This is the pipetting step. It is done by either manual or
electronic dispensing systems. The tips used must be
compatible with the pipette. Multichannel pipettes can
be used for addition of common reagents like conjugates,
substrates, stop solution, etc. The advantage of electronic
dispensing system is that errors are minimized. During
pipetting some bubbles may be formed in the well. They
should be burst using a pin. Different pins should be used
for breaking different wells, as usage of same pin may lead
It is time period during which antigen combines with
antibody or enzyme reacts with substrate. There are two
types of incubation—stationary and rotatory incubation.
In stationary—incubation, mixing takes place through
diffusion of reagents. Because stationary incubation
relies on diffusion of molecules, the role of temperature
becomes extremely critical. To ensure complete
This leads to increased contact between analyte and the
capture/adsorbed reactant. Rotation gives additional
kinetic energy to the system and hence, the reaction is less
It is actually a dilution process to optimally dilute the
original solution without stripping off the bound/capture
protein. It is one of the critical steps in ELISA. The optimal
dilution step requires 3–5 cycles. Less than 3 cycles will
leave behind residual proteins in the wells. The volume
of wash solution dispensed per well should be high
enough to cover the entire surface coated with antigen/
antibody. The entire well must be filled during the wash
cycle. Enough care is needed to prevent well-to-well
overflowing of wash solution. During washing, more
specifically in aspiration step, it is recommended to leave
a small amount of wash buffer in the wells. This creates
a film on the well and thus, prevents denaturation due
to drying effect. The liquid used to wash wells is usually
buffered (PBS) in order to maintain isotonicity, since
most Ag-Ab reactions are optimal under such conditions.
Tap water is not recommended, since tap water varies
greatly in composition (pH, molarity and so on).
The estimation of color can be done either visually (for
rapid tests, Western blots, etc.) or using an ELISA reader.
It is an instrument to measure the optical density and
give the interpretation according to the program. The
instrument can be programmed to do calculation and
print the results. In case of coated tubes, the measurement
is done by an analyzer (Fig. 22.17).
Despite advances in the design of immunoassays, the
problems of unwanted interference have yet to be
completely overcome. An ideal immunoassay should have
¾ The immunochemical reaction behavior should be
identical and uniform for both the reference (standard/
calibrator) preparation and the analyte in the sample
¾ The immunochemical reaction of the reagent is uniform
¾ The immunochemical method is well standardized to
ensure that the size of measurement signal is caused
only by the antigen-antibody reaction
¾ For macromolecules, the results declared in arbitrary
units (IU—International Units), the conversion to (SI)
units is not constant and depends on many factors.
Interference may be defined as “the effect of a substance
present in an analytical system which causes a deviation of
the measured value from the true value, usually expressed
as concentration or activity.”
The IFCC (International Federation of Clinical
Chemistry) offers the following definition: “Analytical
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