We can now predict the development of type 1A diabetes (immune mediated diabetes) such that large trials of prevention (to date unsuccessful) are underway. Type 1A diabetes occurs in approximately 1/300 children of the general US population, 1/20 first degree relatives of a patient with type 1 diabetes and ½ twins (identical to proband with type 1 diabetes). Multiple genes conferring susceptibility have been defined with by far the most important within the major histocompatibility complex (41% of the 48% of familial aggregation currently genetically defined related to MHC class II genes) [2
]. It is now possible to predict appreciable genetic risk even in the general population with HLA class II typing. The persistent expression of >=2 of three “classic” islet autoantibodies (autoantibodies to GAD65, IA-2 or insulin) is associated with almost complete progression to diabetes given long enough follow up, and a fourth major islet autoantigen has recently been discovered [3
]. Hundreds of thousands of individuals have been screened for islet autoantibodies and such screening forms the basis for preventive trials.
The discovery of “cytoplasmic” islet cell autoantibodies with immunohistochemical staining of frozen sections of normal human pancreas initiated the utilization of anti-islet autoantibodies and was rapidly followed by the realization that type 1A diabetes is a chronic autoimmune disorder that could be predicted [4
]. A series of workshops improved the measurement of cytoplasmic islet cell autoantibodies but also highlighted difficulties in standardization of an assay dependent upon “observer” and pancreatic tissue sections. The discovery that insulin autoantibodies were present prior to insulin injections that induce insulin antibodies initiated the studies of what has been termed “biochemical autoantibodies” [6
]. The first assays demonstrating insulin autoantibodies utilized 125
I labeled insulin in a fluid phase radioassay (The discovery of the radioassay by Berson and Yalow utilized insulin antibodies). Shortly after the report of insulin autoantibodies at the onset of diabetes prior to insulin therapy dozens of laboratories set up assays and an immediate controversy developed as to whether insulin autoantibodies predicted diabetes. Remarkably clear results from an insulin autoantibody workshop ended the debate [7
]. Fluid phase radioassays detected insulin autoantibodies of prediabetic and new onset patients while ELISA assays only detected antibodies induced by insulin injection. The first insulin autoantibody assays utilized relatively large volumes of serum and polyethylene glycol precipitation to separate labeled insulin bound to antibody from free insulin. Polyethylene glycol is a non-specific precipitant and factors in sera particularly in cord blood thus could produce low levels of signal. Subsequent assays introduced competition with unlabeled insulin to improve specificity and sensitivity and micro-assays utilizing protein A precipitation were developed [9
]. Insulin autoantibody expression is dramatically age related amongst individuals developing type 1A diabetes with almost all children presenting prior to age 5 have such antibodies but markedly fewer diabetics express insulin autoantibodies at diagnosis after age 12 [10
]. Prospective studies from birth indicate that not infrequently insulin autoantibodies are present years before diagnosis, but then can be absent at diagnosis even though other anti-islet autoantibodies have appeared.
Following the initial application of insulin autoantibody assays, assays for autoantibodies reacting with GAD65 and IA-2 (and related IA-2 beta molecule) autoantibodies were introduced [11
]. These assays were facilitated by the general application of in vitro transcription and translation reticulocyte kits to produce labeled autoantigen with usually 3
H-leucine for 35
S-methionine incorporated into the protein autoantigen. Given a cDNA for a given protein antigen, within a week appropriate labeled autoantigen can be produced and an assay run. Assays can be run in 96-well plates with protein A beads (or isotype specific beads) and filtration to separate bound from free autoantigen. The majority of new assays work the first time they are run, but for those that do not work, perhaps due to improper folding of a protein antigen or lack of essential post-translational modifications of the antigen, it is very difficult to appropriately modify the antigen. Of the three standard assays, the insulin autoantibody assay has remained the most difficult for new laboratories to perform and to standardize. This assay is typically run with a competitive step with unlabeled insulin blocking binding of I-125 insulin to the autoantibody while the GAD65 and IA-2 autoantibody assays do not require such competition. The difficulties with the insulin autoantibody radioassay probably relate to the very low concentrations of insulin autoantibodies, with a very small difference between approximately ½ of positive patients and normal controls. It appears that insulin autoantibodies of man cannot react with insulin bound to plates. Given the presence of these autoantibodies in man, workshops evaluating animal models have concluded that only specific insulin autoantibodies are present in the spontaneous model of diabetes, the NOD mouse. Insulin autoantibody levels are as high in the NOD mouse as the youngest children developing diabetes and follow a classic course, usually decreasing to negative about the time of onset of diabetes.
Evaluation of standard ELISAs for anti-islet autoantibodies in multiple workshops indicated a lack of sensitivity, specificity, or both compared to the fluid phase radioassays. Recently a modified ELISA format for GAD65 autoantibodies that involves fluid phase reactivity of autoantibody with GAD65 performed as well as the best fluid phase radioassays. In this format labeled fluid phase GAD65 bound to antibody that subsequently bound to GAD65 on the ELISA plate [13
The evolution of assay formats in the field of type 1 diabetes illustrates the utility of workshops that occur approximately every two years with evaluation of 100 control sera and 50 sera from patients with new onset diabetes, as well as sera to evaluate assay reproducibility. In general groups are allowed to utilize any methodology they want and it is the comparative results that rapidly determine which methodologies are accepted. The appearance of “biochemical” autoantibodies is used as an intermediate phenotype in studies of the genetics and environmental factors preceding diabetes as well as entry criteria for preventive and new onset trials. Even the recent discovery of a fourth major islet autoantigen, there likely exist additional specific islet autoantibodies, given some rare sera that are positive on sections of pancreas but negative for the radioassays. In contrast the cytoplasmic ICA test cannot detect insulin autoantibodies, and misses most low positive GAD65 and IA-2 autoantibodies.
There is a subset of individuals with single anti-islet autoantibodies, particularly GAD65 and insulin that do not progress to diabetes with more than ten years of follow up. It appears that with a single autoantibody risk of diabetes for relatives of patients is only approximately 20%. In contrast the great majority with >=2 autoantibodies progress. Recent evidence indicates that the affinity of “single” anti-insulin autoantibodies in young children that do not predict diabetes are considerably lower than the insulin autoantibodies associated with diabetes risk. In young children insulin autoantibodies are usually the first to appear [14
It is likely that the assay formats utilized in type 1 diabetes and the ability to develop highly predictive assays (99% specificity) with multiple autoantigens should be applicable to many fields. We would suggest that an essential feature is not to assume that lack of prediction by an assay reflects the basic biology rather than dynamics of the assay and that high affinity autoantibodies reacting with native antigens not bound to solid phases with autoantibody specific reagents (e.g., Protein A) should be reference assays.
This is a field where the ELISA assays are very good and excellent radioassays are available for comparison. Measurement of IgA antibodies against tissue transglutaminase (TG) is the single best autoantibody assay for the prediction of celiac disease, and quantitative levels of the autoantibody reflect degree of intestinal injury. Changing the substrate from guinea pig TG to human recombinant TG led to significant improvements in the performance of this assay [15
]. This autoantibody assay is typically performed in the solid phase, but is also performed in the fluid phase using radioactive detection methods. Tiberti et al proposed that the fluid phase radiobinding assay was superior to standard ELISA assays but larger and direct comparisons to improved ELISA assays is needed [16
]. A head-to-head comparison of a radioassay with 5 commercial ELISAs showed that marked qualitative differences were obvious amongst all of the assays, even though increasing the cutoff for positivity uniformly improved the predictive value for celiac disease [17
]. Since quantitative assessment of TG levels is important, this study underscored the need for standardization of the assays amongst all commercial and research laboratories. A TG autoantibody workshop designed after the diabetes autoantibody workshops is currently underway with the aim of comparison and improvement of the transglutaminase autoantibody assay amongst commercial and research laboratories, with the future goal of standardization.
Part of the underlying pathogenesis of celiac disease involves specific deamidation of amino acids in ingested gliadin proteins to enhance immunogenicity. A new antibody assay against specific previously-deamidated gliadin peptides (DGP) has recently become available, as a marked improvement over the original anti-gliadin antibodies. Both IgA and IgG antibodies against DGP are measured by standard ELISA, and our examination of these antibodies demonstrate that the anti-DGP antibodies parallel (quantitatively) the course of anti-TG autoantibodies (measured by radioassay) as they are followed prospectively over time through serial measurements in children at risk for celiac disease. Anti-DGP antibodies become undetectable sooner than anti-TG autoantibodies when treatment with a gluten-free diet is instituted, which could either reflect the removal of the offending antigen in a patient’s diet, or due to differences in sensitivity between the two assays. Further study into the DGP antibody is important since it likely reflects the specific immune response against exogenous antigen, namely gliadin, rather than the autoimmune response of TG. It remains to be determined whether the appearance of multiple antibodies to indicate celiac autoimmunity can someday improve the predictive value for intestinal lesions in celiac disease, to replace intestinal biopsy as the future “gold standard.”
Open symbols indicate anti-TG autoantibodies. Closed symbols indicate anti-DGP antibodies. Dashed line indicates cutoff for positivity for both assays.