A sample pad with a bed volume of minimum retention

capacity facilitates transfer of the entire specimen

dispensed. This not only ensures minimal wastage of

specimen but also the excess specimen can be used to

wash away unbound conjugate from the test region for

better visualization of results.

Thus, use of sample pad that allows incorporation of

buffer salts, stabilizers and HBR, to a large extent eliminates

variation in pH, ionic concentration and interference of

heterophillic antibodies.

What is the Role of Soak Pad in Membrane-based

Rapid Diagnostic Test?

Use of a soak pad with high bed volume is preferred

in Rapid Diagnostic Tests because the total volume of

specimen that enters the test assay can be increased. This

increased volume can be used to dislodge the conjugate

as well as wash away the unbound/unreacted conjugate

from the test region contributing to clearer background

and better visualization of results.

Why do “Faint Ghost Bands” Appear at the Test

Region if the Device is Left Out on the Worktable?

A common phenomenon observed in the device format is

appearance of faint ghost bands at the test region after some

time. After completion of the test, if the device is exposed to

warm ambient temperatures, evaporation occurs from the

result window. Due to evaportion, the excess sample along

with unreacted/unbound conjugate from the soak pad

flows back to reaction area. This unreacted or unbound

conjugate may then get deposited on the test band resulting

in appearance of a “Faint Ghost Band” after sometime

(Fig. 22.8).

Results must be recorded at the end of the recommended

reaction time for correct interpretation.

FIGS 22.7A and B: A. Band appearance due to avid antibodies. B. Band

appearance due to less avid antibodies

FIG. 22.8: Appearance of “Faint Ghost Band”

572 Concise Book of Medical Laboratory Technology: Methods and Interpretations How do We Interpret “Broken Bands” at the Test/

Control Region?

To prevent evaporation of the specimen from the test

window, the membrane of the device is laminated with

the help of a thin transparent tape. Sometimes, during the

process of lamination, air pockets may be formed between

the membrane and the tape. These air pockets prevent

uniform sample flow, which may result in appearance of

broken bands at the test/control region.

However, appearance of even a broken band at the test

region indicates positive results.

In the following section, we shall discuss the role of

hCG as a marker for diagnosing pregnancy and certain

conditions that may give discrepant results.

Excess Sample Volume Dispensed

Adding excess sample in no way improves the performance

of the test. The excess sample added, cannot be absorbed

by the sample pad and thus flows out through the sides

of the device. Sometimes, the excess sample may flow out

along with the conjugate. The amount of the conjugate left

in the device is insufficient to perform the assay, leading to

invalid results. Secondly, once the specimen flows through

the device, the soak pad cannot retain the excess volume

of the sample, which then may flow out through the sides

of the device or may also flow back to the membrane along

with unreacted/unbound conjugate. This unreacted/

unbound conjugate may then deposit onto the membrane

resulting in apparently discrepant results.

ENZYME IMMUNOASSAY

Introduction

An immunoassay can be defined as a qualitative or

quantitative assay, which relies on the reaction between

an antigen and its specific antibody. The antigen being

bound is called “ligand” and the antibody is the “binder”

of the ligand. Enzyme labeled conjugates were introduced

first in 1966 for localization of antigens in tissues, as an

alternative for fluorescent conjugates. In 1971, enzymelabeled antigens and antibodies were developed as

serological reagents for assay of antibodies and antigens.

Their versatility, sensitivity, simplicity, economy and

absence of radiation hazard have made EIAs the most

widely used procedure in clinical serology. The availability

of test kits and facility of automation have added to their

popularity.

The enzyme-linked immunosorbent assay (ELISA),

[Enzyme immunoassay (EIA) or solid-phase immunosorbent

assay (SPIA)] is a sensitive laboratory method used to detect

the presence of antigens (Ag) or antibodies (Ab) of interest in

a wide variety biological sample.

Many variations in the methodology of the ELISA

have evolved since its development in the 1960s, but the

basic concept is still the immunological detection and

quantitation of single or multiple Ag or Ab in a patient

sample (usually serum).

Classification of ELISA

ELISA can be classified in different ways (Fig. 22.16):

Direct ELISA

Direct ELISA is the most basic of ELISA configurations. It is

used to detect an Ag after it has been attached to the solid

phase (e.g. a membrane or dipstick). An Ab conjugated with

a label (e.g. HRPO, AP, FITC) is then incubated with the

captured antigen. After washing off excess conjugate and

incubating with a substrate and chromogen, the presence

of an expected color indicates a specific Ab-Ag interaction.

The conjugate could be a commercial preparation specific

for the Ag of interest, or an in-house conjugated monoclonal

or polyclonal Ab, or even patient serum (Fig. 22.9).

Indirect ELISA

This is extensively used for the detection and/or titration

of specific antibodies from serum samples. The specificity

of the assay is directed by the antigen on the solid phase,

which may be highly purified and characterized. The first,

or primary Ab is incubated with the Ag, and then the

excess is washed off. A second or secondary Ab conjugate

is then incubated with the samples. The excess is again

removed by washing. For color to develop, a primary

Ab that is specific for the Ag must have been present

in the sample (e.g. human serum, CSF or saliva). This

indicates a positive reaction. It is important, during assay

FIG. 22.9: Direct ELISA

Serology/Immunology 573

optimization, to ensure that the secondary Ab does not

bind nonspecifically to the Ag preparation or impurities

within it, nor to the solid phase (Fig. 22.10).

Capture ELISA

Antigen Capture

In this, more specific approach, a capturing Ab is adsorbed

onto the solid phase. The capture antibody may be the

reagent to be tested (e.g. the titer of a patients immune

response to a known Ag). However, the Ab may be a

standard reagent and the antigen the unknown (as when a

patient’s serum is being investigated). The same stringent

optimization is required as for indirect ELISA. This will

ensure that the Ab does not cross-react in the absence of

Ag, or nonspecifically binds to the solid phase. It is also

important, when detecting the Ag, to use Ab from different

animal species to prevent same-species Ab binding (e.g.

a polyclonal rabbit capture Ab will capture a monoclonal

conjugate if it was raised in rabbits. This will produce a

positive result in the absence of Ag) (Fig. 22.11).

Antibody Capture

In this approach, a capturing Ab is adsorbed onto the solid

phase. The Ab is designed to capture a class of human

Ab (e.g. IgG, IgA or IgM). Next, the sample is applied,

containing the Ab under investigation. After washing,

an Ag specific for the Ab is added and finally an anti-Ag

conjugate provides the signal (Fig. 22.12).

Another approach is to coat antigen on the solid surface.

The antibody (from the sample) binds with it. After washing

an anti-antibody (antibody against antibody) conjugated

with enzyme is added.

Competitive ELISA

This implies that two reactants are trying to bind to a

third. Proper competition assays involve the simultaneous

addition of two competitors. It can be of various types.

Direct Antibody Competition

In this, the solid phase is coated with antigen. The labeled

and unlabeled antibodies both compete for the limited

binding sites for the antigen (Fig. 22.13).

Direct Antigen Competition

This is same as above except that the solid phase is coated