IgG to Bovine Serum Are Not Increased in Children With IDDM Sten-A. Ivarsson, Majvi U. L. Mansson, and Irene L. Jakobsson

IgG antibodies to bovine (BSA) were Method. Human specific IgG antibodies against BSA (B-2518, Sigma, St. Louis, MO) were measured by enzyme-linked immunosorbent assay measured in 91 serum samples from children 4-17 years using Maxisorp F96 (Nunc, Roskilde, Denmark) microtiter plates with of age with newly diagnosed insulin-dependent diabetes certification. Pooled sera from 120 healthy donors (male-to-female mellitus (IDDM). A total of 80 healthy 12-year-old chil- ratio 1:1) were used as a reference. Adult control sera with low/negative dren served as control subjects. BSA values >3 or low or high positive values of specific IgG antibodies against BSA SDs were obtained in 6 IDDM patients and 3 control were obtained from the laboratory staff. Horseradish peroxidase (HRP)- subjects (P = 0.50), and values >2 SDs were obtained in conjugated goat anti-human IgG (Fc-specific; Sigma A-6029) together 12 IDDM patients and 4 control subjects (P = 0.11). In with o-phenylenediamine dichloride (OPD) (Sigma P-3804) was used for conclusion, IgG antibodies to BSA were not significantly detection. All chemicals included were of at least analytical grade. A MilliQ UF Plus water purification system (Millipore, Bedford, MA) was increased at onset of diabetes. Diabetes 44:1349-1350, used. 1995 Assay procedure. BSA-coated microtiter plates (10 |xg/ml) were freshly prepared for each assay and were incubated overnight in a 0.1 mol/1 Na-bicarbonate/carbonate buffer (pH 9.6). Manual rinse was repeated four times with a phosphate-buffered saline (PBS)-buffer (pH 7.2) enetic susceptibility is known to be an important containing Tween-20 (lg/1) at 22°C between all incubation steps. All sera included were diluted 1:100 in this PBS-Tween. The same PBS-buffer factor in the pathogenesis of insulin-dependent served as a blank. Duplicate blanks as well as duplicate patient and diabetes mellitus (IDDM) (1). Individuals having control samples were incubated for 90 min on a rotating platform. specific human leukocyte antigens located on HRP-conjugated antibody (1:4000) was incubated likewise for 60 min, chromosomG e 6 are at increased risk of developing IDDM after which OPD was used as a developer for 15 min, the reaction being stopped by adding a 4 mol/1 H2SO4 solution. Absorbance was read at (1,2). However, in addition to genetic susceptibility, an dual wavelength, optical density (OD) 490 with a reference value of OD environmental trigger factor or factors must initiate the 650 (Milenia, Los Angeles, CA). The estimated OD value was divided by disease, because concordance in identical twins is only the blood donor reference value, and the antibodies were expressed in 35-55% (3,4). The first evidence of a possible link between units (U). The intra-assay coefficient of variation (CV) was <3%, and the interassay CV was 5-8%. IDDM and dietary cow's milk was derived from observations Statistical analysis Fishers's exact test (two-tailed) was used for by Elliott (New Zealand), who noted that although IDDM was statistical analysis of the data. absent in Western Samoa, when Samoans emigrated to New Zealand their offspring born in the new homeland were RESULTS characterized by an IDDM incidence almost comparable to The mean BSA IgG antibody level was 1.67 ± 0.16 U (mean that among other New Zealanders of non-Maori origin (4a). ± SE; range 0.2-7.7) among patients with newly diagnosed This observation was supported by findings in animal exper- IDDM, as compared with 1.42 ± 0.10 U (range 0.5-5.2) iments that showed the addition of cow's milk to rat among approximately age-matched children. BSA antibody food to increase the incidence of diabetes among diabetes- values >3 SD were obtained for 6 IDDM patients and 3 prone BB rats (5). Recently, a constituent of cow's milk, control subjects and values >2 SD for 12 IDDM patients and (BSA), has been implicated as the 4 control subjects, both differences being nonsignificant (P = possible trigger of IDDM (6). Accordingly, we decided to 0.50 and 0.11, respectively). study pre-insulin therapy IgG antibodies to BSA in a group of children with new-onset IDDM. DISCUSSION RESEARCH DESIGN AND METHODS IDDM is an autoimmune disorder in which the etiologic role of environmental agents has been difficult to define. Children Patients. In a group of 91 newly diagnosed IDDM patients, aged 4-17 years (mean 10.7), serum samples were collected before insulin therapy with IDDM are characterized by a higher frequency of was instituted and were stored at — 20°C. Children under the age of 3 autoantibodies. In a recent study, Karjalainen et al. (6) found years were excluded. Eighty healthy 12-year-old children served as the presence of anti-BSA antibodies to be strongly correlated control subjects. with IDDM. Although their method and ours are similar, results of the two studies differ. The discrepancy can perhaps From the Departments of Experimental Research (M.U.L.M.) and Pediatrics be explained by ethnic genetic differences between the (S.-A.I., I.L.J.), University of Lund, University Hospitals of Malmo and Lund, Finnish and Swedish populations. Some countries with the Sweden. lowest prevalences of breast-feeding at 3 months of age have Address correspondence and reprint requests to Sten -A. Ivarsson, Department of Pediatrics, University Hospital, Malmo, 205 02 Malmo, Sweden. been found to have the highest incidences of IDDM, and Received for publication 23 May 1995 and accepted 10 August 1995. several studies have shown a relationship between short BSA, bovine serum albumin; CV, coefficient of variation; HRP, horseradish peroxidase; IDDM, insulin-dependent diabetes mellitus; OD, optical density; OPD, duration of breast-feeding and development of IDDM, which o-phenylenediamine dichloride; PBS, phosphate-buffered saline. suggest that an early introduction of cow's milk protein may

DIABETES, VOL. 44, NOVEMBER 1995 1349 ANTIBODIES TO BSA be a risk factor (7,8). This interpretation derives support from REFERENCES the strong correlation found between a population's incidence 1. Rimoin DL, Rotter JI: The genetics of diabetes mellitus. In Immunology of IDDM and its annual milk consumption (r = 0.96) (9,10). in Diabetes. Andreani D, Dimario U, Federlin KF, Heding LG, Eds. London, Kimpton, 1984, p. 45-62 During the decade from 1960 to 1970, the incidence of 2. Eisenbarth GS: Type I diabetes mellitus: a chronic autoimmune disease. breast-feeding in Sweden was the lowest ever. During the N Engl J Med 314:1360-1368, 1986 following decade, 1970-1980, the incidence of IDDM in 3. Olmos P, Hern RA, Heaton DA, Millward BA, Risley D, Pyke DA, Leslie children increased and was sustained in spite of a concom- RDG: The significance of the concordance rate for type 1 (insulin- dependent) diabetes in identical twins. Diabetologia 33:747-750, 1988 itant and still ongoing increase in breast-feeding. These 4. Barnett AH, Eff C, Leslie RDG, Pyke DA: Diabetes in identical twins: a findings are inconsistent with the hypothesis of Karjalainen study of 200 pairs. Diabetologia 20:87-93, 1981 et al. (6) that avoidance of cow's milk protein during the first 4a. Elliot RB: Epidemiology of diabetes in Polynesia and New Zealand. few months of life may reduce the risk of IDDM and might be Pedia.tr Adolesc Endocrlnol 21:66-71, 1992 5. Elliott RB, Martin JM: Dietary protein: a trigger of insulin-dependent more relevant than reduced overall duration of exposure to diabetes in the BB rat? Diabetologia 26:297-299, 1984 cow's milk. However, it is still possible that maternally 6. Karjalainen J, Martin JM, Knip M, Ilonen J, Robinson BH, Savilahti E, ingested cow's milk protein transferred via breast-feeding Akerblom HK, Dosch HM: A bovine albumin peptide as a possible may play a part (11). Bovine (3-lactoglobulin was found to be trigger of insulin-dependent diabetes mellitus. N Engl, J Med 327:302- 307, 1992 present in in 40% of mothers ingesting cow's milk 7. Borch-Johnsen K, Zachau-Christiansen B, Mandrup-Poulsen T, Joner G, products, the range of concentrations being 5-800 |xg/l Christy M, Kastrup K, Nerup J: Relation between breast-feeding and breast milk (12). incidence rates of insulin-dependent diabetes mellitus: a hypothesis. Lancet 2:1083-1086, 1984 Despite the moderate but statistically significant relation- 8. Mayer EJ, Hammar RF, Gay EC, LeZotte DC, Savitz DA, Klingensmith GJ: ship (odds ratio >1.5) found to exist between early introduc- Reduced risk for IDDM among breast-fed children. Diabetes 37:1625- tion of cow's milk and the development of IDDM, no 1632, 1988 9. Scott FW: Cow's milk and insulin-dependent diabetes mellitus: is there a significant increase in anti-BSA antibodies has been found in relationship? Am J Clin Nutr 51:489-491, 1990 conjunction with new-onset IDDM, either by us in children or 10. Dahl-Jorgensen K, Joner G, Hanssen KF: Relationship between cow's by Atkinson et al. (13) among adults. milk consumption and incidence of IDDM in childhood. Diabetes Care 14:1081-1083, 1991 11. Gerstein HC: Cow's milk exposure type I diabetes mellitus: a critical ACKNOWLEDGMENTS overview of the clinical literature. Diabetes Care 17:13-19, 1994 12. Axelsson I, Jakobsson, I, Lindberg T, Benediktsson B: Bovine beta This study was supported by grants from the Medical Fac- lactoglobulin in the human milk: a longitudinal study during the whole ulty, University of Lund, the Health Services Administration, lactation period. Acta Paedatr Scand 75:702-707, 1986 Malmo, Barndiabetesfonden, the Malmo Diabetes Associa- 13. Atkinson MA, Bowman MA, Kao KJ, Campbell L, Dush PJ, Shah SC, tion, the Novo Nordisk Foundation, the Lions Club Interna- Simell O, Maclaren NK: Lack of immune responsiveness to bovine serum albumin in insulin-dependent diabetes. N Engl J Med 329:1853- tional District 101, and the Albert Palsson Foundation. 1858, 1993

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