Flow Cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 Diabetes Mellitus

The first measurable sign of arising autoimmunity in Type 1 Diabetes Mellitus is the detection of autoantibodies against beta-cell antigens, such as glutamic acid decarboxylase (GAD65). GAD65 autoantibodies (GADA) are usually measured by Radioligand Binding Assay (RBA). The aim of this work was to develop protocols of Flow Cytometric microsphere-based immunoassays (FloCMIA) which involved glutamic acid decarboxylase fused to thioredoxin (TrxGAD65) adsorbed on polystyrene microspheres. Detection of bound GADA was accomplished by the use of anti-human IgG-Alexa Fluor 488 (Protocol A), anti-human IgG-biotin and streptavidindichlorotriazinyl aminofluorescein (DTAF) (Protocol B) or TrxGAD65-biotin and streptavidin- DTAF (Protocol C). Serum samples obtained from 46 patients assayed for routine autoantibodies at Servicios Tecnológicos de Alto Nivel (STAN-CONICET) were analyzed by RBA, ELISA and three alternative FloCMIA designs. Protocol C exhibited the highest specificity (97.8%) and sensitivity (97.4%) and a wide dynamic range (1.00-134.40 SDs). Samples obtained from 40 new-onset diabetic patients were also analyzed to further evaluate the performance of protocol C. The latter protocol showed a sensitivity of 58.6% and a prevalence of 47.5%. Two patients resulted positive only by FloCMIA protocol C and its SDs were higher than RBA and ELISA, showing a significantly wide dynamic range. In conclusion, FloCMIA proved to be highly sensitive and specific, requiring a low sample volume; it is environmentally adequate, innovative and it represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA; making it applicable to most medium-complexity laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Ureta, Daniela Beatriz. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "Profesor R. A. Margni"; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Cientiâ­ficas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral "profesor R. A. Margni"; Argentin

Surface Plasmon Resonance Reveals a Different Pattern of Proinsulin Autoantibodies Concentration and Affinity in Diabetic Patients

Type 1 diabetes mellitus (DM) is characterized by autoimmune aggression against pancreatic beta cells resulting in absolute deficiency of insulin secretion. The first detectable sign of emerging autoimmunity during the preclinical asymptomatic period is the appearance of diabetes-related autoantibodies. In children at risk for type 1 DM, high-affinity Insulin autoantibodies reactive to proinsulin, are associated with diabetes risk. Autoantibodies are usually measured by radioligand binding assay (RBA) that provides quasi-quantitative values reflecting potency (product between concentration and affinity) of specific autoantibodies. Aiming to improve the characterization of the specific humoral immune response, we selected surface plasmon resonance (SPR) as an alternative method to measure proinsulin autoantibodies (PAA). This novel technology has allowed real time detection of antibodies interaction and kinetic analysis. Herein, we have employed SPR to characterize the PAA present in sera from 28 childhood-onset (mean age 8.31±4.20) and 23 adult-onset diabetic patients (≥65 years old, BMI<30) in terms of concentration and affinity. When evaluating comparatively samples from both groups, childhood-onset diabetic patients presented lower PAA concentrations and higher affinities (median 67.12×10−9 M and 3.50×107 M−1, respectively) than the adults (median 167.4×10−9 M and 0.84×107 M−1, respectively). These results are consistent with those from the reference method RBA (Standard Deviation score median 9.49 for childhood-onset group and 5.04 for adult-onset group) where the binding can be directly related to the intrinsic affinity of the antibody, suggesting that there is a different etiopathogenic pathway between both types of clinical presentation of the disease. This technology has shown to be a useful tool for the characterization of PAAs parameters as an alternative to radioimmunoassay, with high versatility and reproducibility associated to low occupational and environmental risk. However, this technology is not eligible for routine marker screening, but this is a powerful technique for a fine description of the thermodynamic parameters of antigen-antibody interaction

Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus

Abstract Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65 kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In order to produce reliable immunochemical tests for large scale screening of autoimmune DM, large amounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65 using the baculovirus expression system in two species of larvae, Rachiplusia nu and Spodoptera frugiperda. GAD65 was identified at the expected molecular weight, properly expressed with high yield and purity in both larvae species and presenting appropriate enzymatic activity. The immunochemical ability of recombinant GAD65 obtained from both larvae to compete with [35S]GAD65 was assessed qualitatively by incubating GADA-positive patients’ sera in the presence of 1 μM of the recombinant enzyme. All sera tested became virtually negative after incubation with antigen excess. Besides, radiometric quantitative competition assays with GADA-positive patients’ sera were performed by adding recombinant GAD65 (0.62 nM–1.4 µM). All dose response curves showed immunochemical identity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed using S. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specificity. GAD65 could be expressed in insect larvae, being S. frugiperda the best choice due to its high yield and purity. The development of a cost effective immunoassay for the detection of GADA was also afforded

Characterization of insulin antibodies by Surface Plasmon Resonance in two clinical cases: brittle diabetes and insulin autoimmune syndrome.

In this study, the characterization of insulin (auto)antibodies has been described, mainly in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by Surface Plasmon Resonance (SPR) in two particular clinical cases of individuals with severe episodes of impaired glycemia. Subject 1 suffers from brittle diabetes associated with circulating insulin antibodies (IA) due to insulin treatment. Subject 2 has insulin autoantibodies (IAA) associated with hypoglycemia in spite of not being diabetic and not having ever received exogenous insulin therapy. After conventional screening for IA/IAA by radioligand binding assay (RBA), we further characterized IA/IAA in sera of both patients in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by means of SPR technology. In both cases, q values were higher and Ka values were lower than those obtained in type 1 diabetic patients, suggesting that IA/IAA:insulin immunocomplexes could be responsible for the uncontrolled glycemia. Moreover, subject 1 had a predominat IgG1 response and subject 2 had an IgG3 response. In conclusion, SPR technology is useful for the complete characterization of IA/IAA which can be used in special cases where the simple positive/negative determination is not enough to achieve a detailed description of the disease fisiopathology

Calibration curve.

<p>(A) Sensorgram and (B) Standard curve of resonance units (RU) vs. antibody concentration as determined with an anti-human-PI polyclonal serum. The concentration values of polyclonal antibody used in the standard curve were: 75 nM, 37.50 nM, 18.75 nM, 9.38 nM, 6.69 nM, 2.34 nM, 1.17 nM and 0.59 nM.</p

Dose-response curves obtained by using rabbit anti-human-PI serum and standard PI in a fluid phase displacement experiment incubated until reaching equilibrium.

<p>The parameter 1/K₀ was calculated by interpolation at (B/F)₀/2 in the upper graphic [B/F = f(log F)]. In the lower graphic representing B/F = f(log dose) the (B/F)₀/2 intercepts the abscisa axis at 1/K₀+q₀/2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033574#pone.0033574-Berzofsky2" target="_blank">[21]</a>.</p

Correlation between K_a obtained using TrxPI or PI in SPR assays.

<p>K_a results were achieved by SPR using either TrxPI or PI as the immobilized antigen on sensor chip. The regression slope was 0.86±0.07 and the correlation coefficient (r²) was 0.80.</p

Representative sensorgrams.

<p>Panel (A): sera from a childhood-onset diabetic patient and panel (B): sera from an adult-onset diabetic patient.</p

RBA and SPR results.

<p>(A) SD scores, (B) concentration (q) and (C) affinity constant (K_a) obtained from 28 newly diagnosed type 1 diabetic patients and 23 adult-onset diabetic patients. All the parameters differed significantly between both groups of patients (p<0.05).</p