.3

M. The precipitate, collected by centrifugation, is re-suspended in and
dialyzed extensively against the standard buffer without NaCl. An
eventual precipitate is removed by centrifugation.

This sample (in 10 ml aliquots) is passed through a Sephadex G-200

column (5 x100 cm); the enzyme elutes just after the void volume.

The pooled fractions are applied to a DEAE Sephadex A-50 (not

DEAE-cellulose) column (4 x 25 cm) equilibrated with standard
buffer (without NaCl).

The enzyme is then eluted with a standard buffer-NaC1 gradient,

using a 500 ml closed mixing chamber containing the standard buffer,
and adding drop wise by gravity a 2% NaCl solution. The yield of
practically pure enzyme is about 400 mg

Catalytic properties of P-galactosidase

BGase follows Michaelis-Menten kinetics and kcat˂ k-l so that Km =

Ks. However, additional enzyme complexes may be distinguished as
follows.

Formula 2
Where P2 is the free galactose and PI is either glucose or an aglyconic
group. The enzyme displays a wide tolerance to the structure of PI; the
Km value for its natural substrate, lactose, (3.85 x10-3M) is higher than
for o-NPG and p-NPG (9.5 x 10-4 M, respectively.

The D-pyranoside is essential and the pH optimum of activity is 7.2

7.7. Acceptor alcohols (methanol, glycerol, 2-ME, Tris) stimulate the
cleavage of o-NPG, but not of other substrates. Heavy metals, organo-
mercuric compounds, chelating agents (EDTA, citrate), prevent the
inhibitory effect of 2-ME which in that case is an excellent activator
of the enzyme. Studies on these parameters in solid-phase EIA are
altogether lacking.