The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (Nl). Cells are re-suspended to 2-5x106 cells/ml with hbss in a 50-ml conical tube. The phosphatase, if used, is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs.
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0747 As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In the present invention, a "polynucleotide fragment" refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in Clone ID NO:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide. 01274 The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background ofthe Invention, Detailed Description, and Examples is hereby incoorated herein by reference. Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder.
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A typical source of EBV is the culture supernatant of the B95-8 cell line (atcc #VR-1492). Plasmid DNA is islated and confirmed by restriction analysis. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The amplified promoter and targeting sequences are digested and ligated together.
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The reactions are analyzed on either 8 polyacrylamide gels.5 agarose gels. Clones positive in elisa are further characterized by PCR fingerprinting (see,.g., International Application. The culture is incubated for 1 hour at 37 C without shaking and then for a further hour at 37C with shaking.
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A preferred embodiment of the invention is the detection and diagnosis of a disease or disorder ofthe testes associated with aberrant expression of a testicular antigen in an animal, preferably a mammal and most preferably a human. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, ppicz, pgapz, pgapzalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, CA).
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