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Tumor marker protein and antibodies thereto for cancer risk assessment or diagnosis

  • US 5,310,653 A
  • Filed: 02/02/1993
  • Issued: 05/10/1994
  • Est. Priority Date: 10/24/1989
  • Status: Expired due to Term
First Claim
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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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