Addition

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

to carry over.

Incubation

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

reaction, longer incubation time is recommended. Rotatory incubation ensures complete mixing of reagents.

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

dependent on temperature.

Wash

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

Serology/Immunology 579

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).

Estimation

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).

Interferences in Immunoassays

Despite advances in the design of immunoassays, the

problems of unwanted interference have yet to be

completely overcome. An ideal immunoassay should have

the following attributes:

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

from batch to batch

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.

Definition of Interference

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

interference is the systematic error of measurement

caused by a sample component, which does not, by itself,