Immunoglobulin Fraction Research Articles

B-090 Efficient Hydrophilic Surface Coating with Low Concentrations of Bovine Gamma Globulins

Abstract Background Bovine Gamma Globulin (BGG) is a high purity immunoglobulin fraction of bovine serum. BGG is used as a blocker to prevent non-specific binding in immunoassays and, due to the mixture of immunoglobulins, its blocking capabilities differ from traditional blockers like Bovine Serum Albumin (BSA). Our objective was to evaluate the adsorption characteristics of BGG to In vitro diagnostic relevant hydrophilic surfaces compared to BSA. A bead-based immunoassay was utilized to quantify the mass of protein coating a bead surface and atomic force microscopy (AFM) to analyze physical properties of the protein-surface layer. Methods The mass of BGG bound to hydrophilic beads (Dynabeads M-270 Carboxylic Acid) was determined across varying concentrations ranging from 0.1% to 1.0% in pH 7.0 phosphate buffer. Coated beads were heated in 1x SDS loading dye to dissociate bound protein, then ran on a nonreducing SDS-PAGE gel. The total protein was measured using densitometry against a standard curve, using ImageJ, and analyzed with GraphPad Prism. This methodology was also used for BSA. AFM was used to image a topographical view of BGG binding to a silica surface. Samples were prepared at concentrations from 0.1% to 1.0% in phosphate buffer at pH 7.0. Followed by a series of washes to remove non-bound molecules, the surface was allowed to dry, then scratched with an AFM cantilever. The surface was then analyzed for roughness and thickness of BGG adsorption for each concentration. Results BGG and BSA have notable differences in their coating properties at a concentration of 1.0%. In the bead assay, the protein-surface binding is enhanced ∼4.8-fold for BGG compared to BSA. Interestingly, even at 0.1% BGG bound ∼0.8-fold more than BSA at 1.0%. Despite a 6-fold increase in mass bound to the bead surface with 1.0% BGG compared to 0.1%, AFM revealed a 1.3-fold and 1.8-fold decrease in height and roughness at the lower concentration, while maintaining effective coating. These results suggest that higher BGG concentrations result in thicker, rougher surface coating. This could lead to steric hindrance and obstruction of binding sites, affecting assay performance. Therefore, a lower concentration of BGG may lead to better surface coating and prevent reduced sensitivity and accuracy within immunoassays. Conclusions The bead binding assay demonstrates that BGG displays surface adsorption with comparable or improved results with lower concentrations compared to BSA. At 0.1%, BGG exhibits effective binding to a hydrophilic surface equivalent to that achieved by BSA at 1.0%. Additionally, AFM data suggest that even at 0.1%, BGG effectively coats the silica surface and potentially reduces steric hinderance compared to BGG at 1%. While BSA remains a reliable choice for many applications, our findings suggest that BGG can be an effective blocker for immunoassay applications. The choice of a suitable blocker varies depending on factors such as assay high background, sensitivity, and specificity. By tailoring the choice of blocker to the specific requirements of the assay, researchers can optimize assay performance by improving the accuracy and the reliability of the assay.

Efficient Enzymatic Hydrolysis of the Whey Protein Fraction from Bovine Milk

Studies were conducted to optimize the hydrolysis of the major whey proteins with the serine proteases Alcalase and Protozyme, and of the immunoglobulin fraction of colostrum with Alcalase. The protein-peptide composition of the enzymatic hydrolysates of whey and colostrum was determined using high-performance liquid chromatography and electrophoresis data under denaturing conditions. The hydrolysis conditions, in conjunction with the application of a heat treatment of the substrate, were designed to enhance the efficiency of whey proteolysis using Protozyme. An increase in the proteolysis degree of the native immunoglobulin fraction of colostrum with Alcalase was also achieved. The results of the experimental work are intended for the production of a hypoallergenic protein component derived from bovine milk

Serum-Derived Bovine Immunoglobulin/Protein Isolate Bind Aflatoxins B2 and G1

Aflatoxins are highly toxic biproducts of mold growth which commonly contaminate agricultural commodities, including food and feed. The United States Centers for Disease Control and Prevention estimates approximately half of people worldwide are chronically exposed to aflatoxins through their regular food consumption. The gut serves as both the initial target of toxicity and the location of absorption, leading to systemic exposure. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a purified protein powder enriched in immunoglobulins IgG (55%), IgM (5%), and IgA (1%) used to help manage gut enteropathy. SBI binds a variety of pro-inflammatory antigens and decreases inflammatory markers in the gut by neutralizing or preventing translocation of antigen across the intestinal barrier. The objective of this study is to determine if the immunoglobulins in SBI bind to aflatoxins. Due to the protective effects of SBI, we hypothesize SBI and a highly purified immunoglobulin fraction (>95%), b ovine gamma globulin (BGG), can bind to aflatoxin B2 (AFB2) and aflatoxin G1 (AFG1). Fluorescence spectroscopy was used to quantify the binding of aflatoxins to SBI. Previous work has shown the binding of aflatoxins to tryptophan containing proteins, such as bovine serum albumin, reduces the fluorescence of the protein’s tryptophan residue. The same principle works for SBI, upon excitation at 280 nm, the emission spectra of SBI exhibits a peak at 370 nm which is decreased upon aflatoxin binding. Either SBI (5 μM) or BGG (5 μM) was titrated with an increasing concentration of either AFB2 or AFG1 (0-40 μM). The protein solution was excited at 280 nm and the emission spectra was recorded at 350-450 nm with quenching quantified at 370 nm. The fluorescence from SBI or BGG was reduced in a concentration-dependent manner as the concentration of AFB2 or AFG1 increased. The quenching constant (Ksv) was calculated using the Stern-Volmer equation, while a derivative from the equation was used to calculate the binding constant (Kb). All protein-aflatoxin pairs tested showed binding with Ksv values ranging from 0.9–1.2 x 104 M−1 (R2=0.99) and Kb values ranging from 2.5 x 104 to 4.21 x 105, with SBI-AFB2 exhibiting the tightest binding. Further, the Gibbs free energy for each pair was -6.0 to -7.7 kcal/mol, indicating favorable binding. The quenching of both protein solutions indicates structural and conformational changes in the native structures of both SBI and BGG, likely caused by the interaction of the immunoglobulins with the aflatoxin. The quenching of BGG suggests immunoglobulins in SBI play a significant role for the observed phenomenon, though other serum proteins within SBI could contribute to the quenched fluorescence. Future work will examine if the binding of the aflatoxins by SBI can prevent or mitigate aflatoxin toxicity in the gut. Proliant Health and Biologicals. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adaptation of Stadier's apparatus for electrophoresis of main milk proteins

The object of research is a Stadier-type apparatus for analytical electrophoresis of proteins. In the milk proteins research, in addition, there is a need to carry out serial express analyzes of their various groups, as well as the isolation of individual homogeneous fractions. The dimensions of working chambers for analytical, express, and micro preparative electrophoresis of caseins and milk whey proteins were proposed to solve this task. For each type of electrophoresis, different chambers and formers are used without changing the design of the apparatus. The apparatus is suitable for electrophoretic systems used for the analysis of milk proteins. Analysis of casein in the anodic system of a homogeneous polyacrylamide gel in the presence of urea allows identification of the main fractions: αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, αS2-CN-13P, β-CN-5P, ϰ-CN-1P and three β-casein fragments f(29-209), f(106-209) and f(108-209). Express electrophoresis in the presence of urea reveals four fractions of caseins: αS1-CN, αS2-CN, β-CN, and ϰ-CN. The analysis of whey proteins in the Davis native disc electrophoresis system allows identification of β-Lg A, β-Lg B, α-La, BSA fractions, and a group of immunoglobulin fractions. The express electrophoregram differs by a common band A and B variants of β-Lg. Due to an adequate selection of electrophoretic systems, it is possible to identify semi-quantitatively all the main fractions of milk proteins under analytical or express mode. The adapted apparatus also makes it possible to conduct micro preparative electrophoresis and obtain the main fractions of milk proteins. In this case, the yield of electrophoretically pure proteins is: β-CN-5P (23±5 %), β-Lg (A+B) (27±6 %), α-La (11±3 %), and purified groups of αS1-CN-8P+αS1-CN-9P (25±6 %), αS2-CN-(10-13P) (6±1.5 %) and ϰ-CN-1P (7±2 %). The apparatus could be used at enterprises producing dairy protein products

Antirotaviral activity of dairy byproducts enriched in fractions from hyperimmune bovine colostrum: the effect of thermal and high hydrostatic pressure treatments.

Nowadays, rotaviruses remain a major health burden, especially in developing countries, and strategies complementary to vaccination are needed. In this view, dairy fractions have attracted great scientific interest, due to their high content of bioactive compounds. The objective of this study was to evaluate the antiviral activity of whey and buttermilk enriched in proteins from hyperimmune bovine colostrum (HBC) against rotavirus. The enriched fractions were spray-dried and subsequently tested for their neutralizing activity against the bovine rotavirus WC3 strain in vitro, using differentiated Caco-2/TC7 cells. The highest antirotaviral activity was observed when whey and buttermilk were enriched in purified immunoglobulin G (IgG), showing complete rotavirus neutralization at concentrations of 3 and 6 mg mL-1 for whey and buttermilk, respectively. Additionally, the use of a crude immunoglobulin fraction also gave satisfactory results. The inhibitory activities of all samples significantly decreased after the application of heat, except for the IgG-enriched buttermilk which showed a slight increase of activity following the application of short-time treatments (75 or 85 °C for 20 s). This sample also showed a significant increase of activity (13%) after the application of low-intensity high hydrostatic pressure treatment (400 MPa for 5 min). The maximum loss of bioactivity was observed at 600 MPa for 10 min (31 and 20% for whey- and buttermilk-based formulas, respectively). This study provides relevant information on the potential of whey, buttermilk, and HBC to be part of functional products as complementary strategies to combat rotavirus infections.

Monoclonal Gammopathy of Undetermined Significance: A Comprehensive Review.

Monoclonal Gammopathy of Undetermined Significance (MGUS) is an asymptomatic premalignant plasma cell dyscrasia with a predominate rise of the IgG immunoglobulin fraction without end-organ damage, often diagnosed incidentally. Despite its progression into various subsequent forms of hematological malignancies, MGUS remains underdiagnosed. A literature search was conducted using the Medline, Cochrane, Embase, and Google Scholar databases, including articles published until December 2022. Keywords used encompassed "Monoclonal Gammopathy of Undetermined Significance," "Plasma Cell dyscrasia," "Monoclonal gammopathy of renal significance," and "IgM Monoclonal gammopathy of Undetermined Significance," This study aimed to conduct a critical review to update knowledge regarding the pathophysiology, risk factors, clinical features, diagnostic protocols, complications, and current and novel treatments for MGUS. We recommend a multidisciplinary approach to manage MGUS due to the complexity of the illness's etiology, diagnosis, and therapy. This comprehensive review also highlights future prospects, such as developing screening protocols for at-risk populations, prevention of disease progression by early diagnosis through genome-wide association studies, and management using Daratumumab and NSAIDs.

Emerging care management strategies for severe asthma: integrating biologic therapy.

Severe asthma accounts for 5% to 10% of asthma cases. Despite this, patients with uncontrolled severe asthma consume significant healthcare resources. Approximately 40% of patients with severe asthma have uncontrolled disease. Treatment guidelines lack strong recommendations because of the heterogeneity of the disease. Together, prescribers and pharmacists can personalize treatment plans with biologics, using patient phenotypes to maximize disease control. Biomarkers such as fraction of exhaled nitric oxide levels, immunoglobulin E, and serum periostin levels help identify inflammation type and stratify patients by endotype. Currently, there are 5 biologics that treat eosinophilic inflammation and 1 newer biologic that treats both eosinophilic inflammation and noninflammatory non-eosinophilic asthma. Healthcare professionals need to consider phenotype and endotype to individualize patient treatment. As medications and hospitalizations are the largest drivers of asthma-related costs to health systems, choosing optimal therapies to reduce uncontrolled symptoms and exacerbations curbs cost. Successful disease management strategies are employed by managed care organizations that have a collaborative team to ensure patient access to biologics.

Efficient and Sustainable Platform for Preparation of a High-Quality Immunoglobulin G as an Urgent Treatment Option During Emerging Virus Outbreaks.

During the pre-vaccine era of the COVID-19 pandemic convalescent plasma has once again emerged as a major potential therapeutic form of passive immunization that in specific cases still represents irreplaceable treatment option. There is a growing concern that variable concentration of neutralizing antibodies, present in convalescent plasma which originates from different donors, apparently affects its effectiveness. The drawback can be overcome through the downstream process of immunoglobulin fraction purification into a standardized product of improved safety and efficacy. All modern procedures are quite lengthy processes. They are also based on fractionation of large plasma quantities whose collection is not attainable during an epidemic. When outbreaks of infectious diseases are occurring more frequently, there is a great need for a more sustainable production approach that would be goal-oriented towards assuring easily and readily available immunoglobulin of therapeutic relevance. We propose a refinement strategy for the IgG preparation achieved through simplification and reduction of the processing steps. It was designed as a small but scalable process to offer an immediately available treatment option that would simultaneously be harmonized with an increased availability of convalescent plasma over the viral outbreak time-course. Concerning the ongoing pandemic status of the COVID-19, the proof of concept was demonstrated on anti-SARS-CoV-2 convalescent plasma but is likely applicable to any other type depending on the current needs. It was guided by the idea of persistent keeping of IgG molecules in the solution, so that protection of their native structure could be assured. Our manufacturing procedure provided a high-quality IgG product of above the average recovery whose composition profile was analyzed by mass spectrometry as quality control check. It was proved free from IgA and IgM as mediators of adverse transfusion reactions, as well as of any other residual impurities, since only IgG fragments were identified. The proportion of S protein-specific IgGs remained unchanged relative to the convalescent plasma. Undisturbed IgG subclass composition was accomplished as well. However, the fractionation principle affected the final product’s capacity to neutralize wild-type SARS-CoV-2 infectivity, reducing it by half. Decrease in neutralization potency significantly correlated with the amount of IgM in the starting material.

Coagulopathy and the humoral response against viral proteins in patients at different stages of COVID-19.

Patients with severe coronavirus disease 2019 (COVID-19) often present with coagulopathies and have high titres of circulating antibodies against viral proteins. Herein, we evaluated the association between D-dimer and circulating immunoglobulin levels against viral proteins in patients at different clinical stages of COVID-19. For this, we performed a cross-sectional study involving patients of the first wave of COVID-19 clinically classified as oligosymptomatic (n = 22), severe (n = 30), cured (n = 27) and non-infected (n = 9). Next, we measured in the plasma samples the total and fraction of immunoglobulins against the nucleoprotein (NP) and the receptor-binding domain (RBD) of the spike proteins by enzyme-linked immunosorbent assay (ELISA) assays. Patients with severe disease had a coagulation disorder with high levels of D-dimer as well as circulating IgG against the NP but not the RBD compared to other groups of patients. In addition, high levels of D-dimer and IgG against the NP and RBD were associated with disease severity among the patients in this study. Our data suggest that IgG against NP and RBD participates in the worsening of COVID-19. Although the humoral response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is partially understood, and more efforts are needed to clarify gaps in the knowledge of this process.

The concentrations of immunoglobulins in bovine colostrum determined by the gold standard method are genetically correlated with their near-infrared prediction

BackgroundThe quality of colostrum administered to calves is based on its concentration in immunoglobulins G (IgG, g/L). Immunoglobulins A (IgA) and M (IgM) are also present but at a lower level. The gold standard reference analysis for these traits, radial immunodiffusion, is time-consuming and expensive. In order to define breeding strategies that are aimed at improving colostrum quality in dairy cattle, a large amount of data is needed, and the use of indicator traits would be beneficial. In the study presented here, we explored the heritabilities of reference (radial immunodiffusion) and near infrared-predicted IgG, IgA, and IgM concentrations and estimated their genetic correlations. First, the colostrum of 765 Holstein cows from nine herds was sampled to perform a reference analysis and the near-infrared spectra (400–2500 nm) were stored. We used a calibration set (28% of the initial samples) that was representative of the herds and cow parity orders to develop prediction equations for IgG, IgA, and IgM concentrations. Finally, these traits were predicted in the validation set (72% of the initial samples) to estimate genetic parameters for the predictions. Genetic correlations between reference and predicted values of each trait were estimated through bivariate linear animal models.ResultsThe three near-infrared-predicted immunoglobulin fractions were genetically correlated with their reference value. In particular, the reference and predicted IgG concentrations were strongly correlated at both the genetic (0.854 ± 0.314) and phenotypic level (0.767 ± 0.019). Weaker associations were observed for IgA and IgM concentrations, which were predicted with lower accuracy compared to IgG. Simulation analyses suggested that improving colostrum quality by selective breeding in Holstein cattle based on near-infrared predicted colostrum immunoglobulins concentrations is feasible. In addition, less than 10 mL of colostrum are needed for spectra acquisition and thus implementation of such analyses is possible in the near future.ConclusionsThe concentrations of colostrum immunoglobulins can be predicted from near-infrared spectra and the genetic correlation between the reference and the predicted traits is positive and favourable, in spite of the large standard errors of the estimates. Near-infrared spectroscopy can be exploited in selective breeding of dairy cattle to improve colostral immunoglobulins concentration.

Process steps for the fractionation of immunoglobulin (Ig) G depleted of IgA, isoagglutinins, and devoid of in vitro thrombogenicity.

Plasma-derived immunoglobulins (IgG) are essential medicines that are in worldwide shortage, especially in low- and middle-income countries. Optimised manufacturing processes can increase supply. We evaluated various new process steps for IgG fractionation. A crude, worst-case, IgG intermediate obtained by caprylic acid fractionation of cryoprecipitate-poor plasma was used as starting experimental material. It was processed inline by Fractogel® (Merck) TMAE anion-exchanger to deplete IgA and IgM, Eshmuno® P (Merck) anti-A and anti-B affinity chromatography to remove anti-A and anti-B isoagglutinins, 0.3% TnBP-1% Triton X-100 (S/D) treatment, C18 chromatography for removal of S/D agents, and single-pass tangential flow filtration (SPTFF) concentration to 20%. Quality, safety, and recovery were evaluated at small and pilot scales to assess purity, removal of IgA, IgM isoagglutinins, S/D agents, thrombogenic factors, and lack of toxicity in a cell model. The starting IgG intermediate contained approximately 90% IgG, IgA, and IgM and 10% albumin. Fractogel® TMAE, equilibrated in 25 mM sodium acetate-pH 6.0 and loaded with up to 225 mg of IgG/mL, could remove IgA and IgM, with over 94% IgG recovery with preserved sub-class distribution in the flow-through. Sequential Eshmuno®-P anti-A and anti-B columns efficiently removed isoagglutinins. The C18 packing, used at up to 17 mL of S/D-IgG solution per mL, removed TnBP and Triton X-100 to less than 1 and 2 ppm, respectively. The 20% purified IgG was devoid of activated factor XI and thrombin generation activity. This purification sequence yields a >99% pure, 20% (v/v) IgG product, depleted of IgA, isoagglutinins, and thrombogenic markers, and should be implementable on various IgG intermediates to help improve the supply of immunoglobulins.

Effectiveness of visible – Near infrared spectroscopy coupled with simulated annealing partial least squares analysis to predict immunoglobulins G, A, and M concentration in bovine colostrum

Visible - near infrared spectroscopy coupled with variable selection using simulated annealing PLS regression was tested to predict immunoglobulin fractions (g/L) of bovine colostrum, namely IgG, IgA and IgM. Immunoglobulins were quantified in 678 samples using the gold standard radial immunodiffusion. Samples were divided in calibration (50%) and validation (50%) datasets. Maximum number of selected variables were limited to 200 and root mean squared error in cross validation (RMSECV) was used as loss function. Performance of the final model developed using the calibration dataset was assessed on the validation dataset. Overall, simulated annealing PLS improved validation RMSECV compared to ordinary PLS regression by 3% to 17%. The present study demonstrated the effectiveness of the calibration model for accurate quantification of IgG, the most abundant immunoglobulin of bovine colostrum (RMSECV = 13.28 g/L; R2 = 0.83). These outcomes could be useful to assess colostrum quality intended for animal and human usage.

A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry

Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 – 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum.

Defining type 2 asthma and patients eligible for dupilumab in Italy: a biomarker-based analysis

BackgroundAsthma is a chronic disease characterized by airway hyperresponsiveness, inflammation and mucus production. In Type 2 asthma, two phenotypic components are often co-expressed (eosinophilic and allergic). Elevated biomarker levels, such as eosinophils (EOS), fraction of exhaled nitric oxide (FeNO) and immunoglobulin E (IgE), are key clinical indicators of Type 2 inflammation. Dupilumab has been recently approved for the treatment of uncontrolled severe Type 2 asthma. Type 2 asthma includes allergic and/or eosinophilic phenotypes. The aim of this analysis was to estimate the dupilumab-eligible population in Italy and characterize it by expected biomarker status.MethodsA 4-step approach was carried out to calculate dupilumab-eligible population. The approach consisted in: (1) estimating the total number of asthma patients in Italy (using 2016–2017 Italian-adapted Global Initiative for Asthma -GINA- guidelines); (2) estimating the number of severe asthma patients with poorly controlled or uncontrolled disease (using the findings of two recent administrative claim analyses conducted in Italy); (3) stratifying the severe uncontrolled population by biomarker levels (EOS, FeNO and IgE) according to the outcomes of the QUEST trial (a clinical study assessing the efficacy of dupilumab in patients with uncontrolled moderate-to-severe asthma; NCT02414854); (4) identifying the sub-populations of severe uncontrolled asthma patients characterised by raised blood EOS and/or FeNO level (thus indicated to receive dupilumab).ResultsAccording to these estimates, about 3.3 million asthmatic patients live in Italy (6.10% of the population). Of them, almost 20 thousand (N = 19,960) have uncontrolled severe asthma. Dupilumab-eligible patients would be N = 15,988, corresponding to 80.1% of the total uncontrolled severe population. Most of these patients (89.3%; N = 14,271) have at least an increase of EOS level, while slightly more than half (51.9%; N = 8,303) have raised levels of both biomarkers. Increased FeNO levels without increased EOS are observed less frequently (N = 1,717; 10.7% of the eligible population).ConclusionsThere is a strong rationale for testing all asthma biomarkers during diagnosis and disease follow-up. Given the large availability and the limited costs, these tests are cost-effective tools to detect severe Type 2 asthma, stratify patients by phenotype, and drive appropriate treatment decisions.

A Novel Apilic Antivenom to Treat Massive, Africanized Honeybee Attacks: A Preclinical Study from the Lethality to Some Biochemical and Pharmacological Activities Neutralization.

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab’)2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25–0.5 μL of intravenous Apilic antivenom/μg honeybee venom. In vivo injection of 0.1–1 μg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 μg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.

Role of Immunoglobulin M and A Antibodies in the Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and -RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin isotypes capable of blocking infection.MethodsWe studied spike- and RBD-specific immunoglobulin isotypes in convalescent and acute plasma/serum samples using a multiplex bead assay. We also determined virus neutralization activities in plasma and serum samples, and purified immunoglobulin fractions using a vesicular stomatitis pseudovirus assay.ResultsSpike- and RBD-specific immunoglobulin (Ig) M, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions’ neutralization potency. IgA also exhibited neutralizing activity, but with lower potency.ConclusionIgG, IgM, and IgA are critical components of convalescent plasma used for treatment of coronavirus disease 2019 (COVID-19).

PRS50 Defining Type 2 Asthma and Patients Eligible to Dupilumab in Italy: A Biomarker-Based Analysis

Background Asthma is a chronic disease characterized by airway hyperresponsiveness, inflammation and mucus production. In Type 2 asthma, two phenotypic components are often co-expressed (eosinophilic and allergic). Elevated biomarker levels, such as eosinophils (EOS), fraction of exhaled nitric oxide (FeNO) and immunoglobulin E (IgE), are key clinical indicators of Type 2 inflammation. Dupilumab has been recently approved for the treatment of uncontrolled severe Type 2 asthma. Type 2 asthma includes allergic and/or eosinophilic phenotypes. The aim of this analysis was to estimate the dupilumab-eligible population in Italy and characterize it by expected biomarker status. Methods A 4-step approach was carried out to calculate dupilumab-eligible population. The approach consisted in: (1) estimating the total number of asthma patients in Italy (using 2016-2017 Italian-adapted Global Initiative for Asthma -GINA- guidelines); (2) estimating the number of severe asthma patients with poorly controlled or uncontrolled disease (using the findings of two recent administrative claim analyses conducted in Italy); (3) stratifying the severe uncontrolled population by biomarker levels (EOS, FeNO and IgE) according to the outcomes of the QUEST trial (a clinical study assessing the efficacy of dupilumab in patients with uncontrolled moderate-to-severe asthma; NCT02414854); (4) identifying the sub-populations of severe uncontrolled asthma patients characterised by raised blood EOS and/or FeNO level (thus indicated to receive dupilumab). Results According to these estimates, about 3.3 million asthmatic patients live in Italy (6.10% of the population). Of them, almost 20 thousand (N = 19,960) have uncontrolled severe asthma. Dupilumab-eligible patients would be N = 15,988, corresponding to 80.1% of the total uncontrolled severe population. Most of these patients (89.3%; N = 14,271) have at least an increase of EOS level, while slightly more than half (51.9%; N = 8,303) have raised levels of both biomarkers. Increased FeNO levels without increased EOS are observed less frequently (N = 1,717; 10.7% of the eligible population). Conclusions There is a strong rationale for testing all asthma biomarkers during diagnosis and disease follow-up. Given the large availability and the limited costs, these tests are cost-effective tools to detect severe Type 2 asthma, stratify patients by phenotype, and drive appropriate treatment decisions.

Changes in immunological parameters in ultramarathon runners depending on the duration of the load.

In recent decades, there has been an increasing interest in long-distance running, not only to the classic marathon distances but also to an ultramarathon -100 km, 250 km, and multi-day races. One of the most reactive body systems in conditions of prolonged muscular loads is the immune system. The work aimed to investigate the changes in immunological parameters in runners for extra-long distances of various durations. We examined 73 male athletes specializing in long-distance and ultra-long-distance running. The design of the survey included the study of immunological status during the marathon race, 100 km run, and six-day run. We studied the dynamics of leukocyte formula; subpopulations of lymphocytes; fractions of immunoglobulins: IgA, IgM, IgG; phagocytic activity. During the marathon run, athletes' immunological changes are manifested by the development of neutrophilic leukocytosis with a decrease in the phagocytic activity of neutrophils. There was a sharp increase in the NLR and decrease in the LMR. A 100-km race caused a decrease in the absolute and relative number of T-lymphocytes. NLR increased almost ten times, and LMR, on the contrary, decreased nearly five times. On the first day of the 6-days run, there is a sharp increase in NLR and a decrease in LMR, after which there is a relative stabilization of both indicators. Changes in immunological parameters during jogging are closely related to the duration of the load. The most pronounced changes are observed on the part of neutrophils and lymphocytes.

Isolation of Heavy Chain Antibodies from Camelus Bactrianus Serum

Objectives: The IgG antibodies from species of Camelidae are named heavy-chain antibodies that lack L-chains of typical antibodies. The antigen-binding site of the dromedary HCAb is located in the single-chain domain referred to as the nanobody. Nanobodies are distinguished from other conventional antibodies by their unique properties, which lead to numerous biopharmaceutical and medicinal applications. Therefore, this study aimed to isolate IgG protein by isoforms from the blood serum of Mongolia’s two-humped camels and determine the contents of isomers of the antibody to enable further C. bactrianus nanobody research. Methods: IgG subclasses were separated by two different affinity chromatography steps using the ÄKTA Prime fast protein liquid chromatography method. The immunoglobulin fractions were determined using assays by the Bradford method and absorbance at 280 nm. The purity of IgG fractions was verified by the SDS-PAGE electrophoresis method. Results: Camel blood serum IgG subclasses were isolated by Protein A and G affinity chromatography columns, and the ratios of IgG1, IgG2, and IgG3 isotypes were 40.5 ± 4.5 kDa, 24.0 ± 8.0 kDa, and 35.5 ± 5.1 kDa, respectively. IgG2 and IgG3 subclass heavy chain bands on SDS-PAGE showed different molecular weights: 45 kDa and 43 kDa. Conclusions: Our findings suggest that IgG protein isotypes in the domestic Camelus Bactrianus serum that we identified were statistically different from C.Dromedarius serum.

P0463KIDNEY INVOLVEMENT IN PARAPROTEINEMIC DISEASES - ANALYSIS OF THE SINGLE CENTER REGISTER OF KIDNEY BIOPSIES

Abstract Background and Aims Kidneys are often damaged in paraproteinemic conditions. Paraproteins are monoclonal immunoglobulins or immunoglobulin fractions that are produced by a clonal population of B- or plasma cell lineage and can cause a variety of histological patterns of kidney injury, such as light chain (AL) amyloidosis or light chain cast nephropathy (LCCN). Monoclonal gammopathy of renal significance (MGRS) represents a group of disorders in which a monoclonal immunoglobulin secreted by B- or plasma cell clone causes renal damage. By definition, these disorders do not meet diagnostic criteria for overt, symptomatic multiple myeloma or a lymphoproliferative disorder, but in contrast to monoclonal gammopathy of undetermined significance (MGUS) there is evidence of end-organ damage that can warrant therapy. Method All patients with paraproteinemic kidney disease were identified by retrospective review of the Hospital Register of kidney biopsies done at Department of Nephrology and Dialysis, in Dubrava University Hospital, Zagreb, from 2009 until 2018. Every kidney biopsy was analyzed by light, immunofluorescent and electron microscopy. Laboratory findings, including serum protein electrophoresis, serum free light chain level and immunofixation of serum proteins, were done for every patient. Clinical and histologic features of patients and features of underlying hematological conditions were analyzed. Results We identified 47 patients (3,28% of all biopsies that were done in that period) with kidney disease with clear hematological background. The mean patients' age at the time of the biopsy was 63 years and 27 of them were females. Two patients had signs of direct infiltration of kidneys with malignant lymphomic cells (non-Hodgkin lymphoma) and were excluded from the analysis. Clinical presentation of the patients at the time of biopsy were: proteinuria in 85% of patients, full nephrotic syndrome in 55%, azotemia in 66% of patients (80% had acute kidney injury of unclear etiology) and hematuria in 12,7%. Most common histologic patterns of kidney injury were AL amyloidosis (45%) and LCCN (30%) but additionally 7 different histological patterns were found: light chain depostion disease, light chain proxymal tubulopathy, fibrillary and imunotactoid glomerulopathy, proliferative glomerulonephritis with monoclonal immunoglobulin deposition, crioglobulinemic glomerulopathy type I and tubulointerstitial damage caused by immunoglobulin deposition. Figure 1 shows main features of patients with AL amyloidosis and cast LCCN. Conclusion Kidney disease can be initial presentation of an underlying paraproteinemia and, as our data showed, can clinicaly present with acute kidney injury, nephrotic or subnephrotic range proteinuria or full nephrotic syndrome. Variety of histologic patterns of kidney injury were described and AL amyloidosis and LCCN were the most common histological findings. Detailed hematologic workup should follow kidney biopsy in order to determine the exact nature and extension of the disease and therefore the most appropriate therapy.