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Annals of Clinical & Laboratory Science 35:121-130 (2005)
© 2005 Association of Clinical Scientists

Methodologic Factors Affect the Measurement of Anti-basal Ganglia Antibodies

Christopher A. Rippel1, John J. Hong1, Dustin Y. Yoon1, Phillip N. Williams1 and Harvey S. Singer1,2
1 Departments of Neurology and 2 Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland

Address correspondence to Harvey S. Singer M.D., Division of Pediatric Neurology, Johns Hopkins Hospital, Jefferson Street Building 124, 600 N. Wolfe Street, Baltimore, MD 21287-1000, USA; tel 410 955 7212; fax 410 614 2297; e-mail:hsinger{at}jhmi.edu.


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 Acknowledgements
 References
 
An autoimmune etiology has been proposed for a variety of movement disorders, making the detection of autoantibodies a high investigative priority. Recognizing the existence of different methodologic approaches to identify these antibodies, we sought to investigate the effects of tissue preparation, antibody selection, and Western immunoblot detection methods on outcome. ELISA and immunoblotting studies were performed in healthy controls evaluating non-pathogenic autoantibodies. Our results indicate that enhanced data can be obtained by using fresh, rather than frozen, postmortem tissue homogenates for Western immunoblots and enzyme-linked immunosorbent assays and support the use of electrochemiluminescent detection for Western immunoblots. Molecular localization is significantly affected by the selected standard. Removal of lipids from homogenates does not affect anti-basal ganglia antibody (ABGA) results. Methodological variables should be taken into consideration when performing and interpreting neuroimmunological assays using sera or isolated IgG.

(received 12 January 2005; accepted 31 January 2005)

Keywords: autoantibodies, ELISA, immunological methods, Western immunoblots


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 Acknowledgements
 References
 
Numerous pediatric movement disorders have been hypothesized to have an autoimmune etiology. This list includes disorders such as Sydenham’s chorea (SC) [1], pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS) [2], dystonia [3], paroxysmal dyskinesias [4], and myoclonus [5]. In each of these entities, the underlying pathology hypothetically involves an immune-mediated mechanism with molecular mimicry. It is proposed that antibodies produced against group A ß-hemolytic streptococci (GABHS) cross-react with neuronal tissue in specific brain regions, which results in movement abnormalities. Experimental affirmation of autoimmunity in each of these clinical disorders, however, requires the identification of autoantibodies and the presence of immunoglobulins at the pathological site.

Antineuronal antibodies (ANAb), specifically anti-basal ganglia antibodies (ABGA), have been measured in children by a variety of techniques including immunofluorescent (IF) histochemistry, enzyme-linked immunosorbent assays (ELISA), and Western immunoblot analyses. Because immunofluorescent histochemical studies are by nature subjective and affected by the presence of autofluorescing lipofuscins in brain tissue, ELISA and Western immunoblotting techniques have been extensively used to assess ABGA in patients with PANDAS, SC, and Tourette syndrome (TS) and age and sex-matched controls. The results of these studies have been inconsistent. ELISA data in children diagnosed with PANDAS using an antigen substrate of delipidated (lipids removed) brain homogenate obtained from frozen human basal ganglia showed elevated levels of ANAb in affected patients compared to controls [6], whereas a second study using supernatant and pellet fractions from fresh human postmortem caudate and putamen (without lipids removed) showed no differences between patients and controls [7].

Additional discrepancies are also noted with Western immunoblotting techniques, which permit the detection of autoantibody activity against specific brain epitopes. In this instance, frozen basal ganglia tissue and a colorimetric (alkaline phosphatase) assay identified only a few bands in sera from controls, but significantly more bands in poststreptococcal patients with motor abnormalities [6]. The authors suggested that molecular weight bands at 60, 45, and 40 kDa are commonly detected in their patients with movement disorders but not in controls. In contrast, investigators using fresh post-mortem tissue and an electrochemiluminescence (ECL) detection system identified multiple bands in controls and subjects with SC and PANDAS [7,8].

The goal of this study is to provide an objective assessment of current methods for measuring ANAb, including ELISA and Western immunoblotting, in order to determine the preferred methodology. Although each method uses specific protocols, we hypothesized that final results could be influenced by the use of serum or IgG, condition of tissue (fresh or frozen), method of tissue preparation (with or without removal of lipids, ie, delipidation), and the method of Western immunoblotting detection (ECL or colorimetric detection). Since differences in results of studies using sera from disease-oriented cohorts could be affected by methodology, changes in clinical phenotype, or duration of the disease process, we have focused primarily on sera from healthy individuals. Autoantibodies against basal ganglia are present in the sera of healthy individuals, suggesting that they might not always be pathogenic [7–14]

The importance of methodological issues is heightened by an ever-expanding list of proposed poststreptococcal autoimmune disorders that require laboratory confirmation [3–5,15–17]. An additional motivation for this investigation is the recent expansion of ABGA studies from pathophysiologic characterization of disease to the suggestion that ANAb testing can be a potentially useful diagnostic tool in poststreptococcal movement disorders [6].


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 Acknowledgements
 References
 
Subjects.  Sera from 10 healthy adults (8 men, 2 women; mean age 27.0 yr, 9 with an age range of 22–35 yr and one age 63 yr) were used in this study. No individual had a personal or family history of movement disorders, SC, PANDAS, TS, ADHD, or obsessive-compulsive disorder. Informed consent was obtained from all participants in this study.

Laboratory investigation.  The presence of antibodies against human basal ganglia tissue was assessed by ELISA and Western immunoblotting (ECL and colorimetric methods). All ELISA and Western immunoblot assays were performed with the following 4 different tissue preparations: fresh, fresh-delipidated, frozen, and frozen-delipidated. ELISA was performed with use of 2 different antibodies (whole serum and isolated IgG) from each of the 10 control subjects. Western immunoblots were performed with 2 antibodies for each control subject; but also included 2 different detection methods (ECL and colorimetric). Laboratory personnel were unaware of the patients’ diagnoses, and statistical comparisons were not performed until all investigations were completed.

Tissue samples.  Tissues were obtained from the Johns Hopkins Hospital Neuropathology Division. Fresh caudate tissue used for ELISA and Western immunoblotting was obtained from a 76-yr-old man who died of a heart problem and had no evidence of neurological disease. The postmortem interval was unknown. Frozen caudate used for ELISA and Western immunoblotting was from a 72-yr-old man who died from unknown cause and had no evidence of neurological disease (postmortem interval, 20 hr).

Supernatant fraction.  Fresh and frozen human caudate (2.5 g of tissue/10 ml Tris) were homogenized in 50 mM Tris with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO) containing protease inhibitors (1 µg/ml of aprotinin, 10 µg/ml of leupeptin, 10 µg/ml of pepstatin, and 1 mM phenylmethylsulfonyl fluoride) in a Teflon-glass homogenizer on ice. Homogenized tissue was centrifuged at 12,000 x g for 30 min at 4°C with an Avanti J-30 I centrifuge (Beckman, Fullerton, CA). The supernatant fraction was collected and aliquots were stored at –80°C. Protein concentrations were measured by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL).

Delipidation of supernatant fractions.  Lipids were removed from the supernatant fractions obtained from fresh and frozen caudate supernatant preparations with the use of diisopropyl ether, according to previously described methods [18,19].

IgG isolation.  IgG was isolated from a 2-ml volume of each control serum with a Protein A Antibody Purification Kit (Sigma-Aldrich, St. Louis, MO). Purification resulted in a final IgG eluate of 5 ml. Concentrations of IgG in serum and IgG isolates were assayed in the Johns Hopkins Hospital Immunology Laboratory by rate nephelometry using Beckman/Coulter IMMAGE System.

Caudate antibody ELISA.  ELISA was performed on the 10 control samples (sera and IgG) with the 4 different homogenate samples. ELISA plate wells were coated with 150 µl of homogenate protein (2.5 µg/ml) in phosphate-buffered saline (PBS) and incubated at 4°C overnight. The plates were then washed for 3 cycles in PBS containing 0.05% Triton X-100 (PBS-T). Each well was blocked with 5% nonfat Carnation dry milk in PBS for 1.5 hr at room temperature. The plates were again washed for 3 cycles in PBS-T. Isolated IgG and whole serum for each of the 10 controls was added as the primary antibody. To determine a specific antibody signal, several positive and negative samples were titrated via serial dilutions to determine optical densities. Serum was diluted 1:250 in PBS; IgG solution was used as isolated from the Protein A Antibody Purification Kit. Tissue blank values were determined for each tissue type by substituting PBS in place of primary antibody. ELISA plate wells were incubated with 100 µl of primary antibody for 1.5 hr at room temperature with agitation. The plates were again washed for 3 cycles in PBS-T. Secondary antibody (sheep anti-human IgG conjugated with horseradish peroxidase, Amersham Biosciences, Arlington Heights, IL) diluted 1:3000 in PBS was added to each well and incubated for 1 hr at room temperature with agitation. The plates were again washed for 3 cycles in PBS-T. Antibody-antigen interaction was assessed by reaction with 100 µl of tetramethylbenzidine (TMB) substrate solution (Vector Laboratories, Burlingame, CA) for 5 min. Reactions were stopped with 50 µl of 1 M H3PO4 per well. The ELISA optical densities were obtained immediately with an automated EMAX microplate reader (Molecular Devices Sunnyvale, CA) at 450 nm. All samples were assayed in triplicate.

Western immunoblotting.  Western immunoblotting was performed on the 10 control samples (sera and IgG) by using the 4 tissue homogenate samples and 2 different detection methods (ECL and colorimetric). We examined all variations of tissue type, primary antibody, and detection method for each of the 10 controls. A total of 30 µg of brain tissue protein per sample (calculated from concentration values obtained through BCA assays) with 0.5 µg of sodium dodecyl sulfate (Life Technologies, Grand Island, NY) and 0.5 µg of dithiothreitol (Fisher Scientific, Fair Lawn, NJ) were denatured at 100°C for 5 min, subjected to electrophoresis in 10% acrylamide ready-gels (Bio-Rad, Hercules, CA), and transferred to 0.45-µm nitrocellulose at 100 V (Schleicher & Schuell Protran, Dassel, Germany) for 80 min. The nitro-cellulose was then incubated overnight at 4°C in blocking solution (5% Carnation nonfat milk dissolved in Tris-buffered saline containing 0.1% Tween 20) with agitation. The nitrocellulose was then washed 3 times in TBS-T for 10 min per wash and exposed to primary antibody diluted with 1% milk in TBS-T for 90 min at room temperature with agitation. Primary antibodies were sera and IgG isolated from controls. To obtain optimal sera signal, immunoblotting images were compared using sera diluted at 1:250 and 1:300. Since preliminary ScanPack analyses showed no significant differences between these dilutions, the final assayed sera were diluted to 1:300; isolated IgG solutions were diluted 1:50. The nitrocellulose was then washed 3 times for 10 min per wash in TBS-T.

For band detection via the ECL method, blots were exposed to secondary antibody, horseradish peroxidase-conjugated sheep anti-human IgG (Amersham Biosciences) diluted 1:3000 with 1% milk in TBS-T, and agitated for 60 min. After washing 3 times for 10 min per wash with TBS-T, the membranes were developed with ECL reagents (Amersham Biosciences) according to the vendor’s protocol. The blots were exposed to Denville Blue Bio Films (Denville Scientific, Metuchen, NJ) for 30 sec. Films were digitally scanned for analysis with ScanPack software as described below.

For band detection by the colorimetric method, blots were exposed to secondary antibody, anti-human IgG alkaline phosphatase conjugate (Promega, Madison, WI), diluted 1:3000 with 1% milk in TBS-T, and agitated for 60 min. After washing 3 times for 10 min per wash with TBS-T, the membranes were developed in 30 ml of Western Blue alkaline phosphatase substrate (Promega) for 3 min. Blots were washed in distilled water and images were captured using a digital scanner and evaluated with ScanPacK software as described below.

To investigate potential discrepant positions of molecular weight bands among different standards, 3 pre-stained SDS-PAGE standards (Bio-Rad Broad Range #161-0318, Bio-Rad Precision Plus Protein #161-0374, and Invitrogen #LC5925) were compared to determine the location of the molecular weights of each band. The 3 standards were electrophoresed on the same gel, transferred, and digitized and analyzed by the hardware and software as described in the protocol. ScanPack assigned molecular weights to the bands, measured at their center, based on absolute markers specific to the particular standard and extrapolated the locations of 25, 36, 50, 75, and 100 kDa bands.

Western immunoblotting data acquisition.  Western blot data were acquired by digitization of blots using a color flat-bed transparent scanner (Microtek ScanMaker 4, Carson, CA). Digital images were analyzed with ScanPack software (Biometra, Gottingen, Germany), which created densitometric data of the blots showing the gray-intensity values (8-bit gray values) vs Rf values for each band. ScanPack evaluated densitographic data files to assign a relative peak area (a larger peak area corresponds to a band of higher density) and molecular weight for each band in the blots. For simplicity in data presentation, bands within a range of ±1 kDa of a given molecular weight were counted at that molecular weight. For example, bands from 59 to 61 kDa were considered to be 60 kDa. For each possible experimental combination, the number of peaks observed and the total area of peaks were recorded.

ELISA and Western immunoblot statistical analysis.  Specific ELISA optical density readings (total reading minus tissue blank) were determined for each sample prior to statistical analysis. Because the sera and IgG preparations contained different IgG levels, and concentrations were not determined until after studies were performed, only post-hoc comparisons of primary antibodies were deemed appropriate for ELISA data. ELISA-specific optical density data were analyzed by a repeated measures ANOVA test for significant differences among the 4 different tissue preparations for both sera and IgG.

Western blot data (peak number and total peak area) were evaluated to determine the acceptability of using a parametric model. Western blot peak number and total peak area data were then analyzed by using the generalized estimating equation (repeated measures analysis) model [20–22] to test for statistically significant differences among the 4 different tissue preparations, between the 2 primary antibody types, and between the 2 detection methods. Antibody type, tissue preparation method, and detection method were treated as within-subjects factors. The selected method of analysis utilizes pooled comparisons to allow valid comparison of 2 methodological variables to each other by correcting for variations due to other methodological variables independent of that comparison. Since the choice of primary antibody (serum or IgG) did not affect Western immunoblots (see Results), pooled comparisons were used for appropriate data presentation and analysis. Thus, for example, data across all 4 tissue types (fresh, fresh-delipidated, frozen, frozen-delipidated) and across both antibody types can be analyzed for independent comparisons of ECL to colorimetric detection. Recognizing the possibility of an outlier effect due to age, results from the 63 yr-old control were included only after determining that all of his values fell within ±1 SD of the mean.


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 Acknowledgements
 References
 
ELISA assays.  Specific optical densities (total optical density minus optical density of tissue blank, mean ± SD) for the 10 controls assayed using the 4 caudate tissue preparations (fresh, fresh-delipidated, frozen, and frozen-delipidated) and 2 primary antibody sources (sera and IgG) are presented in Table 1Go. IgG ELISA optical densities were consistently higher than those for serum. The post-hoc analysis of IgG concentrations in sera and IgG isolates revealed that, on average, IgG isolate contained 3.6 times more IgG antibody than did the whole sera (IgG isolate 11900 µg/ml; whole sera 3350 µg/ml).


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  		Table 1. Specific (total minus blank) ELISA optical densities (mean ± SD) obtained with 4 caudate tissue preparations (fresh, delipidated fresh, frozen, delipidated frozen), and 2 antibody types (serum and IgG).
 
Results of comparisons across tissue type are shown for both serum and IgG primary antibodies in Table 2Go. Observed trends in the comparison of fresh and frozen preparations differed, depending on the primary antibody used. ELISA with serum showed consistently higher optical densities for all fresh homogenates compared to all frozen homogenates. In contrast, ELISA with IgG revealed consistently higher optical densities for all frozen homogenates compared to all fresh homogenates. Removal of lipids had no effect on ELISA optical densities, with the exception of serum against frozen tissue (delipidated frozen tissue was 0.062 optical density units lower than frozen, p = 0.007).


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  		Table 2. Specific ELISA optical density (total minus blank) comparisons among 4 caudate tissue preparations with serum primary antibodies; and among 4 tissue preparations with IgG primary antibodies.
 
Western immunoblotting assays.  Western immunoblots of 3 molecular weight standards are shown in Fig. 1Go. Extrapolated data from ScanPack-derived standard curves show that molecular weight bands varied markedly as determined by each of the 3 standards, with greater discrepancy at higher molecular weights. Molecular weight values presented in the results were based on the Bio-Rad Precision Plus Protein #161-0374 standard.



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  		Fig. 1. Western immunoblots of SDS-Page standards from Bio-Rad Broad Range #161-0318, Bio-Rad Precision Plus Protein #161-0374, and Invitrogen #LC5925. Locations of specific molecular weights determined from ScanPack derived standards are indicated. All numbers are kDa.

 
Fig. 2Go shows immunoblots obtained in the presence of primary antibodies (sera and IgG) from a single control subject against the tissue preparations by the 2 different detection methods (ECL and colorimetric). Similar patterns of bands were identified in all 10 control subjects. Visual inspection of Western immunoblots reveals no apparent differences between delipidated and non-delipidated homogenate samples, regardless of primary antibody or visualization technique. Multiple bands were identified with all tissue preparations and detection methods. Bands at 53 and 23 kDa were visualized using only secondary antibody (ie, blanks, no sera or IgG) suggesting they represent binding to IgG contained within the brain preparation. Blots were analyzed for the presence of bands at several specific molecular weights in the 10 control sera (Table 3Go). With the ECL detection method, bands were identified against fresh caudate and delipidated fresh caudate in 80% at 60 kDa, 80% at 40 kDa, and 100% at 35 kDa, whereas with frozen and delipidated frozen caudate bands occurred in 70, 25, and 40%, respectively. In general, similar results were obtained with the use of sera by the colorimetric method and with the use of IgG as the primary antibody.



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  		Fig. 2. Representative Western immunoblots obtained using primary antibodies (serum and IgG) from a single control subject assayed against 4 caudate tissue preparations and 2 detection methods (ECL and colorimetric); Fr = fresh tissue; FrD = delipidated fresh tissue; Fz = frozen tissue; FzD = delipidated frozen tissue.

 

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  		Table 3. Number of control sera (n = 10) in which antigens were identified against specific molecular weight epitopes in caudate tissue preparations (fresh, fresh-delipidated, frozen, frozen-delipidated) by 2 different Western immunoblotting detection methods.
 
In order to characterize patterns of binding, a repeated measures analysis was performed to compare the number of bands (peaks on ScanPack) (Table 4Go) as well as the band density (total area under the peaks on ScanPack) (Table 5Go). Frozen tissue showed significantly fewer bands than did fresh tissue: fresh had 4 more bands than frozen, p <0.001; fresh had 4 more bands than delipidated frozen, p <0.001; delipidated-fresh had 4 more bands than frozen, p = 0.003; and delipidated fresh had 4 more bands than delipidated frozen, p = 0.003. The number of bands was not significantly affected by the choice of primary antibody, detection method, or removal of lipids.


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  		Table 4. Comparison of number of peaks (bands) on Western immunoblots for 2 antibodies, 4 different caudate tissue preparations, and 2 detection methods.
 

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  		Table 5. Comparison of Western blot peak areas (density units) for 2 antibodies, 4 different caudate tissue preparations, and 2 detection methods.
 
The results of the Western blot repeated measures analysis of peak area for the comparison of fresh to frozen tissue paralleled those for peak number. Thus, fresh tissue gave consistently higher values than frozen tissue: fresh 26,582 units higher than frozen, p <0.001; fresh 28,207 units higher than delipidated frozen, p <0.001; delipidated fresh 29,279 units higher than frozen, p = 0.002; and delipidated fresh 30,904 units higher than delipidated frozen, p = 0.002. Peak areas, in contrast to peak numbers, were significantly lower when assayed by the colorimetric method than by ECL detection; colorimetric blots were reduced by 37,672 units, p <0.001. Peak area values were not significantly affected either by the use of serum or IgG or by the removal of lipids.


   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
Acknowledgements
 References
 
The ability to analyze ANAb both quantitatively and qualitatively in a reproducible manner is an essential first step towards the ultimate goal of detecting autoimmune etiologies in neurological disorders. Three different well-accepted techniques (ELISA, indirect immunofluorescence, and Western immunoblotting) are available, but there are major discrepancies in results applying these protocols to clinical populations. Since studies in a disease cohort might be negatively affected by severity, duration, comorbidity, or therapy, we chose to perform our comparison studies in a healthy, non-medicated control population. Our approach was to identify and evaluate several methodologies used by researchers studying pediatric movement disorders and to apply these in the study of control sera.

Tissue selection and preparation.  Experimental variables pertaining to tissue selection in published studies include the source of tissue, age of tissue donor, postmortem interval for human tissue, and method of preparation and storage. Because of the difficulty in obtaining fresh postmortem tissue homogenates, it has been suggested that alternate tissue preparations (eg, frozen brain) be considered for use in ANAb studies [23]. Nevertheless, the present study’s findings indicate that fresh tissue is preferable. Western immunoblots against fresh tissue consistently showed increased binding activity in terms of both numbers of bands (peak number) and density of bands (peak area). ELISA optical density readings with sera were also higher when fresh rather than frozen tissue was used. Why the opposite trend was observed with IgG as the antibody source is unknown. In regard to delipidation of tissue, since anti-myelin antibodies have been identified in a variety of CNS diseases [24], it is possible that ABGA assay specificity might be improved by removal of lipids. Our results, however, suggest that removal of lipids from tissue samples prior to assays generally adds neither specificity nor sensitivity to Western immunoblot or ELISA. This study did not evaluate potential alternative sources of epitopes such as fetal neuronal tissue, neuroblastoma cell lines, or rodent striatum. Furthermore, since soluble extracts may exclude significant membrane antigens, it is possible that whole tissue or membrane preparations may be preferred over soluble protein extracts [25].

Western blot methods and interpretations.  Although ELISA has been suggested to offer specificity that is comparable to Western immunoblotting [18], immunoblots offer distinct advantages by identifying reactivity against brain epitopes of specific molecular weight and providing the ability to follow bands longitudinally. The 2 detection methods used in this study (colorimetric and ECL) yielded consistent findings in terms of the number and location of bands identified, but differed in the density of bands. Although both detection methods yielded acceptable results, the significant difference in band density between the 2 detection methods suggests that the ECL method may allow for more sensitive quantification. Reports have highlighted the association of pediatric movement disorders with epitopes at molecular weights, eg, 40, 45, and 60 kDa. Our findings, however, raise several potential concerns about assigning specific bands to disease cohorts. First, the molecular weight assigned to a band can be influenced by the accuracy of the purchased standard. Secondly, numerous bands, including those at 40 and 60 kDa, are identified in control sera. Lastly, secondary antibody can in and of itself identify non-specific IgG contained within tissue. An additional issue not addressed in this study is the best methodology to interpret Western immunoblot assays. The most common approach, as utilized in this report, is direct visual observation. In contrast, other investigators attempting to identify differences between control and disease cohorts have used a statistical discriminant analysis technique that calculates and compares mean binding patterns and, if they are discrepant, determines which molecular weights contribute to these differences [7,8,26,27]. Reports with this approach in patients with SC have indicated that epitopes other than 60, 45, and 40 kDa are significant contributors to differences between patients and controls [7,8].

Antibody selection.  Most published ANAb studies have used sera as the primary antibody source, although others have preferred isolated IgG antibodies [28]. Although our results demonstrate the suitability of IgG for ELISA studies, since the concentrations of IgG in sera and IgG isolates differed we are unable to provide a definitive conclusion as to which antibody source is preferable. Higher optical density readings in the IgG ELISA appear to be related to the greater amounts of IgG that were assayed, rather than an inherent benefit to using isolated antibody extracts. Since the secondary antibody (conjugated sheep anti-human IgG) reacts against the entire human antibody and not just gamma heavy or light chains, higher absorbance levels are unlikely to be influenced by competition under conditions of antibody excess. IF studies in proposed poststreptococcal conditions have, to date, used undiluted serum [1,29,30], diluted serum [18,19], or both diluted and undiluted serum [31] as the primary antibody. To our knowledge, no published studies have investigated the use of isolated IgG for IF studies.

Limitations of the study.  Potential shortcomings of this study include the selection of tissue from only a single region of the brain, the inability to evaluate fresh and frozen homogenates from the same postmortem individual, the relatively small number of subjects, and the use of sera from subjects without neuroimmunologic disorders. Additionally, the quantification of immunoglobulin concentrations in IgG isolates and whole sera prior to performing the assays would have permitted evaluations of antibody dilutions containing equal IgG concentrations. The authors also recognize that disease-specific ABGA may have characteristics that differ from those present in the healthy controls.

The goal of this study was not to criticize existing approaches or data, but rather to contribute constructively to the ongoing studies concerning ANAb disorders. We hope that the clarification of methodological variables will benefit researchers who are exploring the pathophysiological mechanisms of ANAb disorders. Since scientists are increasingly presented with neurological disorders of possible autoimmune etiology, we hope to make the medical community aware that variations in methodology can significantly affect the results.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 Acknowledgements
References
 
Supported in part by funding from NIH Grant R01 MH61940. We thank Richard Skolasky of the Johns Hopkins University Department of Neurology for statistical analysis.


   References
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 Acknowledgements
 References
 

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