light scattering techniques, and particle counters.

Enzyme-Multiplied Immunoassay Technique (EMIT)

In the EMIT, the analyte is covalently bound to the enzyme

in spatial proximity to the active site; and consequently,

the formation of the antibody-antigen complex inactivates

the enzyme; addition of hapten results in a reduction of

this inactivation. Over a limited range, the enzyme activity

is approximately proportionate to analyte concentration.

This method has been widely employed for therapeutic

drug monitoring.

Apoenzyme Reconstitution Immunoassay System

(ARIS)

If, however, the antigen is covalently bound to the prosthetic

group of an enzyme such as glucose oxidase and an aliquot

of the coupled antigen to flavin–adenine dinucleotide is

added to determine an analyte, free antibodies prevent the

reconstitution of the enzyme. The concentration of the free

antibody naturally depends on the analyte concentration

in the sample. Similar to the EMIT technique, the ARIS is

used in automatic analyzer systems in clinical chemistry.

Fluorophore-Labeled Homogeneous Immunoassay

(FLHIA)

At first glance, fluorescent labeling appears to have a

much higher detection strength compared to colorimetric

detection, but this is not the case. First, the affinity constant

generally limits the detection strength of a process.

Second, fluorophores are exposed to many influences,

such as quenching by impurities, or even adsorption

of the fluorophore molecule. However, the fact that the

detection can be repeated is advantageous, whereas a

chemical reaction is irreversible.

Homogeneous Fluorescence Polarization Immunoassay

(FPIA)

Direct observation of the formation of a hapten-

(fluorescent labeled) antibody complex is also possible in

polarized light. The presence of free hapten reduces the

antibody-tracer complex concentration, and the degree of

polarization is lowered. The detection strength of this test

is in the µmol/L range and thus not yet high enough for

environmental analysis.

Microparticle Enzyme Immunoassay (MEIA)

There are a number of variations in this method. The

enzyme-labeled binder binds to the analyte, which in

turn is bound to binder-coated microparticles. Initially,

free in solution during the foregoing chemical reactions,

the microparticles are immobilized on glass fiber, and

the complex of primary binder (capture), ligand (analyte)

and labeled binder (conjugate) is exposed to substrate,

producing a colored product.

Heterogeneous ELISA

This requires separation of free and bound label. Most

ELISAs described above, fall into this category.

Based on the functional results ELISA can be classified

as shown in Figure 22.16.

Quantitative ELISA

In this type the concentration of the analyte is measured

and expressed in standardized units (ng/dL for T3, ng/mL

for PSA). Standards are run and graph is plotted against

which the concentration of the analyte is estimated, e.g.

T3, T4, TSH, FSH, LH, etc.

Semi-quantitative ELISA

In this type, the controls are used (positive control,

negative control, cut-off control). An arbitrary unit is given

to express the concentration (EU/mL). Graph may or may

not be used, e.g. TORCH, ANA, etc.

Qualitative ELISA

In this type, the controls are used and is formula based. No

graphs are required, e.g. HIV, HBsAg, etc.

ELISA: Practical Aspects

The different components of ELISA are packed together. This

is commonly known as “Kit”. The components are as follows:

Solid Surface

It can be a microwell, coated tube or bead. This can be

compared to a plate on which the reaction takes place. The

Serology/Immunology 577

microwell can be breakable or unbreakable. The coated

tubes may be of polystyrene or polypropylene in nature.

The solid phase may be coated with antigen, antibody

or streptavidin. The choice of solid phase influences the

measurement of optical density. In the case of Microwell,

it is measured with an ELISA reader; and in coated tube it

is measured by an analyzer.

The process of fixing onto the solid phase is called

“adsorption” and is commonly called coating. Most

proteins adsorb to plastic surfaces, probably, as a result

of hydrophobic interactions between nonpolar protein

structures and plastic matrix. There may be nonspecific

binding of unwanted proteins in available free sites.

This can be avoided by adding “immunologically inert”

proteins so as to block the free sites. These blocking agents

may be added during the coating process.

Calibrators/Controls

They are references against which the value of the analyte

in the sample is estimated. An important fact is that

immunoassays do not actually measure the analyte. They

can only provide a quantitative estimate of concentration

by direct comparison with standard/calibrator material.

The Features of an Ideal Calibrator

¾ A prerequisite for standardization is that the standard/

calibrator and analyte are identical

¾ The calibrator should contain the analyte in a form

identical to that found in the sample

¾ Calibrators should ideally be prepared by using a base

material identical to that in the test sample

¾ For clinical applications, human serum is the preferred

base matrix.

References

The matrix of a calibrator needs to behave in a similar way

to the sample matrix.

For assay of hormones that are bound to serum protein,

it is hard to use any other matrix other than human serum.

A prerequisite for standardization is that the standard/

calibrator and analyte are identical. In other words, the

calibrator should contain the analyte in the form identical

to that found in the sample.

Conjugate

It is the binder in the immunoassay system. The analyte in

the sample may compete (in case of competitive ELISA) or

bind with (in sandwich ELISA) the conjugate. It is either

an antigen or antibody tagged with an enzyme (depending

upon what it is being detected). The conjugate should have

certain characteristics:

¾ The enzyme must be capable of binding to an antigen

or antibody (the enzyme will react with the substrate

to give color)

¾ Should be stable at typical assay temperature

¾ Should be stable when stored at 2 to 8°C

¾ It must undergo only low-grade inactivation of reagent

and enzyme

FIG. 22.16: Classification of ELISA

578 Concise Book of Medical Laboratory Technology: Methods and Interpretations ¾ Long-term stability without loss of immunological and

enzymatic activities.

Most Commonly Used Enzymes in Immunoassays

Alkaline phosphatase, horseradish peroxidase, acetylcholinesterase, carbonic anhydrase, glucose oxidase, glucamylase, glucose-6 phosphate dehydrogenase, lysozyme,

malate dehydrogenase.

Substrate

The confirmation of an antigen-antibody reaction is done by

a suitable indicator. In ELISA this is done by the substrate. The

substrate reacts with the enzyme (in the conjugate) to give a

colored end product. The intensity of the colored product

is directly/inversely proportional to the antigen-antibody

reaction (in turn to the presence/absence or concentration

of the analyte in the sample). The colored end product may

be soluble which is measured colorimetrically. This is mainly

used in quantitative immunoassays. The end product may

also be insoluble which is measured visually. It is suitable

for dot blot assays. The end product remains as a permanent

record (e.g. Western blot strips, Rapid test cartridges, etc.).

The substrate should have the following features:

¾ It should be able to produce intense colored end product

¾ Fast reaction rate or rate of conversion of substrate to

end product

¾ Ability to produce a broad range of colored end

product in a given time depending upon the amount of

conjugate (analyte) it has reacted with.

The Commonly Used Substrates

TMB (tetra-methyl benzidine), OPD (o-phenlyenediamine), DAB [diaminobenzidine (with enzyme

HRP)] and BCIP (5-bromo 4-chloro 3-indolyl phosphate),

[NBT (Nitroblue tetrazolium)] (with enzyme alkaline

phosphatase).

The factors affecting the performance of substrate are:

temperature, pH, buffer composition, etc.

Stop Solution

The enzyme substrate reaction needs to be stopped to

measure the optical density of the end product. The stop

solution acts by destroying the enzyme component. The

commonly used stop solutions are 1N HCl, 4N H2SO4,

NaOH.

Steps in ELISA

There are multiple steps involved in an ELISA procedure.

They can be grouped as follows:

Dilution

This is the first step. The reagents like conjugate, controls,

sample diluent, wash buffer, stop solution, substrate, etc.

are mixed in required proportions. In some cases, sample

may also be diluted in given ratios before adding them

in the well. Proper calculation of dilution ratios should

be made. It is advisable to prepare slight excess of the

quantity required to avoid pipetting errors. In some cases,

the dilution itself will have excess volumes to offset the

pipetting errors.