Analytical sensitivity refers to intra assay precision, whereas
functional sensitivity refers to inter assay precision.
Rapid Immunochromatographic Techniques
Perspective on Membrane-based Rapid
The need for a rapid, reliable, simple, sensitive in vitro
diagnostic assay for use at point-of-care, have lead to
the commercialization of in vitro Rapid Diagnostic Tests
based on the principle of immunochromatography.
Rapid Diagnostic Tests are membrane-based
immunoassays that allow visual detection of an analyte
in liquid specimens. In clinical assays, specimens such
as urine, whole blood, serum or plasma, saliva and other
What are the Principles of Membrane-based Rapid
Currently available Rapid Diagnostic Tests comprise of a
base membrane such as nitrocellulose. A detector reagent
(antigen/antibody-indicator complex) specific to the
analyte, impregnated at one end of the membrane. A capture
reagent is coated on the membrane at the test region.
When the specimen is added to the sample pad,
it rapidly flows through the conjugate pad. Analyte if
present in the specimen, binds to the detector reagent.
As the specimen passes over the test band to which the
capture reagent is coated, the analyte-detector reagent
complex is immobilized. A colored band proportional to
the amount of analyte present in the sample, develops.
The excess unbound detector reagent moves further up
the membrane and is immobilized at the control band.
What are the Components of Membrane-based
Rapid Diagnostic Tests and how are they
Rapid Diagnostic Test consists of (Fig. 22.4)
in the conjugate pad but remains unbound
3. Test band: Coated on nitrocellulose membrane;
4. Control band: Usually antidetector antibodies coated
on the membrane, served to validate the test results
Currently, immunochromatography tests are available
in two formats; “lateral flow” and “transverse flow or flow
through”. The lateral flow formats are available in device
or dipstick format. The lateral flow formats are commonly
employed where rapid detection of pregnancy, drug abuse,
infectious disease or parasitology is required, and serve
as qualitative screening assay at laboratories, physician’s
office or at homes due to their simplicity and ease of
performance. The flow through format is less common
as the assay requires greater operator involvement.
However, some of these assays enable semi-quantitative
estimation of the analyte by visual comparison with an inbuilt reference.
Regardless of the format used, the desired specificity,
sensitivity and assay performance depends upon reliable
formulation and proper assay assembly.
What are the Limitations and Effects of Various
Components on the Performance of Membrane
This section highlights the role of various components of
Rapid Diagnostic Tests and their effect on attaining the
desired performance characteristics.
How does the Nitrocellulose Membrane Affect the
Sensitivity of Rapid Diagnostic Tests?
Rapid Diagnostic Tests are fabricated on a solid support
membrane, usually made of nitrocellulose. Membranes
employed in Rapid Diagnostic Tests are porous.
Depending upon the porosity, some membranes are
better suited for applications with certain specimens than
others. This is because, the pore size of the membrane
has significant effect on the capture reagent binding
properties and the lateral flow rate. The combined effects
of these two phenomena in turn determine the sensitivity
and performance of the test assay.
Pore Size and Capture Reagent Binding Properties
It has been observed that as the pore size decreased the
effective surface area available for binding of capture
reagent increases. Greater effective surface area available
for binding, results in optimal coating of the capture
reagent, which is essential for attaining the desired
Pore Size and Lateral Flow Rate
It has been observed that as the pore size increases, the
lateral flow rate increases. However, slower flow rate
increases the effective concentration (concentration
required for interaction) of the analyte, since a slower
flow rate allows the analyte and the capture reagent
to be in close proximity for a longer times. As it is well
known, immunological reactions are time-dependent and
prolonged exposure of the analyte with the capture reagent
allows better interaction and thus, results in increased
sensitivity. The flow rate is important when the analyte is
present in low concentrations, such as borderline samples.
The relationship between lateral flow rate and effective
Thus, it is important to optimize the membranes such
that Rapid Diagnostic Tests can achieve rapid results
which are also reliable and accurate.
Why are Colloidal Gold Sol Particles Commonly
Employed in the Detector Reagent in Membranebased Rapid Diagnostic Tests?
Interpretation of results in Rapid Diagnostic Tests depends
upon the development of a signal at the stipulated time.
FIG. 22.4: Construction of rapid diagnostic tests
the indicator. The indicator imparts color to the signal,
enabling visual interpretation of results.
Colored latex particles, colloidal gold sol particles, dyes,
enzymes and carbon particles are some of the indicator
used in immunochromatographic assays. However,
stability, protein-binding properties, and particles’ size are
immunochromatographic assays is the colloidal gold sol
Colloidal Gold Sol Particles as Indicator
Homogeneous colloidal gold sol particles are inert and
can couple with antibody/antigen, which is stable in
dry as well as in liquid forms. All the above-mentioned
parameters are determined by the particles’ shape and
Effect of Shape of Colloidal Gold Sol Particles on
Colloidal gold sol particles have a net negative charge
called “zeta potential”. This zeta potential maintains the
be spherical in shape, since, this shape allows uniform
distribution of zeta potential at the surface. In case of
nonhomogeneous particles, the zeta potential is not
uniformly distributed, thus the particles may come together
to form aggregates. These aggregates may permanently
get impregnated into the conjugate pad, or during the
test assay may deposit on the nitrocellulose membrane
leading to discrepant results. Such nonhomogeneous
colloidal gold is usually blue/black in color.
Effect of Shape of Colloidal Gold Sol Particles on
Spherical, homogeneous colloidal gold sol particles also
allow uniform coating of the detector reagent at their
surface. Whereas non-homogeneous colloidal gold sol
particles do not allow uniform coating of detector reagent,
resulting in decreased assay sensitivity and specificity.
Effect of Size on Color of Colloidal Gold Sol Particles
It has been observed that as the colloidal gold sol particles
increase in size, the color turns from light pink to cherry
red to red-purple to blue-black to gray-black. Darker
colored particles are preferred in Rapid Diagnostic Tests
since darker colors allow easy interpretations of results.
However, as the colloidal gold sol particles increase in
size, these particles are less stable and aggregate together.
Secondly, due to the steric hindrance, the larger colloidal
gold sol particles tend to dwarf the coated antigen/antibody
making interaction with the analyte difficult (Fig. 22.5).
Ideally, the colloidal gold sol used in immunochromatographic assay is ~40 nm in size and imparts a
cherry red color, which enables optimal visualization of
results against a clear white background and is stable in
dry and liquid forms. However, purple colored colloidal
gold sol particles if properly stabilized, can also be used in
Why are Variations in Band Appearance Commonly
Observed in Membrane-based Rapid Diagnostic
Tests Employed for Antigen Detection?
The sensitivity/specificity of Rapid Diagnostic Tests
primarily depends upon the detector and capture reagent
pair. Ideally, the detector reagent should be specific to one
epitope of the analyte and the capture reagent specific
to another epitope of the same analyte, thereby enabling
two-site sandwich immunoassay. To illustrate the same,
FIG. 22.5: Graph of particle’s size v/s signal color of colloidal gold sol
FIG. 22.6: Two-site sandwich immunoassay
For higher analyte sensitivity, manufacturers of
commercial Rapid Diagnostic Tests for antigen detection
depend on the use of various combinations of capture
reagents at the test and control band. Avid capture reagents
have a high affinity for the analyte. When the sample
containing the analyte reaches the avid capture reagent
at the best band, due to high affinity, the avid reagent at
the edge of the band captures most of the analyte. Thus,
resulting in a distinct thin colored line at the edge of the
On the other hand, use of less avid capture reagent
(lesser affinity for the analyte) results in capture of the
analyte uniformly across the test or control band. Thus,
broader bands are generated by less avid antibodies.
Variations in band appearance in different assays is
due to use of varying avidity of the antibodies at the test/
What is the Role of Sample Pad in Membranebased Rapid Diagnostic Tests?
Rapid Diagnostic Tests enable detection of the analyte in
several specimens such as urine, whole blood, serum or
plasma. However, the pH, viscosity, ionic concentraction,
turbidity, and total protein content may vary from specimen
to specimen. Variations in these factors can cause alterations
in the colloidal gold particles or the capture reagent
leading to non-specific results. For example, highly turbid
specimens can cause invalid results since the particles from
the specimen may block the membrane preventing sample
flow. Urine specimen becomes acidic on storage due to
bacterial growth. Due to a shift in the pH, the colloidal
gold particles come together to form aggregates which may
interfere in the performance of the test.
Rapid Diagnostic Tests incorporating serum as
specimen may give false-positive results due to the
presence of heterophillic antibodies. These antibodies
have multispecificity and bind the capture reagent to
the detector reagent leading to false positive results. Use
for Rapid Diagnostic Tests incorporating heterophillic
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