Davies, D. F., Rees. B. W. G.. Johnson. A. P., Elwood. P. C. &. Abernethy, M. ( 1974) ... ROGER HARRISON. Depurtment of Biochemistry, University of Bath, Bath.
I008
BIOCHEMICAL SOCIETY TRANSACTIONS
60 48 -
50
8
-2 -Ba
+
40
v1
c-”
30
22
20 10 0
0-25
26-50
Xanthine oxidase was, therefore, purified from bovine milk [9] and used as antigen in an e.1.i.s.a. for IgM anti(xanthine oxidase) antibodies. Comparison of serum samples from 5 0 male patients with MI ( 5 6 6.68 years, mean f s.D.) with those from 50 male controls (53.24 f 7.27 years, mean fs.D.) showed that antibody levels were significantly (Plo0
Level of IgM anti-XO (as % o f std serum)
Fig. I . Levels of IgM anti-(xanthine oxiduse) antibodies in seriim sumples from patients with M I und from controls Serum samples were taken from patients within 24 h of admission to a coronary care unit and stored at - 80°C, before e.1.i.s.a. for IgM anti-(xanthine oxidase) antibodies I l O ] . On the basis of clinical data, collected later, these patients were divided into 50 men with MI and 50 age-matched men with no present or past history of coronary heart disease. MI was defined on the basis of clinical history, serial electroradiograms and cardiac enzymes. Antibody levels in the 50 MI patients ( m ) and 50 controls ( m ) are expressed as percentages of a standard (std) pooled human serum, and were compared by using the Kolmogorov-Smirnov twotailed test [ 1 I]. XO, xanthine oxidase.
I . Davies, D. F.. Ilavies. J. R. & Richards. M. A. (1909) J. Arherosclcv. H e s . 9. 103- I07 2. Davies, D. F., Rees. B. W. G.. Johnson. A. P., Elwood. P. C. & Abernethy, M. ( 1974) Lancer i, 10 12- I 0 I4 3. Toivanen, A., Viljanen, M. K. & Savilahti, E. ( 1975) Lancer ii, 205-207 4. Scott, B. B., Swinburne, M. L., McGuffin, P. & Losowsky, M. S. ( 1976) Lancet ii, 125- I26 5. Gibney, M. J., Gallagher, P. J., Sharratt. G. P.. Benning, H. S., Taylor, T. G. & Pitts, J. M. ( 1 980) Arherosclerosis 37. IS I - IS5 6. Davies, D. F., Rees. B. W. G. & Davies, P. T. G . ( 1980) Lancer i, 1190-1 191
BMFGM was prepared from fresh bovine milk [8] and subjected to SDS/PAGE, giving some nine major Coomassie Blue-staining protein bands. The separated proteins were transferred on to nitrocellulose and immunoblotted with pooled human sera, using standard procedures and goat anti(human immunoglobulins) IgG conjugated to horseradish peroxidase as second antibody. Human immunoglobulins were found to bind primarily to polypeptides with molecular masses 1 5 0 kDa and 145 kDa, corresponding t o the enzyme xanthine oxidase, a major protein component of BMFGM.
’
7. Bryson, S. (1989)Ph.D. Thesis, University of Bath, U.K. 8. Mather. 1. H. & Keenan, T. W. (1975) J. Memhr. Rid. 21, 65-85 9. Nakamura, M. & Yamazaki. I. (1982) J. Hiochcw. 92, 1279-1286 1 0 . Benboubetra, M. ( 1989)Ph.D. Thesis, University of Bath, U.K. 1 1. Siegel, S. ( 1956) Nonparamerric Sruhrics for the Hehovioural Sciences, pp. 127- I 36, McCiraw Hill, New York Received 6 March 1990
Human monoclonal antibodies to xanthine oxidase MUSTAPHA BENBOUBETRA, NlKKI AlNGE and ROGER HARRISON Depurtment of Biochemistry, University of Bath, Bath BA2 7AY, U . K . Antibodies to whole dried cows’ milk are present in most humans and their levels have been reported to be raised in patients with myocardial infarction [ I , 21. These antibodies have been shown to be directed primarily to the membrane surrounding fat globules in milk (the bovine milk fat globule membrane, BMFGM) 13, 41 and, more specifically, to the enzyme xanthine oxidase 14, 51. a major protein component of BMFGM 161. Anti-xanthine oxidase antibodies have been shown by enzyme-linked immunosorbent assay (e.l.i.s.a.), to contain IgG, IgM and IgA 14, 5, 71. Levels o f IgM anti(xanthine oxidase) antibodies have been shown t o be elevated in MI patients compared with controls 14, 5.81. Abbreviations used: BMFGM, bovine milk fat globule membrane; e.1.i.s.a.. enzyme-linked immunosorbent assay; EBV. Epstein-Barr virus.
It is clearly of interest to learn more about the origin and possible pathogenic role of human anti-(xanthine oxidase) antibodies and, to this end, we have used human peripheral blood lymphocytes to generate monoclonal anti-(xanthine) oxidase) antibodies for further study. Initially, attempts were made to generate human monoclonal antibodies by fusion of human peripheral blood lymphocytes with mouse myeloma X63-Ag8.653 cells. Human peripheral blood lymphocytes were incubated with Epstein-Barr virus (EBV ) in the presence o f phytohaemagglutinin A, before fusion with mouse X63-Ag8.653 cells as described by Thompson et ul. [9].Heterohybridomas wcre screened for anti-BMFGM antibodies, using a n c.1.i.s.a. IS], before cloning positive cells by limiting dilution [ 101. In five separate experiments, heterohybridomas were obtained, which continued to produce anti-BMFGM antibodies for several weeks, but which progressively ceased such production after repeated cloning, probably because of loss of human chromosomes. In view of these difficulties, human peripheral blood lymphocytes were screened and cloned following treatment
634th MEETING, BATH
I009
with EBV and phytohaemagglutinin A, but without fusion [ I 1, 121. In three experiments, 2-3 weeks after the first cloning (at 20 cells/well) 26%, 18”/0and 20% of wells were positive for anti-BMFGM antibodies. Corresponding values after the second cloning (at 1 cell/well) were 19%, 1O0/,, 14%);after the third cloning, 42%, 61%, 66% and after the fourth cloning, 59%. 63%, 76%. After the fourth cloning stage, in the second and third experiments, 15 of the fastestgrowing, most strongly producing clones were expanded in 25 ml T flasks and the supernatants were used for further studies. These clones, which continued to grow and produce antibodies for several months, could be frozen and thawed without loss of antibody production. Of the 15 clones, 10 were shown by e.1.i.s.a. and dotimmunobinding assay [ 13, 14) to produce IgG class antibodies. two, IgM antibodies. and three apparently gave positive responses for both IgG and IgM. T h e supernatants from all 15 clones were positive for anti-(xanthine oxidase) antibodies in an e.1.i.s.a. [ 51 which, additionally, showed that two of the three ambiguous clones, mentioned above, produced only IgM class anti-(xanthine oxidase) antibodies. Anti-(xanthine oxidase) antibodies have been affinity purified from three clones in yields (per ml of supernatant) of 33.6 pg (IgM), 50. I pg (IgM) and 105.6 p g (IgG), respectively. These purified anti-(xanthine oxidase) antibodies will be used to quantify thc standard e.1.i.s.a. assay [ 5 I in terms o f protein concentration. They will also be used, in competitive e.1.i.s.a.s. to distinguish between bovine o r human enzyme as
immunogen, an answer to a question which has major implications for the study of ischaemic heart disease. I . Ilavies. 11. F.. Davies. J. K. & Richards. M. A. ( I V h Y ) .I. A/herodi,r.Hcs.9. 103- I07 2. Ihvies, D. F., Johnson. A. P.. Kees. B. W. G., Elwood. P. C, & Aherncthy. M. ( 1974) / . t r r r c ~i. 10 1 2 - 10 I4 3. Ilavies. D. F.. Reex. €3. W. G. & Ihvies. 1’. 7.. G. ( I 982) / t i / . /.cth. 12.22-24 4. Rryson, S.( 1989) Ph.1). Thesis. University o f Hnth. U.K. 5 . Benboubetra. M. ( 1989) Ph.1). Thesis, University o f Bath, U.K. 6. Briley. S. M. & Eisenthal. R. ( 1975) Hioc./rom. J . 147, 41 7-423 7. Ng. Y. L. E., Lewis. W. H. P. & Chui, S. H. ( 1990) h f d l h . .Sc.i. 47.30-35 8. Benboubetra. M.. Harrison. K. & Thomas. K. I). ( I 990) KOchem. .So(,. 7icrri.s. 18. 1007- I008 9. Thompson. K. M.. Hough, D. W.. Maddison. I? J.. Melamcd. M. D. & Hughes Jones. N. ( 1986) J. /mmrrrro/. h f i 4 o d s . 94. 7-12 10. Thompson. K. M. ( I 986) Ph.11. Thesis. University of’ Bath.
U.K. I I . Winger. L., Winger. C., Shastry. P.. Kussell, A. & Longenecker, M. ( 1983) /’roc. Nor/. Acad .Sc,i. lJ..S.A.80. 4484-4488 12. Makowski, F,. Joffe. M. 1. & Kittenberg. M. 13. (I980) J . /mmrrtio/. hfc~t/ioc/c. 90. 85-87 13. Hawkes, R.. Niday, E. & Gordon, J. ( I 982j A d N i o d i i m 119. 142-147 14. Holmberg. I).. Ivarx. F. & Edlund, T, ( 1083) .I. /mmrrrio/. hfe/hot/.s 6 I . 9- 16 Received 7 March 1990
Involvement of polyamines in pancreatic growth induced by dietary soyabean, lectin or trypsin inhibitors G . G R A N T , S . BARDOCZ, D.S.BROWN, W. B. WATT, J. C. STEWART and A. PUSZTAI Roweti Research Institute, Ruckshurn, Aherdeen AR2 (ISR, Scotland, U.K. Pancreatic growth is induced by feeding rats with diets containing raw soyabean o r trypsin inhibitors (Kunitz and Bowman-Birk) o r lectin [ 1-31. Polyamines (putrescine, spermidine, spermine) appear to have a role in this enlargement and, indeed, a rapid increase in polyamine content is evident in the pancreas before the appearance of any signifiAbbreviations used: ODC, ornithine decarboxylase; DFMO, adifluoromethylornithine; SAT. spermine N’-acetyltransferase.
cant hypertrophic o r hyperplastic changes [3J.T h e aim of the present study was to determine the mechanism by which these polyamines were accumulated in the pancreas. Pancreatic enlargement was induced, ;IS previously described [ 31. by feeding o f rats with a raw soyabean-hased diet for 7 days. Furthermore, this growth coincided with an increase in the pancreas pol yaminc (primarily spermidine) content (Table I ) . Over 780 nmol o f polyamines were accumulated in the pancreas within 24 h. However. the activity of ornithine decarboxylase (ODC),the rate-limiting enzyme of polyamine synthesis de iiovo 141, detected in the pancreas during this period was only 3.0 f 0.5 nmol o f putrescine/h per pancreas and thus insufficient to generate the additional pol yamines. ~~
~
lable I . 1’atic.reer.s(tit~iolll’crt?~~reii.s) (itid serum (pmol/ml)polynmine Inels iir rats fkd on (r .soyirhenrr-ha.sed die/ (7,yldiryper rut)
Kesults are means f S.E.M. Diets contained 100 g o f protein/kg from either soyabean or lactalhumin (control) 121. Initial rat weights were 85 f 4 g. Rats were killed 2 h after being fed 2 g of diet. Food intake at 6 and 12 h was 4 and 6 g, respectively. Samples were analysed for polyamines [ 1 I ] . *Significantly different from 0 h ( I >
