A Novel Micro-assay for Insulin Autoantibodies

Journal of Autoimmunity (1997) 10, 473–478

A Novel Micro-assay for Insulin Autoantibodies Alistair J. K. Williams1, Polly J. Bingley1, Ezio Bonifacio2, Jerry P. Palmer3 and Edwin A. M. Gale1 1

Diabetes and Metabolism, Department of Medicine, University of Bristol, Bristol BS10 5NB, UK 2 Istituto Scientifico San Raffaele, Milan, Italy 3 Department of Veterans Affairs, Puget Sound Health Care System and Department of Medicine, University of Washington, Seattle, USA Received 27 November 1996 Accepted 20 June 1997 Key words: Type 1 (insulin-dependent) diabetes mellitus, insulin autoantibodies, autoantibodies, prediction

Insulin autoantibodies (IAA) are established markers of Type 1 diabetes and are widely used for the prediction of this disease. Standard assays require relatively large serum volumes for reliable measurement of IAA, limiting their use in young children. We have developed a novel small volume assay which is suitable for screening large numbers of samples. For reasons of economy we have adopted a two-stage strategy in which all samples are screened for insulin binding and those with raised levels are quantified in an assay using competitive displacement. Using this assay 126 out of 241 (52%) newly diagnosed IDDM patients (median age 10.2, range 1.3–20.7 years) had IAA levels above the 99th centile of 2860 schoolchildren (median age 11.3, range 9.0–13.8 years), including 81 out of 117 (69%) patients below the age of 10 years. The assay compared well overall when measuring IAA in direct comparison with a conventional assay. We conclude that reliable measurement of IAA is possible on less than 50 ìl of serum using this novel assay and that this should facilitate large scale screening, particularly in young children. © 1997 Academic Press Limited

Introduction

We now report a novel IAA assay which has a sensitivity and specificity comparable with established assays, which requires small sample volumes, can easily be automated, and can be used for high throughput testing in all age groups, including young children.

Insulin autoantibodies (IAA) were first identified in the sera of newly diagnosed insulin-naive patients with IDDM by Palmer et al. [1], who proposed that they were immune markers of â-cell damage. Since then IAA have been demonstrated before the onset of diabetes in twins [2], first-degree relatives of IDDM patients [3], ICA-positive polyendocrine patients [4, 5], and schoolchildren [6]. The prevalence of IAA is inversely correlated with age at diagnosis [7, 8], and since they are present in more than 50% of young children at diagnosis, they are particularly useful for disease prediction in this age group. Most laboratories measuring IAA use adaptations of the original radiobinding assay developed by Kurtz et al. [9]. These methods require relatively large serum volumes to achieve precise measurement of antibody levels, which limits large scale evaluation of the role of IAA in the prediction of IDDM. Simple and relatively sensitive ELISAs have been described [10], but these often detect insulin binding in non-diabetes related sera [11]. There is, therefore, a need for a fluid-phase assay that requires small serum volumes and is simple enough to allow screening of large numbers of samples.

Subjects Serum samples were collected during 1989–1990 from 2860 school-children living in the Oxford region of the UK. The median age of the children was 11.3 years, range 9.0–13.8 years [12]. Serum samples from 241 patients with insulin-dependent diabetes diagnosed between 1985 and 1995 before or within 1 week of starting insulin treatment from the same region were also studied. The median age of the patients was 10.2 years, range 1.3–20.7 years. Sera used for comparison of the micro-assay with the conventional method included 51 subjects with newly diagnosed IDDM and 101 healthy controls circulated for the first IDS Combined Autoantibody Workshop (included with the kind permission of Dr G. S. Eisenbarth) and 184 unselected sera from the routine chemical pathology laboratory of the Department of Veterans Affairs, Puget Sound Health Care System, University of Washington, Seattle, WA, which included sera from 17 insulin-treated patients.

Correspondence to: Prof E. A. M. Gale, Medical School Unit, Southmead Hospital, Bristol BS10 5NB, UK. Fax: +44-117-9595336. 473 0896-8411/97/050473+06 r25.00/0/au970154

© 1997 Academic Press Limited

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Figure 1. Plot showing the proportion of schoolchildren (C) and newly diagnosed IDDM patients (◆) above a particular level of insulin binding in the screening assay. The threshold above which samples are assayed in the competition assay is shown by the dotted line.

Methods

and the precipitate transferred to 1.2 ml tubes held in a 96-well reusable tube holder (Micronics, Lelystad, Netherlands). The tubes were counted in a gamma counter (NE1600, Nuclear Enterprises Ltd, Reading, Berks, UK) for 10 min and bound counts for each sample were calculated after subtraction of background counts. Results were expressed in arbitrary units derived from a standard curve constructed from nine doubling dilutions of a serum from a patient with long-standing IDDM in normal human serum taken from a non-diabetic volunteer. The curve spanned the range from 100 units (undiluted) to 0.39 units (one in 256). A logarithmic curve fitting algorithm was used (Excel, Microsoft Corporation, Redmond, WA, USA). Four quality-control sera were included in each assay and these were obtained from a long-standing insulin treated patient (high control), islet cell antibody-positive first-degree relatives of IDDM patients (medium and low controls) and from a healthy volunteer (negative control).

Strategy Competition assay Specificity of binding is conventionally assessed by incubating all sera with label in the presence or absence of cold insulin. This is necessary, since in some sera there is binding of 125I-labelled insulin that is not displaced by the addition of excess cold human insulin and this type of binding is not associated with IDDM [5, 13]. Since the micro-assay is intended for screening large numbers of sera, an alternative strategy was adopted for reasons of economy. All samples were screened for insulin binding in a screening assay, and those having a level above 0.4 units were assayed in a competition assay with competitive displacement by cold insulin. The threshold for including samples in the competition assay was selected to maximize sensitivity in cases while requiring less than 6% of samples from healthy schoolchildren to be tested for competitive displacement (Figure 1).

Screening assay Serum (5 ìl) was added to duplicate wells in a deepwell microtitre plate (Beckman Instruments (UK) Ltd, High Wycombe, Bucks, UK) on ice, followed by 15,000 cpm A14-125I-labelled human insulin (>2000 Ci/mmol) (Amersham International plc, Little Chalfont, Bucks, UK) diluted in 25 ìl 50 mmol/l Tris buffer pH 8.0 containing 1% vol/vol Tween-20 (TBT). The plate was centrifuged briefly at 500×g, mixed, and incubated at 4°C for 72 h. Immune complexes were precipitated by adding 10 ìl of preswollen protein A-sepharose gel per well (equivalent to 2.5 mg dry gel) (Pharmacia Biotech AB, Uppsala, Sweden) in 50 ìl TBT and incubating on an orbital shaker for 90 min at 4°C. The immune complexes were washed by the addition of 600 ìl ice-cold TBT using an AM527 Handiwash (Dynatech Laboratories Ltd, Billingshurst, West Sussex, UK), followed by centrifugation at 500×g for 3 min at 4°C and aspiration of the supernatant. After five washes, 100 ìl TBT was added to each well

The competitive assay was performed using the same method as the screening assay, but further duplicate wells of each sample were incubated with 15,000 cpm 125 I-labelled insulin diluted in 25 ìl TBT buffer containing 8 units/ml unlabelled human insulin (Humulin, Lilly, Basingstoke, Hants, UK). Specific bound counts were calculated for each sample by subtracting the counts of the tubes with excess unlabelled insulin from those with label alone, and converted into arbitrary units as described above, including an additional standard at 0.2 units.

Conventional radiobinding assay The method has been previously described [5] and involves an acid charcoal extraction to remove endogenous insulin. Aliquots of each extract were assayed with and without the addition of cold human insulin. Samples were tested in duplicate and results expressed as percentage of binding (cpm bound/cpm total). Displaced percentage binding was calculated as the percentage binding obtained when sera were incubated with and without cold insulin (Ä% binding). The interassay coefficient of variation (CV) over 25 assays was 9% at both 2.2 Ä% binding and 29.2 Ä% binding. The assay has been shown to have a validity, consistency, specificity, and sensitivity of 100% in recent IDW- and IDF-sponsored IAA proficiency serum exchanges.

Data analysis Differences in antibody prevalence according to age and gender were analysed by the chi-squared test. The sensitivity and specificity of IAA were analysed as receiver operator characteristic (ROC) plots [14]. In this study we have used the ROC plot to relate

Novel micro-assay for insulin autoantibodies

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Figure 2. Standard curves from 10 consecutive screening assays. In each assay r 2 >0.99.

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0.04±0.05 0.46±0.16 1.3±0.21 313±82

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The negative control serum was obtained from a healthy volunteer, the low and medium controls from ICA-positive first-degree relatives of IDDM patients and the high control from an insulin-treated patient.

antibody prevalence in cases (i.e. sensitivity) to the prevalence in the background population at progressively increasing thresholds over the whole range of antibody measurement.

Results Figure 2 shows the curves from 10 consecutive screening assays. For each assay the binomial curve fit gave r2 >0.99. The inter-assay precision of the screening assay and of the competitive IAA assay for the four control sera are shown in Table 1. The percentage binding obtained for the lowest standard (0.39 units) was above that of the negative control in all assays. 157 schoolchildren (5.5%) and 172 (71%) IDDM patients had insulin binding levels>0.4 units, and these were reassayed in the competition assay; the

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IAA (units)

Table 1. Inter-assay precision of the screening and competition steps of the micro-assay

99th centile

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Figure 3. IAA results for the 2860 schoolchildren (C) and 241 newly diagnosed IDDM patients (•). The numbers in boxes represent those children (2789 schoolchildren and 75 IDDM patients) with IAA levels below the lowest standard (0.2 units).

distribution of insulin autoantibody levels in the schoolchildren and cases, following screening and retesting, are shown in Figure 3. The upper centile of the distribution in schoolchildren corresponded to IAA levels>0.86 units after testing in the competition assay. IAA levels>0.86 units after competitive displacement were found in 126 (52%) patients, including 81 out of 117 (69%) patients aged less than 10 years at diagnosis and 45 out of 124 (36%) of those aged 10–20 years (P<0.0001) (Figure 4). The prevalence was similar in males (76 out of 144, 53%) and females (50 out of 97, 52%).

Comparison with the conventional assay Figure 5A shows IAA levels obtained for the IDS workshop samples with the micro-assay against those obtained with the conventional assay. In order to overcome the difficulty of comparing the assays using laboratory reference ranges derived from different control populations, the 99th centile of the 101 work-

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Figure 4. Receiver operator characteristic (ROC) curve for IAA results from 2860 school-children and 241 cases, above the 97.5th centile of the results obtained in the schoolchildren. ROC curves for results in children above and below age 10 years are also shown. Sensitivity is calculated as the proportion of cases with antibody levels greater than or equal to each threshold, and background prevalence as the proportion of schoolchildren with antibody levels greater than or equal to the same threshold. Superior overall performance of IAA in young children was demonstrated by a shift of the curve towards the top left-hand corner of the graph. —–, <10 years; —–, all; - - -, >10 years.

shop control sera was used to define the threshold for raised antibody levels when comparing the two methods. Of the 51 IDDM patients in the combined antibody workshop, 26 were found to have IAA levels above the 99th centile of the workshop controls with the micro-assay or the conventional assay. IAA levels below these thresholds were found in 125 samples (25 IDDM patients and 100 controls) by both assays. Ten samples (nine IDDM patients and one control) were above the 99th centile in both assays, and a further 17 IDDM samples were above this threshold with the micro-assay. Figure 5B shows the IAA levels obtained for the unselected sera with the micro-assay against those obtained with the conventional assay. Both the microassay and the conventional assay found nine (eight insulin-treated) of the unselected samples to have IAA levels above the 99th centile of the workshop controls. Four samples were found to have IAA levels greater than the 99th centile in only one assay; two samples with the conventional assay (one insulin-treated) and two samples with the micro-assay (two insulintreated). IAA below these thresholds was found in 171 samples (six insulin-treated) by both assays.

Discussion Insulin autoantibody levels are inversely related to age at diagnosis of IDDM [7, 8], being most prevalent in ICA-positive relatives below age 10 years [15]. This association with childhood suggests that they will prove essential as markers of early anti-islet autoimmunity in young children. Established IAA assays

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Figure 5. Plot of IAA levels for the micro-assay (units) against levels obtained with the conventional assay (Ä% binding) for (A) 152 IDS combined antibody workshop samples and (B) 184 unselected sera from a routine chemical pathology laboratory. Only samples with levels above the 99th centile of the 101 workshop controls in either assay are shown. A further 125 workshop samples and 171 unselected samples were found to have IAA below this level in both laboratories. Dotted lines show thresholds for antibody levels equivalent to the 99th centile of the 101 controls included in the combined antibody workshop. (◆) represents newly diagnosed IDDM patients (A) or insulin-treated patients (B) and (e) controls (A) or non-insulin treated patients (B).

offer good sensitivity and specificity but require large serum volumes, a disadvantage for repeated testing in infants and small children. Studies of the early natural history of islet autoimmunity in the general population, and any intervention trials which result, will require large numbers of children to be tested using high throughput assays. The new micro-assay for measuring insulin autoantibodies described here has relatively high sensitivity and specificity, and can be performed on less than 50 ìl of serum. This micro-assay for insulin autoantibodies is simple, robust, and technically undemanding and uses a format similar to that of assays measuring autoantibodies to glutamate decarboxylase and protein tyrosine phosphatase IA-2 [16–18]. It can achieve relatively high throughput, since the 96-well plate format facilitates automation of the pipetting and washing steps and the method can be adapted for counting either in a gamma counter or scintillation counter. It is therefore applicable to large-scale population screening and will facilitate repeated testing of small children. The assay detects insulin auto-

Novel micro-assay for insulin autoantibodies

antibodies in 53% of patients with newly diagnosed IDDM, including 69% of patients less than 10 years of age, compared with 1% of schoolchild samples. Moreover, within the defined set of sera included in the combined antibody workshop, results obtained with the micro-assay were comparable to those obtained using a conventional method for IAA determination. Our method differs from the conventional method in three major respects. First, a smaller volume of serum is used; most established assays require at least five times the amount of serum. Our results show that sensitivity has been maintained in spite of the use of small serum volumes. Second, there is no acid charcoal extraction step to remove bound insulin prior to antibody measurement, raising the possibility that high levels of circulating insulin may interfere with antibody measurement. Extraction is not, however, feasible with the small volumes of serum used for this method, and the additional complexity involved would reduce sample throughput. Measurement could potentially be improved by testing fasting samples [3], but we developed the assay using random samples since this makes the method much more applicable for general use, and found that the assay achieved good discrimination between cases and schoolchildren under these conditions. This level of discrimination without extraction may be the result of the small volume of serum used, since the amount of radiolabelled insulin added is equivalent to more than 50 mU/l in the 5 ìl sample, a relatively high physiological level, which may reduce the impact of variations in serum insulin concentrations. It is interesting to note that other established assays using much larger serum volumes do not always remove insulin prior to testing. The third major difference in the micro-assay is the use of protein A-sepharose rather than polyethylene glycol (PEG) precipitation to separate bound from free radiolabelled insulin. Protein A-sepharose has previously been shown to be effective in removing insulin antibodies from plasma prior to measurement of free insulin [19], and free insulin measured in extracted samples correlates well with results obtained after PEG extraction. The use of protein A-sepharose rather than PEG extraction may also discriminate non-specific insulin binding in cord blood from that due to autoantibodies [20]. Despite the comparable sensitivities and concordance between the micro-assay and the conventional assay, some discrepancies were found. Sera from 17 new-onset IDDM patients included in the combined autoantibody workshop had elevated levels in the micro-assay only (Figure 5B). Six of these sera and one from an additional patient, however, had IAA levels above the mean±3 SD threshold of the conventional assay established from 92 local control samples. The serum from one insulin-treated patient had high insulin binding in the conventional assay but little binding in the micro-assay (Figure 5B). This discrepancy may be explained by the use of protein A which, unlike protein G, shows only weak binding to the human IgG3 subclass [21] which can contribute to insulin binding [22]. Elevated insulin binding was found when this serum was reassayed with protein

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G-sepharose (data not shown), consistent with the presence of IgG3 insulin antibodies. The use of protein G alone or in combination with protein A-sepharose may therefore be warranted. In conclusion, we have developed an assay for IAA that achieves sensitivity and specificity equivalent to established assays, but uses much less serum. This should allow fuller evaluation of the role of IAA in risk assessment, both alone and in combination with other islet autoantibodies. The assay should prove particularly useful in young ‘at-risk’ individuals, in whom the benefits of successful intervention may be greatest, and will also facilitate longitudinal studies in infancy to improve our understanding of the early pathogenesis of IDDM.

Acknowledgements We would like to thank Marcia Bruley and Regina Park for technical assistance and Pam Sawtell for co-ordinating the collection of samples. PJB is funded by the Juvenile Diabetes Foundation. The BartsOxford Study is supported by the British Diabetic Association and this project was supported in part by the Medical Research Service of the Department of Veterans Affairs and grants from the National Institute of Health DK02456 and DK17047.

References 1. Palmer J.P., Asplin C.M., Clemons P., Lyen K., Tatpati O., Raghu P.K., Paquette T.L. 1983. Insulin antibodies in insulin dependent diabetes before insulin treatment. Science 222: 1337–1339 2. Soeldner J.S., Tuttleman M., Srikanta S., Ganda O.P., Eisenbarth G.S. 1985. Insulin-dependent diabetes mellitus and autoimmunity: islet-cell autoantibodies, insulin autoantibodies, and beta-cell failure. N. Engl. J. Med. 313: 893–894 3. Vardi P., Dib S.A., Tuttleman M., Connelly J.E., Grindbergs M., Radizabeh A., Riley W.J., Maclaren N.K., Eisenbarth G.S., Soeldner J.S. 1987. Competitive insulin autoantibody assay. Prospective evaluation of subjects at high risk for development of type 1 diabetes mellitus. Diabetes 36: 1286–1291 4. Bosi E., Becker F., Bonifacio E., Wagner R., Collins P., Gale E.A.M., Bottazzo G.F. 1991. Progression to type 1 diabetes in autoimmune endocrine patients with islet cell antibodies. Diabetes 40: 977–984 5. Hegewald M.J., Schoenfeld S.L., McCulloch D.K., Greenbaum C.J., Klaff L.J., Palmer J.P. 1992. Increased specificity and sensitivity of insulin antibody measurements in autoimmune thyroid disease and type 1 diabetes. J. Immunol. Methods 154: 61–68 6. Schatz D., Krischer J., Horne G., Riley W., Spillar R., Silverstein J., Winter W., Muir A., Derovanesian D., Shah S., Malone J., Maclaren N. 1994. Islet cell antibodies predict insulin-dependent diabetes in United States school age children as powerfully as in unaffected relatives. J. Clin. Invest. 93: 2403–2407 7. Arslanian S.A., Becker D.J., Rabin B., Atchison R., Eberhardt M., Cavender D., Dorman J., Drash A.L. 1985. Correlates of insulin antibodies in newly

A. J. K. Williams et al.

478

8.

9.

10.

11.

12.

13.

14.

15.

diagnosed children with insulin-dependent diabetes before insulin therapy. Diabetes 34: 926–930 Vardi P., Ziegler A.G., Mathews J.H., Dib S., Keller R.J., Ricker A.T., Wolfsdorf J.I., Herskowitz R.D., Rabizadeh A., Eisenbarth G.S., Soeldner J.S. 1988. Concentration of insulin autoantibodies at onset of Type 1 diabetes: inverse correlation with age. Diabetes Care 11: 736–739 Kurtz A.B., Mathews J.A., Mustaffa B.E., Daggett P.R., Nabarro J.D.N. 1980. Decrease of antibodies to insulin, proinsulin and contaminating hormones after changing treatment from conventional beef to purified pork insulin. Diabetologia 18: 147–150 Wilkin T.J., Nicholson S., Casey C. 1985. A microenzyme linked immunosorbent assay for insulin antibodies in serum. J. Immunol. Methods 76: 185–194 Greenbaum C.J., Palmer J.P., Kuglin B., Kolb H., and participating laboratories. 1992. Insulin autoantibodies measured by radioimmunoassay methodology are more related to insulin-dependent diabetes mellitus than those measured by enzyme-linked immunosorbent assay: results of the fourth international workshop on the standardization of insulin autoantibody measurement. J. Clin. Endocrinol. Metab. 74: 1040–1044 Bingley P.J., Bonifacio E., Shattock M., Gillmor H.A., Sawtell P.A., Dunger D.B., Scott R.D.M., Bottazzo G.F., Gale E.A.M. 1993. Can islet cell antibodies predict IDDM in the general population? Diabetes Care 16: 45–50 Kurtz A.B., DiSilvio L., Bosi E. 1988. The determination of detection limits for insulin antibody assays. Diabetologia 31: 395–399 Zweig M.H., Campbell G. 1993. Receiver-Operating Characteristic (ROC) plots. A fundamental evaluation tool in clinical medicine. Clin. Chem. 39: 561–577 Bingley P.J., for the ICARUS Group. 1996. Interactions of age, islet cell antibodies, insulin autoantibodies and

16.

17.

18.

19.

20.

21.

22.

first phase insulin response in predicting risk of progression to IDDM in ICA positive relatives: the ICARUS dataset. Diabetes 45: 1720–1728 Petersen J.S., Hejnaes K.R., Moody A., Karlsen A.E., Marshall M.O., Hoier-Madsen M., Boel E., Michelsen B.K., Dyrberg T. 1994. Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay. Diabetes 43: 459–467 Bonifacio E., Lampasona V., Genovese S., Ferrari M., Bosi E. 1995. Identification of protein tyrosine phosphatase-like IA2 (islet cell antigen 512) as the insulin-dependent diabetes-related 37/40K autoantigen and a target of islet-cell antibodies. J. Immunol. 155: 5419–5426 Payton M.A., Hawkes C.J., Christie M.R. 1995. Relationship of the 37,000- and the 40,000-Mr tryptic fragments of islet antigens in insulin-dependent diabetes to the protein tyrosine phosphatase-like molecule IA-2 (ICA512). J. Clin. Invest. 96: 1506–1511 Chevenne D., Valade F., Bridel M.-P., Rigal O., Demellier J.-F., Sachs C., Porquet D. 1991. Protein A-Sepharose used to measure free insulin in plasma. Clin. Chem. 37: 64–67 Bilbao J.R., Calvo B., Urrutia I., Linares A., Castano L. 1997. Anti-insulin activity in normal newborn cord-blood serum. Absence of IgG-mediated insulin binding. Diabetes 46: 713–716 Kronvall G., Williams R.C. 1969. Differences in anti-protein A activity among IgG subgroups. J. Immunol. 103: 828–833 Koch M., Francois-Gerard C., Sodoyez-Goffaux F., Sodoyez J.-C. 1986. Semi-quantitative assessment of anti-insulin total IgG and IgG sub-classes in insulin-immunized patients using a highly sensitive immunochemical micromethod. Diabetologia 29: 720–726