Biochemistry – COLOURIMETRIC ASSAYS

Immunoblot Assay

● Using a micropipette, apply the stated volume of serum onto a nitrocellulose membrane. The spot
must be as small & concentrated as possible so that its color can be clearly observed in subsequent
steps.
● Incubate the nitrocellulose membrane in blotto. Blotto contains known proteins like albumin which
bind to the remaining areas of the membrane not bound to antigens in the serum.
● Incubate the nitrocellulose membrane in wash buffer. This is to remove any unbound proteins from
the excess blotto.
● Remove the nitrocellulose membrane using forceps, taking care not to touch the membrane with
your fingers.
● Drain the excess wash buffer from the membrane by touching the corner of the membrane to a dry
paper towel.
● Incubate the nitrocellulose membrane in a solution of antibody 1 for specific binding to the
complementary antigen on the membrane.
● Incubate the nitrocellulose membrane in wash buffer to remove any non-specific binding.

● Remove the nitrocellulose membrane using forceps, taking care not to touch the membrane with
your fingers.
● Drain the excess wash buffer from the membrane by touching the corner of the membrane to a dry
paper towel.
● Incubate the nitrocellulose membrane in a solution of antibody-enzyme conjugate (usually Ig-
horseradish peroxidase) to allow the antibody domain of the conjugate to bind to any Antibody 1 on
the membrane.
● Incubate the nitrocellulose membrane in wash buffer to remove any non-specific binding.

● Remove the nitrocellulose membrane using forceps, taking care not to touch the membrane with
your fingers.
● Drain the excess wash buffer from the membrane by touching the corner of the membrane to a dry
paper towel.
● Incubate the nitrocellulose membrane in the substrate solution (peroxide substrate). The enzymatic
domain of the conjugate catalyzes the hydrolysis of the substrate to give an observable precipitate.
● A colored dot (formation of precipitate) indicates a positive result. The absence of such a colored
dot indicates a negative result.

ELISA Assay
● You will be instructed to prepare serial dilutions of a standard solution of antibodies. You will then
add volumes of an antigen stock solution, sample serum(s) & the diluted antibody solutions into two
or more separate rows of a microtiter plate.
● Remember to orientate the plate correctly using the numbers along the top edge of the plate &
letters along the left edge of the plate. This is important is filling the correct wells in subsequent
steps, otherwise you will get a negative/zero reading if you fill the wrong wells as the
spectrophotometer is only programmed to read certain wells.
● Basic sequence of steps for preparing the wells in the microtiter plate: Add antigen/serum &
incubate for binding to wells; wash with buffer to remove unbound antigen; add blocking agent &
incubate to bind to the remaining areas of the membrane not bound to antigens in the serum; wash
with buffer to remove unbound blocking agent; add diluted primary antibody solutions into
respective wells & incubate for binding to antigen; wash with buffer to remove non-specific binding;
add diluted secondary antibody solutions into respective wells & incubate for binding to primary
antibody; wash with buffer to remove non-specific binding; add substrate & incubate for color
development in wells; add stop solution to wells to stop the reaction; use of pre-programmed
spectrophotometer to read absorbance of solutions in the wells.
● Every time you are instructed to empty the wells, invert the plate over the sink in one swift, vertical
downward motion. Do not tilt the plate diagonally to empty the wells as this will result in cross-
contamination of wells.
● Every time you are instructed to dry the wells, invert the plate & pat dry over paper towels.

Principles

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