3'-Fucosyllactose (Fucal-"3Gal/31-"4Glc) was synthesized as described previously5, converted to the p-isothiocyanate-phenethylamine (PIP) derivative and coupled with bovine serum albumin (BSA) according to Smith et a/.6. The product (3'-fucosyllactose-PIP-BSA), which contained 20 mols of 3'-fucosyllactose per mol of BSA, was injected into a rabbit. The resulting antiserum formed a sharp precipitation line with the antigen, but not with BSA, lactose-PIP-BSA, 4'-fucosyllac-tose-PIP-BSA nor with 6'-fucosyllactose-PIP-BSA (Fig. 1). Furthermore, these BSA derivatives without the Fuco:l-"3Gal linkage were unreactive to the antiserum even in a binding assay, in which Sepharose coupled with the derivatives were stained with the antiserum by indirect immunofluorescence technique used in Fig. 2. Fig. 1 Ouchterlony double-diffusion analysis on the specificity of the rabbit antiserum against 3'-fucosyllactose-PIP-BSA. The antigen (0.4 mg) was mixed with Freund's complete adjuvant and injected into the footpads of a rabbit. After 3 and 5 weeks, the rabbit received two booster injections using half the amount of antigen. The rabbit was bled 10 days after the last immunization. The double-diffusion analysis was performed in 1% agar gel containing 2% Triton X-100, 0.15 M NaCl, 50 jxg ml"1 of phenyl-methysulphonyl fluoride and 0.05% sodium azide in 0.01 M Tris-HC1 buffer, pH 7.6, on a slide glass. The central well contained the antiserum and the peripheral wells contained 20 jjig each of the following materials: a, 3'-fucosyllactose-PIP-BSA, b, 4'-fucosyllactose-PIP-BSA, c, 6'-fucosyllactose-PIP-BSA, d, lac-tose-PIP-BSA, e, BSA. Preparation of 4'-fucosyllactose and 6'-fucosyllactose has been described elsewhere12. Carbohydrate contents of the PIP-BSA derivatives were determined by the phenol-H2SO4 reaction13, and were found to be 20 mols per mol of BSA in 3'-fucosyllactose-PIP-BSA, 25 mols per mol of BSA in the 4'-fucosyl compound and 23 mols per mol of BSA in the 6'-fucosyl compound.Fig. 2 Reaction of the serial tissue sections from a mouse uterus with the anti-Fucal-"3Gal serum or with UEA-1. The organs were taken from a 3-month-old female 129/SV mouse, quick frozen and sectioned in a cryostat. The section was fixed for 1 min with acetone, a, Immunofluorescence staining with the antiserum showing the presence of the antigen in the squamous epithelium of the uterine cervix. The section was incubated with the antiserum diluted 20-fold with phosphate-buffered saline and then with FITC-conjugated F(ab')2 fragment of goat anti-rabbit IgG (Cap-pel), and was observed by a fluorescence microscope using epi-illumination (Olympus, Model BH-RFL-LB). x80. Very similar results were also obtained using BALB/c, ICR and ddY mice. No staining was observed in control experiments where rabbit non-immune serum, rabbit anti-BSA or rabbit anti-lactose-PIP-BSA was used instead of the specific antiserum. b, Staining with FITC-UEA-1. The tissue sections were incubated with 25 u.1 of FITC-UEA-1 at 1 mgmF1. x80. The reactivity of the antiserum to normal cells was also studied. Frozen sections of organs of adult mice were incubated with the diluted antiserum, and then fluorescein isothiocyanate (FITC)-labelled F(ab')2 fragment of goat anti-rabbit IgG. We found that the antiserum stained squamous epithelium of the uterine cervix (Fig. 2a), ciliated border of the oviduct, epithelium of the bronchus, ependyma lining the ventricles of the brain and spermatocytes of the testis. The following organs were negative for the antigen: small intestine, proximal and distal colons, liver, spleen, kidney, epididymis, ovary and endometrium. The extremely restricted distribution of the antigen in the mouse suggests that the antiserum does not cross-react with H antigen whose determinant is Fuca 1 -" 2Gal linkage7 or with SSEA-1 whose determinant involves Fucal->3GlcNAc linkage2'3. SSEA-1 is known to be present in the epididymis and in the endometrium but not in squamous epithelium of the uterine cervix. It is also present in a portion of epithelial cells of the oviduct but not in ciliated border of the oviduct8. H antigen detected by staining with FITC-conjugated Ulex europaeus agglutinin 1 (UEA-1) was expressed in a variety of the adult tissues including endometrium of the uterus (Fig. 26), small intestine, proximal and distal colons. Furthermore, the present antiserum did not stain erythrocytes of a number of human subjects whose blood types included H and Lea. The failure to react with Lea erythrocytes excluded the possibility that the antiserum cross-reacts with Fuca 1 -" 4GlcNAc linkage7. From all these results, we propose that the antiserum specifically recognized Fuco;l->3Gal linkage. The antigen recognized by the antiserum was termed as FG3 antigen. The antigen was detected in human colonic adenocarcinoma (Fig. 3a). The staining reaction was inhibited by 0.2 M 3'-fucosyllactose, but not by 0.2 M lactose, 0.2 M fucose, nor by lOmgmP1 BSA. So far frozen sections of 17 human colonic adenocarcinoma have been examined. Of these, seven cases expressed the antigen in intracytoplasmic granules (Fig. 3a) and five cases expressed it along the cell apex. As the antigen was present not only on the luminal surface of the carcinoma but also in the invasive nests of the carcinoma in deep layers of intestinal wall (Fig. 3a), we considered that the antigen was produced by the carcinomas themselves. Only tumour cells within the colonic tissue section were positive for the antigen (Fig. 36). Antiserum against lactose-PIP-BSA did not react with the tumour region. FG3 antigen was undetectable in tissue sections of normal human duodenum, liver, gallbladder, pancreas, lung, thyroid, thymus, lymph node, muscle, connective tissue and blood vessels. Although further studies are needed to examine the distribution of the antigen in other normal tissues and also in other neoplastic conditions, the tumour-specific expression of the antigen in human colon is of significant interest. Fig. 3 Immunofluorescent staining of a human colonic adenocarcinoma using the anti-Fucal-"3Gal serum. The tissue section was prepared from surgically removed specimen of a human colon adenocarcinoma of 44-year-old female Japanese patient (Blood group B type). Staining was performed as described in the legend of Fig. 2. a, Cancerous lesion, x 160. b, Non-neoplastic mucosa of this patient, x 80.Fig. 4 Immunofluorescent staining of F9 embryonal carcinoma cells using the anti-Fucal-"3Gal serum. F9 cells were cultured on a glass coverslip in Dulbecco's modified minimum essential medium containing 15% fetal calf serum. Cells on the coverslip were stained as described in the Fig. 2 legend, except that acetone treatment was omitted, a, Specific staining, x 120. b, Control staining using non-immune serum, x 120. c, Phase contrast microscopic observation of the cells shown in a. x 120. d, Phase contrast microscopic observation of the cells shown in b. x 120. As stem cells of teratocarcinoma are known to express unusual fucosyl glycopeptides9'10, we examined the expression of FG3 antigen in a stem cell line, F9 and found it (Fig. 4). The antigen was also detected on stem cells of teratocarcinoma OTT6050 and on another stem cell line, STT-M. Therefore, some embryonic antigens expressed on teratocarcinoma stem cells may contain this unusual fucosyl linkage.However, the Fucal-"3Gal linkage is not found in gly-coproteins and glycolipids isolated from diverse sources, but its presence has been reported in mixtures of higher homologues of human milk oligosaccharides11. The present results suggest that the unusual fucosyl linkage is expressed in certain cancerous and embryonic tissues. It is possible that such carbohydrate linkages are involved in intercellular communication during early embryonic life but are expressed aberrantly in certain cancerous cells. We thank Miss Kumiko Sato for secretarial assistance.