Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-05-30
2000-04-18
Eyler, Yvonne
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 723, 435810, 435975, 530324, 530325, 530326, 536 235, 536 231, G01N 3353, G01N 33574, A61K 3800, C07H 2104
Patent
active
060513845
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method of detecting p53-specific antibodies in body fluids. It further relates to a kit useful therefor. Also, the invention relates to p53 fragments and DNA sequences coding for them, said fragments comprising binding regions for p53-specific antibodies, as well as to methods of preparing them.
Eukaryotic cells are known to express a protein identified as p53 (p53). This protein is known in terms of its primary structure and comprises 393 amino acids (see Lamb, P. and Crawford, L. V., Mol. Cell. Biol., Vol. 6 (1986), 1379-1386). Increased expression of p53 is found in many tumor diseases. This may be accompanied by the presence of specific anti-p53 antibodies. A variety of methods for the detection of such antibodies have been reported. For instance, p53-containing cell extracts are radioactively labelled in vivo and immunoprecipitated with sara from patients (see de Fromentel, C.C. et al., Int. J. Cancer 39 (1987), 185-189). Also, anti-p53 antibodies are coated on supports where p53 is adsorbed from cell extracts and added to sera from patients. Bound anti-p53 antibodies are detected with iodine-coupled protein A (see Crawford, L. V. et al., Mol. Biol. Med. 2 (1984), 261-272).
In those methods of detection radioactive substances are employed. This means higher safety risk and thus limited utility, especially in hospitals. Further, as the in vivo labelling of cells is expensive and requires extensive equipment, this method is useful only to a limited extent in large-scale, serial experiments. Also, the use of cell extracts frequently leads to unspecific antibody binding.
Therefore, it is the object of the present invention to provide a method of detecting p53-specific antibodies that does not involve the aforementioned drawbacks.
In accordance with the invention, this object is achieved by a method in which support-bound p53 and/or support-bound fragments thereof comprising binding regions for p53-specific antibodies are incubated with body fluids and the specific antibodies (a) bound to p53 and/or fragments thereof are allowed to react labelled antibodies (c) directed against antibodies (b), with the labelling being non-radioactive in either case.
The term "p53" includes a p53 protein comprising a wild-type sequence. Such a protein can be isolated from cells such as HepG2 (see Knowles, B. et al., Science 209 (1990), 497-499). p53 may also have a sequence other than one of the wild type. Such a sequence may include additions, deletions and/or substitutions of one or more amino acids. Further, p53 may be part of a fusion protein. Such a protein, like any other p53, can be isolated from cells expressing said protein after genetic engineering. Such cells include both prokaryotic and eukaryotic cells. Examples of the former include E. coli strains such as BL21 (see Studier, F. W. et al., Methods in Enzymology 185 (1990), 60-89), while the latter are exemplified especially by mammal, yeast and insect cells. Genetic engineering includes conventional methods known in the art whereby nucleic acids having particular sequences are prepared, inserted in cells and expressed. Anybody skilled in the art is aware of the materials such as vectors and conditions useful in those methods (see, egg., Maniatis, T. et al., Cold Spring Harbor Laboratory, 1982).
According to the invention, a p53 cDNA from HepG2 cells (see above) is subjected to reverse transcriptase in a conventional manner and amplified in a common PCR process. The cDNA is compared to published data (egg., EMBL gene library) in terms of its sequence and inserted in the known vector pet 3d to give the recombinant vector pet 92/2. This vector is transformed to the bacterial strain BL21 (see above) to express p53. p53 induction is achieved by adding IPTG. The bacteria are sedimented and subjected to lysozyme and DNase I treatment after freezing and thawing. The lysates are incubated with urea solutions having different concentrations, and p53 is released after centrifugation and obtained in pure form by polyacrylamide gel electrophoresis
REFERENCES:
patent: 5652115 (1997-07-01), Marks et al.
Harlow et al, "Antibodies: A laboratory Manual" Cold Spring Harbor Laboray, Chapter 14, pp.555-566 and 590, 1988.
Harris et al, "Molecular basis for heterogeneity of the human p53 protein" Mol. Cell. Biol., vol. 6, No. 12, p. 4650-4656, Dec. 1986.
Lamb et al, "Characterization of the human p53 gene", Mol. Cell Biology, vol. 6, No. 5, p. 1379-1385, May 1986.
Vojtesek et al, "An immunochemical analysis of the human nuclear phosphorprotein p53", J. of Immunol. Methods, vol. 151, p. 237-244, Jun. 1992.
Hassapoglidou et al, Antibodies to the p53 Tumor Suppressor Gene Product Quantified in Cancer Patient Serum With a Time-Resolved Immunofluormetric Technique, Dec. 1992.
Klein Ralf
Schranz Peter
Tessmer Claudia
Volkmann Martin
Zentgraf Hanswalter
Deutsches Krebsforschungszentrum Stiftung des Offentlichen Recht
Eyler Yvonne
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