Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

Hammarström, Sten; Engvall, Eva; Johansson, Bengt; Svensson, Sigfrid; Sundblad, Göran; Goldstein, Irwin J.

doi:10.1073/pnas.72.4.1528

Cited by 94 publications

(39 citation statements)

References 31 publications

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“…Already accurate double antibody sandwich and competitive sandwich ELISA methods for AFP have been described by several groups of workers (Belanger et al, 1973(Belanger et al, , 1976Hevey et al, 1976;Masseyeff et al, 1976;Kirkpatrick et al, 1977) and the method is in routine use in Nice, France. Carcinoembryonic antigen levels are perhaps of less clinical importance at present but there is a growing demand for assays, and ELISA has been successfully used by Engvall and collaborators (Engvall, 1977;Engvall and Carlsson, 1976;Engvall and Perlmann, 1975;Hammarstrom et al, 1975).…”

Section: Oncofetal Proteinsmentioning

confidence: 99%

Enzyme immunoassays with special reference to ELISA techniques.

Voller¹,

Bartlett²,

Bidwell³

1978

J Clin Pathol

639

256

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Section: Oncofetal Proteinsmentioning

confidence: 99%

Enzyme immunoassays with special reference to ELISA techniques.

Voller¹,

Bartlett²,

Bidwell³

1978

J Clin Pathol

639

256

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“…They did not contain detectable albumin or AFP at the 0.1% level. Carcinoembryonic antigen (CEA) was purified from colon cancer metastases as described (16). Bovine serum albumin (BSA) was from Pentex, bovine hemoglobin from Sigma, and bovine fetuin from Gibco.…”

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confidence: 99%

Immunological crossreaction between alpha-fetoprotein and albumin.

Ruoslahti¹,

Engvall²

1976

Proc. Natl. Acad. Sci. U.S.A.

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(16). Bovine serum albumin (BSA) was from Pentex, bovine hemoglobin from Sigma, and bovine fetuin from Gibco.The samples to be reduced and alkylated (17) were dissolved at a concentration of about 10 mg/ml in 0.1 M Tris-HCI buffer (pH 8.2) containing 8 M urea. After deaeration with nitrogen, dithiothreitol was added to a final concentration of 10 mM. After 3 hr at room temperature under nitrogen, the reaction mixture was cooled on ice and recrystallized iodoacetamide was added to make the final concentration 20 mM. After another 15 min the sample was dialyzed against distilled water and lyophilized. Phosphate-buffered saline containing 0.05% gelatin, 0.02% sodium azide, and 0.1% Triton X-100 (PBS-GTX) with urea added to it to give a 2 M solution was used to dissolve the dry proteins, some of which were poorly soluble in ordinary buffers. In some cases the reduced and carboxamidomethylated (RC-) protein was diluted directly into this solution for testing without dialysis and lyophilization. HAFP labeled with 1251 was reduced and alkylated similarly and freed of reactants by gel filtration on Sephadex G-15 equilibriated with PBS-GTX.Antisera. Anti-HAFP sera have been described (6,8). Using a similar schedule, we produced one antiserum each to RC-HSA and RC-BSA and two to RC-HAFP in rabbits. The dose used per injection was 5 mg (RC-HSA, RC-BSA) or 0.5-2 mg (RC-HAFP) of protein suspended in PBS and given in complete Freund's adjuvant. A precipitating antiserum to RC-CEA was obtained similarly, using 0.25 mg of RC-CEA per injection.Immunological Tests. Immunodiffusion was performed on plates cast in a conventional buffer and soaked overnight in PBS-GTX prior to use. Radioimmunoassays were performed using the double antibody technique as described (4). The first and second antibody were added in the form of an IgG fraction in assays where native albumin was tested for crossreactivity with AFP. The diluent buffer used throughout was PBS-GTX. RESULTSLack of Crossreaction between Native AFP and Albumin. Antisera produced against highly purified HAFP do not react with albumin in immunodiffusion (ref. 15, our unpublished results). Radioimmunoassay also failed to reveal any crossreactivity between native HAFP and native HSA or RC-HSA. RC-HAFP was also almost completely devoid of activity in the usual AFP assay (Fig. 1).

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“…The fact that the antigenic activity of CEA is not altered upon extensive degradation of the carbohydrate moiety (Hammarstrom et al, 1975) appears to rule out the possibility that exterior sugar chains significantly affect the affinity of the molecule by steric hindrance. It seems reasonable however to postulate subtle differences in the structure of the antigenic determinant or variations in the amount of carbohydrate-rich non-CEA glycoprotein to account for the different activities of the CEA fractions.…”

Section: Resultsmentioning

confidence: 99%

Carcinoembryonic antigen: isolation of a sub-fraction with high specific activity

Rogers¹,

Searle²,

Bagshawe³

1976

Br J Cancer

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Summary.-Four sub-fractions of carcinoembryonic antigen have been obtained by chromatography of conventional CEA on Con A-Sepharose and their immunoreactive contents have been determined. Comparative studies have shown that a fraction eluted with 2% methyl glucoside (CEA 2B) had the highest activity, with a potency (60 u/,ug) twice that of unfractionated CEA although appreciable activity was also found in the other fractions. The amino acid composition of CEA 2B is similar to that reported for conventional CEA but there is a lower content of neutral hexoses and a comparatively high content of N-acetylglucosamine. Experiments with a pool of sera from 11 patients with colonic cancer, which had been fractionated on Con A-Sepharose, have shown that nearly all the CEA activity was contained in a fraction eluted with 2% methyl glucoside. A convenient method of isolating CEA with high specific activity directly from perchloric acid extracts of tumour tissue is also described.

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Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

Abstract: The carbohydrate moiety of carcinoembryonic antigen could be sequentially degraded by repeated cycles of periodate oxidation, reduction, and mild acid hydrolysis (Smith degradation

Cited by 94 publications

References 31 publications

Enzyme immunoassays with special reference to ELISA techniques.

Enzyme immunoassays with special reference to ELISA techniques.

Immunological crossreaction between alpha-fetoprotein and albumin.

Carcinoembryonic antigen: isolation of a sub-fraction with high specific activity

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