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A reverse enzyme-linked immunosorbent assay (ELISA) modified from a prostate antigen (PA) assay previously reported has been developed to measure circulating PA-binding globulin (PABG). Serum specimens taken from normal males, normal females, male patients with nonprostatic cancers, patients with benign prostatic hypertrophy, and patients with various stages of prostate cancer were analyzed for PABG. Results revealed that only patients with an advanced stage of prostatic cancer exhibited an elevated level of PABG. PABG was then isolated from prostatic cancer patients' serum by PA affinity chromatography. Upon immunodiffusion and immunoelectrophoresis, this PABG preparation reacted with purified PA, anti-PA xenoantibodies and anti-human IgG. By the immunoperoxidase technique, PABG stained positively in prostatic ductal epithelial cells and negatively in all other tissues examined. An additional PABG preparation, which reacted with anti-PA and anti-human IgG and not with purified PA, was also isolated by an anti-PA affinity chromatography. These PABG preparations were separately subjected to high-performance liquid chromatography for further purification. Three major protein peaks at Mr of greater than 240K, 150K, and 34K were obtained. These results demonstrated that circulating IgG antibody reactive with PA was present in patients with metastatic cancer of the prostate, and this in part was in complexed form with PA and was specifically reactive with ductal epithelial elements of the prostate.

A pancreas cancer-associated antigen (PCAA) was identified and isolated from ascites fluid of human pancreatic cancer. Purified PCAA was homogeneous as determined by polyacrylamide gel electrophoresis. PCAA was a glycoprotein with a molecular weight of approximately 1,000,000 and consisted of 20% carbohydrates and 80% peptides, had an isoelectric point of 4.7, and migrated to alpha 2-beta region. It possessed a sedimentation coefficient of 14S and appeared to be a fibrous or fibroglobular protein. Immunoreactivity of PCAA was sensitive to proteolytic enzymes, perchloric acid, KSCN, glycine-HCl at pH 2.5, urea and lithium diiodosalicylate; and insensitive to neuraminidase or beta-glucosidase. Immunohistochemical technique revealed that PCAA was located in the cytoplasm of ductal epithelial cells of malignant pancreas. Using heteroantiserum raised against purified PCAA, horseradish peroxidase and CNBr-activated Sepharose 4B, an enzyme-immunoassay (EIA) for circulating PCAA has been developed. From a group of 40 healthy blood donors, an upper limit of 16.2 micrograms of PCAA/ml of serum has been tentatively determined. An elevated PCAA was shown in 67% (29/43) of patients with pancreas cancer, as well as in 30% (11/36) of lung cancer patients, 27% (10/37) of colonic cancer patients, and in 16% (6/36) of breast cancer patients. The reactive antigen in sera of these cancers was shown to be immunologically identical. PCAA also was detected in extracts of various human tissues, particularly pancreatic tumors, colonic tumors, and in a normal colon. Further, PCAA exhibited heterogeneity in molecular weight, isoelectric point, and electrophoretic mobility.

Pieural effusion from a patient with adenocarcinoma of the breast was precipitated by 50% ammonium sulfate and chromatographed on Sephadex G-200. The resulting elution profile consisted of three protein peaks. Fractions were monitored by immunodiffusion and assayed for [125I]C1q binding activity (C1q B.A.). The first protein peak (eluted at the void volume) showed appreciable Clq B.A. and contained immunoglobulin G (IgG) and complement 3 (C3). These data suggested that antigen-antibody complexes were present in this macromolecular peak. IgG and C3 were also present in the second peak of Sephadex G-200 gel filtration while the third peak contained the albumin fraction. The known tumor-associated antigens, carcinoembryonic antigen, alpha-fetoprotein and ferritin, were not detected in any of the protein fractions as determined fay immunodiffusion analysis. The first Sephadex protein peak which contained high molecular weight IgG was chromatographed on Sepharose 6B. The second protein peak obtained by Sepharose filtration contained IgG and C3 and was further applied to a column of protein A-Septtarose. Proteins which bound to the immobilized protein A were dissociated with 2.5 M KSCN and chromatographed on Sepharose CL-6B under dissociating conditions. IgM rheumatoid factor, IgG and four putative antigens were obtained by these dissociation and chromatographic procedures. The isolated IgG exhibited a molecular weight of monomeric IgG and bound to saline extracts of malignant tissue at a greater amount titan to normal or benign breast extracts (p

Levels of Clq binding activity have been measured in the sera of patients with benign and malignant breast disease. Cancer patients showed significantly higher binding activity than patients with gross fibrocystic disease. Clq binding levels were not related to levels of carcinoembryonic antigen, or rheumatoid factor. The Clq reactive material was identified as being precipitable with Protein A, and predominantly eluted in gel filtration fractions with a molecular weight of approximately 1 x 10(6). These studies indicate that Clq binding level may be of use in the differential diagnosis of benign and malignant breast disease.

Ductal carcinoma antigen (DCA), as recognized by monoclonal antibody (MAb) F36/22, was purified in high yield and to homogeneity from malignant effusions by means of physicochemical techniques. Fractionation procedures were monitored by immunoenzymometric assays. Preparations purified over 2,000-fold were obtained through acid-extraction, lectin-chromatographies and gel filtration steps. Purified antigen demonstrated a single component upon electrophoretic examination. This material had a high molecular weight and high immunoreactivity. Neuaminidase treatments failed to perturb the antigenicity of the component, indicating an absence of sialic acid residues at the antibody-combining site. Further, extensive proteolytic digestion effected the release of heterogeneous glycopeptides which retained immunologic activity, suggesting the presence of carbohydrate at the active site. Immunologically, DCA was compared with other tumor-associated mucin-like gly-coproteins which have been detected in the circulation of patients. Results indicated no cross-reactivity between DCA and other mucins, including CA 19–9 and CA-125. Further, no antigenic relationship was noted for purified carcinoembryonic antigen (CEA), also a heavily-glycosylated structure. These data thus suggest that immunological monitoring of disease may be approached using a selected panel of antigenically distinct reagents.

A prostate-specific antigen, distinct from acid phosphatase, was identified by immunologic procedures in prostate tissues (normal, benign hypertrophic, and cancerous) and seminal plasma, as well as in sera of patients with prostatic cancer and of nude mice bearing human prostatic tumor. This antigen was shown by immunoperoxidase staining to be confined to epithelial cells comprising the prostatic ductal elements. Prostate antigen was purified from prostatic tissue and seminal plasma, and it was shown to have a molecular weight of 33,000-34,000 with no subunit component. The isoelectric point of purified antigen was around 6.9, though several unpurified isomers with different isoelectric points also were observed. Serum-borne prostate antigen showed a molecular weight of 90,000-100,000 but it exhibited a molecular weight of 36,000 in the presence of sodium dodecyl sulfate. A sandwich-type, peroxidase-linked immunosorbent assay capable of detecting 0.1 ng of the antigen per milliliter of blood was developed. With this technique, serum level of the antigen was found to increase in patients with prostatic cancer as compared with normal males. The prostate-specific antigen can be a useful marker for detection of prostatic cancer.

Hybridoma culture F5 has been developed which secretes monoclonal antibody (McAb) directed to an epitope of a prostatic glycoprotein of Mr 34 kD (Prostate Antigen, PA). Tissue levels of PA have been evaluated using a competitive-binding enzyme-immunoassay based upon the inhibition of McAb binding activity to purified antigen. Results indicated the specific occurrence of high antigen concentrations in extracts prepared from prostatic tissues. The antigenicity of epitope F5 is resistant to tissue fixation and embedding protocols, and has been demonstrated upon immunoperoxidase staining procedures. Immunoperoxidase data strongly indicate that McAb F5 possesses a singular specificity towards prostatic epithelial cells. Other tissues, whether normal or cancerous, fail to express this determinant. Specimens examined included epithelial and nonepithelial tissues along with a panel of carcinomas and sarcomas. The antibody was able to detect tumor cells at extra-prostatic sites and represents a powerful probe for the detection and differential diagnosis of metastatic cancer of the prostate.

Soluble immune complexes were studied by a newly developed procedure which consisted of precipitation at 2.5% polyethylene glycol, binding to immobilized Protein A, and subjecting to isoelectric focusing (pH 3 to 10). In a model system, bovine serum albumin: anti-bovine serum albumin complexes bound to immobilized Protein A were desorbed from the Protein A matrix and dissociated into antigen and antibody. The antigen and antibody components were separated based upon differences in isoelectric points. By this approach, IgG-type immunoglobulins and putative antigens can be detected from biological fluid of human specimens.

Pieces of human prostatic tissue (approximately 1 mm3) were encapsulated in XM-50 Amicon hollow fibers and either implanted into the testis of an adult male rat or placed in culture. The protein synthetic capacity of such tissue pieces removed 1-42 days later was monitored by TCA precipitation, SDS-PAGE, and autoradiography. The results showed that tissue pieces retained their functional protein synthetic elements after this procedure. A newly developed solid-phase enzyme immunoassay for human prostatic antigen (PA), described in this report, was used to monitor PA levels in the serum of the recipient host or culture media. In some instances PA was detected 1-2 weeks postimplantation, a result that implies maintenance of functional secretory elements in vivo. Finally, morphology of tissue pieces 1-2 weeks postimplantation sometimes showed the presence of ductal epithelia and stromal elements in distribution patterns typical of those seen in fresh biopsy samples. We conclude that the function and structure of prostatic tissue implanted intrastesticularly compares favorably with that maintained in conventional explant culture. As such, the hollow fiber method offers promise for a new way of monitoring hormonal and/or chemotherapy testing of human prostatic tissue.

Murine monoclonal antibody F36/22 was generated against the human breast carcinoma cell line MCF-7. The antibody reacts strongly with selected carcinoma cell lines and insignificantly against cell lines of mesenchymal derivation. No reactivity versus normal blood elements, bone marrow components or lymph nodes has been detected. Antigen is highly expressed in primary malignant tumors of ductal lineage to the virtual exclusion of lymphomas, sarcomas and non-ductal carcinomas, hence the designation Ductal Carcinoma Antigen. A small number of normal ductal structures also produce the antigen, although at greatly reduced levels. The antigen is found in the circulation of breast cancer patients where its exists as a high molecular weight glycoprotein. Purified antigen exhibits high density and a significant amount of carbohydrate content, thus resembling a mucin-like structure. The antibody-combining region also represents a carbohydrate component which is released upon alkaline-borohydride cleavage. The antigen behaves as an effective target in vitro for antibody dependent cytotoxicity as mediated both with complement and effector cells. Further, the passive administration of monoclonal antibody F36/22 results in a rapid decrease in the volume of human tumor xenografts as grown on athymic mice. The usefulness of this murine system as a pre-clinical model for human immunotherapies is under study.

According to the American Cancer Society, this year an estimated 57,000 new cases of prostate cancer (1979) will be diagnosed among American males (American Cancer Society, 1979). The Society also estimates that 20,000 lives will be lost to this form of cancer. One of the major reasons for this high mortality is that many cases are not detected until after metastases have occurred (Gittes and Chu, 1976). Therefore, markers that may serve as aids for early diagnosis of this disease and in monitoring the effectiveness of therapies have long been investigated. The purpose of this chapter is to review the prostate tumor markers that have been shown to be of significant value in diagnosis and treatment of this disease. It will include new information for old markers such as prostatic acid phosphatase, and data on new markers under investigation, such as prostate antigen. Other known markers that have been shown to be associated with cancer of the prostate are also described briefly.

A pancreas-specific antigen was identified by immunologic techniques and purified from saline extract of human pancreas. The purified pancreas-specific antigen was shown to be homogeneous by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. It had a molecular weight of 44000 as estimated by gel filtration or sodium dodecyl sulfate-gel electrophoresis, and a sedimentation coefficient of 3.4 S as analyzed by sucrose gradient centrifugation. Pancreas-specific antigen possessed an isoelectric point of 4.9 and migrated to alpha-beta region upon immunoelectrophoresis. By colorimetric assay procedures, pancreas-specific antigen exhibited no enzyme activity, such as amylase, protease, esterase, lipase, acid phosphatase, alkaline phosphatase peroxidase, deoxyribonuclease or ribonuclease. Immunoreactivity of pancreas-specific antigen was sensitive to proteolytic enzymes, perchloric acid and high temperature (70 degrees C, 10 min); but insensitive to neuraminidase or beta-glucosidase. Immunohistochemical staining revealed that pancreas-specific antigen was located in acinar cells of human pancreas. In addition, a higher concentration of pancreas-specific antigen was detected in pancreatic juice than in the saline extract of pancreas. This newly identified pancreas-specific antigen, therefore, may be a useful marker protein in physiological studies of pancreas and pancreatic secretion.

Hybridoma clones were established by fusing spleen cells from mice immunized with purified breast cancer-related immune complexes to drug-resistant nonproducing myelomas. Hybridoma cultures were evaluated for antibody production using immunofluorescence, radioimmunoassay and immunoperoxidase techniques. One antibody demonstrated strong reactivity against breast tumor cells (BT-20) in culture and also was found to bind ductal epithelial cells within sections of human mammary tissue. Antigen was detected within the soluble fraction of breast tumors with molecular weight greater than 500 K. The possible relationship between antigen detected in breast tumor cells and in immune-complexed form is discussed.

Monoclonal antibody (McAb) F36/22, raised against a human breast tumor line, identifies an antigen found in the circulation of cancer patients. Antigen was purified from malignant effusions using McAb-affinity chromatography followed by adsorption-desorption from immobilized wheat germ lectin. Electrophoretic analysis demonstrated the isolation of a single high mol. wt glycoprotein exhibiting an isoionic point near pH 4.2 and a density of approx. 1.45 g/ml. Although highly reactive with wheat germ lectin, a negligible or weak interaction was observed with concanavalin A, lentil lectin and peanut agglutinin. The antigen was immune-precipitable, indicating the occurrence of multiple McAb-binding sites, and was resistant to heat and acid treatments. Antigenicity was not perturbed following protease or neuraminidase treatments, but was affected upon exposure to alkaline conditions. Taken together, these data suggest that McAb F36/22 recognizes a high mol. wt component occurring in circulation as a mucin-like glycoprotein.

Thin sections of aldehyde fixed Cyperus alternifolius root tips were stained with uranyl acetate, washed with EDTA, and poststained with lead citrate to selectively stain ribonucleic acid (RNA) containing structures. As previously shown by other investigators RNA containing structures appeared electron dense, while nuclear regions known to contain DNA appeared less electron dense or “bleached”. RNase treatment removed all nuclear structures staining for RNA; light microscopic cytochemistry (Azure B) was performed on 1-3 micron sections of plastic embedded material to confirm the enzymatic removal of RNA.Granular appearing fibrils with indistinct boundaries were observed at the periphery of bleached metaphase chromosomes (Fig. 1). These fibrils are similar to the ribonucleoprotein perichromatin fibrils observed by Monneron and Bernhard in interphase nuclei. Nucleolar remnants were also observed associated with metaphase chromosomes.