Tumor marker protein and antibodies thereto for cancer risk assessment or diagnosis
First Claim
1. A process for preparing an immunogen of a tumor-associated protein comprising the steps of:
- (a) collecting from plasma, tumor cytosol or ascitic fluid of carcinoma-bearing mammals or from culture medium in which cancer cells were grown, the protein fraction which is precipitated from plasma, tumor, cytosol or ascitic fluid between 30% and 60% saturation of an aqueous ammonium sulfate solution or from culture medium at 90% saturation of an aqueous ammonium sulfate solution, respectively;
(b) chromatographing the protein fraction on a molecular sieving column and collecting a protein fraction having a molecular weight in the range of about 50 to 90 kilodaltons;
(c) applying the collected chromatographed protein fraction to a high performance liquid chromatography (HPLC) phenyl hydrophobic interaction column and allowing protein to bind to the column;
(d) eluting the bound protein on the phenyl hydrophobic interaction column with a buffer comprising about 16% ammonium sulfate;
(e) collecting the first distinct protein peak eluted from the column;
(f) electrophoresing the collected protein;
(g) transblotting the electrophoresed protein to a nitro-cellulose sheet;
(h) cutting from the nitro-cellulose sheet that portion which contains a band of protein corresponding to about 65 kD.
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Abstract
A tumor-associated marker protein was purified and antibodies thereto developed for cancer diagnosis and assessment of cancer risk associated with the long-term use of synthetic steroid hormones, both contraceptive and non-contraceptive, and other drugs that exhibit tumor promotional properties. The marker protein and antibodies thereto provided are interspecies immunologically cross-reactive.
In summary, the marker p65 tumor-associated factor of the present invention has the following characteristics:
(a) binds substantially completely to a phenyl hydrophobic interaction column in a buffer containing 20% ammonium sulfate and eluted at ca. 16% ammonium sulfate;
(b) localized primarily in the nuclear envelopes with only small amounts present in the cytoplasm from where is released to the blood circulation in vivo or cell culture medium in vitro;
(c) induced in normal, adult tissues by chemical carcinogens (initiators) but not by tumor promoters, the carcinogen-induced production being enhanced by the latter.
Also disclosed herein are processes for purifying the 65 kDa tumor marker from plasma, tumor cytosol or ascitic fluid of carcinoma bearing animals; processes for producing antisera and purified antibody preparations to the 65 kDa tumor marker; and methods using antibody to the 65 kDa to diagnose or assess the likelihood of cancer.
72 Citations
10 Claims
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1. A process for preparing an immunogen of a tumor-associated protein comprising the steps of:
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(a) collecting from plasma, tumor cytosol or ascitic fluid of carcinoma-bearing mammals or from culture medium in which cancer cells were grown, the protein fraction which is precipitated from plasma, tumor, cytosol or ascitic fluid between 30% and 60% saturation of an aqueous ammonium sulfate solution or from culture medium at 90% saturation of an aqueous ammonium sulfate solution, respectively; (b) chromatographing the protein fraction on a molecular sieving column and collecting a protein fraction having a molecular weight in the range of about 50 to 90 kilodaltons; (c) applying the collected chromatographed protein fraction to a high performance liquid chromatography (HPLC) phenyl hydrophobic interaction column and allowing protein to bind to the column; (d) eluting the bound protein on the phenyl hydrophobic interaction column with a buffer comprising about 16% ammonium sulfate; (e) collecting the first distinct protein peak eluted from the column; (f) electrophoresing the collected protein; (g) transblotting the electrophoresed protein to a nitro-cellulose sheet; (h) cutting from the nitro-cellulose sheet that portion which contains a band of protein corresponding to about 65 kD. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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Specification