Rapid screening test for detecting hepatitis-associated antigen.

Hepatitis-associated antigen can be detected within 2 hours by using an electrophoretic technique and cellulose acetate membranes saturated with antibody. The speed of the technique now allows testing of blood intended for transfusion on the day of collection, and the sensitivity of the method compares favorably with standard immunodiffusion.

The zinc glycinate marker in human colon carcinoma.

In an attempt to identify new tumor markers in human colon carcinoma, we produced antisera in rabbits tolerant to normal human tissue antigens and immunized with zinc glycinate-treated extracts of liver metastases from a colon carcinoma. These antisera reacted with carcinoembryonic antigen and with an additional component present in the tumor extracts but not detected in the extracts of normal tissues. The new component, the zinc glycinate marker (ZGM), had an alpha2 mobility on immunoelectrophoresis, was soluble in 1 M perchloric acid, and had a molecular weight of approximately 2X10(6), as indicated by its elution pattern on Sepharose 6-B chromatography. It differed from alpha fetoprotein, nonspecific cross-reacting antigens (NCA, NGP, or CCA III), ferritin-like molecules, and blood group substances A, B, H, Lewis a, and Lewis b. The ZGM was similarly identified in saline or zinc glycinate extracts of 11 of 23 carcinomas of the colon. With routine hematoxylineosin staining, no histologic differences were apparent between tumors bearing the antigen and those without it.

Tissue localization of zinc glycinate marker and carcinoembryonic antigen by immunofluorescence. I. Preparation of antisera against the zinc glycinate marker.

Antisera against the zinc glycinate marker (ZGM) were produced in New Zealand White rabbits with induced tolerance to normal tissue components and CEA, and in mature, previously uninoculated rabbits for use in immunofluorescent histologic localization of ZGM in colon cancers and other tissues. Analysis of the antisera by immunodiffusion and counterimmunoelectrophoretic techniques showed them to be specific for ZGM when tested with ZGM, carcinoembryonic antigen, normal tissue extracts, or cell elements of normal blood.

Demonstration and immunochemical characterization of carcinoembryonic antigen in human pancreatic juice.

Pancreatic juice collected from 10 patients without evidence of malignant disease of the pancreas or other organs was pooled, extracted, and fractionated by Sepharose 6-B and Sephadex G-200 gel filtration. The carcinoembryonic antigen (CEA) activity in the material was demonstrated and studied by: a) radioimmunoassay, b) competitive binding to antibodies against CEA, c) precipitin inhibition, and d) Ouchterlony analysis. The immunochemical identity of the active material to CEA purified from liver metastases of colon cancer was demonstrated.

Tissue localization of zinc glycinate marker and carcinoembryonic antigen by immunofluorescence. II. Immunofluorescence microscopy.

Preliminary indirect immunofluorescence studies on the zinc glycinate marker (ZGM) were compared with carcinoembryonic antigen (CEA) immunofluorescence, ZGM, detected in 26 of 29 human colon adenocarcinomas, was associated with the epithelial component of the malignant glands. Fluorescence was generally less strong and more granular for ZGM than for CEA and was found in intraglandular spaces, luminal border areas, and cytoplasm. ZGM concentration and tissue localization appeared to be related to tumor differentiation. ZGM was also detected in benign colon mucosae (adjacent to and distant from the carcinomas) from patients with colon carcinoma, but differed from CEA in that it was present in the deep crypt portion only. Gastric, pancreatic, esophageal, and anal adenocarcinomas, as well as benign gastric pyloric and small bowel mucosae had detectable ZGM. CEA, but not ZGM, was observed in 20 nongastrointestinal carcinomas to date. Studies are under way to determine whether ZGM is a marker associated with colon and gastrointestinal adenocarcinoma specifically or undifferentiated crypt cells of the colon and digestive tract in general.

A cell surface glycoprotein expressed by colorectal carcinomas including poorly differentiated, noncarcinoembryonic antigen-producing colorectal tumors.

A monoclonal antibody to a cell surface glycoprotein on human colorectal carcinomas was raised using the undifferentiated colon carcinoma cell line MIP 101 as the immunogen. This antibody, ND4, is an IgG2a which does not cross-react with carcinoembryonic antigen (CEA), non-specific cross-reacting antigen, or blood group substances A, B, and H. Immunoprecipitation using lysates of cells grown in [35S]methionine or [3H]glucosamine and lysates of cells surface labeled with 125I showed binding to a cell surface glycoprotein with a molecular weight of approximately 160,000. Indirect immunofluorescence showed binding to the cell surface of 14 of 15 human colorectal carcinoma cell lines including six of six that do not secrete CEA. Two of seven human noncolorectal carcinoma lines and one of six nonhuman cell lines also bound antibody. Immunoperoxidase staining of formalin-fixed tissues showed prominent antibody binding with 19 of 33 (58%) human colorectal carcinomas, including five of six poorly differentiated tumors, five of 43 (12%) normal colonic mucosal biopsies, and one of 17 (6%) normal noncolonic tissues. One of 11 (9%) noncolonic tumors, a gastric adenocarcinoma, stained with ND4. Preliminary data obtained by a nonquantitative nitrocellulose dot-immunoassay have tentatively identified this glycoprotein in the serum of 15 of 37 (41%) patients with colorectal cancer. Three of the 15 patients had early stage disease and normal CEA levels (less than 2.5 ng/ml). Three patients had circulating antigen detectable preoperatively but not after tumor resection. Only one of 11 (9%) sera samples from normal subjects was positive. The characteristics of ND4 suggest that it may be of value in monitoring patients with colorectal carcinomas who do not have plasma CEA elevations. It may also be of value in the differential diagnosis of metastatic, poorly differentiated adenocarcinomas of unknown primary origin.

Isoelectric focusing in agarose.

We describe here the use of a new agarose for isoelectric focusing of body fluids and tissue proteins. Macromolecular proteins even when significantly greater in size than 1 X 10(6) mol.wt. were easily and rapidly focused.

Direct tissue isoelectric focusing in agarose.

We are isoelectric focusing small amounts of tissues placed directly on electroendosmosis-free agarose gels instead of using the saline extracts of tissue homogenates. More numerous proteins are extracted during isoelectric focusing of the solid tissues than present in the same tissues.

The baton dialyzer.

Macromolecular solutions (such as radioactive materials, sterile solutions, infectious materials) requiring special handling precautions for dialysis may be safely and easily handled using a baton-shaped dialysis device.

Immunocytochemical localization of carcinoembryonic antigen in benign and malignant colorectal tissues. Assessment of diagnostic value.

Immunoperoxidase and immunofluorescence staining for carcinoembryonic antigen (CEA) was performed on paraffin and frozen sections, respectively, of colonic carcinomas (70), liver and lymph node metastases (20), mesenteric nodes (150), mucosa adjacent to carcinoma (40), colonic resection margins (20), normal colon (ten), and colorectal polyps (64) in order to assess its potential diagnostic value. On the basis of this study of the immunocytochemical localization of CEA in colorectal tissues, conclusions were as follows. (1) Localization of CEA to glycocalyx of surface epithelial cells is a normal finding in the colon and is similar in normal colon and mucosa distant and adjacent to infiltrating carcinoma. (2) Although strongly positive cell surface and intraluminal staining frequently correlates with the presence of carcinoma in neoplastic polyps, it is not by itself a reliable diagnostic criterion. (3) Failure to demonstrate CEA in a gland-forming carcinoma makes a diagnosis of colorectal carcinoma unlikely. (4) Poorly differentiated colorectal carcinoma usually contains much less demonstrable surface CEA, but may occasionally stain cytoplasm strongly. (5) Although lymph node micrometastases from colorectal carcinoma are readily demonstrated by immunoperoxidase staining for CEA, screening of hematoxylin and eosin-stained sections by a competent pathologist appears to be adequate for their detection.

Studies of the renin-renin substrate reaction in man; kinetic evidence for inhibition by serum.

Increasing evidence suggests that the renin-renin substrate reaction is regulated by factors other than the concentrations of enzyme and substrate. Partially purified human renin and renin substrate extracted from the plasma of each of eight human subjects were used to construct substrate-velocity curves comparing the rate of substrate cleavage in the whole serum of each individual with the rate in a corresponding system containing purified autologous substrate, or purified substrate plus a small amount of autologous serum. Linear regression analysis of the double reciprocal plots were used to compare the kinetic constants in paired experiments. Maximal reaction velocity (Vmax) was significantly lower (p greater than .05) when the reaction rate was measured in whole serum, in seven of the eight patients, while Km did not differ significantly, suggesting the presence of noncompetitive inhibition by human serum. The mean Michaelis constant in serum was 449 ng/ml while the average native substrate concentration of the five normal subjects was 629 ng/ml, an excess of less than twofold. The data suggest that plasma renin activity in man depends upon the concentrations of inhibitor and substrate, as well as upon the concentration of renin.

Semipreparative isoelectrophoresis of DTPA-conjugated antibody leads to isolation of antibody isoforms with different immunoreactivities.

Diethylenetriaminepentaacetic acid (DTPA)-conjugated purified rabbit anti-human serum albumin antibody was subjected to free-flow, semipreparative isoelectrophoresis using an apparatus that fractionates protein molecules based on isoelectric point (pI). The results indicate that (1) the apparatus was capable of developing linear pH gradients and separating proteins, (2) > or = 86% of the protein was recovered following fractionation, and (3) the protein concentration of the individual fractions varied. Focusing the fractions on an isoelectric slab gel revealed that the pIs of the isoforms were modified. The isoforms were radiolabeled with indium-111, their DTPA to antibody molar ratios determined, and their immunoreactivities evaluated by a solid-phase radioimmunoassay. The results demonstrate that (1) the molar ratio of DTPA to antibody varied among the fractions, and (2) the immunoreactivity of the majority of fractions was higher than that of unfractionated DTPA-antibody conjugate. Semipreparative isoelectric focusing may therefore improve the potential of radioimmunoconjugates in the radio-diagnosis and therapy of disease.