Note: Descriptions are shown in the official language in which they were submitted.
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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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2 ~ 7 ~
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
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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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2~7~
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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-
.
,
2~7~7~
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
18
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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Solid phase ABBOS was also used to assay serum
anti-ABBOS antibodies as described above for use of solid
phase BSA (BSA coated microspheres).
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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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 ~
21
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.
2~7~79~
22
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~
23
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.
.
24 2~ 3~
TABLE 4
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
. .
'' ~ ' ' .
.
2 5 ~ ~ r~
TABLE 5
Table Sensitiviry and specificity of PCFIA and EIA in detecdng an~-BSA antibodies.
. .
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
.
.
~ a r~
26
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 ~ ~
27
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 ~
28
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~
29
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 ~
TABLE 6
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
'
TABLE 7
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~
33
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.
- ' :
'~
34
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-
NH2
(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.
36
TABLE 8
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%
-~
38
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
,
-
39
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
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~_ . ~
