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Original Studies |
Institute of Diabetes Research (H.E.N.) and Institute of Diabetes Research and Academic Hospital Schwabing (A.-G.Z.), Munich, Germany; and Istituto Scientifico San Raffaele (E.B.), Milan, Italy
Address all correspondence and requests for reprints to: Prof. Dr. Anette-G. Ziegler, Institut für Diabetesforschung, Kölner Platz 1, D-80804 Munich, Germany. E-mail: anziegler{at}lrz.uni-muenchen.de
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Control population. Sera of 144 healthy subjects with no family history of type 1 diabetes (median age, 27 yr; range, 1–57) and cord blood of 19 newborns of parents without type 1 diabetes were tested to establish the threshold of positivity for the protein A/G IAA assay.
Offspring of parents with type 1 diabetes. Serum samples from offspring of parents with type 1 diabetes used for measurement of insulin autoantibodies in this study were obtained from the BABYDIAB study (2). This is a prospective study from birth in offspring of parents with type 1 diabetes, and schedules for regular blood sampling at birth, 9 months, 2 yr, 5 yr, and 8 yr of age in all offspring. By August 1998, 1481 samples at birth (including 480 children of diabetic fathers), 1036 at 9 months, 613 at 2 yr, and 156 at 5 yr have been included and tested for autoantibodies to insulin (PEG IAA assay), islet cells (ICA), glutamic acid decarboxylase (GADA), and the protein tyrosine phosphatase IA-2 (IA2A). To determine the prevalence of IAA in cord blood, 247 cord blood samples from offspring of fathers with type 1 diabetes were retested for IAA in the protein A/G-based IAA assay. The distribution of IAA results in these cord blood samples was also compared to those for the 9 month serum samples from 65 offspring of fathers with type 1 diabetes. From the remaining 233 offspring of fathers, insufficient cord blood sample was available for retesting. The ability of the protein A/G IAA assay to detect insulin antibodies in cord blood was validated in 42 newborns of mothers with type 1 diabetes and in 18 newborn/mother pairs. For confirmation of early development of IAA and to compare sensitivity and temporal development of antibody responses for both PEG and protein A/G IAA assays, 2 yr follow-up samples from all BABY-DIAB offspring with elevated PEG IAA confirmed in the same sample (n = 79), and 9 month samples from all offspring with multiple islet autoantibodies (n = 21) were retested in the protein A/G IAA assay.
Samples for comparison of venous sera and whole capillary blood. Venous serum and whole capillary blood samples were collected from 10 first degree relatives of type 1 diabetic patients with islet autoantibodies from the Munich family study, 5 patients with type 1 diabetes, and 19 control subjects.
Methods
Protein A/G IAA assay. The assay of Williams et al. (4) was used with some minor modifications as previously described (5). The interassay CV of the assay was 11%. For whole blood testing, 200 µl capillary blood from the finger-tip or ear-lobe were collected with capillary ethylenediamine tetraacetate tubes (Microvette 200 KE, Sarstedt, Nuernbrecht, Germany) and frozen at -20 C. After thawing, the suspension was thoroughly mixed, and 10 µl whole capillary blood per well were used in the assay without further modifications.
PEG IAA assay. Samples were tested using the conventional radiobinding assay, which is based on PEG precipitation and described by Soeldner et al. (6). Results were expressed as specifically precipitated nanounits bound insulin per mL serum. The threshold for positivity was calculated as 50 nU/mL. The interassay CV of the assay was 10.5%. The assay achieved 100% sensitivity and specificity in the Immunology of Diabetes Workshops proficiency exchanges.
Hemolysis experiment. Two blood samples were drawn from a healthy control subject (age, 31.5 yr; female). One sample was immediately centrifuged, and the serum was collected; the other was frozen for 1 h at -20 C, and then hemolyzed serum was collected after thawing and centrifugation. Hemolyzed serum was diluted with the normal serum so as to obtain serum containing 1%, 7%, 14%, 28%, and 100% hemolysate, and all samples were measured in the PEG and protein A/G IAA assays.
IgM-IAA. IgM-IAA were measured using an assay format similar to that described for the protein A/G assay (5). Sepharose beads coated with monoclonal anti-IgM antibodies were used for the detection of IgM-IAA in place of the protein A/G-Sepharose mix used in the protein A/G assay. Per well, 10 µl Sepharose 4B streptavidin beads (Zymed, San Francisco, CA) were washed with ice-cold PBS (50 mmol/L phosphate buffer and 150 mmol/L NaCl, pH 7.4) and incubated with 10 µl (5 µg) biotinylated monoclonal anti-IgM antibody (PharMingen, San Diego, CA) with rotation at 4 C for 18 h. Subsequently, the coated beads were washed twice in PBS and once in TBT buffer (50 mmol/L Tris and 1% (vol/vol) Tween-20, pH 8.0) and after resuspension in TBT buffer, 50 µl were added to each tube.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Sera from 144 healthy subjects of nondiabetic parents were tested
for protein A/G IAA (Fig. 1
). The median
level of IAA in this group was 0.7 U (range, 0–16.8 U), and the 97.5th
percentile was 3.7 U. The cut-off for positivity was defined as 4.0 U.
All 19 cord blood samples of normal birth had IAA levels below 4 U
(median, 0.8; range, 0–2.4).
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Of 42 cord blood samples from neonates of diabetic mothers, 32
(76%) showed elevated insulin antibodies in the protein A/G assay
(median, 18.9 U; range, 0–267; P <
10-4), and a strong linear correlation (r = 0.8;
P < 10-4) with insulin antibody titers in
the maternal circulation (data not shown) was found in the 18
newborn/mother pairs. None of the 247 cord blood samples from offspring
of fathers with type 1 diabetes, including samples from 5 who
subsequently developed multiple islet autoantibodies, had protein A/G
IAA levels higher than the threshold of 4 U (median, 1.0 U; range,
0–2.8). In offspring of fathers with type 1 diabetes, the distribution
of protein A/G IAA in cord blood did not differ from that in 9 month
samples (median, 1.2 U; range, 0–35), except that 2 offspring had
positive IAA values (Fig. 1
). Both of these offspring also had PEG IAA,
and 1 has developed other islet autoantibodies in subsequent
samples.
Performance of the protein A/G assay in antibody- positive children
Two-year follow-up samples from 79 offspring with IAA greater than
50 nU/mL in the PEG assay were examined in the protein A/G IAA assay
(Figs. 2 and 3). In 21 of these, at least 1 other
islet autoantibody (GADA, IA2A, or ICA) was also present or developed
in later samples (Table 1). Protein A/G
IAA were detected in the 2 yr sample of all of these, and there was a
strong linear correlation in IAA levels between the protein A/G and PEG
assay results in these samples (r2 = 0.95;
P < 10-4). Eight of those with multiple
islet autoantibodies at 2 yr already had PEG IAA in their 9 month
sample, and in 6 of these, IAA were also detected in the protein A/G
IAA assay (cases 1032, 4000, 4005, 4161, 4262, and 5006), whereas case
4050 had a protein A/G IAA level just below the threshold of positivity
(3.9 U). In two other offspring, protein A/G IAA was detected at 9
months in the absence of PEG IAA (cases 3941 and 4125). In 9 of the 21
cases, IAA was detected in both assays before any other islet
autoantibodies, and in only 1 case (no. 1649) did IAA appear later
(Table 1).
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and 3). Follow-up samples after 2
yr of age were available in 32 of these 58 offspring, and in only 6 of
these were PEG IAA found in a follow-up sample; 2 also had protein A/G
IAA (Fig. 2). Hemolysis affects the PEG IAA assay
Because of the discordance between the two IAA assays, discrepant
samples were investigated, and surprisingly, 32 (62%) of the samples
appeared hemolyzed to a level discernible to the naked eye. To further
analyze the influence of hemolysis on measurement of IAA, totally
hemolyzed and nonhemolyzed sera from a healthy control subject were
mixed and tested by the two methods (Fig. 4). Hemolysis had no influence on the
readout of the protein A/G assay, but increased insulin binding in the
PEG IAA assay in a dose-dependent manner, leading to false positive
results (>50 nU/mL) in the sample containing 28% hemolysate and in
the totally hemolyzed sample.
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Measurement of IgM IAA was performed in 14 2-yr follow-up samples who were discrepant for PEG IAA and protein A/G IAA results, but not hemolytic. None of the sera had IgM levels higher than background levels.
Protein A/G IAA measurement in whole capillary blood samples
Correlation of IAA levels measured by the protein A/G assay in
venous sera and whole capillary blood is shown in Fig. 5. Twelve of the patients or first degree
relatives had IAA above 4 U in serum and capillary blood. Three
relatives and 18 of the control subjects were IAA negative in both
samples. One control subject had 0 U IAA in the venous serum sample and
5.4 U in the capillary blood sample. Overall, 97% of the control,
first degree relatives, and patients’ samples were concordant
(r2 = 0.99; P < 10-4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The BABYDIAB study has also shown that in offspring from fathers with type 1 diabetes, neither GADA nor IA2A could be detected at birth, but because of the presence of nonspecific binding factors in cord blood, the presence of IAA at birth in these offspring could not be determined (2). As IAA are the earliest detectable marker of autoimmunity, it was important to examine this to ascertain whether islet autoimmunity and indirectly islet damage is a pre- or a postnatal occurrence. The protein A/G assay was found to reliably detect cord blood IgG insulin antibodies transplacentally acquired in offspring from mothers with type 1 diabetes and, unlike the PEG IAA assay, had a similar distribution of results in cord blood and 9 month serum samples from offspring of fathers with type 1 diabetes. The observation that none of the offspring from father probands had IAA at birth supports a postnatal initiation of islet autoimmunity. Our original findings of ICA at birth in occasional offspring (2) have not been confirmed upon retesting using a different pancreas substrate (unpublished observations), and although the development of islet autoimmunity prenatally cannot be excluded, the BABYDIAB study shows that the autoimmunity in most children who develop type 1 diabetes is clearly postnatal.
After birth, not all IAA detected with the PEG IAA assay were confirmed with the protein A/G assay. Apart from those offspring who had or developed other islet autoantibodies, only few others had IAA that were detected in both the PEG and protein A/G assays, indicating that the detection of confirmed IgG IAA is associated with a likelihood of autoimmunity to spread to other autoantigens and a high risk for disease. Overall, at 2 yr of age, 4.4% of all BABYDIAB offspring have IAA that are detected in both assays. This remains higher than the 2 yr prevalence of GADA (2.9%) and IA2A (2.6%) in these offspring (Ziegler, A.-G., unpublished observations), and although confirmed IAA is found in the absence of GADA, IA2A, or ICA, these other antibodies rarely occur in BABYDIAB offspring in the absence of IAA; indeed, IAA often precedes the appearance of other antibodies.
Discrepancies in IAA measurement between the two assays were frequently attributable to the use of a hemolyzed sample, which was shown to give false positive results in the PEG IAA assay. The observation that hemolysis did not affect the protein A/G IAA assay is important, because in newborns and neonates blood drawing can be difficult, and hemolytic samples are frequently obtained. Moreover, unseparated samples that are sent by post are sometimes affected by hemolysis. This observation is also important because it potentially allows testing of whole blood samples from capillary blood collection. In a limited number of samples we have shown that protein A/G IAA measurement in whole capillary blood samples does give results very comparable to those obtained in serum samples. Measurement in whole capillary blood is advantageous for screening and large population studies, as it simplifies blood collection and avoids the separation of serum or plasma, thereby reducing the cost of sample preparation.
Not all discrepancies between IAA measurements in the two assays could be attributed to hemolysis. It is also unlikely that they are due to the presence of IgM antibodies to insulin, because we could not find such antibodies in discrepant sera. It is not known whether the PEG IAA detected in these sera is true antibody and/or is relevant to the pathogenesis of type 1 diabetes. It could be argued that as most of the discrepancies in nonhemolyzed samples had low PEG IAA levels, the PEG IAA assay is more sensitive than the protein A/G assay for antibody detection. However, most offspring with PEG IAA only in their 2 yr samples did not have PEG IAA and other islet autoantibodies in follow-up samples. Moreover, although two offspring who later developed other islet autoantibodies had weak PEG IAA only at 9 months of age, two others had protein-A/G IAA only at this age, indicating that the sensitivities of both assays for measuring low levels of diabetes-related IAA are similar, but may still be inadequate to detect all insulin autoimmunity. Longer follow-up of the discrepant cases is required before the presence of PEG IAA in the absence of any other islet antibodies can be excluded to represent a risk factor for type 1 diabetes. This will also be true for the detection of protein-A/G IAA only.
In conclusion, this study confirms that insulin-specific IgG autoantibodies are the first islet autoantibodies detectable in the majority of children who develop diabetes-associated autoimmunity, that these antibodies appear after birth, and that they can be reliably detected using a protein A/G radiobinding assay. Based upon the observations of this and previous studies, testing for IAA at 2 yr of age with the protein A/G IAA assay on whole capillary blood samples is likely to be a sensitive screening strategy for the early detection of first degree relatives of patients who will develop diabetes in childhood. Specificity may require the confirmation of IAA using the PEG-IAA assay and follow-up for the detection of other islet autoantibodies.
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Acknowledgments |
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Footnotes |
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Received September 30, 1998.
Revised December 16, 1998.
Accepted January 5, 1999.
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) are strongly correlated between the two assays
(r2 
0.95; P <
10-4). Only a few samples with PEG IAA in the absence of
other islet autoantibodies (large and small circles)
were confirmed in the protein A/G IAA assay. Samples represented with a
small circle are those in which hemolysis was
discernible to the naked eye.
), 10 relatives with islet autoimmunity, and 5 patients with type 1
diabetes (
). The upper limit for positivity is indicated as a
dotted line. Regression analysis gave an r2
of 0.99 (P < 10-4).