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Color
Immunochromatographic
Assay (CICA),
 TEST
FORMAT
DESCRIPTIONS
CLINICAL
LABORATORY
ANALYZERS
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Color Immunochromatographic
Assay (CICA), |
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Immunochromatographic,
Lateral Flow or Strip Tests Development Ideas
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About the
Technology:
Immunochromatographic assays, also called lateral flow tests or
simply strip tests, have been around for some time. They are a
logical extension of the technology used in latex agglutination
tests, the first of which was developed in 1956 by Singer and
Plotz.1 The benefits of immunochromatographic tests include:
1. User-friendly format.
2. Very short time to get test result.
3. Long-term stability over a wide range of climates.
4. Relatively inexpensive to make.
These features make strip tests ideal for applications such as
home testing, rapid point of care testing, and testing in the
field for various environmental and agricultural analytes. In
addition, they provide reliable testing that might not otherwise
be available to third world countries.
The principle behind the test is straightforward, and will be
discussed in greater depth in a subsequent section. Basically, any
ligand that can be bound to a visually detectable solid support,
such as dyed microspheres, can be tested for qualitatively, and in
many cases even semi-quantitatively. Some of the more common
lateral flow tests currently on the market are tests for
pregnancy, Strep throat, and Chlamydia. These are examples of
conditions for which a quantitative assay is not necessary.
Reaction Schemes:
The two predominant approaches to the tests are the
Non-Competitive (or direct) and Competitive (or competitive
inhibition) reaction schemes. These can best be explained
graphically, as shown in Figures 1 and 2:
Direct (Double Antibody
Sandwich) Reaction Scheme
This format is used when testing for larger analytes with multiple
antigenic sites, such as LH, hCG, and HIV. In this case, less than
an excess of sample analyte is desired, so that some of the
microspheres will not be captured at the capture line, and will
continue to flow toward the second line of immobilized antibodies,
the control line. This is species-specific anti-immunoglobulin
antibodies, specific for the conjugate antibodies on the
microspheres.
Competitive Reaction Scheme:
This is used most often when testing for small molecules with
single antigenic determinants, which cannot bind to two antibodies
simultaneously. If this format is chosen, it is important to pay
close attention to the amount of antibody bound to the
microspheres, in relation to the amount of free antigen in the
sample. If the sample does not contain an excess of free antigen,
some of the microspheres will bind at the capture line, giving a
weak signal, and making the test result ambiguous.
Typically, the membranes used to hold the antibodies in place are
made up of primarily hydrophobic materials. Both the microspheres
used as the solid phase supports and the conjugate antibodies are
hydrophobic, and their interaction with the membrane allows them
to be effectively dried onto the membrane. These hydrophobic
interactions are very reliable, so much so, that getting the
hydrophobically bound antibody/microsphere complexes to enter into
the mobile phase upon sample introduction can be difficult. One
variation to the above reaction schemes which has been proposed is
the "Boulders in a Stream" approach2.
This gets around the problem of protein-coated microspheres
sticking to the membrane non-specifically by using a membrane that
is inert, and does not bind antibodies. This makes migration of
the mobile phase antibodies very efficient and reliable. The
capture antibodies, rather than being physically bound by the
membrane, are attached to large microspheres, which will be held
in place physically, rather than chemically, as the sample passes
by, much like boulders in a stream. This can be used for both of
the above-mentioned reaction schemes, and is diagrammed below
(Fig. 3):
These principles are well-documented in the literature, and appear
very straightforward. However, in order to maximize efficiency and
minimize development and production costs, there are some
guidelines which, if followed, could possibly reduce some of the
hurdles normally associated with the development of a new
technology.
Materials:
Antibodies - three types:
1. Stationary Phase
a. Capture Line Antibodies
b. Control Line Antibodies
2. Mobile Phase
a. Conjugate Antibodies (Antibodies on dyed microspheres, to which
the sample analyte will bind initially)
(If you are testing for small molecules using the competitive
binding format, you will also need purified antigen, or an
antigen/carrier molecule (BSA) conjugate, for attachment to test
lines.)
Membranes - dependent upon the approach that you choose, as
previously mentioned. Some options for this include:
1. Nitrocellulose (High Protein Binding)
2. Cellulose Acetate (Low Protein Binding)
3. Glass Fiber Membranes (Non-Protein Binding)
Membrane manufacturers generally offer a wide variety of material
types and pore sizes, so it is a good idea to investigate several
options before deciding which specifications most closely match
your test objectives.
Microspheres - several sizes and polymers to choose from.
Conjugate antibody or antigen is attached, and microspheres
migrate down the membrane upon introduction of your sample. Some
hints to choosing an appropriate particle are as follows:
1. Optimal flow rate is achieved by choosing microspheres 1/10 the
pore size of the membrane through which they will migrate, or
smaller.
2. Optimal colors for visualization in various types of samples:
a. Whole Blood: Black or Dark Blue
b. Serum: Bright Red or Bright Blue
c. Urine: Green, Blue, Red, or Black
d. Saliva: Any Dark Color
e. Cerebral Spinal Fluid: Any Dark Color
3. To minimize hindered flow caused by the inherent hydrophobic
interactions between membrane and particle (in the case of a
hydrophobic membrane), pretreatment of the membrane with a
substance that will maintain a small distance between the
microspheres and the membrane, yet which can be easily rehydrated,
is often helpful (Fig. 5). Examples of substances commonly used
are sucrose, various water soluble inert polymers, and
surfactants. The idea is to choose a substance that is stable in
dry form, yet dissolves easily upon rewetting to allow the
antibody bound microspheres to flow easily through the membrane
upon addition of the sample. (A sample procedure for doing this is
included later in this text.)
In addition to treating the
membranes, the microspheres themselves can also be pre-treated
with surfactants, synthetic or protein-based blockers. If done
correctly, this can also help to reduce the problem of reagent
mobility upon sample introduction. Much work has been done in
developing optimum mixtures of these various polymers, detergents,
and blockers.
Reaction Kinetics:
Now that we have looked at the principles behind these tests and
some specifics regarding their manufacture, let's consider some of
the factors involved in choosing the appropriate raw materials.
Test developers are often concerned with reaction kinetics. A
faster test will not only be more attractive commercially, but
often will be more accurate. On the other hand, the test must
proceed slowly enough that antibody/antigen reactions are able to
occur. Some principles that govern the kinetics of
immunochromatographic assays are as follows:
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