Reverse Phase Proteomic Array Profiling of Asparagine Synthetase Expression in Newly Diagnosed Acute Myeloid Leukemia.

Asparaginase-based therapy is a cornerstone in acute lymphoblastic leukemia (ALL) treatment, capitalizing on the methylation status of the asparagine synthetase (ASNS) gene, which renders ALL cells reliant on extracellular asparagine. Contrastingly, ASNS expression in acute myeloid leukemia (AML) has not been thoroughly investigated, despite studies suggesting that AML with chromosome 7/7q deletions might have reduced ASNS levels. Here, we leverage reverse phase protein arrays to measure ASNS expression in 810 AML patients and assess its impact on outcomes. We find that AML with inv(16) has the lowest overall ASNS expression. While AML with deletion 7/7q had ASNS levels slightly lower than those of AML without deletion 7/7q, this observation was not significant. Low ASNS expression correlated with improved overall survival (46 versus 54 weeks, respectively, p = 0.011), whereas higher ASNS levels were associated with better response to venetoclax-based therapy. Protein correlation analysis demonstrated association between ASNS and proteins involved in methylation and DNA repair. In conclusion, while ASNS expression was not lower in patients with deletion 7/7q as initially predicted, ASNS levels were highly variable across AML patients. Further studies are needed to assess whether patients with low ASNS expression are susceptible to asparaginase-based therapy due to their inability to augment compensatory ASNS expression upon asparagine depletion.

Alterations of mannosylated glycopatterns recognized by Hippeastrum hybrid lectin in saliva of patients with lung cancer.

OBJECTIVES: Lung cancer (LC) is the malignant tumor with the highest mortality rate worldwide, and precise early diagnosis can improve patient prognosis. The purpose of this study was to investigate whether alterations in the glycopatterns recognized by the Hippeastrum hybrid lectin (HHL) in salivary proteins are associated with the development of LC. MATERIALS AND METHODS: First, we collected saliva samples from LC (15 lung adenocarcinoma (ADC); 15 squamous cell carcinoma (SCC); 15 small cell lung cancer (SCLC)) and 15 benign pulmonary disease (BPD) for high-throughput detection of abundance levels of HHL-recognized glycopatterns using protein microarrays, and then validated the pooled samples from each group with lectin blotting analysis. Finally, the N-glycan profiles of salivary glycoproteins isolated from the pooled samples using HHL-magnetic particle conjugates were characterized separately using MALDI-TOF/TOF-MS. RESULTS: The results showed that the abundance level of glycopatterns recognized by HHL in salivary proteins was elevated in LC compared to BPD. The proportion of mannosylated N-glycans was notably higher in ADC (31.7%), SCC (39.0%), and SCLC (46.6%) compared to BPD (23.3%). CONCLUSIONS: The altered salivary glycopatterns such as oligomannose, Manα1-3Man, or Manα1-6Man N-glycans recognized by HHL might serve as potential biomarkers for the diagnosis of LC patients. CLINICAL RELEVANCE: This study provides crucial information for studying changes in salivary to differentiate between BPD and LC and facilitate the discovery of biomarkers for LC diagnosis based on precise alterations of mannosylated N-glycans in saliva.

Label-free OIRD detection of protein microarrays on high dielectric constant substrate with enhanced intrinsic sensitivity.

Oblique-incidence reflectivity difference (OIRD) is a dielectric constant-sensitive technique and exhibits intriguing applications in label-free and high-throughput detection of protein microarrays. With the outstanding advantage of being compatible with arbitrary substrates, however, the effect of the substrate, particularly its dielectric constant on the OIRD sensitivity has not been fully disclosed. In this paper, for the first time we investigated the dependence of OIRD sensitivity on the dielectric constant of the substrate under top-incident OIRD configuration by combining theoretical modeling and experimental evaluation. Optical modeling suggested that the higher dielectric constant substrate exhibits a higher intrinsic sensitivity. Experimentally, three substrates including glass, fluorine-doped tin oxide (FTO) and silicon (Si) with different dielectric constants were selected as microarray substrates and their detection performances were evaluated. In good agreement with the modeling, high dielectric constant Si-based microarray exhibited the highest sensitivity among three chips, reaching a detection limit of as low as 5 ng mL-1 with streptavidin as the model target. Quantification of captured targets on three chips with on-chip enzyme-linked immunosorbent assay (ELISA) further confirmed that the enhanced performance originates from the high dielectric constant enhanced intrinsic OIRD sensitivity. This work thus provides a new way to OIRD-based label-free microarrays with improved sensitivity.

Changes in serum immunoglobulin G glycosylation patterns for antiphospholipid syndrome patients with lectin microarray.

Antiphospholipid syndrome is a rare autoimmune disease characterized by persistent antiphospholipid antibodies. Immunoglobulin G plays a vital role in disease progression, with its structure and function affected by glycosylation. We aimed to investigate the changes in the serum immunoglobulin G glycosylation pattern in antiphospholipid syndrome patients. We applied lectin microarray on samples from 178 antiphospholipid syndrome patients, 135 disease controls (including Takayasu arteritis, rheumatoid arthritis and cardiovascular disease) and 100 healthy controls. Lectin blots were performed for validation of significant differences. Here, we show an increased immunoglobulin G-binding level of soybean agglutinin (p = 0.047, preferring N-acetylgalactosamine) in antiphospholipid syndrome patients compared with healthy and disease controls. Additionally, the immunoglobulin G from antiphospholipid syndrome patients diagnosed with pregnancy events had lower levels of fucosylation (p = 0.001, recognized by Lotus tetragonolobus) and sialylation (p = 0.030, recognized by Sambucus nigra I) than those with simple thrombotic events. These results suggest the unique serum immunoglobulin G glycosylation profile of antiphospholipid syndrome patients, which may inform future studies to design biomarkers for more accurate diagnosis of antiphospholipid syndrome and even for the prediction of clinical symptoms in patients.

Quantitative Detection of Multiple Cardiovascular Biomarkers by an Antibody Microarray-Based Metal-Enhanced Fluorescence Assay.

Accurate detection of multiple cardiovascular biomarkers is crucial for the timely screening of acute coronary syndrome (ACS) and differential diagnosis from acute aortic syndrome (AAS). Herein, an antibody microarray-based metal-enhanced fluorescence assay (AMMEFA) has been developed to quantitatively detect 7 cardiovascular biomarkers through the formation of a sandwich immunoassay on the poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate)-decorated GNR-modified slide (GNR@P(GMA-HEMA) slide). The AMMEFA exhibits high specificity and sensitivity, the linear ranges span 5 orders of magnitude, and the limits of detection (LODs) of cardiac troponin I (cTnI), heart-type fatty acid binding protein (H-FABP), C-reactive protein (CRP), copeptin, myoglobin, D-Dimer, and N-terminal pro-brain natriuretic peptide (NT-proBNP) reach 0.07, 0.2, 65.7, 0.6, 0.2, 8.3, and 0.3 pg mL-1, respectively. To demonstrate its practicability, the AMMEFA has been applied to quantitatively analyze 7 cardiovascular biomarkers in 140 clinical plasma samples. In addition, the expression levels of cardiovascular biomarkers were analyzed by the least absolute shrinkage and selector operator (LASSO) regression, and the area under receiver operator characteristic curves (AUCs) of healthy donors (HDs), ACS patients, and AAS patients are 0.99, 0.98, and 0.97, respectively.

Definition of the binding specificity of the T7 bacteriophage primase by analysis of a protein binding microarray using a thermodynamic model.

Protein binding microarrays (PBM), SELEX, RNAcompete and chromatin-immunoprecipitation have been intensively used to determine the specificity of nucleic acid binding proteins. While the specificity of proteins with pronounced sequence specificity is straightforward, the determination of the sequence specificity of proteins of modest sequence specificity is more difficult. In this work, an explorative data analysis workflow for nucleic acid binding data was developed that can be used by scientists that want to analyse their binding data. The workflow is based on a regressor realized in scikit-learn, the major machine learning module for the scripting language Python. The regressor is built on a thermodynamic model of nucleic acid binding and describes the sequence specificity with base- and position-specific energies. The regressor was used to determine the binding specificity of the T7 primase. For this, we reanalysed the binding data of the T7 primase obtained with a custom PBM. The binding specificity of the T7 primase agrees with the priming specificity (5'-GTC) and the template (5'-GGGTC) for the preferentially synthesized tetraribonucleotide primer (5'-pppACCC) but is more relaxed. The dominant contribution of two positions in the motif can be explained by the involvement of the initiating and elongating nucleotides for template binding.

Revisiting the antigen markers of vector-borne parasitic diseases identified by immunomics: identification and application to disease control.

INTRODUCTION: Protein microarray is a promising immunomic approach for identifying biomarkers. Based on our previous study that reviewed parasite antigens and recent parasitic omics research, this article expands to include information on vector-borne parasitic diseases (VBPDs), namely, malaria, schistosomiasis, leishmaniasis, babesiosis, trypanosomiasis, lymphatic filariasis, and onchocerciasis. AREAS COVERED: We revisit and systematically summarize antigen markers of vector-borne parasites identified by the immunomic approach and discuss the latest advances in identifying antigens for the rational development of diagnostics and vaccines. The applications and challenges of this approach for VBPD control are also discussed. EXPERT OPINION: The immunomic approach has enabled the identification and/or validation of antigen markers for vaccine development, diagnosis, disease surveillance, and treatment. However, this approach presents several challenges, including limited sample size, variability in antigen expression, false-positive results, complexity of omics data, validation and reproducibility, and heterogeneity of diseases. In addition, antigen involvement in host immune evasion and antigen sensitivity/specificity are major issues in its application. Despite these limitations, this approach remains promising for controlling VBPD. Advances in technology and data analysis methods should continue to improve candidate antigen identification, as well as the use of a multiantigen approach in diagnostic and vaccine development for VBPD control.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase.

Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.

Antibody array-based proteome approach reveals proteins involved in grape seed development.

Grape (Vitis vinifera) is one of the most widely cultivated fruits globally, primarily used for processing and fresh consumption. Seedless grapes are favored by consumers for their convenience, making the study of seedlessness a subject of great interest to scientists. To identify regulators involved in this process in grape, a monoclonal antibody (mAb)-array-based proteomics approach, which contains 21,120 mAbs, was employed for screening proteins/antigens differentially accumulated in grape during development. Differences in antigen signals were detected between seeded and seedless grapes revealing the differential accumulation of 2,587 proteins. After immunoblotting validation, 71 antigens were further immunoprecipitated and identified by mass spectrometry (MS). An in planta protein-protein interaction (PPI) network of those differentially accumulated proteins was established using mAb antibody by immunoprecipitation (IP)-MS, which reveals the alteration of pathways related to carbon metabolism and glycolysis. To validate our result, a seedless-related protein, DUF642 domain-containing protein (VvDUF642), which is functionally uncharacterized in grapes, was ectopically overexpressed in tomato (Solanum lycopersicum "MicroTom") and led to a reduction in seed production. PPI network indicated that VvDUF642 interacts with pectin acetylesterase (VvPAE) in grapes, which was validated by BiFC and Co-IP. As anticipated, overexpression of VvPAE substantially reduced seed production in tomato. Moreover, S. lycopersicum colourless non-ripening expression was altered in VvDUF642- and VvPAE-overexpressing plants. Taken together, we provided a high-throughput method for the identification of proteins involved in the seed formation process. Among those, VvDUF642 and VvPAE are potential targets for breeding seedless grapes and other important fruits in the future.

Harnessing the potential of reverse-phase protein array technology: Advancing precision oncology strategies.

The last few decades have seen remarkable strides in the field of cancer therapy. Precision oncology coupled with comprehensive genomic profiling has become routine clinical practice for solid tumors, the advent of immune checkpoint inhibitors has transformed the landscape of oncology treatment, and the number of cancer drug approvals has continued to increase. Nevertheless, the application of genomics-driven precision oncology has thus far benefited only 10%-20% of cancer patients, leaving the majority without matched treatment options. This limitation underscores the need to explore alternative avenues with regard to selecting patients for targeted therapies. In contrast with genomics-based approaches, proteomics-based strategies offer a more precise understanding of the intricate biological processes driving cancer pathogenesis. This perspective underscores the importance of integrating complementary proteomic analyses into the next phase of precision oncology to establish robust biomarker-drug associations and surmount challenges related to drug resistance. One promising technology in this regard is the reverse-phase protein array (RPPA), which excels in quantitatively detecting protein modifications, even with limited amounts of sample. Its cost-effectiveness and rapid turnaround time further bolster its appeal for application in clinical settings. Here, we review the current status of genomics-driven precision oncology, as well as its limitations, with an emphasis on drug resistance. Subsequently, we explore the application of RPPA technology as a catalyst for advancing precision oncology. Through illustrative examples drawn from clinical trials, we demonstrate its utility for unraveling the molecular mechanisms underlying drug responses and resistance.

Functional proteomics based on protein microarray technology for biomedical research.

This chapter traces a route through Proteomics from its origins to the present day. The different proteomics applications are discussed with a focus on microarray technology. Analytical microarrays, functional microarrays and reverse phase microarrays and their different applications are discussed. Several studies are mentioned where the great versatility of this approach is shown. Finally, the advantages and future challenges of microarray technology are outlined.

Autoantibody Profiling Using Human Autoantigen Protein Array and AlphaScreen.

Autoantibodies that recognize self-antigens are believed to have a close relationship with diseases such as autoimmune diseases, cancer, and lifestyle diseases. Analysis of autoantibodies is essential for investigating pathology mechanisms, diagnosis, and therapeutics of these diseases. We developed an autoantibody profiling assay using a cell-free synthesized protein array and high-throughput screening technology. Our assay system can sensitively detect interaction between recombinant antigen protein and autoantibody and efficiently analyze autoantibody profiling in patients' sera.

Direct competitive assay for HER2 detection in human plasma using Bloch surface wave-based biosensors.

The overexpression and/or amplification of the HER2/neu oncogene has been proposed as a prognostic marker in breast cancer. The detection of the related peptide HER2 remains a grand challenge in cancer diagnosis and for therapeutic decision-making. Here, we used a biosensing device based on Bloch Surface Waves excited on a one-dimensional photonic crystal (1DPC) as valid alternative to standard techniques. The 1DPC was optimized to operate in the visible spectrum and the biosensor optics has been designed to combine label-free and fluorescence operation modes. This feature enables a real-time monitoring of a direct competitive assay using detection mAbs conjugated with quantum dots for an accurate discrimination in fluorescence mode between HER2-positive/negative human plasma samples. Such a competitive assay was implemented using patterned alternating areas where HER2-Fc chimera and reference molecules were bio-conjugated and monitored in a multiplexed way. By combining Label-Free and fluorescence detection analysis, we were able to tune the parameters of the assay and provide an HER2 detection in human plasma in less than 20 min, allowing for a cost-effective assay and rapid turnaround time. The proposed approach offers a promising technique capable of performing combined label-free and fluorescence detection for both diagnosis and therapeutic monitoring of diseases.

Antibody Profiling of Dengue Severities Using Flavivirus Protein Microarrays.

Dengue is a viral disease transmitted by Aedes aegypti mosquitoes. According to the World Health Organization, about half of the world's population is at risk of dengue. There are four serotypes of the dengue virus. After infection with one serotype, it will be immune to such a serotype. However, subsequent infection with other serotypes will increase the risk of severe outcomes, e.g., dengue hemorrhagic fever, dengue shock syndrome, and even death. Since severe dengue is challenging to predict and lacks molecular markers, we aim to build a multiplexed Flavivirus protein microarray (Flaviarray) that includes all of the common Flaviviruses to profile the humoral immunity and cross-reactivity in the dengue patients with different outcomes. The Flaviarrays we fabricated contained 17 Flavivirus antigens with high reproducibility (R-square = 0.96) and low detection limits (172-214 pg). We collected serums from healthy subjects (n = 36) and dengue patients within 7 days after symptom onset (mild dengue (n = 21), hospitalized nonsevere dengue (n = 29), and severe dengue (n = 36)). After profiling the serum antibodies using Flaviarrays, we found that patients with severe dengue showed higher IgG levels against multiple Flavivirus antigens. With logistic regression, we found groups of markers with high performance in distinguishing dengue patients from healthy controls as well as hospitalized from mild cases (AUC > 0.9). We further reported some single markers that were suitable to separate dengue patients from healthy controls (AUC > 0.9) and hospitalized from mild outcomes (AUC > 0.8). Together, Flaviarray is a valuable tool to profile antibody specificities, uncover novel markers for decision-making, and shed some light on early preventions and treatments.

Role of Serum Inflammatory Cytokines in Sepsis Rats Following BMSCs Transplantation: Protein Microarray Analysis.

Bone marrow stromal cells (BMSCs) have emerged as a potential therapy for sepsis, yet the underlying mechanisms remain unclear. In this study, we investigated the effects of BMSCs on serum inflammatory cytokines in a rat model of lipopolysaccharide (LPS)-induced sepsis. Sepsis was induced by intravenous injection of LPS, followed by transplantation of BMSCs. We monitored survival rates for 72 h and evaluated organ functions, histopathological changes, and cytokines expression. Sepsis rats showed decreased levels of white blood cells, platelets, lymphocyte ratio, and oxygen partial pressure, along with increased levels of neutrophil ratio, carbon dioxide partial pressure, lactic acid, alanine aminotransferase, and aspartate aminotransferase. Histologically, lung, intestine, and liver tissues exhibited congestion, edema, and infiltration of inflammatory cells. However, after BMSCs treatment, there was improvement in organ functions, histopathological injuries, and survival rates. Protein microarray analysis revealed significant changes in the expression of 12 out of 34 inflammatory cytokines. These findings were confirmed by enzyme-linked immunosorbent assay. Pro-inflammatory factors, such as interleukin-1ß (IL-1ß), IL-1α, tumor necrosis factor-α (TNF-α), tissue inhibitor of metal protease 1 (TIMP-1), matrix metalloproteinase 8 (MMP-8), Leptin, and L-selectin were upregulated in sepsis, whereas anti-inflammatory and growth factors, including IL-4, ß-nerve growth factor (ß-NGF), ciliary neurotrophic factor (CNTF), interferon γ (IFN-γ), and Activin A were downregulated. BMSCs transplantation led to a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory and growth factors. We summarized relevant molecular signaling pathways that resulted from cytokines in BMSCs for treating sepsis. Our results illustrated that BMSCs could promote tissue repair and improve organ functions and survival rates in sepsis through modulating cytokine networks.

Comprehensive autoantibody profiling in systemic autoimmunity by a highly-sensitive multiplex protein array.

Comprehensive autoantibody evaluation is essential for the management of autoimmune disorders. However, conventional methods suffer from poor sensitivity, low throughput, or limited availability. Here, using a proteome-wide human cDNA library, we developed a novel multiplex protein assay (autoantibody array assay; A-Cube) covering 65 antigens of 43 autoantibodies that are associated with systemic sclerosis (SSc) and polymyositis/dermatomyositis (PM/DM). The performance of A-Cube was validated against immunoprecipitation and established enzyme-linked immunosorbent assay. Further, through an evaluation of serum samples from 357 SSc and 172 PM/DM patients, A-Cube meticulously illustrated a diverse autoantibody landscape in these diseases. The wide coverage and high sensitivity of A-Cube also allowed the overlap and correlation analysis between multiple autoantibodies. Lastly, reviewing the cases with distinct autoantibody profiles by A-Cube underscored the importance of thorough autoantibody detection. Together, these data highlighted the utility of A-Cube as well as the clinical relevance of autoantibody profiles in SSc and PM/DM.

Two Decades of Arrayed Imaging Reflectometry for Sensitive, High-Throughput Biosensing.

Arrayed imaging reflectometry (AIR), first introduced in 2004, is a thin-film interference sensor technique that optimizes optical properties (angle of incidence, polarization, substrate refractive index, and thickness) to create a condition of total destructive interference at the surface of a silicon substrate. The advantages of AIR are its sensitivity, dynamic range, multiplex capability, and high-throughput compatibility. AIR has been used for the detection of antibodies against coronaviruses, influenza viruses, Staphylococcus aureus, and human autoantigens. It has also shown utility in detection of cytokines, with sensitivity comparable to bead-based and ELISA assays. Not limited to antibodies or antigens, mixed aptamer and protein arrays as well as glycan arrays have been employed in AIR for differentiating influenza strains. Mixed arrays using direct and competitive inhibition assays have enabled simultaneous measurement of cytokines and small molecules. Finally, AIR has also been used to measure affinity constants, kinetic and at equilibrium. In this review, we give an overview of AIR biosensing technologies and present the latest AIR advances.

Profiling humoral responses to COVID-19 immunization in Kawasaki disease using SARS-CoV-2 variant protein microarrays.

Kawasaki disease (KD) is a form of acute systemic vasculitis syndrome that predominantly occurs in children under the age of 5 years. Its etiology has been postulated due to not only genetic factors but also the presence of foreign antigens or infectious agents. To evaluate possible associations between Kawasaki disease (KD) and COVID-19, we investigated humoral responses of KD patients against S-protein variants with SARS-CoV-2 variant protein microarrays. In this study, plasma from a cohort of KD (N = 90) and non-KD control (non-KD) (N = 69) subjects in categories of unvaccinated-uninfected (pre-pandemic), SARS-CoV-2 infected (10-100 days after infection), and 1-dose, 2-dose, and 3-dose BNT162b2 vaccinated (10-100 days after vaccination) was collected. The principal outcomes were non-KD-KD differences for each category in terms of anti-human/anti-His for binding antibodies and neutralizing percentage for surrogate neutralizing antibodies. Binding antibodies against spikes were lower in the KD subjects with 1-dose of BNT162b2, and mean differences were significant for the P.1 S-protein (non-KD-KD, 3401; 95% CI, 289.0 to 6512; P = 0.0252), B.1.617.2 S-protein (non-KD-KD, 4652; 95% CI, 215.8 to 9087; P = 0.0351) and B.1.617.3 S-protein (non-KD-KD, 4874; 95% CI, 31.41 to 9716; P = 0.0477). Neutralizing antibodies against spikes were higher in the KD subjects with 1-dose of BNT162b2, and mean percentage differences were significant for the 1-dose BNT162b2 B.1.617.3 S-protein (non-KD-KD, -22.89%; 95% CI, -45.08 to -0.6965; P = 0.0399), B.1.1.529 S-protein (non-KD-KD, -25.96%; 95% CI, -50.53 to -1.376; P = 0.0333), BA.2.12.1 S-protein (non-KD-KD, -27.83%; 95% CI, -52.55 to -3.115; P = 0.0195), BA.4 S-protein (non-KD-KD, -28.47%; 95% CI, -53.59 to -3.342; P = 0.0184), and BA.5 S-protein (non-KD-KD, -30.42%; 95% CI, -54.98 to -5.869; P = 0.0077). In conclusion, we have found that KD patients have a comparable immunization response to healthy individuals to SARS-CoV-2 infection and COVID-19 immunization.

Utility of Protein Microarrays for Detection of Classified and Novel Antibodies in Autoimmune Neurologic Disease.

BACKGROUND AND OBJECTIVES: Neural antibodies are detected by tissue-based indirect immunofluorescence assay (IFA) in Mayo Clinic's Neuroimmunology Laboratory practice, but the process of characterizing and validating novel antibodies is lengthy. We report our assessment of human protein arrays. METHODS: Assessment of arrays (81% human proteome coverage) was undertaken using diverse known positive samples (17 serum and 14 CSF). Samples from patients with novel neural antibodies were reflexed from IFA to arrays. Confirmatory assays were cell-based (CBA) or line blot. Epitope mapping was undertaken using phage display immunoprecipitation sequencing (PhiPSeq). RESULTS: Control positive samples known to be reactive with linear epitopes of intracellular antigens (e.g., ANNA-1 [anti-Hu]) were readily identified by arrays in 20 of 21 samples. By contrast, 10 positive controls known to be enriched with antibodies against cell surface protein conformational epitopes (e.g., GluN1 subunit of NMDA-R) were indistinguishable from background signal. Three antibodies, previously characterized by other investigators (but unclassified in our laboratory), were unmasked in 4 patients using arrays (July-December 2022): Neurexin-3α, 1 patient; regulator of gene protein signaling (RGS)8, 1 patient; and seizure-related homolog like 2 (SEZ6L2), 2 patients. All were accompanied by previously reported phenotypes (encephalitis, 1; cerebellar ataxia, 3). Patient 1 had subacute onset of seizures and encephalopathy. Neurexin-3α ranked high in CSF (second ranked neural protein) but low in serum (660th overall). Neurexin-3α CBA was positive in both samples. Patient 2 presented with rapidly progressive cerebellar ataxia. RGS8 ranked the highest neural protein in available CSF sample by array (third overall). RGS8-specific line blot was positive. Patients 3 and 4 had rapidly progressive cerebellar ataxia. SEZ6L2 was the highest ranked neural antigen by arrays in all samples (CSF, 1, serum, 2; Patient 3, ranked 9th overall in CSF, 11th in serum; Patient 4, 6th overall in serum]). By PhIPSeq, diverse neurexin-3α epitopes (including cell surface) were detected in CSF from patient 1, but no SEZ6L2 peptides were detected for serum or CSF samples from Patient 3. DISCUSSION: Individualized autoimmune neurologic diagnoses may be accelerated using protein arrays. They are optimal for detection of intracellular antigen-reactive antibodies, though certain cell surface-directed antibodies (neurexin-3α and SEZ6L2) may also be detected.