Even though most thyroid subjects are undiagnosed due to nonspecific symptoms, universal screening for thyroid disease is not recommended for the general population. In this study, our motive is to showcase the early appearance of thyroid autoantibody, anti-TPO, prior to the onset of thyroid hormone disruption; hence the addition of anti-TPO in conjunction with traditional thyroid markers TSH and FT4 would aid to reduce the long-term morbidity and associated health concerns. Here, a total of 4581 subjects were tested multiple times for TSH, FT4, anti-TPO, and anti-Tg and followed up for 2 years. We streamlined our subjects into two groups, A1 (euthyroid at first visit, but converted to subclinical/overt hypothyroidism in follow-up visits) and A2 (euthyroid at first visit, but converted to hyperthyroidism in follow-up visits). According to our results, 73% of hypothyroid subjects (from group A1) and 68.6% of hyperthyroid subjects (from group A2) had anti-TPO 252 (±33) and 277 (±151) days prior to the onset of the thyroid dysfunction, respectively. Both subclinical/overt hypothyroidism and hyperthyroidism showed a significantly higher percentage of subjects who had anti-TPO prior to the onset of thyroid dysfunction compared to the combined control group. However, there was no significant difference in the subjects who had anti-Tg earlier than the control group. Further assessment showed that only anti-TPO could be used as a standalone marker but not anti-Tg. Our results showcase that anti-TPO appear prior to the onset of thyroid hormone dysfunction; hence testing anti-TPO in conjunction with TSH would greatly aid to identify potentially risk individuals and prevent long-term morbidity.

Background Antibody diagnostics play an important role in disease detection and can potentially aid in monitoring of the immune responses to see if an individual has developed immunity. Developing high throughput diagnostics which does not involve handling of infectious material becomes imperative in the case of pandemics such as the recent outbreak of SARS-CoV2. Methods A protein microarray technology was used to detect the plurality of antibody response to four novel antigens namely S1 glycoprotein, Receptor binding domain (RBD), S2 glycoprotein and Nucleoprotein of the novel coronavirus named SARS-CoV2 using serum samples. A DBS card was additionally used to compare its performance with a venipuncture-based serum separator tube (SST) draw. Results The three main subclasses of antibodies IgM, IgA and IgG were analyzed to see the variations in immune responses in the affected population and compared to their microbial RT-PCR based NP swab results. The clinical sensitivity and specificity were determined to be 99.67% and 99.77%. In the matrix comparison study, which would enable patients to test without risk of transmitting the virus, DBS (Dried Blood Spot) matched with higher than 98% accuracy to a venipuncture-based SST collection. Conclusion Multiplex testing enables higher sensitivity and specificity which is essential while establishing exposure on a population scale. This flexible platform along with a discrete collection methodology would be crucial and broadly useful to scale up testing in current and future pandemics. Minimum sample volume that can be collected using DBS cards can be processed in this multiplex pillar plate format enabling the capacity to provide the reliability of high throughput analyzers while having the ease of collection similar to rapid tests.

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BackgroundMost antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods.MethodsUsing a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes.ResultsUsing a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity.ConclusionsThese novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis.

Current serological immunoassays have inherent limitations for certain infectious diseases such as Lyme disease, a bacterial infection caused by Borrelia burgdorferi in North America. Here we report a novel method of manufacturing high-density multiplexed protein microarrays with the capacity to detect low levels of antibodies accurately from small blood volumes in a fully automated system. A panel of multiple serological markers for Lyme disease are measured using a protein microarray system, Lyme Immunochip, in a single step but interpreted adhering to the standard two-tiered testing algorithm (enzyme immunoassay followed by Western blot). Furthermore, an enhanced IgM assay was supplemented to improve the test’s detection sensitivity for early Lyme disease. With a training cohort (n = 40) and a blinded validation cohort (n = 90) acquired from CDC, the Lyme Immunochip identified a higher proportion of Lyme disease patients than the two-tiered testing (82.4% vs 70.6% in the training set, 66.7% vs 60.0% in the validation set, respectively). Additionally, the Immunochip improved sensitivity to 100% while having a lower specificity of 95.2% using a set of investigational antigens which are being further evaluated with a large cohort of blinded samples from the CDC and Columbia University. This universal microarray platform provides an unprecedented opportunity to resolve a broad range of issues with diagnostic tests, including multiplexing, workflow simplicity, and reduced turnaround time and cost.

Introduction. Individuals with one autoimmune disease are at risk of developing a second autoimmune disease, but the pathogenesis or the sequential occurrence of multiple autoimmune diseases has not been established yet. In this study, we explored the association and sequential occurrence of antibodies in thyroid disease and systemic autoimmune disease subjects. We evaluated thyroid hormones, thyroid-stimulating hormone (TSH), free thyroxine (FT4), thyroid autoantibodies, anti-thyroperoxidase (anti-TPO), and anti-thyroglobulin (Tg) to comprehend the association with systemic autoimmune autoantibodies, anti-nuclear antibodies (ANA), and autoantibodies to extractable nuclear antigens (ENA) in subjects with thyroid-related symptoms. Methods. A total of 14825 subjects with thyroid-related symptoms were tested at Vibrant America Clinical Laboratory for thyroid markers (TSH, FT4, anti-TPO, and anti-Tg) and an autoimmune panel (ANA panel and ENA-11 profile) from March 2016 to May 2018. Thyroid-positive (based on TSH and FT4 levels), anti-TPO-positive, and anti-Tg-positive subjects were assessed for the prevalence of ANA and anti-ENA antibodies. A 2-year follow-up study was conducted to assess the sequential order of appearance of autoimmune markers in thyroid and systemic autoimmune diseases. Results. In the retrospective analysis, 343/1671 (20.5%), 2037/11235 (18.1%), and 1658/9349 (17.7%) of thyroid+, anti-TPO+, and anti-Tg+ subjects were found to be seropositive for ANA. Anti-ENA was detected in a higher prevalence than ANA with 475/1671 (28.4%), 3063/11235 (27.3%), and 2511/9349 (26.9%) in the same groups of subjects, respectively. Our results are found to be much higher than the reported prevalence of anti-ENA in general population. During the 2-year follow-up study, anti-TPO appeared significantly earlier than ANA and anti-ENA in an average of 253 (±139) and 227 (±127) days, respectively. Conclusions. A high prevalence of anti-ENA and ANA was found to be coexisting with autoimmune thyroid disease subjects, with anti-TPO occurring prior to the onset of ANA and anti-ENA. Therefore, frequent follow-ups and evaluation of ANA and anti-ENA in subjects with anti-TPO positivity would be beneficial in early detection of other systemic autoimmune diseases.

Background: Antibody diagnostics play an important role in disease detection and can potentially aid in monitoring of the immune responses to see if an individual has developed immunity. Developing high throughput diagnostics which does not involve handling of infectious material becomes imperative in the case of pandemics such as the recent outbreak of SARS-CoV2. Methods:A protein microarray technology was used to detect the plurality of antibody response to four novel antigens namely S1 glycoprotein, Receptor binding domain (RBD), S2 glycoprotein and Nucleoprotein of the novel coronavirus named SARS-CoV2 using serum samples. A DBS card was additionally used to compare its performance with a venipuncture-based serum separator tube (SST) draw. Results:The three main subclasses of antibodies IgM, IgA and IgG were analyzed to see the variations in immune responses in the affected population and compared to their microbial RT-PCR based NP swab results. The clinical sensitivity and specificity were determined to be 98.1% and 98.6%. In the matrix comparison study, which would enable patients to test without risk of transmitting the virus, DBS matched with higher than 98% accuracy to a venipuncture-based SST collection. Conclusion:Multiplex testing enables higher sensitivity and specificity which is essential while establishing exposure on a population scale. This flexible platform along with a discrete collection methodology would be crucial and broadly useful to scale up testing in current and future pandemics.Minimum sample volume that can be collected using DBS cards can be processed in this multiplex pillar plate format enabling the capacity to provide the reliability of high throughput analyzers while having the ease of collection similar to rapid tests.

Micronutrients are involved in various vital cellular metabolic processes including thyroid hormone metabolism. This study aimed to investigate the correlation between serum levels of micronutrients and their effects on thyroid parameters. The correlation of serum levels of micronutrients and thyroid markers was studied in a group of 387 healthy individuals tested for thyroid markers (T4, T3, FT4, FT3, TSH, anti-TPO, RT3, and anti-Tg) and their micronutrient profile at Vibrant America Clinical Laboratory. The subjects were rationalized into three groups (deficient, normal, or excess levels of micronutrients), and the levels of their thyroid markers were compared. According to our results, deficiency of vitamin B2, B12, and B9 and Vit-D25[OH] ( p < 0.05 ) significantly affected thyroid functioning. Other elemental micronutrients such as calcium, copper, choline, iron, and zinc ( p < 0.05 ) have a significant correlation with serum levels of free T3. Amino acids asparagine (r = 0.1765, p < 0.001 ) and serine (r = 0.1186, p < 0.05 ) were found to have a strong positive correlation with TSH. Valine, leucine, and arginine ( p < 0.05 ) also exhibited a significant positive correlation with serum levels of T4 and FT4. No other significant correlations were observed with other micronutrients. Our study suggests strong evidence for the association of the levels of micronutrients with thyroid markers with a special note on the effect of serum levels of certain amino acids.