Representation of epitopes on colon-specific antigen-p defined by monoclonal antibodies.

Colon-specific antigen-p (CSAp) is a large molecular-sized protein restricted to normal and neoplastic gastrointestinal tissues and to some mucinous ovarian tumors. Murine monoclonal antibodies (MAbs) were raised against CSAp that was affinity purified with goat polyclonal antibodies from GW-39 human colonic carcinoma xenografts or against the CSAp-producing colon cancer cell line SW-948. Two of the MAbs, designated Mu-2 and Mu-4, recognized a CSAp determinant containing sialic acid, and this epitope was also expressed on bovine submaxillary mucin (BSM). Blocking experiments demonstrated that the Mu-2 and Mu-4 MAbs recognized different determinants. A third MAb, Mu-3, did not cross-react with BSM, but unlike Mu-2 and Mu-4, it did react with human saliva. Reactivity of Mu-3 with saliva did not correlate with major blood group and Lewis-related secretory blood group substances in saliva. This reactivity was not related to sialylated Lewis activity. The fourth antibody, Mu-1, appeared to react with a conformational determinant since its epitope was destroyed by heat treatment or thiol reduction. Enzyme immunoassays have demonstrated that all four epitopes may be expressed on one molecular species.

A simple and sensitive nonradioactive method for the detection of urinary human chorionic gonadotropin and diagnosis of early human pregnancy. II. Single-unit test.

A simple, sensitive, and reliable single-unit nonradioactive method for the detection of human chorionic gonadotropin (hCG) in concentrated urine and the diagnosis of early pregnancy is reported. This unit, presently termed the Ayerst pregnancy test kit (APTK), consists of four components: a sampler-filter paper cone, an ultrafilter-concentrator to which a vial holder is attached, a support stand with a mirror, and an immunologic reagent vial. In the APTK, 5 to 6 ml of urine were sampled, filtered, and concentrated, and the hCG in the retentate was detected by Ayerst immunologic reagents [APTK(AY)] and by the Pregnosticon "All In" [APTK(P)]. Some of the unconcentrated urine samples (0.1 ml) were also tested in hemagglutination inhibition tests (HIT) using Ayerst [HIG(AY)] and Pregnosticon "All In" [HIT(P)] reagents. Urine samples from pregnant, nonpregnant (ovulating and nonovulating), perimenopausal, and menopausal women were tested. It was found that the APTK(AY) and APTK(P) were significantly more sensitive and reliable than the HIT(AY) and HIT(P) in detecting low levels of urinary hCG for early diagnosis of pregnancy. The sensitivity and specificity of the APTK(AY) were better than those of the APTK(P). The APTK(AY) give significantly more correct positive and negative results than the other tests performed simultaneously. The APTK(AY) is simpler and safer than the serum radioimmunoassays and radioreceptor assay presently used to detect low levels of hCG for the early diagnosis of pregnancy and other hCG-producing states.

Physicochemical approach to the purification of human alpha1-fetoprotein from the ascites fluid of a hepatoma-bearing patient.

A method for the purification of human alpha1-fetoprotein from the ascites fluid of a hepatoma-bearing patient is described that is capable of yielding large quantities of pure alpha1-fetoprotein within a relatively short period of time. The technique is based entirely on the physicochemical properties of the alpha1-fetoprotein molecule and uses sequential purification steps: ion-exchange chromatography on DEAE-Sephadex A-50, molecular-sieve chromatography on Sephadex G-200, negative-affinity chromatography on Sepharose-Blue Dextran, positivepaffinity chromatography on concanavalin A-Sepharose and, finally, molecular-sieve chromatography on Sephadex G-100. The efficiency of the entire procedure in its present form is 15% of the alpha1-fetoprotein activity of the starting preparation from ascites fluid. The purity of the final product was shown by polyacrylamide gel electrophoresis, radioimmunoelectrophoresis, and determinations of the NH2-terminal and COOH-terminal amino acid residues of the alphs1-fetoprotein isolated. Amino acid analysis of the final product revealed a composition very similar to those reported for alpha-fetoprotein preparations that have been previously isolated by the use of immunochemical technology.

The radioimmunoassay of circulating carcinoembryonic antigen of the human digestive system.

A radioimmunoassay has been developed for determining the serum levels of carcinoembryonic antigen of the human digestive system in patients with cancer of the colon and rectum. The assay is simple to perform and has a high degree of reproducibility and specificity. The test detects a concentration of 2.5 ng of carcinoembryonic antigen per ml of serum and this has provided the first demonstration of a circulating tumor-specific antigen in the sera of cancer patients.

Physicochemical studies of the carcinoembryonic antigens of the human digestive system.

A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.