Immunological studies of thymine dimer quantitation.
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Date
1992
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Abstract
Ultraviolet irradiation of DNA induces the formation of a
number of mutagenic lesions. The most prolific of these is
the cis-syn thymine dimer (formed maximally at 260 nm) and
this has been implicated in the reaction pathways that lead
to ultraviolet-induced carcinogenesis.
In order that the molecular events underlying these
neoplastic events be understood, it is imperative that the
thymine dimers formed in ultraviolet-irradiated thymine
containing systems be quantitated. In this laboratory,
dimer quantitation is performed using reverse phase high
performance liquid chromatography (HPLC) with ultraviolet
(DV) detection and the data obtained has allowed a kinetic
mechanism for lesion formation to be proposed.
Such studies have used in vitro thymine containing
substrates (aqueous thymine, thymidine, thymidylyl-3',5'thymidine,
calf thymus DNA and pUC19 plasmid DNA) to
generate the thymine dimer using DV irradiation. with the
planned extension of this research to in vivo cellular
systems (where DNA and hence thymine concentrations are
intrinsically less than those of in vitro systems), a more
sensitive technique for thymine dimer quantitation is
required.
An immunological approach to providing this technique was
chosen. Here, DV-irradiated DNA was injected into rabbits
whose immune system mounted a ' response (i.e. antibody
production) to the DV-DNA antigen. Blood was drawn from the
rabbits at regular intervals to obtain the antibodies. The
technique of immunoblotting was chosen and developed to
allow detection of the thymine dimer antigen. This involved
the reaction between the UV-DNA antigen, the primary
antibody (generated by the rabbit) and a secondary antibody
conjugated to an enzyme, all of which were immobilized on
a commercially available membrane system.
Detection and quantitation of the immune complex
immobilized on the membrane was performed using the
technique of enhanced chemiluminescence. Upon addition of
a chemiluminescent substrate (luminol) to the immune
complex, the horseradish peroxidase enzyme catalysed the
reaction of luminol, with one of the products being light
of 425 nm to 430 nm. This light impinged on a luminescence
film which was developed and printed using standard
photographic techniques. The use of dilutions of the
primary antibody in the immunoblotting protocol with
enhanced chemiluminescent detection, allowed correlations
of antibody dilutions with UV-DNA antigen to be made.
This immunoblotting technique with enhanced
chemiluminescent detection has been used successfully in
detecting thymine dimer lesion formation at levels
currently above the detection limit of the HPLC. It has
also been used successfully in detecting and quantitating
thymine dimers at levels undetectable by the HPLC. To this
end it has proved to be 4000 to 8000 times more sensitive
than the chromatographic technique.
Any immunological technique requires that the antibody of
interest be purified and characterized. Here, purification
of the crude serum was performed using the classical
technique of ammonium sulphate precipitation of proteins.
As an alternative technique, affinity chromatography was
performed on the crude serum using a Memsep 1000 affinity
chromatography cartridge attached to a preparative HPLC
system. Chromatographic data illustrating this purification
are given. Characterization of the DV-DNA antigen was
performed by considering the specificity of the antibody
response in the laboratory animal.
Support for the kinetic mechanisms previously proposed for
pyrimidine dimer formation in DNA is also given in this
work. Calf thymus DNA was irradiated and dimer yields
obtained by immunoblotting. These were used in the computer
programme CAKE together with the previously determined rate
constants to determine simulated dimer yields. A good
agreement between experimental and simulated data indicated
the validity of the mechanism at a DNA concentration of
0.025 mg/ml.
Description
Thesis (M.Sc.)-University of Natal, Durban, 1992.
Keywords
Thymine., DNA., Photochemistry., Immunoassay., Theses--Chemistry.