CA2074790A1 - Methods for detecting pre-clinical iddm - Google Patents

Abstract

ABSTRACT

Methods are provided for detecting IDDM and pre-clinical IDDM. Novel peptides are provided for use in these methods.

Description

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METHODS FOR DETECTING PRE-CLINICAL IDDM

This invention relates to methods for detecting autoimmune diseases and pre-clinical autoimmune diseases.

In particular, it relates to methods for detecting insulin dependent diabetes mellitus (IDDM) and pre-clinical IDDM. Peptide fragments are provided for use in these methods.

Backaround Epidemiological evidence in man t4, 20-22] and data from animal feeding studies [13, 23-25] have suggested a diabetogenic effect of dietary cow milk proteins. Supportive serological findings have been identified in animals [12,13] and humans [15-17]
associating immunity to cow's milk proteins and Type 1 diabetes. The most direct evidence for a pathogenic link between cow's milk proteins and diabetes comes from a family study in Finland, where exclusive breast-feeding for the first 3-4 months of life was found to protect from later development of diabetes [22].
Most of these studies did not identify a specific cow milk protein or explain the near global increase in diabetes incidence despite emphasis on breast-feeding. However, these latter observations [22]
are consistent with the view that in humans (as in diabetes-prone rats[3]) a diabetes associated immune response to bovine serum albumin ~BSA) or to peptide fragments or portions thereof is triggered in the early post-natal period [1,26].
An existing detection method for pre-clinical IDDM is based on detection of islet cell antibodies ~.

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[36-38]. Unfortunately, this is a very difficult and laborious (2-day) assay which requires manual processing, visual judgment of results with its inherent inaccuracies. There are only a few centers in any country which can perform this assay according to international standards. Up to 20% of the population and 30-50% of IDDM family members have such antibodies. The test predicts clinical IDDM in only a subset of cases and is positive at diagnosis in only 80%.
Animal work with BB diabetic rats showed detectable antibodies to bovine serum albumin in IDDM and in some animals without IDDM but with some histological islet changes (Martin et al., (October, 1991), Ann. Med., V. 23, p.447). Since not all animals showing such changes progress to overt IDDM, these studies did not indicate that detection of antibodies to bovine serum albumin could be predictive of progression to IDDM.

Until the work of the present inventors, no convenient clinical method with sufficient predictive and discriminatory ability was available to screen human subjects and detect pre-clinical IDDM.

Figures Figure 1: (A)Serum IgG anti-BSA Antibody Concentrations at Diagnosis of Insulin-Dependent Diabetes in 142 Children (hatched bars) and 79 Normal Children (black bars). The distribution of serum IgM(C) and IgA(B) anti-BSA Antibody Concentrations is shown in the lower panelafter normalization by smoothing.

Figure 2: Measurements of total and ABBOS-Specific Anti-BSA Antibodies in 17 Diabetic Patients. The black bars represent patient anti-BSA levels, the white bars the levels remaining after removal of ABBOS-specific antibodies. The horizontal bar indicates the mean anti-.

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BSA concentrations in 17 normal children (upper line) andthe concentrations after removal of anti-ABBOS antibodies in the same sera.

Figure 3:(A-D) A: Distribution of moderately- (light grey bars), high- (grey bars), and very highly elevated -(black bars) IgG-anti-bovine serum albumin antibodies in 40 diabetic children as determined by particle concentration fluoroimmunoassay (PCFIA). B: PCFIA
standard curve for a serum pool (from diabetic children) containing 12.3 KfU/~l IgG- and 4.2 KfU/~l IgA-anti-BSA
antibodies: binding competition with increasing amounts of BSA and ovalbumin/Tween-20. C: Anti-BSA standard curves for enzyme immunoassay (EIA). D: Binding competition with increasing amounts of free ovalbumin/Tween-20 for IgG-() and IgA-(~) anti-BSA
antibodies, as well as with increasing amounts of free BSA for IgG-(~)and IgA- (-)anti-BSA antibodies, KfU=kilo fluorescence units.

Figure 4: Mean levels (~SEM) of anti-BSA antibodies in Type 1 diabetic- and matched control children as detected by particle concentration fluoroimmunoassay (PCFIA, upper panels) and enzyme immunoassay (EIA, lower panels).
Difference between diabetic and control children:
PCFIA:IgG, p<0.0001; IgA, p<0.001. EIA: IgA, p<0.01.

Figure 5 - Correlation between the levels of anti-BSA
antibodies as determined by enzyme immunoassay (EIA) and ' .

2~7~0 particle concentration fluoroimmunoassay (PCFIA) in diabetic- and control children. Shaded areas represent BSA-antibody levels considered as "non-elevated" ( ~ ) negative for BSA antibodies by PCFIA, (~) negative for BSA antibodies by EIA). A: IgG in diabetic children, n=40, r,0.28, p=0.09; B: IgA in diabetic children, n=40, r,=0/11, p=0.48; C: IgG in control children n=179, r,=0.02, p=1.0; D: IgA in control children, n=179, r,=-0.05, p=1Ø Correlation coefficients were determined by Spearman's rank correlation.

DESCRIPTION OF THE INVENTION

The measurement of autoimmune disease-associated antibody levels specific for proteins or protein fragments derived from common dietary sources as well as measurement of T lymphocyte sensitization to such fragments by the methods in the invention provide a unique and new clinical and investigational tool for 1.) the diagnosis of early, pre-clinical disease as prerequisite for the development and use of disease delaying or preventative therapies;
2.) the differential diagnosis of autoimmune patients at first clinical presentation;

3.) the monitoring of disease course and effects of therapy.
The present inventors were the first to show by a prospective study that by determining human serum levels of antibodies to bovine serum albumin or to certain natural or synthetic peptide fragments thereof by a method to be described, one can detect those individuals who will develop IDDM.

In accordance with one embodiment of the invention, a method is provided for detecting IDDM or pre-clinical IDDM by measuring serum antibodies to bovine serum albumin (BSA) by a particle concentration 2 a ~

fluoroimmunoassay (PCFIA) technique as described in Examples 1 and 3. The relevant antibodies are detected by their binding to particle-bound BSA.

Other methods have been used to detect anti-BSA
antibodies in serum, for example ELISA techniques, as described in Example 2. It can be seen from example 2 that the antibodies detected by the ELISA method do not provide the discrimination required for reliable diagnosis of diabetes or pre-diabetes.

Particle concentration fluoroimmunoassay detected elevated IgG-anti-bovine serum antibodies in all diabetic children, enzyme immunoassay in 25% (p<0.0001).
Fluoroimmunoassay detected elevated levels in 2.2% and enzyme immunoassay in 10% of control children (p<0.002).
Elevated IgA-anti-bovine serum albumin antibodies in patients were slightly more often detected by fluoroimmunoassay than by enzyme immunoassay, while in control children enzyme immunoassays detected elevated levels three times more often (p<0.01). Values measured in either assay showed overall no correlation in either patient (IgG: r,-0.28; IgA: r,-0.11) or control sera (IgG: r, 0.02; IgA: r,--0.05). Fluoroimmunoassay for IgG
was 100% disease-sensitive (enzyme immunoassay: 25%, p<0.0001) and more disease-specific (IgG; p<0.02).

Our findings demonstrate that these assay techniques thus detected distinct subsets of anti-bovine serum albumin antibodies with little (IgG) or some (IgA) overlap. In fluoroimmunoassay procedures, antigen:antibody binding occurs within 1-2 min while hours are allowed in an enzyme immunoassay. Antibodies with high on-off binding rates typical for immune response following hyperimmunization are therefore measured preferentially by particle concentration fluoroimmunoassay and it is these antibodies which appear 6 2 ~
to be associated with diabetes. These observations emphasize the need for epidemiological surveys to validate immunoassay procedures used for clinical purposes.

Comparison of the amino acid sequences of various serum albumin proteins, including human and bovine proteins, suggested to the inventors to focus on the region between amino-acids 138-166, the region of greatest divergence between human and bovine (Glerum et al., (1988), Diabetes Research, vol. 10, p. 103).

In accordance with a further embodiment of the invention, various novel peptides within this region have been synthesised, as described in Example 4.

It has been found that the bulk of the diagnostic antibodies detected by PCFIA as described above bind to peptide CS2185, ABBOS, as described in Example 1.

The PCFIA assay described above may be performed using particle-bound BSA-peptides such as particle-bound AB80S instead of particle-bound BSA.
The ABBOS peptide can be used to detect up to 90% of IDDM-associated anti-BSA antibodies even when modified at the C-terminus as described in Example 4.

These peptides may be fragments of the natural BSA protein or synthetic peptides prepared by a suitable technique. Such techniques will be known to those .
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skilled in the art and include chemical synthesis and recombinant techniques.

Analogues of these peptides which retain their ability to bind to IDDM-associated anti-BSA antibodies are included within the scope of the invention.

In accordance with a further embodiment of the invention, a method is provided for detecting in IDDM
patients and in pre-clinical IDDM subjects sensitised T
lymphocytes which specifically recognise and proliferate in response to peptide fragments of BSA, including ABBOS
and CS2267.

This detection method can be used to detect IDDM, pre-clinical IDDM and also to detect reactivation of the ~ cell-attacking immune process in patients after ~cell or islet transplantation.

In accordance with a further embodiment of the invention, a peptide is provided, CS2267, which binds specifically to the sensitised T lymphocytes present in IDDM and pre-clinical IDDM patients.

By coupling a peptide such as CS2267 to toxic compounds, immunotoxins can be prepared which are directed to and can destroy the specific T lymphocytes which mediate ~ cell destruction in IDDM, providing a means of arresting the disease process.

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Example 1 - Detection of anti-BSA antibodies by PCFIA

Patient Populations: Blood samples were obtained from 142 Finnish children (83 males, 59 females, mean (+SD) age 8.4+4.3 years) with newly diagnosed insulin-dependent diabetes mellitus. Fifty patients had diabetic ketoacidosis, 48 diabetic ketosis only, and the remainder hypoglycemia alone. All were continuously dependent on at least one daily injection of human insulin and had increasing insulin dependence after diagnosis. We also studied 79 age-, sex- and region-matched children admitted for minor surgery (42 males, 37 females, mean age 8.4+3.1 yr), and from 300 adult Toronto blood donors.
Blood samples were obtained from the patients before the first insulin injection and 3 to 4 months later and, in a random subset of 44 patients, 1 to 2 years later. The serum samples from the two groups of children were sent coded to Toronto.

Clinical assessment included history and measurements of insulin and islet cell autoantibodies identified either by indirect immunofluorescence or complement fixation test . Sample volumes were insufficient for full titration of the earliest samples, islet cell antibody results are therefore expressed as positive or negative. The HLA-A, -B, -C, DR.Dw haplotypes of all patients were determined as described~.

Measurement of anti-BSA Antibodies Anti-BSA antibodies were measured by particle concentration fluoro-immuno assay (PCFIA ) as described in Dosch et al, (1988) "Characteristics of Particle Concentration Fluorescence Immunoassay (PCFIA): Novel Alternatives to ELISA and RIA," in Proceedings of 1987 .

Pandex Symposium on Particle Concentration Fluorescence Immunoassay.

96 - well unidirectional flow vacuum filtration plates were used for the assay and phase separation procedures were carried out by the robotic Screen Machine~
instrument (IDEXX, Portland, Me., U.S.A.) which is programmable for reagent additions, timed incubations, phase separations, washings and measurements of particle bound fluoresceinated secondary antibody, as described in Dosch et al, (1990), Int. Immunol. Vol. 2, p.833 and Cheung et al. (1991), J. Biol. Chem., Vol. 266, p 8667.

Two-hundred microlitres of BSA [Grade V, Sigma Chemical Co., St. Louis, Mo., U.S.A., 10% in phosphate-buffered saline (PBS; 40g NaCl, lg KCl, lg KH2PO4, 5.75g Na2HPO4, o.5g CaCl2, 0.5g MgCl2/5 litres distilled water, pH 7.2)] was coupled covalently (100 ~l of 10 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide) onto 400 ~1 (5% stock; IDEXX) carboxylated polystyrene beads (diameter 0.75 ~m). Subsequently, 10% Tween-20 in 1.0%
ovalbumin-PBS was used as blocking agent. Concentrations down to 1% Tween-20 in 0.1% ovalbumin-PBS may be used as ~blocking agent. After repeated washings, beads were -25 stored in 1% Tween-20-PBS. Over a period of 9 months the activity of the beads remained unchanged. Ovalbumin was obtained from Sigma.

Twenty microlitres of test serum dilutions 30 (1:100-1:1,000) were added to microwells containing 20 ~l of 1:20 diluted BSA-coated microspheres (initial 2.5%
weight/volume). Up to ten plates were inserted into the Screen-Machine for programmed phase separations, washings and addition (100 ng/well) of affinity purified, custom BSA-free fluorescein-conjugated goat anti-human IgG, IgA, 2 ~
and IgM (Fc-fragment specific, BioCan, Mississauga, Ontario, Canada). Drying and precipitation of serum protein was avoided by short (1 min) incubation, phase separation and washing procedures at low (5mm Hg) vacuum pressure. Prior to reading, wells were vacuum dried for 1 min and fluroescence emission (472/512nm) was read under high vacuum from the concentrated particle cake at the bottom of the well. Kinetics of the system have been published (Dosch et al. (1988) above). We have processed up to 3,000 replicate samples per day per instrument per operator.

A calibrated pool of serum from diabetic patents was used as the standard in each plate. This standard contained 12.3 kilofuorescence Units (KfU) IgG
anti-BSA antibodies per microliter, 4.2KfU/~l IgA and 4.0 KfU/~l IgM anti-BSA antibodies. The anti-BSA assays had a sensitivity of 1.0(IgG, IgA) and 10.0 (IgM) ng/ml, the intraassay-and interassay coefficients of variation were 8.9% and 9.8%, respectively. Addition of BSA (but not ovalbumin) blocked antibody binding in a dose-dependent fashion. Anti-BSA antibody concentrations exceeding the mean plus 2 SD in the 79 normal children were defined as elevated.
Data Analysis: Antibody concentrations as expressed as kilofluorescence units per microliter relative to the standard serum pool. The results were analyzed using Chi-square statistics, parametric one-way analysis of variance, and Student's unpaired t-test for normally distributed values. The distribution of anti-BSA concentrations was normal for each isotype. In the ' .

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case of skewed distribution, the Mann-Whitney-U test and Spearman's rank-correlation test were used(r,). Anti-BSA
antibody concentrations after diagnosis were evaluated using a paired test.

Anti-BSA Antibodies: The serum IgG anti-BSA
antibody concentrations in the diabetic patients were considerably higher than those in the normal children (Figure 1), the mean concentration being almost seven fold higher (Table, 1,P<0.001, Table 2). The elevated serum IgG anti-BSA concentrations in the diabetic patients did not reflect generalized immune responses against nutritional antigens, since the patients and the normal children had similar serum concentrations of IgG
antibodies to the major cow milk proteins casein and ~-lactoglobulin (Table 1).

The mean serum concentration of IgA anti-BSA
antibodies was higher in the diabetic patients than in the normal children (P~0.0001, Table 1), but the values overlapped (Figure 1). Two thirds of the diabetic patients (and two normal children) had elevated : concentrations (P<0.0001, Table 2). Consistent with the young age of the diabetic patients, the patients who had IgA anti-BSA antibodies were older than those with low antibody concentrations (10.1+3.4 vs. 6.0+4.6 yr;
p<o. ooOl) .

The mean serum concentration of IgM anti-BSA
antibodies in the diabetic patients were slightly lower than those in the normal children (P~0.05, Table 1).
Less than 1 percent of patients had elevanted . .

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concentrations compared with 8 percent of normal children (P<O.O1, Table 2, Figure 1).

No diabetic patient or normal child had IgD
anti-BSA antibodies, and in a random subset of patients no IgE anti-BSA antibodies were detected.

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While low concentrations of (mainly IgM) anti-BSA antibodies were detected both in the diabetic and normal children, high concentrations of IgG and IgA anti-BSA antibodies were found only in the diabetic children.
The latter findings indicated the existence of a close (100%) association between BSA-specific immune responses and the clinical expression of insulin-dependent diabetes. Consistent with an active, antigen-driven immune response against BSA in diabetes, there was a significant correlation between IgM and IgG anti-BSA
concentrations (r,=0.77; P<0.0001).

The concentration of long lived IgG anti-BSA
antibodies remained in the diabetic patients 3 to 4 months after diagnosis, the concentration of the short lived IgA anti-BSA antibodies was lower (P<0.001, Table 3). In the 44 patients studied 1 to 2 years after diagnosis, the concentration of all three types of anti-BSA antibodies was lower (P<0.001), reaching normal levels in most patients (IgG: 27 patients, IgA: 43 patients, Table 2). These results are consistent with the decline in antigenic stimulation by beta cell p69 13~14.

Studies of Specificity: Additional studies were done using serum from 44 diabetic children and 44 normal children. IgG antibodies to bovine milk casein (Sigma) and ~-lactoblogulin (Sigma) were measured using coated microspheres as described for BSA. The ABBOS
peptide (BSA sequence position 152-168) and the homologous region of rat serum albumin (ABRAS peptide) were synthesized with a C-terminal cysteine residue not present in the natural sequence, and the C-terminal cysteine was biotinylated. Solid phase ABBOS and solid phase ABRAS were prepared by binding the biotinylated ; 35 peptide to streptavidin coupled to carboxylated polystyrene microspheres, as described by Dosch et al.
(1988) above. 20~1 (0.5% w/v) of ABBOS-or ABRAS-.

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conjugated microspheres were mixed with 3~1 patient or normal serum in a final volume of 0.3 or 3 ml. After incubation at 4C for 15 minutes, the mixtures were centrifuged and 20~1 of the supernatant was used for measurement of residual anti-BSA antibodies.

Anti-ABBOS Antibodies: Measurement of anti-BSA
antibodies before (Fig. 2, black bars) and after exposure of serum to solid phase ABBOS peptide (white bars) was done to determine the proporation of anti-BSA antibodies that specifically bound the ABBOS region of BSA.

These studies were done using serum from 44 diabetic patients with high, moderate and realtively low anti-BSA concentrations and an average near the overall mean (Fig. 2, 17 representative samples). The concentration of IgG anti-BSA antibodies decreased by two-thirds (range 30 to 70%) after reaction of serum with ABBOS peptide. Similarly, a large proportion of the IgA
and IgM anti-BSA antibodies (23 to 71%) were ABBOS-specific (Table 3), delineating a severe bias for this short sequence that represents less than two percent of the BSA molecule. The amount of anti-BSA antibody removed by incubation of serum with the ABRAS peptide was within normal assay variation (-10%).

The decrease in anti-BSA antibody concentrations after diagnosis was initiated by the disappearnce of ABBOS-specific antibodies (90%), P<0.0001). By 1 to 2 years after diagnosis only 7 of the 44 patients studied had anti-BSA antibodies with specificity for the ABBOS peptide and 17 of the 44 patients had slightly elevated anti-BSA concentrations.
Serum samples from the 17 normal children with the highest anti-BSA concentrations were studied similarly ':' . - :.
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. , 2~7~79a after incubation of their serum with the solid phase ABBOS peptide. The anti-BSA antibody concentrations were not significantly reduced in the absorbed serum, and only 2 serum samples contained detectable IgG or IgA anti-ABBOS antibodies, respectively. In 300 adult blooddonors from Toronto, the range and mean of IgG anti-BSA
antibody concentrations were similar to that in the 79 normal Finnish children (Table 3), and IgG anti-ABBOS
antibodies were found in three percent of the samples.
Anti-BSA Antibodies and Disease Markers: No relationships were found between the concentration of anti-BSA or anti-ABBOS antibodies and the severity of disease presentation (blood glucose, HbAI, serum C-peptide concentrations), the duration of symptoms before diagnosis, or the severity of diabetic ketosis or acidosis. The specificity, concentrations and isotype distribution of the antibodies were similar in multiplex and simplex families.
At the time of diagnosis 78% of the patients were islet cell antibody positive, 58% had complement-fixing islet cell-, and 47% had insulin autoantibodies.
Niether anti-BSA concentrations, isotype diversity nor specifity were associated with the presence or absence of islet cell- or insulin autoantibodies.

Children heterozygous for HLA-DR3/4 or -Dw3/4 initially had more severe diabetes (higher blood glucose and HbA~ and lower serum C-peptide concentrations) than those negative for such haplotype combinations, but the frequencies or concentrations of insulin- and islet cell autoantibodies were similar in both haplotype groups.
The concentrations of BSA/ABBOS antibodies were comparable among the diabetic children with or without HLA-DR3/4 or -Dw3/4, as well as in those with -DR3/x, -DR4/x and -Dw4/x.

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Monoclonal antibodies binding both BSA and ABBOS (anti-BSA/ABBOS antibodies) were generated from Epstein-Barr virus transformed B lymphocytes of a newly diagnosed diabetic patient ~. Monoclonal antibodies as well as polyclonal rat anti-ABBOS antiseral4 gave negative reactions for insulin and islet cell autoantibodies.
Conversely, addition of up to 1000 and 100 ~g of BSA or ABBOS peptide respectively did not alter the results of assays for islet-cell antibodies and insulin autoantibodies in the serum of all 15 diabetic patients tested.

Example 2 - Comparison of PCFIA and ELISA

Patients: Forty Finnish diabetic children (22 males, mean + SD age 6.2+4.5 years, range 0.9-15.5 years) were randomly selected from the patient population of Example 1 for assay comparison and contained a typical range of elevated levels of BSA antibodies (Fig. 3A).
Samples were drawn at the time of diagnosis of diabetes, and selected sera were sent coded back to the laboratory in Finland to be analysed by EIA. Control subjects comprised 179 age- and sex-matched non-diabetic Finnish children (98 males, mean + SD age 6.2+3.6 years, range 0.9-15.9 years) Anti-BSA antibodies in patient and control sera were measured by PCFIA as described in Example 1.

An in-house (positive) standard serum pool from diabetic children was used and a standard curved prepared in every plate (Fig. 3B). Competition experiments showed ,. , , , , :
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, 2~790 satisfactory specificity for BSA. Free BSA blocked antibody binding in a dose-dependent fashion, whereas neither ovalbumin nor Tween-20 nor both together were able to displace antibody (Fig. 3D). Results are expressed as kilo fluorescence units (KfU) per microlitre based on instrument gain (5x), serum dilution and assay volume as derived from the standard containing 12,300 KfU/ml IgG- and 4,200 KfU/ml IgA-anti-BSA antibodies.
Due to the linearity of fluorescence emission energy the values were normally distributed. Elevated antibody levels were defined to exceed the mean level in control subjects plus 2 SD.

Enyme Linked Immunosorbent Assay (ELISA) The serum samples assayed by PCFIA as described above were also assayed by an enzyme linked immunosorbent technique for anti-BSA antibodies, a conventional three-layer solid phase procedure modified from Tainio et al.
(1988), Acta Paediatr Scand., vol. 77, p.807.

The method employs polystyrene Microstrip wells ($absystems, Helsinki, Finland) that are processed in an automatic EIA analyser (Auto-EIA II; Labsystems, Helsinki, Finland), which can process up to three plates (66 samples) per day. The plates were coated with 2 ~g/ml (100 ~l) BSA (A-4378; Sigma) in 0.1 mol/1 PBS-5 mol/lNaN3 (pH 7.4) overnight at room temperature. After washing with PBS-NaN3, the wells were saturated with 1%
gelatin-PBS-NaN3 for 1 h at 37C and stored at +4C until used.

Serum samples were diluted 1:40 in 0.5%
gelatin-PBS-NaN3 prepared in 0.05% Tween-20. Three replicates of 100~1 of serum dilutions were plated, two in coated wells and one in a non-coated well. After a 60 min incubation at 37C wells were washed (4x). Diluted 2 ~

alkaline phosphatase conjugated anti-human IgG- or -IgA
(cat. no. 67806 and 67808; Orion Diagnostica, Espoo, Finland) was added and incubated for 60 min followed by four washes as before. After 45 min incubation of substrate [pNPP (p-nitrophenyl phosphate) 2mgtml in DEA
(N-N-diethylaniline)-buffer] the reaction was stopped with 1 mol/1 NaOH. Absorbances (optical density 405nm) in non-coated wells were subtracted from test values.

Intra-assay and inter-assay variations of the assay were 9.3% and 15.8%, respectively. For serum assays, serial dilutions of a BSA-antibody positive standard were run on each plate (Fig. 3C) and the results expressed as percent binding of the standard serum.
Values for both IgG- and IgA were skewed despite log-transformation, and thus the limit for positivity was set at the 90th percentile of the values in control subjects.
This limit was selected after examining a series of cut-off values as giving the highest sensitivity with acceptable specificity. Sera having an absorbance of 3.9% for IgG and 14.2% for IgA of the standard were considered as positive.

Stat~stical analvsis Statistical analysis was performed using cross-tabulation, Chi-Square statistics and Student's unpaired t-test in the case of normally distributed variables.
Since the distribution of BSA antibody levels obtained by EIA was skewed despite transformation, the difference between diabetic and control children was evaluated by a Mann-Whitney U-test. Mann-Whitney U-test and Spearman's rank correlation test were used to compare antibody levels between EIA and PCFIA. Sensitivity and specificity of the assays was determined, and the results evaluated by cross-tabulation with chi-square statistics.
Results are presented as means + SEM.

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The 40 sera from diabetic children contained a range of elevated IgG-anti-BSAK~ antibodies; however, only 25% were found positive by EIA (Table 4, p<0.0001).
Conversely, IgG-anti-BSA~A antibodies in control subjects were elevated more frequently than those detected by PCFIA (Table 4, p<0.0002). Elevated IgA-anti-BSA
antibodies in diabetic children were found in 50% and 42%
(p=NS), however only 3.3% and 10% of control children were positive in PCFIA and EIA, respectively (Table 4, p<0.01). These results demonstrated that PCFIA but not EIA preferentially detects disease-associated BSA
antibodies in children with Type 1 diabetes. In contrast, EIA shows preference for detection of antibodies more prevalent in the general population.
Neither procedure detected all BSA antibodies.

BSA antibody levels in diabetic children are shown in Figure 4. There was a significant difference in ; the levels of both IgG-, and IgA-anti-BSA antibodies between diabetic and control children when determined by PCFIA (p<0.0001 and P<0.001). In contrast, the levels of IgG-anti-BSAaA antibodies were roughly similar in diabetic and control children. IgA-anti-BSA~A antibodies were higher in diabetic children, but the difference (p<0.01) was less prominent than in PCFIA (p<0.001).
These findings suggest a quantitative difference in the subsets of antibodies detected by PCFIA, and this difference distinguishes diabetic and control children.

Individual PCFIA- and EIA values are compared in Figure 5 for diabetic and control children. Shaded areas indicate the levels considered as negative (see above). The correlation between PCFIA and EIA was very poor (-0.05sr~S0.28, O.O9~p~0.1). In diabetic subjects only a subset of sera (IgG:-20% and IgA: -32%) gave relatively low or high anti-BSA values in both assays i.e. showed correlation. Moreover, among control 21~7479~

subjects only one IgA sample (0.3%) was positive in both assays.

BSA-antibodies in control sera bound significantly more frequently in EIA than in PCFIA (IgG:
p<0.002 and IgA: p<0.01, Table 4) and only one out the 18 control subjects positive for IgG- or IgA-anti-BSA~A was positive by PCFIA. On the other hand, of the four IgG-and six IgA- positive control sera detected by PCFIA, only one was positive by EIA (Fig. 5). Therefore, most disease-associated BSA-antibodies were only detected by PCFIA. The sensitivity of PCFIA in detecting disease-associated IgG anti-BSA antibodies was excellent when compared to EIA (100% vs 25%, Table 5; p<0.0001) and the disease-specificity was higher for PCFIA (Table 5;
p<0.02). For the IgA isotype assay results were comparable with respect to disease-sensitivity, but in EIA this was at the cost of assay specificity (p~0.05).
These findings cuggest that PCFIA and EIA preferentially detect different subsets of BSA antibodies with no (IgG) or some overlap (IgA). Antibodies detected in EIA are not associated with Type 1 diabetes but are found commonly in the general population.

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Table . Frequency of elevated and-BSA antibodies in diabedc (n-~O) and control children (n=179) as defincd by pamcle conccntration fluoroimmunoassay (PCE;IA) and by enzyrne immuno-assay (EIA).
Iso~pe Padents Positivc Posidve Control Subjccts n (%) n (9~O) PCFIA EIA P PCFIA EIA P

IgG 4oa (100%) lOb (2S%) ~0.0001 4 (2.2%) 18 (109O) ~0.002 IgA 20a (50%) l?a (42%) NS 6 (3.3%) 18 (10%) <0.01 Fcr diffcrcncc bcn~rccn andbody positivc padcnts and control subjcc~ using PCFLA and EIA a p~O.OOOl, b p~O.OS

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Table Sensitiviry and specificity of PCFIA and EIA in detecdng an~-BSA antibodies.
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Scnsidvity (%) Specificin~r (9~O) PCE;IA ElA P PCFL~ EIA P

IgG 100 25 ~0.0001 98 90 ~0.02 IgA 50 42 NS 97 90 <0.05 .

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In order to compare the PCFIA assay procedure to the more commonly available EIA, we analysed a large number of samples using both techniques. The comparison revealed unequivocal differences with diabetes-associated anti-BSA molecules detected almost exclusively by PCFIA.
Both, levels and frequency of positive responses among diabetic children were significantly higher in PCFIA than EIA. The wide scatter of antibody levels in EIA for both patients or control subjects caused major overlap between the groups and made differences statistically insignificant. A large proportion of samples were below the detection limit of EIA, causing considerable skewness for measurement of both isotypes examined.

PCFIA detected BSA antibodies in children with diabetes, whereas only a few non-diabetic children were positive. In contrast, only a small proportion of the antibodies were disease-associated in EIA, and levels were elevated more often in non-diabetic subjects. With ; 20 only one out the 18 controls positive for IgG- or IgA-anti-BSA~A elevated by PCFIA as well, the dichotomy was clear between antibody subsets detected in either procedure. Interestingly, the single elevated IgG and IgA values detected in both procedures derived from the same serum sample, suggesting that the host determines the choice of antibody species utilized in the common immune response to dietary BSA.

The BSA molecule consists of 608 amino acids and there are several areas where the sequence differs from human serum albumin. One of those is the described ABBOS (pre-BSA position 153-169 [2]).

As seen in example 1, most of the diabetes-associated antibodies detected by PCFIA in diabeticchildren are directed against this epitope, whereas in non-diabetic control subjects the major epitopes are s~ 7 ~ ~

different, with less than 3% of donors able to recognize ABBOS. Since the ABBOS epitope is immunologically cross-reactive with a (beta-cell) autoantigen, p69 [1,3], the poor immunogenicity of this epitope in the general population is not surprising and clearly identifies the diabetic population. We have tentatively linked this principal difference to efficient antigen presentation of ABBOS by diabetes-associated MHC class II molecules coupled with a delay in oral (or mucosal) tolerance development in diabetic subjects [1,3]: our focus on the latter was triggered by the report that the single highest marker of diabetes risk (DQ~ non-ASP57) also marks susceptibility for IgA deficiency, a regulatory abnormality of mucosal immunity [27,28].
An antigen such as BSA has several epitopes which can induce a wide spectrum of high and low affinity antibodies. Our results are very reminiscent of the observation that insulin-autoantibodies (IAA) detected by EIA are poorly disease-associated [29] and have a low predictive value compared to fluid-phase radiobinding assays (RIA) [30-32]. EIA has been characterized by low sensitivity and an unacceptably high rate of false positives [29], similar to the results obtained in this study. EIA detects mainly low affinity IAA that have high binding capacity, whereas RIA detects high affinity, low binding capacity and strongly disease-associated subset(s) of IAA [32]. The same could apply to a fluid-phase assay such as PCFIA, in which accessibility to the epitopes may be different from EIA. The same epitopes may not be available in PCFIA and EIA due to different binding procedures. Disease-associated epitopes may not be accessible if bound to EIA plate [33]. On the other hand, an excess of adhesive antigens on EIA-plate surface may bias binding of non-disease-associated, low affinity antibodies with high binding capacity as reported for IAA
in healthy blood donors [34].

2 ~

The striking lack of correlation between the two assay systems is less suggestive of gradual differences in average antibody affinity, but indicates absolute distinctions in the quality of antibodies detected. Maturation of an antigen drive (hyper-) immune response produces an antibody repertoire that is not only of high affinity but also favors immunoglobulins with fast binding kinetics [35], i.e. antibodies characterized by high on:off antigen binding rates and release required, for example, for rapid opsonization of pathogens and re-utilization of antibody. We speculate that the combination of large-surface area of antigen-conjugated microspheres, consequent ease of antigen accessibility and the fast dynamics of PCFIA (1 mi binding periods) all contribute to the preferential detection of antibodies with a high on:off binding rate.
The observations presented here emphasize the importance of clinical validation for serological assay procedures which rarely cover all possible immunoglobulin repertoires able to associate with a given antigen.

Example 3 Detection of pre-clinical IDDM by PCFIA
~.
Patients and Cases: The samples in this study were obtained as a subset of the blood samples employed in Example 1 which included over 90~ of all families with a newly diagnosed child with IDDM ("INDEX CASE") over a period of several years (the study is still ongoing).
Blood samples were taken from siblings of index ceases at the time of diagnosis for the latter and every six-twelve months later. Over 700 families were enrolled and 19 siblings of index cases turned diabetic so far. Our samples represent a subset of 11 of these converters and one hundred siblings which appear healthy (at most 1-2 of these are expected to convert to diabetes). Of all these children there were 1-6 samples available taken at the above interval.

207479~

Serum IgG anti-BSA antibody levels were measured as described in Example 1.

The results are set out in Tables 6 & 7.

Table 6 shows IgG anti-BSA antibody levels present in the very first sample taken from each subject (i.e. when the 'index case' was diagnosed). Diabetes developed in these children 1-5 years later. Table 7 shows IgG anti-BSA antibody levels in a random subset of 100 healthy siblings to the index cases.

- ~

.
. ~ .

2 ~

Elevated levehi are det`illed as levels above the Mean +2 Standard Deviations (SD) found in normal controls. The subset of control sibling samples shown here (last line) is not significantly dit`terent f~om levels observed in matched, random population controls (i.e.
samples from healthy children).

~=_ .__ .
pre- Months to IgG anti-BSA IgG anti-ABBOS
Sample IC DiabetesClinical IDDM (KfU/ml)~ (KfU/ml)~
67()3 Yes _ 3 Years 1 1 . 1 2 6. 1 84()6 Yes 2 Years 4 2 9 4 . 6 . _ 1310~S Yes I Years 2 . 9 1 4 . 3 14512 Yes 2 Years 1 5 . 0 2 __1 0 . 5 _ 253()3 Yes I Years 3 . 9 7 _4 . 1 253()4 Yes 2 Years 5 . 9 1 2 . 9 ~-272()3 Yes 2 Years 1 0 . 0 0 6 . 1 3()703 Yes 4 Years _2 . 1 6 2 . 2 324()3 Yes 4 Years 8 . 4 7 4 . 5 45303 Yes 3 Years 1 0 . 0 1 7 . 3 .. _ . .___ 51605 Yes 5 Years 3 . 3 9 5 . 2 . _ .._ *N=1()0 Unknown Mean: 0.56i0.26¦0.22~0. 13 *Random subset of lQQ healthy siblings to index cases ' 2~7~ -l lgG anti ¦ lgG anti Sample ID BSA(KfU/~I) 5 Sample ID BSA(KfU/~
005 04 0.8 537 03 0.45 ()06 04 1.01 542 04 0.98 015 08 0.47 545 03 0.3 017 03 0.31 55004 _ 0.22 018 03 0.36 558 05 0.89 01903 l 0.93 _ 56303 0.58 019 04 0.24 594 04 0.47 494 04 0.28 ~ 59604 0.34 033 06 1 0.81 I_ 597 04 i 035 03 0.52 597 05 0.62 04306 10.32 ~ 59706 0.95 05004 j0.33 I_ 1 59707 0.39 05005 10.3 ~ 60103 1 0.68 051 05 0.9 I_ 611 04 0.58 053 04 10.4 I_ 1 613 03 j0.54 064 03 0.87 61603 0.73 06603 0.54 61605 0.47 079 03 0.98 635 03 0.85 08407 0.64 63505 0.81 091 04 0.82 637 04 0.67 099 04 0.94 647 04 0.31 105 03 0.36 665 04 0.91 128 05 0.42 _ 684 05 0.49 77004 0.55 _ 69603 0.3 125 03 0.69 66803 0.79 TABLE 7 CC:)~IT ' D
_ lgG anti lgG anti Sample ID BSA(KfU/~I) Sample ID BSA(KfU/~
141 03 0.54 714 03 _ 0.61 14204 0.49 714 04 _ 0.85 14206 0.47 729030.33 167 05 0.43 _ _ 729 05 0.83 191 03 0.71 770040.55 21706_ 0.69 _ 778050.6 684 04 0.63 778 060.32 303 03 0.58 795 030.41 31006 0.35 803 040.38 32203 0.52 821 050.44 359 03 0.48 823 031.07 38605 0.9 825 110.36 387 05 0.22 829 030.1 6 42204 0.54 82903 -0.46 44S 04 1 .1 830 030.38 45205 0.23 830041.06 4550_ 0.31 834040.3 466 03 0.44 852 030.43 473 04 1 .1 3 853 040.98 47305 0.62 856040.52 479 03 0.1 6 859 040.79 _ 48003 0.47 ~61 030.33 501 03 0.43 873 030.06 Mean0.56 SD 0.26 2~7~

Exam~le 4 Detection of BSA-sensitised IDDM-associated T-lymphocytes Patients: Venous blood samples were obtained with informed consent from patients with recent onset IDDM and from healthy controls. T lymphocytes were prepared from the blood samples and T cell proliferation in response to BSA and to various synthetic peptide fragments of BSA was assayed as described in Dosch et al (1990), Int. Immunol., Vol. 2, p. 833, but with substitution of serum free media.

Synthetic peptides were generated on a Pharmacia peptide synthesiser (Pharmacia LTD, Montreal, Quebec) following the manufacturers' recommendations and purified by conventional HPLC techniques.

- ' :
'~

2~7479~

The following synthetic peptides were developed:
ABBOS: ac-Phe-Lys-Ala-Asp-Glu-Lys-Lys-Phe-Trp-Gly-Lys-Tyr-Leu-Tyr-Glu-Ile-Ala-Arg-Arg-Cys-O-NH2 (ABBOS (alias CS2185): FKADEKKFWGKYLYEIARR) ABRAS: ac-Gln-Glu-Asn-Pro-Thr-Ser-Phe-Leu-Cys-His-Tyr-Leu-His-Glu-Val-Ala-Lys-Lys-(ABRAS (no alias): OENPTSFLCHYLHEVARR) CS2270: ac-Tyr-Ala-Asn-Lys-Tyr-Gly-Val-NH2 (YANKYGV) CS2268: ac-Lys-Phe-Trp-Gly-Lys Tyr-NH2 (KFWGKY) CS2267: ac-Glu-Phe-Lys-Ala-Asp-Glu-Lys-Lys-NH2 (EFKADEKK) CS2240: ac-Ile-Glu-Thr-Met-Arg-Glu-Lys-Val-Leu-Thr--Cys-O-NH2 (IETMREKVLT) 20~ ~rl9~

Briefly, heparinized blood is diluted 1:1 with serum-free HL/l medium and mononuclear cells are purified on Ficoll-Hypaque gradients, washed in HL/l, counted and 2x105 are added to 96 well microculture plates in a volume of 200 ~1 HL/l. Control cultures receive either no or a supplement of the pan-T cell mitogen Phytohemagglutinin (l~g). BSA (0.01-10~g) or other test proteins, or 0.1-l~g of a synthetic peptide are added to triplicate test cultures. Cultures are incubated for 8-10 days at 37C
in a humidified atmosphere of 5% C02 in air until pulsed for 6 hrs with l~Ci3HTdR. Cells are then harvested with an automated harvester and incorporated thymidine is measured by scintillation counting. Alternative measures of T cell activation such as CCFA or measurements of IL2 production etc. are equally suited. All data presented here employed the above, standard TdR incorporation procedure.

The results from IDDM patients are shown in Table 8 and those from normal controls in Table 9.

All data are expressed as proliferative responses relative to that induced by PHA t(cpm -experimental/cpm PHA)*100]. A positive response is defined as mean response in unstimulated cells plus 2 SD.
All patients but no controls showed a positive response to at least BSA or ABBOS, usually both, and, where tested, to CS 2267, the minimum T cell stimulatory peptide under our conditions. Occasional small responses were seen to CS2268 but not to CS2270 or CS2240.

2~7~0 SAMPLEID T cell Proliferation (Fr~ction of PHA Reslponse) ABBOS CS2270 CS'26X CS2'67 CS2240 USA ABRAS Cells DM-15-B 34% nd nd nd nd 35% 10% 9%
DM-I~B 33% nd nd nd nd 36% 8% 9%
DM-17-B 41% nd nd nd nd 42% 6% 6%
DM-18-B 37 % nd nd nd nd 27 % 7 % 5%
DM-19-B 14% nd nd nd nd 21% 8% 5%
M-o3 42% nd nd nd nd 30% 10% 10 M-oS 71% nd nd nd nd 70% 31% 26 M-o6 16% 5% 7% 13% 6% 19% 3% 4%
M-07-B 12% 6% 11% 10% 8% 15% 5% 4%
M-08-B 11% 7% 4% 11% 2% 6% 3% 3%
M-09-C 14% 3% 6% 14% 3% 10% 4% 3%
M-10-B 14% 4% 9% 39C/o 4% 12% 4% 3%
M-II-B 25% 9% 9% 74% 9% 27 % 4% 5%
M-12-B 18% 5% 9% 11% 5% 18% 6% 3%
M-ol 8% nd nd nd nd 33% 3% 2%
M-o2 10% nd nd nd nd 18% 7 % 4%
M-04-C 8% nd nd nd nd 12% 3% 4%
MO-01-C 16% nd nd nd nd 16% 6% 6%
MO-02-C 20% nd nd nd nd 13% 5% 10 MO-04-C 27 % nd nd nd nd 28% 8% 8%
MO-05-C 44% nd nd nd nd 44% 7 % 7%
MO-06 15% nd nd nd nd 15% 11% 10 MO07 ¦ 19% nd nd nd nd l19% 6% 6%

TABLE 8 CO~1T'D ~74~

T cell Proliferation (Fraction of PHA Response) SAMPLEID ABBOS I CS2270 I CS2268 ~CS2267 ¦ CS2240 ¦ BSA ¦ ABRAS ¦ Celk MO-08 29%nd nd nd nd 38% 9% 6%
MO-14-B 18%7% 12% 8% nd 20% 6% 6%
MO-17 12%7 % 9% 6% nd 15% 6% 3%
MO-18 lO~o 5% 9% 8% nd 16% 5% 3%
MO-19-B 23%8% 10% 19% nd 18% 5% 8%
MO-21-C 14 % 5% 5% 10% 6% 11% 4% 4%
MO-22-B 20~o 5% 10% 23~o 5% 17% 4% 3%
MO-03-C't 281% ~nd nd nd nd 280%43% 35%
: MO-03 is NOT iwluded n st _ _ _ ~
N: 3Z 13 13 13 32 3Z 32 Mean: 22.47% 5.84% 8.52% 18.95% 5.31% 23.36% 6.81% 6.16%
SD 13.94% 1.66% 2.34% 18.70% 2.24% 13.33% 5.06% 4.42%
Net: 16.77% 0.14% 2.82% 13.25% -0.39% 17.66% 1.11% 0.46%

-~

TABLE 9 ?~ f T Cell Proliferation in Normals (cpm, % of PHA Response) SamplelD ABBOS ICS2270 ICS2268 ICS2267 ICS2240 ¦BSA ¦ABRAS ICellS
MD1 1.3 1.45 1.5 1.26 1.19 1.48 1.57 1.45 JM 3.98 3.84 3.8 3.36 3.52 3.44 3.25 3.1 RC 1 39 nd nd nd nd 2.Z8 Z.56 1.4 MD2 3 . 5 3. 67 3 Z 1 4. 1 2 3 . 7 7 3 .0 3 3 .18 3.05 SL 3.39 3.36 3.06 3.02 3.19 3.13 3.13 2.64 MH Z.75 Z.Z Z 99 3 16 Z.8 Z.Z9 Z.Z7 Z.44 R.T. 2.46 nd nd nd nd 2.24 2.03 2.12 L P 2 3 nd nd nd nd 3.63 3.11 2.32 BM 2 12 nd nd nd nd 2.06 2.09 2.21 T A. 2.71 nd nd nd nd 2.74 2.66 Z.71 T.S. 4.98 nd nd nd nd 3.49 1.84 1.38 S.C. 2.68 nd nd nd nd 3.53 0.54 0.8 N.A. 1.2 nd nd nd nd 1.24 1.08 1.17 H H 1.71 nd nd nd nd 1.81 1.55 1.57 J.P. 3.59 nd nd nd nd 2.74 2.79 3.23 C Z 2.83 nd nd nd nd 2.99 1.84 1.97 J.S. 1.75 nd nd nd nd 1.78 1.83 1.44 V.S. 2.17 nd nd nd nd 2.22 1.81 1.95 D.S. 4.03 nd nd nd nd 3.94 3.6 4.27 J.F. 1.5 nd nd nd nd 1.5 1.53 1.44 I.S. 4.84 nd nd nd nd 4.06 3.7 4.06 R.H. 1.37 nd nd nd nd 1.24 1.15 0.89 A.Q 5.73 nd nd nd nd 6.83 5.75 4.68 E.A. 2.42 nd nd nd nd 1 .94 1.92 2.05 J.A. 3.48 nd nd nd nd 4.95 3.71 3.14 , -TAB LE 9 C ONT ' D
~S3~l~7~q~

T Cell Proliferation in Normals (cpm, % of P~IA Response) .
Sample ID ABBOS CS2270 CS2268 CS2267 CS2240 BSA ABRAS Cells O.C. 4.27 nd nd nd nd _ 4.24 3.2 3.91 L R 6.01 nd nd nd nd 5.57 4.96 4.8 I.F. 2.7 nd nd nd nd 3.07 2.74 3.18 _ ~ nd i~ nd ~ 6.27 j 6.98 6.12 J.L. 2.16 nd nd nd nd 2.21 1.92 2.29 N.L. 2.42 nd nd nd nd 1.74 1.87 2.08 RG 4.03 nd nd nd nd 3.58 3.44 2.9 IM-2 3.29 2.88 3.09 3.15 3.12 2.85 3.09 2.89 SL-2 2.95 2.34 2.44 2.54 2.5 2.84 2.63 2.55 HM 3.55 3.58 3.41 3.59 3.41 3.65 3.4 3.49 3 4.26 3.84 3.9 3.63 4.69 0.57 3.54 i Means: 3.0917 3.0644 3.0378 3.1222 3.0144 3.0914 2.6469 2.6453 SD 1.357 0.913 0.718 0.844 0.794 1.353 1.33 1.191 Net: 0.4417 0.4144 0.3878 0.4722 0.3644 0.4414 -0.003 -0.005 Mn+2SD 5.8049 4.8897 4.4731 4.8097 4.6024 5.7966 5.3079 5.0274 Mn+3SD 7.1616 5.8023 5.1908 5.6535 5.3963 7.1492 6.6384 6.2184 ~ .

.. . . . . . . ~. --. .~
: : ~ . . :
..
.

IDDM patients were found to have sensitized T
lymphocytes which specifically recognize and proliferate in response to peptide CA 2267 while normal controls lacked such sensitized T lymphocytes. Subjects with pre-clinical IDDM as assessed by currently availableindicators showed the same response as IDDM patients.

Having established the predictive power of anti-ABBOS and anti-BSA immunity, the present inventors have also developed an ln vitro system to detect T
lymphocytes which initiate and sustain this immunity until final ~ cell demise.

.

R~:F'EE~.~IOES IN SU''E~;Cl~[PI 2 0 7 4 7 ~ a REFERENCES

1. Todd JA, Aitman TJ, Cornall RJ, et al. Genetic analysis of autoimmune type 1 diabetes melli-tus in mice. Nature 1991; 351: 542-7.

2. Barnett AH, Eff C, Leslie RDG. Diabetes in identical twins. Diabetologial981; 20: 87-93.

3. Drash AL. What do epidemiologic observations tell us about the etiology of insulin depen-dent diabetes mellitus? Schweiz Med Wochenschr 1990; 120: 39-45.

4. Elliott RB, Martin JM. Dietary protein: a trigger of insulin-dependent diabetes in the BB rat?
Diabetologia 1984; 26: 297-9.

5. Daneman D, Fishman L, Clarson C, Martin JM. Dietary triggers of insulin-dependent dia-betes in the BB rat. Diabetes Res 1987; 5: 93-7.

6. Scott FW, Cloutier HE, Souligny J, Riley WJ, Hoorfar J, Brogren CH. Diet and antibody pro-ducdon in the diabetes-prone BB rat. In: Larkins R, Zimmet P, et al., ed. Diabetes 1988.
Preceedings of the 13th International Diabetes federation Congress. Elsevier Science Publishers: Amsterdam 1989: 763-7 .

7. Elliott RB, Reddy SN, Bibby NJ, Kida K. Dietary prevention of diabetes in the non-obese di-abetic mouse. Diabetologia 1988; 30: 62-4.

8. Savilahti E, Akerblom HK, Tainio V-M, Koskimies S. Children with newly diagnosed insulin dependent diabetes mellitus have increased levels of cow's milk antibodies. Diabetes Res 1988; 7: 137-140.

: . ' -c 'okota A, Yarnaguchi Y, Ueda Y, et al. Comparison of islet cell antibodies, islet cell surface antibodies and anti-bovine serum albumin antibodies in type 1 diabetes. Diabetes Res and Clin Pracl990; 9: 211-7.

10. Scott FW. Cow milk and insulin-dependent diabetes mellitus: is there a relationship? Am J
Clin Nutr 1990; 51: 489-91.

11. Pocecco M, Nicoloso F, Tonini G, Presani G, Marinoni S. Increased levels of cow's milk antibodies in children with newly diagnosed insulin-dependent diabetes mellitus (IDDM).
Horm Res 1991; 35: 67.

12. Virtanen SM, Rasanen L, Aro A, et al. Infant feeding in Finnish children less than 7 yr of age with newly diagnosed IDDM. Childhood Diabetes in Finland Study Group. Diabetes Care 1991; 14: 415-7.

13. Martin JM, Trink B, Daneman D, Dosch H-M, Robinson BH. Milk proteins in the etiology of insulin-dependent diabetes mellitus (IDDM). Ann Med 1991; 23: 447-52.

14. Glerum M, Robinson BH, Martin JM. Could bovine serum albumin be the initiating antigen ultimately responsible for the development of insulin dependent diabetes mellitus? Diabetes Res 1989; 10: 103-7.

15. Dosch H-M, Karjalainen J, Morkowsky J, Martin JM, Robinson BH. Nu~itional triggers of IDDM. In: Laron Z, ed. Pediatric and Adolescent Endocrinology. Karger: Basel 1992: in press .

16. Beppu H, Winter WE, Atkinson MA, Maclaren NK, Fujita K, Takahashi H. Bovine albumin antibodies in NOD mice. Diabetes Res 1987; 6: 67-9.

17. Nerup J, Mandrup-Poulsen T, Molvig J, Helqvist S, Wogensen L, Egeberg J. Mechanism of pancreatic ~-cell destruction in Type 1 diabetes. Diabetes Care 1988; 11: 16-23.

. ' .

207~0 Hanenberg H, Kolb-Bachofen V, Kantwerk-Funke G, Kolb H. Macrophage infiltration pre-cedes and is a prerequisite for Iymphocytic insulitis in pancreatic islets of pre-diabetic BB
rats. Diabetologia 1989; 32: 126-34.

19. Ihm S-H, Yoon J-W. Studies on autoimmunity for initiation of ~-cell destruction. VI.
M'acrophages essential for development of ~-cell-specific cytotoxic effectors and insulitis in NOD mice. Diabetes 1990; 39: 1273-8.

20. Gorsuch AN, Spencer KJM, Lister J. The natural history of type 1 (insulin-dependent) dia-betes mellitus: evidence for a long prediabetic period. Lancet 1981; ii: 1363-5.
21. Tarn AC, Thomas JM, Dean BM, Ingram D, Schwarz G, Bottazzo GF. Predicting insulin-dependent diabetes. Lancet 1988; i:845-850.

22. Thivolet C, Beaufrere B, Betuel H, et al. Islet cell and insulin autoantibodies in subjects at high risk for development of Type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1988; 31: 741-6.

23. Karjalainen J, Knip M, Mustonen A, Akerblom HK. Insulin autoantibodies at the clinical manifestation of insulin-dependent diabetes mellitus - A poor predictor of clinical course and antibody response to exogenous insulin. Diabetologia. 1988; 31: 129-133.

24. Karjalainen JK. Islet cell antibodies as predictive markers for IDDM in children with high background incidence of disease. Diabetes 1990; 39: 1144 50.

25. Ilonen J, Surcel HM, Partanen J, Koskimies S, Knip M, Kaar ML. Extended HLA haplo-types in families with insulin-dependent diabetes mellitus in northern Finland. Tissue Antigens 1988; 32: 139-44.

26. Dosch HM, Larn P, Guerin D, Leeder JS. Characteristics of Particle Concentration Fluorescence Immunoassay (PCFIA): Novel alternadves to ELISA and RIA. In: Gembala P-2~7~790 L, ed. Proceedings of the 1987 Pandex Symposium on Particle Concentration FluorescenceImmunoassay. Baxter Healthcare Corporation: Chigago, IL 1988: 5-22 .

27. MacKenzie T, Dosch HM. Clonal and molecular characteristics of the human IgE-commit-ted B cell subset. J Exp Med 1989; 169: 407-30.

28. Bruining GJ, Molenaar J, Tuk CW, Lindeman J, Bruining HA, Marner B. Clinical time-course and characteristics of islet cell cytoplasmic antibodies in childhood diabetes.
Diabetologia 1984; 26: 24-9.

29. Pagano G, Cavallo PP, Cavalot F, et al. Genetic, immunologic, and environmental hetero-geneity of IDDM. Incidence and 12-mo follow-up of an Italian population. Diabetes 1987;
36: 859-63.

30. Walter MH, Dosch H-M, Cox DW. A Deledon Map of the Human Immunoglobulin Heavy Chain Variable Region. J Exp. Med 1991; 174: 335-49.

31. Husby S, Oxelius VA, Teisner B, Jensenius JC, Svehag SE. Humoral immunity to dietary antigens in healthy adults. Occurrence, isotype and IgG subclass distribudon of serum and-bodies to protein andgens. Int Arch Allergy Appl Immunol 1985; 77: 416-22.

32. Kolb H, Dannehl G, Gries FA, Bellmann O. ~rosp~cdve analysis of islet cell andbodies in children with Type 1 (insulin-dependent) diabetes. Diabetologia 1988; 31: 189-194.

33. Karjalainen J, Salmela P, Ilonen J, Surcel HM, Knip M. A comparison of childhood and adult Type 1 diabetes mellitus. N Engl J Med 1989; 320: 881-6.

34. Migliore-Sarnour D, Jolles P. Casein, a prohormone with an immunomoduladng role for the newborn Experimentia 1988; 44: 188-93.

2 ~3 rs1 ~ 7 ~ ~
Baisch JM, Weeks T, Giles R, Hoover M, Stastny P, Capra JD. Analysis of HLA-DQ
genotypes and susceptibility in insulin-dependent diabetes mellitus [see comments]. N Engl J Med 1990; 322: 1836-41.

36. Reijonen H, Silvennoinen KS, Ilonen J, Knip M. Lack of association of T cell receptor beta-chain constant region polymorphism with insulin-dependent diabetes mellitus in Finland.
Clin Exp Immunol 1990; 81: 396-9.

REF~RENOES IN S~2UA~ BRACKETS 2 0 ~ ~ 7 9 0 Reference~
1. Dosch H-M, ~Carjalainen J, Mo~lcows~cy J, Mamn JlM, Robinson BH. (1992) Nutridonal triua~ ~f II)DM. In: Lcvy-Marchall. C, Czcrnichow, P (od~) Pcdialric and.Adole~ccnt Enh~ba~.
Karger: Bascl. p. 202-217 2. Glenum M, Ro~ BH, Mar~ JM (1989) Couldbo_~ bc thc in~
uldmucly ~blc far the dcvclapmcnt of in~lin ~__ _~? ~Re~ ik.
103-107 . .
3. Ma~tin JM, Trinl~ B, Da~niniD, Dosd H-M, Robin~on ~ed~logy ~ ~
of insulin dependent diabetes mellitus (IDDM). Ann Med 23: 447-4S2 4. Elliott RB, Roddy SN, Bibby NJ, Kida K (1988) Die~ypm~tit of d~betu in th~n_ diabetic mouse. Diabetologia 36: 62 64 5. Scott FW, Daneman D, Mardn JM (1988) Evidence for a critic l ~b of ~iet in the deYelapm~t of insulin~ependent diabe~ mellitua Diabete~ Ra 7(4): lS3-lS7 6. Ka~ialainen J, Mudn JM, Knip M et al. (1992) Evidenee for a bo~ine nlbumin pqi~i u candidate trigg~ of Typc 1 diabetes. N Engl J Med 32S: in pr~
7. Dosch HM, Lam P, Guerin D, Lccder JS (1988) Charaeteri~ie~ of partiele eoneentration fluorescence immunoassay (PCE;IA): No~el altema~ive~ to ELISA aod RIA, In: Gembal~ P-L (ed) Procceding~ of the 1987 P~ndex Symposium on P~cle ~_FIuaracenee Imm~y, BaxterHcaltheare Ca~p~adon, Chieago, pp S-22 8. Hui MF, Lam P, Do~ch HM (1989) Propcnia and heterogendty of human fetal p~B eell~
~ansformed by EBV. J ~nmunol 143(8): 2470-2479 9. Dosch HM, Lam P, Hui MF, Hibi T (1989) Coneerted generation of Ig isotype diver~ity in human feul bone malrow. J lmmunol 143: 2464-2469 10. Doseh H-M, Lam P, Hui MF, Hibi T, Cheung RIC (1990) EBV utiL~zes a unique aeti~radon pathway for the transfonnadon of human B cells. ~t Im~nunol 2(Sept. 1990): 833-848 11. Walter MH, Doseh H-M, Cox DW (1991) A deledon map of the human immunoglobulin heavy chain region. J Exp Med 174: 335-349 2~7~0 . Beppu H, Winter WE, Atlcinson MA, Maclaren NK, Fujita K, Talcahashi H (1987) Bovine albumin antibodies in NOD mice. Diabetes Res 6: 67-69 13. Scott FW, Cloutier HE, Souligny J, Riley WJ, Hoorfar J, Brogren CH (1989) Diet and antibody production in thc diabctes-prone BB rat. In: Larlcins R, Zimme~ P, Chisholm D (ed~ Diabetet 1988. Proceedings of the 13th International Diabetes Federation congnss, Elsevier Science PublisherS Amsterdarn, pp 763-767 14. Colman PG, Campbell IL, Kay TWH, Harrison LC (1987) 64 000-M, auto-antigen in type 1 diabetet: cvidence of its location on human isle~ Diabetss 36: 1432-1440 15. Yolcota A, Yamaguchi Y, Ueda Y et al. (1990) Comparison of islet cell antibodies, islet cell surface antibodiet and anti-bovine serurn albumin antibodies in type 1 diabetes. Diabetes Res Clin Prac 9:

16. Savilahd E, Alcerblom HK, Tainio V-M, Koslcimies S (1988) Children with newly diagnosed insulin dependent diabetes mellitus have inc~eascd levels of cow's millc andbodies. Diabe~ Res 7:

17. Pocecco M, Nicoloto F, Tonini G, Pres~i G, MarinoQi S (1991) Incre#ed levels of cow's mill~
andbodies in children with newly diagnosed insulin-dependent diabetes mellitus (lDDM). Horm Res,p67 18. Tainio VM, Savilahd E, Arjomaa P, Salmenpera L, Perheentupa J, Siimes M (1988) Plasma antibodies to cow's miLlc are inrceased by eady weaning and consumpdon of unrnodified millc, but production of plasma IgA and IgM cow's millc antdbodies is stunulated evcn dunng e~cclusively breast-feoding Acta Paediatr Scand 77: 807-811 19. Cheung RK, Dosch H-M (1991) The tyrosine kinase lck is critically involved in the growth transfo~madon of human B lymphocytes. J Biol Chem. 266(14): 8667-8670 20. Borch-Johnsen K, Joner G, Mandrup-Poulsen T et al. (1984) Relation between bnst-feeding and incidence rates of insulin-dependent diabetes mellitus. Lancet II: 353-60 21. Scott FW (1990) Cow milk and insulin-dependent diabetes mellitus: is there a reladonship? Am J
Clin Nutr 51(3): 489-91 2a~r~
. Virtanen SM. R~nen L Aro A et al. (1991) Infant feeding in Fmnish children la~ tbu~lyr of age with newly diagno~cd IDD~ Childhood Diabe~ in Finland Study Group. Diabe~Care 14(5): 415417 23. ElliottRBj ~n lM (1984) Diet~y pro~i~- ~ trig~.of in~tdiabc~ m Diabetolog~ 26(297): 297-299 24. Danem~.-D, Fi~hm~LL Clanon C. M~lM (1987~ Die~rtngg~of in~ul_1 diabetu in the BB raL Diabetes Re~ S(93): 93-97 2S. Scot~ ,M, uGs~ EB (1991? C~n~en~ mmm~: diet ~ an en~me~l I~ __ of insoli~nt diabetu me~li~ Can J Phy~ P~col 6X3): 311-319 26. PilcbcrCC, D~K, ~lliott RB (1991) ICA aol~ obpm e dy cbildl~ Di~aa~:-Pract 14(SuppL 1): 82A
27. Dosch H-M, Hui MF, Doherty PJ (1992) Development of nonnal and abnnal immune function in ma~L Dev ~nunol 2: in pre~.
28. OlenJp O, Smith CE, Ham~tr~n L (1990) Dif~*~Jcitl~ at po i~on S7 of ~DQ
beta chain a~ociatod with susceptibiliq ant _o~ ID I~ deficie~cy. N~re 347: 28!P~
29. Caudwell J, Stojanovs~i C. Colma P (1991) Caud~ agai~t the use of commercid l~ Icit assays for preclinical IDDM sc~eening. Diabetes Res ain Pract 14(Suppl. 1): 89A
30. Kuglin B, Kolb H, Greenbaum C, Maclaren NK, Lernmaric A, Palmer IP (1990) Iho fourth internadonal wo*shop on the standardisation of in~alin autoantibody measurement. Diabetologia 33: 638-639 31. Levy-Marcoal C, Bridel MP, Sodoyez-Goffau~ F et al. (1991) Superioriy of radiobindin~ assay over ELISA for deuction of IAAs in newly diagno#d ype 1 diabetic childrem Diabete~ Care 14(Jan): 61-63 32. Greenbaum GJ, Palmer JP, Kuglin B, Kolb H (1992) Insulin autoantibodies measurcd by RIA
methodology are more relaud to IDDM than those measured by ELISA. J Clin Endocrinol Metab 74: in press 33. Sodoyez-Goffaux F, Koch M, Dozio N, Brandenburg D, Sodoyez J-C (1988) Advantages and pitfalls of radioimmune and enzyme linked immunosorbent assays of insulin andbodies.
Diabetologia 31: 694-702 49 ~7~7~0 34. Sodoycz J-C, Sodoyez-Goffa~Ls F, Koch M, Bouillennc C. Franco~-Ge~d C, Bo~i E (1990) Qonally rc~icted in~ulin autoannbodies in a coharl of 2200 hcaldly bl~od d4noa. Diabe~b~a 33:

35. Foote J, Mil~in C (1991) Kinedc man~radon of an ~mmune ~upo~e. N~e 3S2: S30 532 36 Karjal;lin~n JK. I~let cell alltibodic~ predictive markers for ~DDM in children with high background h~uid~ e of di~ e. Viabe~e~. 199(); 39: 1144-50.
37 Karjalainen J, Knip M, Mu~(onen A, Akerblom HK. Insulin autoantibodies at the clinical manifest;ltion of insulin-dependent diabetes mellitus - A poor predictor of clinical course and antibody re~ponse to e~o~enou~ insulin. Diabetes. 1988; 31: 129-133.
38 Karjalainen J, Knip M, Mustonen A, llonen 1, Akerblom HK. Relation between insulin antibody al1d complelnel1l-fi~il1g isle~ cell antibody at clinical diagnosis of rDDM.
Diabete~. I9X6: 35: 62()-2.

~_ . ~

Claims (8)

1. A method for detecting an autoimmune disease or a pre-clinical autoimmune disease in a mammal comprising obtaining a serum sample from said mammal and determining the level of antibodies to a dietary protein or a fragment thereof in the serum by particle concentration fluoroimmunoassay employing particle-bound dietary protein or a fragment thereof as antigen.

2. A method for detecting pre-clinical insulin dependant diabetes mellitus in a mammal comprising obtaining a serum sample from said mammal and determining the level of antibodies to bovine serum albumin or a fragment thereof in the serum by particle concentration fluoroimmunoassay employing particle-bound bovine serum albumin or a fragment thereof as antigen.

3. A method for detecting insulin dependent diabetes mellitus in a mammal comprising obtaining a serum sample from said mammal and determining the level of antibodies to bovine serum albumin or a fragment thereof in the serum by particle concentration fluoroimmunoassay employing particle-bound bovine serum albumin or a fragment thereof as antigen.

4. A method for detecting an autoimmune disease or a pre-clinical autoimmune disease in a mammal comprising obtaining T lymphocytes from said mammal and determining the proliferative response of said lymphocytes to a dietary protein or a fragment thereof.

5. A method for detecting insulin dependent diabetes mellitus or pre-clinical insulin dependent diabetes mellitus in a mammal comprising obtaining T lymphocytes from said mammal and determining the proliferative response of said lymphocytes to bovine serum albumin or a fragment thereof.

6. A peptide having one of the following amino acid sequences and analogues thereof:
(a) (b) (c) (d) (e) (f) and (g) (h)

7. An isolated DNA comprising a nucleic acid sequence encoding one of the amino acid sequences of claim 6.

8. Use of a peptide in accordance with claim 6 coupled to a cytotoxic compound to reduce or eliminate sensitized T lymphocytes in a human.