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An indirect competitive immunoassay for insulin autoantibodies based on surface plasmon resonance
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2008 (English)In: Biosensors and Bioelectronics, ISSN 0956-5663, Vol. 24, no 4, 876-881Artikel i tidskrift (Refereed) Published
Abstract [en]

We have developed a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D). When measuring trace molecules in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labeled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (4 days to 50 min), while sensitivity is maintained at a level comparable to that offered by RIA.

Place, publisher, year, pages
2008. Vol. 24, no 4, 876-881
Keyword [en]
SPR, Type 1 diabetes, Insulin autoantibodies, Indirect competitive immunoassay, RIA
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:liu:diva-12469 (URN)10.1016/j.bios.2008.07.018 (DOI)oai:DiVA.org:liu-12469 (OAI)
Note
The status of article IV on the day of defence was: Accepted.Available from2008-09-06 Created:2008-09-06 Last updated:2011-01-28Bibliographically approved
In thesis
1. Interaction Studies in Complex Fluids with Optical Biosensors
Open this publication in new window or tab >>Interaction Studies in Complex Fluids with Optical Biosensors
2008 (English)Doktorsavhandling, sammanläggning (Other academic)
Abstract [en]

In this thesis interactions in complex fluids, such as serum and meat juice, were analysed with optical biosensor techniques.

Panels of lectins immobilised on gold surfaces were used for investigation of differences in protein glycosylation pattern in sera and meat juices between various species. The present panel was also used for investigation of global glycosylation changes of serum proteins in type 1 diabetes patients. Biorecognition was evaluated with null ellipsometry and scanning ellipsometry combined with multivariate data analysis techniques (MVDA). Principal component analysis (PCA) showed that the lectin panel enabled discrimination between sera from the different species as well as for the different meat juices. The results also indicate that there is a measurable global alteration in glycosylation pattern of serum proteins in type 1 diabetic patients compared to healthy subjects. Using an artificial neuronal net (ANN), it was also possible to correctly categorise unknown serum samples into their respective class or group. The analytical potential of combining information from lectin panels with multivariate data analysis was thereby demonstrated.

Also, a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D) has been developed. When measuring trace molecules, such as autoantibodies, in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labelled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (from 4 days to 50 min), while sensitivity is maintained at a level comparable to that offered by RIA. Finally, the assay was used in a screening study of newly diagnosed type 1 diabetes patients and non-diabetic subjects.

Linköping: Linköping University Electronic Press, 2008. 70 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1195
Keyword
Type 1 diabetes, Insulin, Lectin panel, Ellipsometry, Meat juice, Serum proteins
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-14694 (URN)978-91-7394-852-5 (ISBN)
Public defence
2008-09-19, Planck, Fysikhuset, Campus Norrköping, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from2008-09-26 Created:2008-09-19 Last updated:2009-04-24Bibliographically approved
2. Autoantibodies related to type 1 diabetes in children
Open this publication in new window or tab >>Autoantibodies related to type 1 diabetes in children
2011 (English)Doktorsavhandling, sammanläggning (Other academic)
Abstract [en]

Type 1 diabetes is an autoimmune disease resulting from destruction of the insulin producing beta cells in the pancreas. The patients need life-long heavy treatment and still complications, both acute and later in life, are common. The incidence of type 1 diabetes has increased rapidly during the last decades, especially among young children. The disease can be predicted by genes predisposing type 1 diabetes, mainly human leukocyte antigen (HLA) genes, together with presence of autoantibodies to beta-cell antigens, where multiple autoantibodies confer the highest risk. A number of immune system intervention trials are now ongoing aiming to halt the progression of the inflammatory process in the beta cells.

This thesis aimed to investigate the prevalence and levels of autoantibodies in healthy children and in children with type 1 diabetes. Another aim was to study different properties of one of these autoantibodies, such as to which epitopes the antibodies bind and the distribution of immunoglobulin (Ig)-G subclasses, after immunomodulatory treatment in children with type 1 diabetes.

We found that positivity to autoantibodies against glutamic acid decarboxylase (GADA) and tyrosine phosphatase like protein islet antigen-2 (IA-2A) was associated with HLA risk genotypes in 5-year old children from the general population. HLA risk genotypes seemed important for persistence of autoantibodies and for development of type 1 diabetes, while emergence of autoantibodies, especially transient autoantibodies, seemed to be more influenced by environmental factors. Improved methods for detection of autoantibodies are needed, for prediction of diabetes and for identification of high-risk individuals suitable for prevention treatments. Therefore, an assay for measurement of insulin autoantibodies (IAA), based on surface plasmon resonance (SPR), was developed. The main advantages of this method are that there is no need for labelling and that it is time-saving compared to the traditionally used radioimmunoassay (RIA), but further development of the method is needed.

Treatment with GAD-alum (Diamyd) in children with type 1 diabetes has shown to preserve residual insulin secretion. This clinical effect was accompanied by an increase in GADA levels. We investigated the epitope reactivity of GADA in both GAD-alum and placebo treated children, and found that binding to one of the tested epitopes was temporarily increased after injection of GAD-alum. This result suggests that the quality of GADA was, to some extent, transiently affected by the treatment. On the other hand, no changes in binding to epitopes associated with stiff person syndrome (SPS) were observed, which together with the lack of change in GAD65 enzyme activity further strengthens the safety of the treatment. We also observed that the distribution of IgG subclasses was changed by GAD-alum treatment, with a lower proportion of IgG1 and higher IgG3 and IgG4. Lower IgG1 and higher IgG4 suggest a temporary switch towards a protective Th2 immune response, which has previously been observed in the same individuals for other immunological markers.

In conclusion, measurement of autoantibodies related to type 1 diabetes is an important tool for studying the autoimmune process in pre-diabetic and type 1 diabetic children. In addition to the use as markers of disease progression, the autoantibodies may be used for studying the effects of immunomodulatory treatments on the humoral immune response.

Linköping: Linköping University Electronic Press, 2011. 116 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1218
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-64593 (URN)978-91-7393-276-9 (ISBN)
Public defence
2011-02-18, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from2011-01-28 Created:2011-01-28 Last updated:2011-01-28Bibliographically approved

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