Delineation of four carcinoembryonic antigen (CEA) related antigens in normal plasma by transblot studies using monoclonal anti-CEA antibodies with different epitope specificities

Antigens related to the carcinoembryonic antigen (CEA) were isolated from normal human plasma by perchloric acid extraction, gel permeation chromatography and immunoaffinity chromatography using a monoclonal antibody with broad specificity and high affinity. The antigens were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The binding of five monoclonal anti-CEA antibodies with different epitope specificities to the immobilized antigens was analyzed. Two antigens with mol. wts of greater than 200,000 and 177,000 bound all five antibodies, and two antigens with mol. wts of 114,000 and 85,000 bound three of the five antibodies. The findings reported indicate that even monoclonal antibodies with high specificity for colonic cancer CEA detect CEA-related antigens in normal human plasma.     

Further comparative studies on chemical properties of carcinoembryonic antigen in tumor tissues and closely related antigens in adult feces and meconium

The chemical structure of carcinoembryonic antigen (CEA) and two closely related antigens, normal fecal antigen-2 (NFA-2) in normal adult feces and nonspecific cross-reacting antigen-2 (NCA-2) in the meconium, were further analyzed comparatively. The NH2-terminal amino acid sequence of NCA-2 was newly determined to position 18 and found to be identical to that so far determined for CEA- and NFA-2. After proteolytic digestion with chymotrypsin or protease V8, the digests of these antigens showed two groups of fragments upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One consisted of the sharply banded fragments which were identical in all antigens and stained only with Coomassie brilliant blue (CBB) (five bands in the range 2500-10,000 daltons for chymotrypsin and 11 bands in the range 8000-35,000 daltons for protease V8, respectively), and the other consisted of the dispersed fragments which had variable mol. wts in the range 10,000-100,000 and were stainable with both CBB and periodic acid-Schiff reagent. Elution profiles of CEA, NFA-2, and NCA-2 from lectin columns, especially from concanavalin A-Sepharose columns, suggested some differences in oligosaccharide chains between them. These results indicate that the fundamental chemical structure of these antigens seems to be very similar to one another and is divided into two parts; an homologous portion(s) which is common to all three antigens and contains no sialylated sugar components, and a heterogeneous portion(s) which is variable among these antigens and contains sialylated sugar components.     

Purification and characterization of a 50,000 mol. wt glycoprotein antigen, and localization of determinants involved in cross-reactivity with carcinoembryonic antigen

A 50,000 mol. wt glycoprotein antigen (50 k) was purified from metastatic colon tumor. The initial purification was monitored by cross-reactivity of the antigen with an unabsorbed antiserum against carcinoembryonic antigen (CEA, Mr 180,000). Six mg of antigen was obtained from 250 g of tissue, with a recovery of 14% and a relative purification of 143-fold. The amino acid composition of 50 k was similar to that of CEA. The carbohydrate content, primarily glucosamine and mannose, totaled about 25%. Immunodiffusion showed that 50 k lacked some of the CEA epitopes, and that normal spleen contained an antigen identical to 50 k. Radioimmunoassays showed that the high avidity anti-CEA antibodies in the unabsorbed antiserum were mainly cross-reactive with 50 k, probably via a similar but not identical antigenic site. The immunochemical properties of 50 k thus correspond to those of the non-specific cross-reacting antigen (NCA). The molecular size and carbohydrate composition reported for various NCA-like antigens have differed, so identity of 50 k an NCA cannot be established on this basis. Immunoreactive fragments of 50 k were prepared by digestion with each of three different proteolytic enzymes, and were purified by high performance liquid chromatography (HPLC). A polypeptide of 20,000-30,000 mol. wt containing about half of the 50 k determinants, was recovered in each case. Experiments with mixtures showed that the three purified fragments all contained the same subset of antigenic determinants. The fragment-localized subset represented most, if not all of the 50 k determinants involved in CEA cross-reactivity. Similarly, a CEA fragment was shown to contain essentially all of the CEA determinants involved in 50 k cross-reactivity. The fragments of 50 k and CEA were quite resistant to further proteolytic digestion, and comparison of a CEA fragment with a 50 k fragment by partial acid hydrolysis and HPLC peptide mapping failed to reveal structural homology to account for the cross-reactivity.     

Identification of a previously unrecognized polypeptide associated with lymphocyte function associated antigen one (LFA-1)

In lymphocyte function associated antigen one (LFA-1) preparations from metabolically labeled lymphocytes we have observed a new polypeptide component of 86-kilodalton additional to the already described - and β-chains. This chain is cosynthesized with the - and β-chains and can be covalently cross-linked with them, resulting in a three-chain complex. This complex is recognized by the H35–89.9 anti-LFA-1 monoclonal antibody. Cleveland peptide mapping analysis indicates that the new chain is structurally different from the - and β-chains of the LFA-1 complex. The chain has been observed in B-cells as well as in T-cells. Labeling properties of the 86-kilodalton chain suggest that this molecule is not exposed on the membrane.