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respectively. This may select for different cell types with different patterns of differentiation, and the differences in xanthine oxidase activities may reflect this rather than relationship to malignancy. (3) Boone (1976) has shown that when Balb/3T3 cells are grown on glass beads and implanted into mice, tumours are formed. Although this may merely represent spontaneous transformation after inoculation, it raises the question of whether Balb/3T3 and 3T3 are genuinely normal lines, or represent some form of pre-neoplastic state. In addition, we have preliminary evidence that xanthine oxidase activity is lowered in o r absent from a spontaneously transformed derivative of Balb/3T3 cells. Finally, in regard to possible clinical relevance, it must be noted that xanthine oxidase is present in significant amounts in humans only in liver and small-intestinal mucosa (Watts et ul., 1965). M. M. C. is grateful to the Medical Research Council of Ireland and to the Irish Cancer Society for financial support. Aaronson, S. A. & Todaro, G. J. (1968) Science 162, 1024-1026 Boone, C. W. (1976) J. CellPhysiol. 89, 757-758 Burton, A. F. & Begg, R. W. (1957) Can. J . Biochein.Physiol. 35, 881-887 Clynes, M. M., Hurley, M . P. & Shannon, M. F. (1979) Biochenr. SOC.Trans. 7,72-74 Duke, E. J., Joyce, P. & Ryan, J. P. (1973)Biockem.J. 131, 187-190 Haddow, A., Horning, E. S., Bergel, F., Timmis, G. M., Osdene, T. S., Bray, R. C., Avis, P. & Brown, A. (1953) Camer Res. Campaign Amw. Rep. 31, 35-38 Haddow, A., de Lamirande, G., Bergel, F., Bray, R. C. &Gilbert, D. A. (1958) Narure(London) 182, 1144-1 I46 Lewin, I., Lewin, R. & Bray, R. C. (1957) Nuritre (London) 180,763-764 Prajda, N., Morris, H. P. & Weber, G. (1976) Cancer Res. 36,4639-4646 Todaro, G. J . &Green, H. (1963) J. CellBiol. 17, 299-313 Watts, R. W. E., Watts, J. E. M . & Seegmiller, J. E. (1965) J. Lab. Clin. Med. 66, 688-697 Weber, G. (1977~)N. Engl. J. Med. 296,486-493 Weber, G. (19776) N. Engl. J. Med. 296,541-551

The Effects of Sera from Cancer Patients on PhytohaemagglutininInduced Lymphocyte Transformation ALAN H. JOHNSON and PATRICK B. COLLINS Division of Biochemistry, Royal College of Surgeons in Ireland, St. Stephen’s Green, Dublin 2, Ireland Mitogen-induced lymphocyte transformation is a n accepted means of measuring, a t least in part, the effectiveness of T-lymphocytes in cell-mediated immune responses. There is considerable evidence for the depression of cell-mediated immune responses in most tumour-bearing patients. In this study we have examined the effect of serum from cancer patients on mitogen-induced lymphocyte transformation and have partially characterized the immunosuppressive factors present in such sera. Lymphocytes from healthy young adults were purified and cultured as described previously (Johnson & Collins, 1977). The cells were cultured in 200pl samples (lo5 cells) in microtitre plates with serum o r serum fractions (10 %, v/v) and lOOng of purified phytohaemagglutinin (Wellcome Laboratories, Beckenham, Kent, U.K.) at 37°C for 48h. C3H]Thymidine (0.5pCi : 1 Ci/2mmol; The Radiochemical Centre, Amersham, Bucks., U.K.) was added to each well 4 h before harvesting. The cancer patients whose sera were used in this study were all anergic and none were receiving cytotoxic therapy. Autologous serum was separated from the samples obtained a t the same time as blood taken for lymphocyte purification. All serum samples were stored at -20°C until required. Human serum albumin was obtained from Behringwerke A.G. (Marburg, Federal Republic of Germany). The fractionation of serum from the patient with the osteogenic sarcoma was essentially as described by Van Oss & Bronson ( I 974). Serum (20mI) was diluted 2.5-fold with

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0 0

00 0

8

e,

00 0 0 0 0

0 0

0

om

0 0

4 0 , A

B

C

D

E

F

G

Fig. 1. Effect of sera from cancer patients on phytohaemagglutinin-inducedlymphocyte transformatfbn Key: A, control (no phytohaemagglutinin); B, autologous serum; C, human serum albumin; sera frotn cancer patients; D, osteogenic sarcoma; E, lymphosarcoma; F, adenocarcinoma of the bowel ; G, bronchogenic carcinoma.

water and was titrated to pH 5.0 with acetic acid and left overnight at 4°C. The precipitated globulins were removed by centrifugation (3,OOOg for 15min) and the euglobulinfree serum was equilibrated at pH 5.0 with 0.03 M-sodium acetate/O.O12~-aceticacid, pH5.0, absorbed to a DEAE-cellulose column (35cmx 1.1 cm) equilibrated with the same buffer. The unadsorbed protein (fraction A) was collected and further fractions were collected by elution with 0.1 M-sodium acetate/O.O4~-aceticacid, pH 5.0 (fraction B), 0.2 M-sodium acetate/0.08 M-acetic acid, pH 5.0 (fraction C), 0.3 M-sodium acetate/ 0.12~-aceticacid, pH5.0 (fraction D), and 0.5M-sodium acetate/0.20~-aceticacid, pH 5.0 (fraction E); lOml fractions were collected and the protein content was monitored by measuring the Azso of the fractions. Protein was determined by the method of Lowry et al. (1951). Peak fractions were pooled and desalted by membrane-partition chromatography using DMO5 Diaflo membranes (Amicon Corp., Lexington, MA, U.S.A.), which retain molecules of molecular weight greater than 500. Further molecular-size fractionation of the pooled samples was performed by using U M 10 membranes, which ratain molecular species in excess of lO000mol.wt. The effects of serum derived from patients with four different malignancies on phytohaemagglutinin-induced lymphocyte transformation, by using lymphocytes from five normal donors in five separate experiments, is shown in Fig. 1. They are compared with those obtained by using autologous serum and human serum albumin. All of the cancer sera are immunosuppressive, the most inhibitory being that obtained from the patient with osteogenic sarcoma. In the presence of this serum only approximately one-third of the [3H]thymidine incorporation obtained with autologous serum was observed. This serum was fractionated by DEAE-cellulose chromatography. The effects of the five major column fractions on phytohaemagglutinin-induced lymphocyte transformation were investigated together with those of albumin and unfractionated serum. These 1979

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Table 1. Efects of the serum fractions from a patient with osteogenic sarcoma on phytohaemagglutinin-inducedlymphocyte transformation

For details of the fractions see the text. Results are means+s.E.M., for four experiments. Protein fraction [3H]Thymidine incorporation (c.P. m./105 lymphocytes) (5mgimU Human serum albumin 8647 ? 172 Unfractionated serum 4226 k 227 DEAE-cellulose fraction A 7870 f 1068 B 7846k1156 C 3750+ 1301 D 5847k 1556 E 3175 k 948

results are summarized in Table 1 . The unfractioned serum results in approx. 50% decrease in [3H]thymidine uptake as compared with the effects of albumin. The immunosuppressive activity is primarily confined to fractions C and E, although some inhibition was also observed with fraction D. Membrane-partition chromatography of fractions C , D and E indicated that the immunosuppressive activity is of mol.wt. greater than 1oOOo. These fractions require further characterization. This work is supported by a grant from the Irish Cancer Society. Johnson, A. H. & Collins, P. B. (1977) Biochenz. Soc. Trans. 5 , 1728-1730 Lowry, 0.H., Rosebrough, N., Farr, A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Van OSS,C. J. & Bronson, P. M. (1974) Prep. Biochem. 4, 127-139

Enzyme-Immunoassay of Anti-(Bovine Insulin) Antibodies MARGARET E. TELFORD*t and G. BRlAN W l S D O M t *Northern Area Laboratory, Cushendall Road, Ballymena, N . Ireland, U.K., and ?Department of Biochemistry, The Queen's University of Belfast, Belfast BT9 7BL, N . Ireland, U.K. Enzyme-immunoassay has been shown to be a sensitive, reproducible and convenient method of measuring specific antibodies (Schuurs & Van Weeman, 1977). We applied the enzyme-immunoassay method of Engvall & Perlmann (1972) to the measurement of antibodies against bovine insulin in human serum samples from four groups; (1) nondiabetic patients; (2) diabetics who had never been treated with bovine insulin; (3) diabetics being treated with bovine insulin and showing no signs of reaction or resistance; (4) diabetics being treated with bovine insulin who showed reactions to the hormone or resistance to the therapy (manifested by a n insulin requirement of more than 200i.u. per day). Insulin from bovine pancreas (24i.uJrnl) was used as antigen. Rabbit anti-(human IgG*) antibodies were purified by immunoadsorption on a column of human IgG attached to Ultrogel AcA 34 (Guesdon & Avrameas, 1976) and conjugated to highly purified alkaline phosphatase (EC 3.1.3.1) with glutaraldehyde (Engvall & Perlmann, 1972). All dilutions and washings were made in 0.02~-sodiumphosphate buffer, pH7.2 ( O . ~ M - N ~ , H P Oadjusted , to pH7.2 with 0 . 2 ~ - N a H , P oand ~ then diluted to 0.02M with water) containing 0.15 M-NaCI and 0.05 % (v/v) Tween 20, except the conjugate

* IgG, immunoglobulin G. VOl. I