Antibasal ganglia antibodies and PANDAS

We read with interest the article by Singer and colleagues1 in which they investigated the presence of antibasal ganglia antibodies (ABGA) in serum from 15 children with pediatric autoimmune neuropsychiatric disorders (PANDAS) and 15 age-matched, healthy controls. The authors used enzymelinked immunosorbent assay (ELISA) and Western immunoblotting (WB) methods to detect ABGA against supernatant, pellet, and synaptosomal preparations from human adult postmortem basal ganglia. On the WB analysis, they observed a difference between patients and controls only for the caudate supernatant fraction, with numerous determinants of different molecular weight contributing to the difference. The individual antigens showed low discriminant power on multivariate analysis of densitometric data of WB, therefore, failing to identify highly specific reactivities as discriminant for patients with poststreptococcal neuropsychiatric disorders. In addition, ELISA measurements did not differentiate between the two groups. In contrast with the authors’ findings, our group recently has reported2 a significant difference in ABGA detection, using both an ELISA and WB, between a cohort of 40 children presenting with movement disorders associated with streptococcal infection and a control cohort of 190 children, subdivided into different clinical groups (other neurological diseases, uncomplicated streptococcal infections, and autoimmune diseases). In our study, WB showed higher discriminant power, with a sensitivity of 93% and a specificity of 94.7% in detecting poststreptococcal movement disorders. Antibody reactivity was present to specific antigens with a defined molecular weight, rather than as a polyspecific response. The same specific antibody reactivity was reported previously by our group in different cohorts of patients with Sydenham’s chorea (SC)3 and Tourette syndrome.4 The same accuracy was found in the subgroup of patients who fulfilled the working criteria5 for PANDAS. ELISA was also discriminant between patients and controls, but with lower specificity and sensitivity than WB. We believe that important differences in the techniques used by Singer and colleagues1 and our group might account for the discrepancies in the results. First, during tissue extraction, Singer and coworkers collect and store the supernatant protein fraction directly after centrifugation of the homogenate at 12,000 g for 30 minutes. At this stage, the supernatant is likely to contain a high concentration of lipids and lipophilic proteins (mainly originating from myelin), mixed with other proteins, which would affect the purity of the protein extract. In our technique, we perform lipid extraction, using di-isopropyl ether, and then recentrifuge the homogenate at a lower speed, to minimize contamination with lipid components and increase the concentration of the protein fraction. The elimination of the lipidic component may significantly enhance the specificity of the assay, because antibodies against myelin components constitute a nonspecific finding in several diseases of the central nervous system.6 Second, while presenting their WB method, the authors do not explain how they prepared their proteins for electrophoresis separation. Specifically, there is no mention of the detergent used, whether they have heated the proteins, and whether they have pretreated them with a reducing agent, such as -mercapto-ethanol or dithiothreitol. These steps are important to adequately separate the proteins by molecular weight with polyacrylamide gel electrophoresis. Heating and chemical reduction dissolve the tertiary and secondary structure of the proteins, allowing a homogenous binding of the anionic detergent to the linear polypeptides.7 Third, Singer and colleagues separate proteins by gel electrophoresis using a constant acrylamide concentration (10%), whereas Church and associates use a 4 to 12% gradient acrylamide gel, possibly obtaining a different pattern of separation. Finally, the WB detection method used by the authors is enhanced chemiluminescence (ECL), whereas our group favors colorimetric detection. Although ECL is the most sensitive method of detection for various immunoblotting techniques, in our hands, it has a lower specificity, as it tends to produce a large number of responses in both patients and controls. This nonspecific binding would limit the overall discriminatory power of the assay. Colorimetric analysis is undoubtedly a less sensitive method, but it improves the specificity and reproducibility of the assay. In conclusion, we agree with Singer and colleagues that the evidence implicating autoimmunity in the pathophysiology of poststreptococcal neuropsychiatric disorders is inconclusive at present. Documenting cross-reactivity between specific brain autoantigens and streptococcal antigens is an important aim in this regard. We would, therefore, strongly advocate studies to compare assays with the primary aim of standardizing the techniques used to detect ABGA.