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 the 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 streptavidin-dichlorotriazinyl 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 those of 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 represents a cost-effective alternative to traditional GADA determination by RBA and/or ELISA, making it applicable to most medium-complexity laboratories.

Probing protein conformation with a minimal photochemical reagent.

3H-diazirine (3H-DZN), a photoreactive gas similar in size to water, was used to probe the topography of the surface and inner space of proteins. On photolysis 3H-DZN generates 3H-methylene carbene, which reacts unselectively with its molecular cage, inserting even into C-H bonds. Labeling of bovine alpha-lactalbumin (alpha-LA, MW: 14,200) with 1 mM (3)H-DZN yielded 0.0041 mol CH2/mol of protein, in agreement with the expectation for an unspecific surface-labeling phenomenon. The cooperative urea-induced unfolding of alpha-LA, as monitored by the extent of 3H-methylene labeling, agrees with that measured by circular dichroism spectroscopy in the far and near ultraviolet regions. At 8 M urea, the unfolded state U was labeled 25-30% more than the native state N primarily because of the increase in the accessible surface area (ASA) of the protein occurring upon unfolding. However, this result lies below the approximately 100% increment expected from theoretical estimates of ASA of state U. Among other factors, most likely the existence of a residual structure in U, that involves helices H2 and H4 of the alpha subdomain, might account for this fact, as shown by a comparative analysis of peptide labeling patterns of N and U samples. In this paper, we demonstrate the usefulness of the 3H-methylene labeling method to monitor conformational transitions and map solvent accessibility along the polypeptide sequence, thus opening the possibility of outlining structural features of nonnative states (i.e., denatured states, molten globule). We anticipate that this technique also would help to identify ligand binding and oligomerization sites in proteins.

Experimentally approaching the solvent-accessible surface area of a protein: insights into the acid molten globule of bovine alpha-lactalbumin.

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein alpha-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH(2)) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of alpha-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH(2) labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH(2) labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.

Assessing native and non-native conformational states of a protein by methylene carbene labeling: the case of Bacillus licheniformis beta-lactamase.

Much knowledge of protein folding can be derived from the examination of the nature and size of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemical modification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labeling of Bacillus licheniformis beta-lactamase (BL-betaL) with 1 mM 3H-diazirine yielded 8.3 x 10(-3) mol CH2/mol protein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfolded state U in 7 M urea was labeled 60% more than the native state N. This result lies well below the increment of ASA expected from theoretical estimates and points to the presence of residual organization in state U and/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I was demonstrated from two sequential transitions occurring at 1.5-3.0 M and 3.5-6.5 M urea. This technique shows a close correlation with optical probes most sensitive to changes in tertiary structure, a statement supported by the fact that the largest change occurs along the N-I portion of the N-I-U transition and along the acid pH-induced N-A transition. In the latter case, state A is labeled 70% more than state N, an increment consistent with the loosening of tight interactions in the core of the protein. Fragmentation of labeled BL-betaL into peptides provides a sequential map of solvent accessibility. Thus, amino acid residues pertaining to the Omega-loop and to helices alpha5 and alpha6 line the major cavity of the protein, that is big enough to lodge the diazirine reagent. Methylene labeling, by introducing an original (and perhaps unique) experimental measurement of ASA, enlightens subtle aspects of complex transitions and makes possible a comparative structural characterization of the native as well as non-native states.