To establish its complete amino acid sequence we describe in this paper the results of sequence analysis on fragments derived from tetra-S-carboxymethyl-(CMA) and tetra-S-aminoethyl-(AEA) derivatives by chymotryptic, thermolytic and tryptic digestions. Parts of this work were published previously in Russian2-6). ExperimentalMaterials and Reagents Actinoxanthin was isolated from the culture filtrate of Actinomyces globisporus 1131 via ammonium sulfate precipitation at 4-5°C followed by trichloroacetic acid precipitation of accompanied impurities , decolourizing by Dowex I x 2 (acetate form), chromatography using DEAE Sephadex A-50 (acetate form) and gel filtration on Sephadex G-75 as described earlier1). Analytical polyacrylamide gel electrophoresis was carried out using ORSTEIN-DAVIS System1). Homogeneity was shown by the following criteria: single band on polyacrylamide gel disc electrophoresis at pH 6.5, single symmetric peak by analytical column chromatography on Sephadex G-75, single NH,-terminal alanine by dansyl procedure , single C-terminal glycine by tritium-labeling procedure and amino acid analysis.Porcine trypsin was obtained from Novo (Denmark); chymotrypsin (crystallized) , thermolysin (3 x crystallized, 40 % Na-acetate, Ca-acetate), carboxypeptidases A and B (DFP-treated, in suspension) were obtained from Worthington Biochemical Corp., Freehold, NY, U.S.A.; leucine aminopeptidase was supplied by Serva Feinbiochemica Co., Heidelberg, FRG. All the enzymes were used without further purification.

Tetrasaccharide Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc is known as carbohydrate determinant of cancer- and AIDS-associated antigen Lewisy (Ley). Synthetic antigen to generate mouse monoclonal antibodies (MAbs) directed to Ley was prepared and constructed as a spacer-armed tetrasaccharide coupled with lipophilized polymer, Ley-PAA-PE, where PAA is a 30-kD polyacrylamide and PE is phosphatidylethanolamine. An efficient immune response was provided by using Ley-PAA-PE adsorbed on Salmonella minnesota. Positive hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) using Ley-PAA as a coating agent. An inhibitory version of the same test system showed absolute specificity of two MAbs: only hapten Ley and Ley-PAA were strong inhibitors, in contrast to Leb, tri- and disaccharidic fragments of the mentioned tetrasaccharides, as well as their PAA-conjugates. MAbs obtained against synthetic antigen specifically stained the Ley (+) cell line A431.

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