Nonspecific Antibody Research Articles

Prevalence of comorbid autoimmune diseases and antibodies in newly diagnosed multiple sclerosis patients

Abstract Background Diagnosing multiple sclerosis (MS) is challenging due to diverse symptoms and the absence of specific biomarkers. Concurrent autoimmune diseases (AID) or non-specific antibodies further complicate diagnosis, progression monitoring, and management. Data on AID prevalence in MS patients are sparse. This study aims to identify concurrent AIDs alongside MS. Methods In this retrospective single-center study, we analyzed patient records at our university hospital from 2010 to 2017, focusing on cases suspected of inflammatory demyelinating disease. The 2017 McDonald criteria were applied. Additionally, we measured neurofilament light (NfL) levels from available CSF samples in our biobank. Results We identified a total of 315 patients, of whom 66% were women. In total, 13.7% of all patients had concurrent AID, while 20.3% had isolated antibody findings without AID. The most common AID was autoimmune thyroiditis (8.9%), followed by chronic inflammatory skin diseases (1.6%), arthritis (1%), type 1 diabetes (1%), Sjögren’s syndrome (0.6%), and inflammatory bowel diseases (0.6%). Cardiolipin antibodies were the most frequent isolated antibody finding (8.6%). Our data showed that, from the perspective of the initial demyelinating event, neither comorbid AID nor isolated antibodies significantly influenced relapses or MS progression over a median follow-up of 9 months. Standard CSF parameters and NfL levels were similar between the groups at the time of MS diagnosis. Conclusion Our study shows that AIDs, particularly autoimmune thyroiditis, frequently occur at the onset of MS. The proportion of AIDs commonly treated with immunomodulatory therapy in our cohort was similar to that observed in the general population. Comorbid AID did not affect NfL levels, indicating similar disease activity. Future research should explore new AID emergence during the course of MS, especially considering the increased incidence of rheumatic diseases later in life.

Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP.

Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases.

Synthesis and evaluation of bifunctional DFO2K: a modular chelator with ideal properties for zirconium-89 chelation.

The synthesis and evaluation of the newest generation of our DFO2 chelator family-DFO2K-is described. DFO2K was designed with a simple synthetic route to access different bifunctional derivatives, with each derivative having similar metal ion coordination spheres and high denticity (up to 12 coordinate) to ensure stable coordination of zirconium-89. The high denticity could potentially enhance stability with other large oxophilic radiometals. Zirconium-89 is the most popular radionuclide to pair with large macromolecules such as antibodies (immunoPET) for positron emission tomography applications. Although clinically successful, the stability of the "gold standard" chelator desferrioxamine B (DFO) can be improved as significant bone uptake is observed in animal models, despite no obvious stability issues in humans. Following the synthesis of DFO2K we assessed its radiolabeling efficiency with zirconium-89 and compared with DFO, which revealed rapid and nearly identical radiolabeling kinetics to DFO. The resultant [89Zr]Zr-DFO2K complex showed improved stability over [89Zr]Zr-DFO in different in vitro stability assays such as hydroxyapatite and 1000-fold molar excess EDTA challenges. Furthermore, biodistribution studies of the non-bifunctional chelators in healthy mice showed that [89Zr]Zr-DFO2K had a similar distribution profile and clearance to [89Zr]Zr-DFO. The bifunctional derivative p-SCN-Ph-DFO2K was conjugated to a non-specific human IgG antibody and evaluated after 2 weeks circulating in healthy female CD1 mice. Mice administered [89Zr]Zr-DFO2K-IgG showed substantially lower bone uptake in PET-CT images than [89Zr]Zr-DFO-IgG, with PET ROI data and ex vivo biodistribution revealing a statistically significantly lower bone uptake for DFO2K. Overall, owing to its high denticity, ease of synthesis, improved solubility over DFO2 and DFO2p, and stable chelation of zirconium-89, DFO2K appears to be an improved alternative chelator to DFO for zirconium-89 chelation.

Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Techniques for improving the specificity of sandwich enzyme-linked immunosorbent assay-based drug screening.

ELISA assays are commonly used for drug screening by racing laboratories but are known to suffer from limited specificity. Inaccurate ELISA screening results are typically produced by non-specific antibody interactions or by the retention of chromogenic material in the sample well due to sample degradation. While confirmation of drug positives can be achieved by mass spectrometry, the follow-up of inaccurate ELISA screening results represents an unnecessary cost in staff time and reagents. This is particularly true in the case of rhEPO screening using sandwich ELISA assays, where the confirmation method requires up to 3days to perform. While most racing laboratories purchase commercial ELISA kits, these products can be customised to provide increased specificity for enhanced screening of positive samples. The specificity of commercial sandwich ELISA kits can be improved by a variety of mechanisms including the addition of competing analyte specific antibodies, substitution of capture antibodies or by performing ELISA analysis with and without capture antibodies. Non-specific signals in difficult matrices such as canine urine can also be reduced by the addition of BSA solutions to the ELISA plate prior to the addition of samples.

Rare coincidence: Macro-thyroid-stimulating hormone and multiple manufacturer-specific interferences in thyroid hormone immunoassays.

Immunoassays are widely used for laboratory assessment of endocrine functions including thyroid hormones. While usually adequate for patient evaluation, they are known to potentially suffer from interference from a variety of factors. We report the case of a 44year-old male patient without clinical symptoms of thyroid disease who presented for specialist evaluation after pathological thyroid function tests prompted a transferal by his primary care practitioner. Thyroid function tests showed discrepant results across immunoassays and platforms of different manufacturers. Polyethylene glycol precipitation prompted the diagnosis of macro-thyroid-stimulating hormone, while heterophilic and non-specific antibody blocking reagents proved ineffective in eliminating the interference in thyroid-stimulating hormone, free triiodothyronine and free thyroxine measurements. Further assessment ruled out a diagnosis of familial dysalbuminemic hyperthyroxinemia, leaving an exclusion diagnosis of manufacturer-specific interference in free triiodothyronine and free thyroxine assays due to unknown factors. Both clinicians and laboratory specialists must be aware of potential interference in immunoassays which otherwise might be misleading, potentially triggering unnecessary (invasive) follow-up procedures or therapeutic interventions. Close communication is required for successful troubleshooting. To our knowledge, no other case of both macro-thyroid-stimulating hormone and manufacturer-specific interference in a single patient has been documented thus far.

Stečena koagulopatija u trogodišnjegdječaka s makrohematurijom– prikaz bolesnika

Acquired coagulopathy is a rare, potentially life-threatening disorder which should be suspected if acute bleeding in patients with a negative previous hemorrhagic diathesis is encountered. This disease arises due to the formation of antibodies against clotting factors in a previously healthy child or adult, which specifically partially or completely neutralize the procoagulant activity of clotting factors or accelerate their removal from the circulating blood, reducing their plasma concentrations and increasing the bleeding tendency. The aforementioned disorder should be confirmed by determining global coagulation times, concentrations of individual clotting factors, and determining the presence of specific or non-specific antibodies. Timely treatment aimed at prompt stopping of bleeding and eradicating the resulting inhibitors will prevent the unfavorable outcome. It is recommended to continue monitoring patients in the outpatient setting with determination of PT and APTT for a year, because in 20% of cases a relapse of the disease occurs, although more often in adults. In this paper, we presented a three-year-old boy in whom we determined prolonged global coagulation times and the presence of non-specific inhibitors of the coagulation factors F II, F IX, F XI and F XII through the diagnostic workup of macrohematuria. The treatment with fresh frozen plasma and corticosteroids resulted in clinical improvement and disease remission. Acquired coagulopathy is a very rare disorder in childhood with scarce literature data. The aim of this paper is to demonstrate the possible causes, clinical course and treatment of this life-threatening disorder in children.

Exploring the possible link between the spike protein immunoglobulin G4 antibodies and cancer progression

Repeated inoculation with messenger RNA (mRNA) vaccines elicits immunoglobulin G4 (IgG4) antibody production. Such an increase in the concentration of specific and non-specific IgG4 antibodies allows the growth of some types of cancer by blocking the activation of effector immune cells. This work proposes the hypothesis that cancer growth may be indirectly promoted by increased concentrations of non-specific IgG4 antibodies by the following mechanisms: 1) IgG4 antibodies can bind to anti-tumor IgG1 antibodies and block their interaction with receptors located on effector cells, thus preventing the destruction of cancer cells, 2) IgG4 can interact with fragment crystallizable gamma receptor IIb (FcγRIIB) inhibitory receptors, thus reducing effector functions of innate immune cells, and 3) targeting of specific epitopes by IgG4 could be oncogenic by inducing the production of a microenvironment that can promote cancer development. This article reviews the supporting literature and suggests several experimental protocols to evaluate this hypothesis in the context of repeated inoculation with mRNA vaccines. Additionally, this work proposes some management options aimed at reducing the unfavorable molecular consequences that could mediate cancer development when encountering high concentrations of IgG4 antibodies.

Designing a Novel di-epitope Diphtheria Vaccine: A Rational Structural Immunoinformatics Approach.

The design of an epitope-based vaccine against diphtheria toxin (DT) originated from the idea that many strong binder epitopes may be structurally located in the depth of DT. Subsequently, many ineffective antibodies may be produced by the presentation of those epitopes to T and B lymphocytes. The other critical issue is the population coverage of a vaccine that has been neglected in traditional vaccines. </P> Objective: Given the issues above, our study aimed to design a peptide-based diphtheria vaccine, considering the issues of unwanted epitopes and population coverage. </P> Methods: The frequencies of pre-determined HLA alleles were listed. A country in which almost all HLA alleles had been determined in almost all geographical distribution was selected. The epitopes within the sequence of diphtheria toxin were predicted by the NetMHCIIPan server based on the selected HLA alleles. Strong binder epitopes on the surface of diphtheria toxin were selected by structural epitope mapping. The epitopes, which cover almost all the human population for each of the HLA alleles in the candidate country, were then selected as epitopebased vaccines. </P> Results: At first, 793 strong binder epitopes were predicted, of which 82 were surface epitopes. Nine surface epitopes whose amino acids had extruding side chains were selected. Finally, 2 epitopes had the most population coverage and were suggested as a di-epitope diphtheria vaccine. The population coverage of the di-epitope vaccine in France and the world was 100 and 99.24 %, respectively. HLA-DP had the most roles in epitope presentation. </P> Conclusion: Our results indicated that 97.75 % of unwanted antibodies (791 epitopes) have been reduced. Achieving two immunodominant surface epitopes confirmed our rational filtration strategy for sequential reduction of unwanted epitopes. Our novel insight may pave a new way to designing novel peptide-based vaccines to avoid producing non-specific antibodies.

Estrogen receptor beta expression and role in cancers

Estrogen drives the growth of some cancers, such as breast cancer, via estrogen receptor alpha (ERα). Estrogen also activates ERβ, but whether ERβ is expressed and has a role in different cancers is debated. The use of nonspecific antibodies has contributed to the confusion, and this review delves into ERβ's controversial role in cancer and focuses on tumor expression that can be supported by non-antibody-dependent assays. We discuss its expression at the transcript level and focus on its potential role in lymphoma, granulosa cell tumors, testicular, and adrenal cancers, emphasizing recent findings and the complexities that necessitate further research.

M6ACali: machine learning-powered calibration for accurate m6A detection in MeRIP-Seq.

We present m6ACali, a novel machine-learning framework aimed at enhancing the accuracy of N6-methyladenosine (m6A) epitranscriptome profiling by reducing the impact of non-specific antibody enrichment in MeRIP-Seq. The calibration model serves as a genomic feature-based classifier that refines the identification of m6A sites, distinguishing those genuinely present from those that can be detected in in-vitro transcribed (IVT) control experiments. We find that m6ACali effectively identifies non-specific binding peaks reported by exomePeak2 and MACS2 in novel MeRIP-Seq datasets without the need for paired IVT controls. The model interpretation revealed that off-target antibody binding sites commonly occur at short exons and short mRNAs, originating from high read coverage regions that share the motif sequence with true m6A sites. We also reveal that the ML strategy can efficiently adjust differentially methylated peaks and other antibody-dependent, base-resolution m6A detection techniques. As a result, m6ACali offers a promising method for the universal enhancement of m6A profiles generated by MeRIP-Seq experiments, elevating the benchmark for omics-level m6A data integration.

Dietary Artemisia arborescens Supplementation Effects on Growth, Oxidative Status, and Immunity of Gilthead Seabream (Sparus aurata L.).

Fish infectious diseases are one of the main constraints of the aquaculture sector. The use of medicinal plants provides a sustainable way of protection using safe, eco-friendly compounds in a more cost-effective way of treatment, compared to antibiotics. The aim of the present study is the assessment of Artemisia arborescens (AA) feed-supplementation effects on gilthead seabream (Sparus aurata). Fish with an average initial body weight of 109.43 ± 3.81 g, were divided into two groups based on AA feed composition (A25 and A50). Following two months of ad libitum feeding, the effect of diets on fish weight and length were measured. Fish serum and mucus were analyzed for non-specific immune parameters (nitric oxide, lysozyme, myeloperoxidase, protease-/anti-protease activity, and complement), antibody responses, oxidative stress (cytochrome P450 1A1, metallothionein), and metabolism markers (total protein, alkaline phosphatase, and glucose). Expression levels of antioxidants (sod1, gpx1), cytokines (il-1b, il-10, tfgb1, and tnfa), hepcidin, and heat shock protein grp75 genes were measured in spleen samples. A results analysis indicated that A. arborescens use as a feed supplement has a compromised positive effect on the growth performance, immune response, and blood parameters of gilthead seabream. Overall, the suitability of A. arborescens as an efficient food supplement for gilthead seabream health improvement was investigated, setting the basis for its application assessment in Mediterranean aquaculture.

Ilex kudingcha’s phenolic-rich fraction inhibits murine squamous cell carcinoma (SCC7) through restoring adaptive immunity and Th1-polarized immune balance in vitro and in vivo

The Ilex kudingcha methanol extract ethanol supernatent (ESIKME) was analyzed for its phenolic compounds using high-performance liquid chromatography. Murine squamous cell carcinoma (SCC)-7 was treated in vitro and in vivo. ESIKME was found abundant in 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid and modulated the immune response toward Th1-polarized immune balance through changing the splenocytes’ Th1/Th2 cytokine secretion profile in vitro. ESIKME had a significant effect against SCC7 cells in vitro. ESIKME administration by daily gavage for 26 days did not cause any apparent toxicity to SCC7-bearing mice. There were relatively lower tumor weights in the medium dose group (KM, 100 mg/kg b.w./day). KM administration slightly restored the B-cell and T-cell immunity through increasing serum non-specific antibody levels and the splenocytes’ proliferative ability in the experimental mice. KM administration tended to increase Th1-polarized immune balance through increasing (IL-2+TNF-⍺)/IL-10 (Th1/Th2) secretion ratios using splenocytes isolated from the experimental mice. Th1/Th2 cytokine secretion ratios by splenocytes were negatively correlated with tumor weights, revealing that ESIKME inhibits SCC7 through restoring adaptive immunity and Th1-polarized immune balance. Our results are the first to prove that ESIKME, which is a phenolic-rich fraction, inhibits SCC7 through partly restoring adaptive immunity and Th1-polarized immune balance in the tumor-bearing mice.

Photoimmuno-antimicrobial therapy for Staphylococcus aureus implant infection.

Implant infections caused by Staphylococcus aureus are responsible for high mortality and morbidity worldwide. Treatment of these infections can be difficult especially when bacterial biofilms are involved. In this study we investigate the potential of infrared photoimmunotherapy to eradicate staphylococcal infection in a mouse model. A monoclonal antibody that targets Wall Teichoic Acid surface components of both S. aureus and its biofilm (4497-IgG1) was conjugated to a photosensitizer (IRDye700DX) and used as photoimmunotherapy in vitro and in vivo in mice with a subcutaneous implant pre-colonized with biofilm of Staphylococcus aureus. A dose of 400 μg and 200 μg of antibody-photosensitizer conjugate 4497-IgG-IRDye700DXwas administered intravenously to two groups of 5 mice. In addition, multiple control groups (vancomycin treated, unconjugated IRDye700DX and IRDye700DX conjugated to a non-specific antibody) were used to verify anti-microbial effects. In vitro results of 4497-IgG-IRDye700DX on pre-colonized (biofilm) implants showed significant (p<0.01) colony-forming units (CFU) reduction at a concentration of 5 μg of the antibody-photosensitizer conjugate. In vivo, treatment with 4497-IgG-IRDye700DX showed no significant CFU reduction at the implant infection. However, tissue around the implant did show a significant CFU reduction with 400 μg 4497-IgG-IRDye700DX compared to control groups (p = 0.037). This study demonstrated the antimicrobial potential of photoimmunotherapy for selectively eliminating S. aureus in vivo. However, using a solid implant instead of a catheter could result in an increased bactericidal effect of 4497-IgG-IRDye700DX and administration locally around an implant (per operative) could become valuable applications in patients that are difficult to treat with conventional methods. We conclude that photoimmunotherapy could be a potential additional therapy in the treatment of implant related infections, but requires further improvement.

Constitutive immunity is influenced by avian influenza virus-induced modification of gut microbiota in Eurasian teal (Anas crecca)

Understanding the dynamics of migrant birds' gut microbial communities is essential for evaluating their ecological interactions, since these birds act as vectors for zoonotic viruses and their gut microbiome may have exceptional relationship with zoonotic viral infection. The Eurasian teal duck Anas crecca traverses continents during migration, combining and providing intercontinental links for avian influenza viruses (AIV) of different origins. The present study aimed to investigate how the AIV infection affects gut microbial composition and evaluate the consequent physiological stress and constitutive immunity of teal birds. Samples were collected from 2 flocks during their migratory stopover in northern Egypt. An important shift in gut microbiota of AIV-infected individuals has been detected by RT-PCR. In healthy teal, firmicutes dominated followed by proteobacteria, while the structure was reversed in infected birds. Infection with AIV significantly increased the stress hormone corticosterone, accompanied by a significant increase in both oxidative stress markers and antioxidants. Constitutive immunity, measured by plasma bactericidal effect against E. coli, the nonspecific natural antibodies, and the mediated complement activation, was reduced in AIV-infected teal birds. Constitutive immunity parameters were proportionally correlated to the firmicutes and inversely to the proteobacteria abundances, but not to the viral positivity. In conclusion, the present study provides initial evidence of the alteration of the gut microbiome in the Eurasian teal Anas crecca by AIV infection and demonstrates that the AIV-induced reduction in constitutive immunity is a consequence of the shift in microbiome composition rather than the virus infection itself or its induced stress.

Surface Modification of Polystyrene with Boronic Acid for Immunoaffinity-Based Cell Enrichment.

Obtaining an enriched and phenotypically pure cell population from heterogeneous cell mixtures is important for diagnostics and biosensing. Existing techniques such as fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require preincubation with antibodies (Ab) and specialized equipment. Cell immunopanning removes the need for preincubation and can be done with no specialized equipment. The majority of the available antibody-mediated analyte capture techniques require a modification to the Abs for binding. In this work, no antibody modification is used because we take advantage of the carbohydrate chain in the Fc region of Ab. We use boronic acid as a cross-linker to bind the Ab to a modified surface. The process allows for functional orientation and cleavable binding of the Ab. In this study, we created an immunoaffinity matrix on polystyrene (PS), an inexpensive and ubiquitous plastic. We observed a 37% increase in Ab binding compared with that of a passive adsorption approach. The method also displayed a more consistent antibody binding with 17 times less variation in Ab loading among replicates than did the passive adsorption approach. Surface topography analysis revealed that a dextran coating reduced nonspecific antibody binding. Elemental analysis (XPS) was used to characterize the surface at different stages and showed that APBA molecules can bind upside-down on the surface. While upside-down antibodies likely remain functional, their elution behavior might differ from those bound in the desired way. Cell capture experiments show that the new surface has 43% better selectivity and 2.4-fold higher capture efficiency compared to a control surface of passively adsorbed Abs. This specific surface chemistry modification will allow the targeted capture of cells or analytes with the option of chemical detachment for further research and characterization.

Improved Specificity of Glutamate Decarboxylase 65 Autoantibody Measurement Using Luciferase-Based Immunoprecipitation System Assays.

Autoantibodies to glutamate decarboxylase (GADA) are widely used in the prediction and classification of type 1 diabetes. GADA radiobinding assays (RBAs) using N-terminally truncated antigens offer improved specificity but radioisotopes limit the high-throughput potential for population screening. Luciferase-based Immunoprecipitation System (LIPS) assays are sensitive and specific alternatives to RBAs with the potential to improve risk stratification. The performance of assays using the Luciferase (Nluc-) conjugated GAD65 constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585) were evaluated in 434 well-characterised sera from recent-onset type 1 diabetes patients and first-degree relatives. Non-radioactive, high-throughput LIPS assays are quicker and require less serum than RBAs. Of 171 relatives previously tested single autoantibody positive for autoantibodies to full-length GAD65 by RBA but had not progressed to diabetes, fewer retested positive by LIPS using either truncated (n=72) or full-length (n=111) antigen. The Nluc-GAD65(96-585) truncation demonstrated the highest specificity in LIPS assays overall but in contrast to RBA, N-terminus truncations did not result in a significant increase in disease-specificity compared with the full-length antigen. This suggests that binding of non-specific antibodies is affected by the conformational changes resulting from addition of the Nluc antigen. Nluc-GAD65(96-585) LIPS assays offer low blood volume, high specificity GADA tests for screening and diagnostics.

Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Affinity Chromatography for Anti-Glucosylated Adhesin Antibody Purification: Depletion of Nonspecific Anti-Protein Antibodies and Antibody Recovery with Unconventional Elution Solutions.

Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein.

Carbon Nanostructured Immunosensing of Anti-SARS-CoV-2 S-Protein Antibodies.

The rampant spread and death rate of the recent coronavirus pandemic related to the SARS-CoV-2 respiratory virus have underscored the critical need for affordable, portable virus diagnostics, particularly in resource-limited settings. Moreover, efficient and timely monitoring of vaccine efficacy is needed to prevent future widespread infections. Electrochemical immunosensing poses an effective alternative to conventional molecular spectroscopic approaches, offering rapid, cost-effective, sensitive, and portable electroanalysis of disease biomarkers and antibodies; however, efforts to improve binding efficiency and sensitivity are still being investigated. Graphene quantum dots (GQDs) in particular have shown promise in improving device sensitivity. This study reports the development of a GQD-functionalized point-of-contamination device leveraging the selective interactions between SARS-CoV-2-specific Spike (S) Protein receptor binding domain (RBD) antigens and IgG anti-SARS-CoV-2-specific S-protein antibodies at screen-printed carbon electrode (SPCE) surfaces. The immunocomplexes formed at the GQD surfaces result in the interruption of the redox reactions that take place in the presence of a redox probe, decreasing the current response. Increased active surface area, conductivity, and binding via EDC/NHS chemistry were achieved due to the nanomaterial inclusion, with 5 nm, blue luminescent GQDs offering the best results. GQD concentration, EDC/NHS ratio, and RBD S-protein incubation time and concentration were optimized for the biosensor, and inter- and intra-screen-printed carbon electrode detection was investigated by calibration studies on multiple and single electrodes. The single electrode used for the entire calibration provided the best results. The label-free immunosensor was able to selectively detect anti-SARS-CoV-2 IgG antibodies between 0.5 and 100 ng/mL in the presence of IgM and other coronavirus antibodies with an excellent regression of 0.9599. A LOD of 2.028 ng/mL was found, offering comparable findings to the literature-reported values. The detection sensitivity of the sensor is further compared to non-specific IgM antibodies. The developed GQD immunosensor was compared to other low-oxygen content carbon nanomaterials, namely (i) carbon quantum dot (CQD), (ii) electrochemically reduced graphene oxide, and (iii) carbon black-functionalized devices. The findings suggest that improved electron transfer kinetics and increased active surface area of the CNs, along with surface oxygen content, aid in the detection of anti-SARS-CoV-2 IgG antibodies. The novel immunosensor suggests a possible application toward monitoring of IgG antibody production in SARS-CoV-2-vaccinated patients to study immune responses, vaccine efficacy, and lifetime to meet the demands for POC analysis in resource-limited settings.