Autoantibodies specific to type 1 diabetes

An autoantibody is an immunoglobulin-type protein, which your immune system (B lymphocytes) produces as a molecular weapon against aggressors. The essential difference compared to a regular antibody lies in its target. Normally, antibodies recognize foreign agents, such as bacteria or viruses. An autoantibody, however, attacks a structure of your own body, such as a protein, an enzyme, a receptor or a component of a cell. This confusion between what is self and what is foreign is the cause of autoimmune diseases [1].

In practice, autoantibodies are useful to you primarily as disease markers. If they appear in your blood, it means your immune system has started a reaction directed against one of your own tissues. In type 1 diabetes, autoantibodies are not responsible for the destruction of the beta cells in the pancreas, which is carried out by cytotoxic T lymphocytes (the ones that kill directly), supported by helper T lymphocytes, macrophages and dendritic cells. The presence of autoantibodies signals that the autoimmune process exists. It also allows type 1 diabetes to be diagnosed even before your blood glucose rises above normal values [1].

Type 1 diabetes is an autoimmune disease. Your immune system mistakenly identifies the beta cells in the pancreas (the ones that produce insulin) as a threat and starts a battle against them [1]. Autoantibodies appear because B lymphocytes (cells of the immune system) are activated and start producing antibodies against structures in the beta cells or even against insulin itself. The exact cause of this faulty reaction is not fully understood. A combination is presumed to exist between a genetic predisposition (especially the HLA, human leukocyte antigen system) and certain environmental triggers. These include viral infections, alterations of the gut microbiota or other influences still insufficiently understood.

The appearance of autoantibodies is not a sudden event. They can appear several months or years before you show the clinical manifestations of type 1 diabetes. The type and the order of appearance vary greatly from one person to another [2]. This long interval between the onset of autoimmunity and the appearance of hyperglycemia explains why type 1 diabetes has preclinical stages. If you are at high risk, autoantibody testing can identify the disease while the beta cells still function nearly normally.

The four autoantibodies internationally recognized as the standardized markers of type 1 diabetes are:

• the anti-insulin antibodies (IAA);
• the anti-glutamic acid decarboxylase antibodies (GADA) — directed especially against the GAD65 isoform (one of the two variants of the enzyme);
• the anti-tyrosine phosphatase antibodies (IA-2A or anti-islet antigen 2);
• the anti-zinc transporter 8 antibodies (ZnT8A) [3].

This is the antibody panel recommended both for presymptomatic screening in people at high risk and for classifying diabetes in adults, when there is suspicion of autoimmune diabetes [4] [5].

Each of the four autoantibodies recognizes a different target in the pancreatic beta cells, which makes the panel complementary. If one antibody comes back negative, the positivity of the others can compensate in establishing the presence of autoimmunity. Of all of them, GADA is the most frequently encountered in adults, while IAA appears earliest in young children. IA-2A signals an increased risk of rapid progression to hyperglycemia, and ZnT8A is useful especially when the other three antibodies are absent [6]. When you are investigating the autoimmune nature of diabetes, evaluating all four autoantibodies together gives you the highest probability of confirming the diagnosis.

GADA antibodies are autoantibodies directed against the enzyme called glutamic acid decarboxylase, which exists in two main forms, GAD65 and GAD67. The clinical tests used in practice detect especially anti-GAD65 antibodies [3]. This enzyme is found in the beta cells of the pancreas, but also in the neurons that produce the neurotransmitter GABA. That is why the GADA level can also be elevated in a few rare neurological conditions, an important point when your result is interpreted.

In the context of type 1 diabetes, GADA is the most frequently identified autoantibody in adults and tends to persist longest in the blood after diagnosis [7]. GADA is the main autoantibody recommended for testing adults with suspected type 1 diabetes, being the first-line investigation. If you already have diabetes and discover only GADA positive, with no other autoantibodies present, progression toward insulin dependence is usually slower, but the pace varies a great deal from one person to another, and the moment when insulin is started is decided by your doctor. This profile is characteristic of the autoimmune form of adult-onset type 1 diabetes called LADA (latent autoimmune diabetes of the adult).

IA-2A antibodies are directed against an enzyme of the tyrosine phosphatase class (also called islet antigen 2 or ICA512), located on the membrane of the secretory granules in beta cells. The secretory granules are structures inside the beta cells that store insulin before it is released into the blood. The destruction of beta cells during the autoimmune process exposes this protein to the immune system, which reacts by producing IA-2A [3].

IA-2A has a particular clinical significance, indicating an independent and increased risk of progression to symptomatic type 1 diabetes (stage 3) [8]. For this reason, people with isolated IA-2A are monitored in the same way as those with several autoantibodies present. IA-2A is more frequent in children and adolescents than in adults. Its combination with GADA, IAA or ZnT8A signals advanced autoimmunity and a high probability of rapid evolution toward clinically manifest hyperglycemia [9].

IAA antibodies are autoantibodies directed against insulin, the hormone produced by the beta cells of the pancreas. Their particularity is that their target is also the final product of the attacked cells, which makes IAA a direct marker of the autoimmune recognition of insulin. In the young child with a genetic predisposition for type 1 diabetes, IAA is usually the first autoantibody to appear in the blood. Sometimes it is present many years before the clinical onset of the disease [2].

An important practical point for you is that IAA cannot be reliably measured after you start insulin treatment. The explanation is that your body naturally produces antibodies against exogenous insulin, and these cannot be distinguished from the actual autoimmune ones. For this reason, IAA testing is useful only before starting insulin therapy, especially in children or in the context of presymptomatic screening for type 1 diabetes [3].

ZnT8A antibodies target a protein called zinc transporter 8, present in the membrane of the insulin granules in beta cells. This transporter has the role of bringing zinc ions into the interior of the granules, and zinc is essential for storing insulin locally in a stable crystallized form. The ZnT8A antibodies show that the immune system has recognized this protein as a target, not that the transporter itself is defective [10].

ZnT8A is the most recently included of the four standardized autoantibodies, having been described in 2007. It is particularly useful clinically as a complementary test. It can happen that your diabetes looks like type 1, but GADA, IA-2A and IAA are negative. In this case, the presence of ZnT8A can confirm the autoimmune nature of your disease [6]. Including ZnT8A in the standard panel has increased the diagnostic sensitivity (the proportion of real cases that the tests detect), reducing the number of cases mistakenly labeled as idiopathic type 1 diabetes or as atypical type 2 diabetes.

The autoantibodies specific to type 1 diabetes have high specificity (they very rarely give positive results in people without the disease). A confirmed positive result indicates, in the vast majority of cases, an autoimmune process directed against the beta cells [6]. The greater the number of positive autoantibodies, the higher the diagnostic value. If you have two or more autoantibodies persistently present, progression toward clinically manifest type 1 diabetes is considered almost certain in the long term. This remains true even if blood glucose is still normal at the time of testing [9].

On the other hand, the sensitivity of a single autoantibody is limited, which is why testing is always done for the whole standardized panel. A single positive result must be confirmed by a second test, ideally in a laboratory participating in the international program for the standardization of autoantibody testing (the Islet Autoantibody Standardization Program, IASP). You can ask the laboratory whether it takes part in this program. This confirmation is necessary because a significant proportion of children with a single autoantibody can revert to negativity on subsequent testing [5]. This retesting prevents both underdiagnosis and the mistaken labeling as type 1 diabetes of a person with a false positive result.

Yes. The presence of a pancreatic autoantibody in your blood is not equivalent to a diagnosis of type 1 diabetes and does not guarantee that the disease will appear. A significant proportion of people who have a single positive autoantibody, especially in childhood, spontaneously revert to negativity on repeat testing, without diabetes ever appearing. Your risk of progression depends on several factors, such as the number of autoantibodies present, their titre (quantity), the age at which they appear, the type of autoantibody and the genetic predisposition [11].

If you persistently have two or more autoantibodies, your risk of progression toward symptomatic type 1 diabetes is very high in the long term. The interval until the appearance of hyperglycemia can, however, be months, years or even decades [9]. This phase of autoimmunity without clinically manifest diabetes is called stage 1 (with normal blood glucose) or stage 2 (with dysglycemia, that is blood glucose values above normal but below the threshold for diabetes) of type 1 diabetes. It allows close surveillance, carried out together with your diabetes specialist. They decide what is monitored (blood glucose and glycated hemoglobin, that is the average blood glucose over the last few months) and at what intervals, and they also interpret the results for you. This follow-up helps you avoid the abrupt onset with diabetic ketoacidosis when hyperglycemia eventually appears, as the figure below shows [12].

Yes, it is possible. A small proportion of patients with type 1 diabetes have no detectable autoantibody at the moment of diagnosis [13]. A negative autoantibody test does not exclude a diagnosis of type 1 diabetes, at any age. The suspicion remains strong especially if you are under 35 years of age (the age threshold used in the guidelines for diagnosing type 1 diabetes in adults) and if you do not have clinical features of type 2 diabetes or of monogenic diabetes (a rare form of diabetes caused by a single altered gene) [14].

In this case the clinical picture, the rapid progression toward insulin dependence and the low level of C-peptide (a marker of the insulin your body produces) point the diagnosis toward type 1 diabetes [5]. Some of these cases could be explained by the technical limits of the tests. The autoantibodies may be present in concentrations that are too small, or directed against structures (antigens) not yet included in the standard panel. This category corresponds to the form called idiopathic type 1 diabetes, in which there is permanent insulinopenia (the body produces practically no insulin at all) and a tendency to ketoacidosis, but without evidence of autoimmunity [15].

Sometimes. The level of autoantibodies can decrease progressively after you receive a diagnosis of type 1 diabetes stage 3, and some can become undetectable on subsequent testing. This phenomenon is explained by antigen depletion. As your beta cells are destroyed almost completely, the source that stimulates the immune system disappears, and the production of autoantibodies decreases [7]. The four standardized autoantibodies, however, behave differently. GADA persists longest, sometimes years or decades after diagnosis, while IA-2A and ZnT8A decrease more rapidly.

In the case of IAA antibodies, the situation is special. After you start insulin therapy, your body produces antibodies against exogenous insulin. These cannot be distinguished from the original autoimmune ones, so the test is no longer interpretable. This dynamic explains why, if you want to clarify the type of diabetes after several years of insulin treatment, the autoantibody panel should not include IAA. However, a negative result does not exclude the autoimmune origin of the disease.

Yes. Research in the field of autoimmunity in type 1 diabetes has identified several autoantibodies that are not yet included in the standardized routine panel but which are being actively studied. Among them are the anti-tetraspanin 7 antibodies (TSPAN7), a protein from the granules of the beta cells related to the IA-2 antigen, and the anti-chromogranin A antibodies. Autoantibodies directed against new structures in the beta cells are also being studied. These structures are generated by changes occurring over time in some proteins (like a premature aging of theirs) [10].

These additional markers have two aims. The first is to identify cases in which type 1 diabetes is present, but the four classic autoantibodies are negative (the apparently idiopathic form). The second is to refine the estimation of the risk of progression in people with already confirmed autoimmunity [15]. For now, these tests remain at the research level. It is likely that in the coming years the list of autoantibodies in current use will broaden. That will be able to offer you a more secure diagnosis and a better assessment of risk.