ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 76

(RIA; see below). The most common methods of iodination result in
the chemical oxidation of to reactive radioactive iodine using
lactoperoxidase (an enzyme found in raw cow's milk) or chloramine-T
(chloramines are compounds that are composed of chlorine and nitro-
gen and, interestingly, are widely used as antiseptics), which couple the
to the phenyl side chains of tyrosine residues. Commercially pre-
pared solid phase iodination reagents such as Pierce Biotechnology's
“IODO-BEADS” are also often used.
Biotin-labeled antibodies
Biotin is one of the most commonly used reagents for generating non-
radioactive probes. Biotin is a water soluble vitamin, vitamin H, a member
of the vitamin B complex. The most visible sign of biotin-deficiency is
thinning of the hair. Biotin can be covalently coupled to proteins, and co-
valently incorporated into DNA. Biotin is detected using avidin (an egg
white protein) or streptavidin, both of which bind with high affinity. Strep-
tavidin is a 60 kDa protein from the bacterium Streptomyces avidinii that
has high affinity for biotin with a (a measurement of the dissocia-
tion constant which is used to reflect the affinity of the interaction be-
tween two molecules) of orders of magnitude higher than most
antibodies bind antigens. Streptavidin can be fluorescently labeled or
conjugated with enzymes, such as alkaline phosphatase or horseradish
peroxidase, and therefore can be used as a second-step reagent for
the detection of biotinylated antibody or nucleic acid probes (in blotting
reactions by chemiluminescence) or proteins detected by biotinylated
antibodies by immunofluorescence. Streptavidin-coated beads are also
used for affinity purification of biotinylated proteins or proteins bound to
biotinylated antibodies.
Enzyme-linked antibodies
Enzymes such as horseradish peroxidase (HRP) and alkaline phos-
phatase (AP) can be covalently attached to antibodies to aid in their
detection. Enzyme-linked antibodies are the reagents that make meth-
ods such as ELISA and immunohistochemistry possible (see below).
For example, HRP (isolated from Amoracia rusticana, or horseradish
root) combines with peroxide to oxidize a variety of substrates, and can
be detected by the production of an insoluble product that is red-brown
in color. Enzymes, especially HRP, are also the label of choice for West-
ern blotting techniques using non-radioactive probes in which the bound
antibodies are detected using enhanced chemiluminescence (ECL) to
detect the enzyme linked antibody. Chemiluminescence is the direct