The universality of immuno-PCR for ultrasensitive antigen detection
| Autor: | Alastair D. Burt, Marian Case, Glenn N. Major, Margaret F. Bassendine |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
DNA damage
DNA polymerase DNA repair Immunoblotting Enzyme-Linked Immunosorbent Assay Biochemistry Polymerase Chain Reaction Sensitivity and Specificity law.invention chemistry.chemical_compound DNA Adducts O(6)-Methylguanine-DNA Methyltransferase law Humans Antigens Polymerase chain reaction Electrophoresis Agar Gel biology DNA Methyltransferases Molecular biology Peptide Fragments chemistry Biotinylation Agarose gel electrophoresis biology.protein Ethidium bromide |
| Zdroj: | Europe PubMed Central |
| ISSN: | 0300-5127 |
| Popis: | Immuno-PCR is a relatively new method for ultrasensitive antigen detection that utilises polymerase chain reaction (PCR) from a secondary antibody-target DNA conjugate to detect the specific binding of a primary antibody to its target antigen [l] . There have been several reports of the use of immuno-PCR, but the universality of a single protocol for low-level detection of a variety of antigens has not been confirmed. We are seeking to measure the very low levels of tissue DNA damage (adducts) that are expected to be formed following incidental human exposure to potentially carcinogenic alkylating agents. Detection and quantitation of such low-level damage is technically difficult. This is the case for the powerfully promutagenic DNA base lesion 06-methylguanine, which may be produced in human DNA following exposure to methylating agents such as the environmentally ubiquitous nitrosamines. To assess the feasibility of using immuno-PCR for detection of very low levels of a DNA adduct and the universality of the method for detecting a variety of different antigens, we established an immuno-PCR protocol using purified rabbit polyclonal antibodies recognising four biochemically diverse antigens: pyruvate dehydrogenase complex (PDC; oligomeric; Mr 8.5 x lo6), the DNA repair enzyme 06-methylguanine-DNA methyltransferase (MGMT; monomeric; Mr 2 1,700), an oligopeptide from the N-terminal end of human MGMT (20-mer; Mr 2,3 10) and a DNA adduct (06-methylguanosine) conjugated to BSA (06-MeG-BSA; Mr 67,600). This protocol is a modification of the method of Zhou et a1 [2], the basic principle of which relies upon detection of antigen-bound antibody by the sequential use of (i) a second antibody-streptavidin conjugate directed against the primary antibody, (ii) a biotinylated target DNA (which will bind to streptavidin with high affinity) and (iii) PCR for the detection of the immobilised target DNA. Our modifications include (i) the biotinylated target DNA for PCR, which was produced using a fragment of the gene for ahydroxynitrile lyase from the Cassava plant. (A BamH 1 restriction enzyme site was added to this fragment for subsequent digestion and fill-in labelling of the 5'-end of the cut DNA using biotinylated-14-dATP and the Klenow fragment of DNA polymerase), (ii) using 96-well polycarbonate plates for thermocycling and (iii) using NeutrAvidin (Pierce) instead of streptavidin. PCR products were analysed by agarose gel electrophoresis, with ethidium bromide staining of amplified DNA. Antigen detection limit was defined by the point of disappearance of the ethidium bromide signal. A standard peroxidase-based sandwich enzyme linked immunosorbent assay (ELISA) was performed with antigen coated on 96 well plates to serve as a comparison for the immunoPCR. Serum IgG was purified by protein A-Sepharose affinity chrom-atography for use in both immuno-PCR and ELISA. |
| Databáze: | OpenAIRE |
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