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Background and objectives To detect HPA-15 alloantibodies, we previously developed a human platelet antigen 15 (HPA-15)-expressing cell line-based modified rapid monoclonal antibody immobilization of platelet antigen (CL-MR-MAIPA) assay. In this study, the protocol was modified for easier performance by introducing the mixed-passive haemagglutination (MPHA) principle. Material and methods In total, 20 samples that tested negative for HPA alloantibodies and eight that tested positive for HPA-15 alloantibodies (two and six positive for HPA-15a and HPA-15b antibodies, respectively) by CL-MR-MAIPA assay were used in this study. HPA-15 cell lines were incubated with serum/plasma and then solubilized. The lysate was transferred to a round-bottom well, which was coated with anti-human CD109 monoclonal antibodies. After incubation and repeated washings, sheep red blood cells, coated with anti-human IgG, were added to the wells. Haemagglutination was assessed the next day. Results The proposed cell line-based immune complex capture-dependent mixed-passive haemagglutination (CL-IC-MPHA) assay consisted of four steps, but required only 2 h to perform, except for overnight incubation for haemagglutination. Two HPA-15a alloantibody samples were reactive only for HPA-15a cells, and six HPA-15b alloantibody samples were reactive only for HPA-15b cells with the CL-IC-MPHA assay. The 20 samples that tested negative for HPA alloantibodies did not react with HPA-15a or HPA-15b cells. These data indicated that the CL-IC-MPHA assay was highly specific and sensitive. Unfortunately, the CL-IC-MPHA assay's analytic sensitivity was twofold to eightfold lower than that of the CL-MR-MAIPA assay. Conclusion A novel, easy-to-perform protocol was successfully developed to detect HPA-15 alloantibodies with high specificity and sensitivity.

Background Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease with unmet therapeutic need in a critical cohort of recalcitrant cases. Immunoadsorption (IA) aims at an immunomodulatory depletion of pathogenic serum mediators and has recently revealed promising clinical results for the treatment of AD. Objective To determine efficacy, sustainability, safety, and clinical impact of IgE selective IA in AD using a single‐use IgE immunoadsorber column. Methods This open‐label pilot study comprised five patients (mean SCORAD 67.9 ± 11.4, range 52.2‐81.9; mean serum IgE level 5904 ± 5945 U/mL, range 1000‐15 600 IU/mL) who underwent IgE‐selective IA. Three patients continued prior therapy with systemic immunosuppressive drugs during IA as an add‐on therapeutic approach. All patients received three courses of IA. The first course consisted of three consecutive daily treatments followed by two sequences with two consecutive applications. All courses were performed on a monthly regimen. Results IA proved efficacy in selectively depleting serum IgE levels in all participants (mean reduction by cycle of 81% ± 12%, range 64%‐93%). It further led to a clinically relevant and sustained improvement of AD with a maximum decline in SCORAD and EASI scores by up to 35% and 52%, respectively, compared to baseline. Scores persisted below baseline for at least 12 weeks beyond the last IA. The intervention was also well tolerated with no severe adverse events during a total of 35 procedures. Conclusion Data of this preliminary trial indicates clinical efficacy, feasibility, safety as well as tolerability of IgE‐selective IA in AD.

Objective: Posterior urethral valve is the most common lower urinary tract obstruction in male children. A high percentage of patients with posterior urethral valve evolve to end‐stage renal disease. Previous studies showed that cytokines, chemokines, and components of the renin–angiotensin system contribute to the renal damage in obstructive uropathies. The authors recently found that urine samples from fetuses with posterior urethral valve have increased levels of inflammatory molecules. The aim of this study was to measure renin–angiotensin system molecules and to investigate their correlation with previously detected inflammatory markers in the same urine samples of fetuses with posterior urethral valve. Methods: Urine samples from 24 fetuses with posterior urethral valve were collected and compared to those from 22 healthy male newborns at the same gestational age (controls). Renin–angiotensin system components levels were measured by enzyme‐linked immunosorbent assay. Results: Fetuses with posterior urethral valve presented increased urinary levels of angiotensin (Ang) I, Ang‐(1‐7) and angiotensin‐converting enzyme 2 in comparison with controls. ACE levels were significantly reduced and Ang II levels were similar in fetuses with posterior urethral valve in comparison with controls. Conclusions: Increased urinary levels of angiotensin‐converting enzyme 2 and of Ang‐(1‐7) in fetuses with posterior urethral valve could represent a regulatory response to the intense inflammatory process triggered by posterior urethral valve. Resumo: Objetivo: A válvula de uretra posterior é a obstrução do trato urinário inferior mais comum em crianças do sexo masculino. Uma alta porcentagem de pacientes com válvula de uretra posterior evolui para doença renal em estágio final. Estudos anteriores mostraram que citocinas, quimiocinas e componentes do sistema renina‐angiotensina contribuem para o dano renal em uropatias obstrutivas. Recentemente, descobrimos que amostras de urina de fetos com válvula de uretra posterior tinham níveis aumentados de moléculas inflamatórias. O objetivo deste estudo foi medir as moléculas de renina‐angiotensina e investigar sua correlação com marcadores inflamatórios previamente detectados nas mesmas amostras de urina de fetos com válvula de uretra posterior. Métodos: Amostras de urina de 24 fetos com válvula de uretra posterior foram coletadas e comparadas com amostras de urina de 22 recém‐nascidos saudáveis de mesma idade gestacional (controles). Os níveis dos componentes de SRA foram medidos por ensaio de imunoabsorção enzimática. Resultados: Os fetos com válvula de uretra posterior apresentaram níveis urinários aumentados de angiotensina (Ang) I, Ang‐(1‐7) e enzima conversora de angiotensina 2 em comparação com os controles. Os níveis de enzima conversora de angiotensina eram significativamente menores e os níveis de Ang II eram semelhantes nos fetos com válvula de uretra posterior em comparação com os controles. Conclusões: O aumento dos níveis urinários de enzima conversora de angiotensina 2 e de Ang‐(1‐7) em fetos com válvula de uretra posterior poderia representar uma resposta regulatória ao intenso processo inflamatório desencadeado pela válvula de uretra posterior. Keywords: Posterior urethral valve, Renin–angiotensin system, Angiotensin II, Angiotensin‐(1‐7), ACE2, Inflammation, Palavras‐chave: Válvula de uretra posterior, Sistema renina‐angiotensina, Angiotensina II, Angiotensina‐(1‐7), ACE2, Inflamação

In this study, we described an ultrasensitive and high-throughput luciferase immunosorbent assay (LISA) for qualitative and quantitative detection of anti-HIV-1 antibody. Anti-HIV antibody in serum or plasma samples was captured by protein A/G-coated microtiter plate and detected with crude cell lysates expressing Nanoluc luciferase (Nluc) enzyme fused with HIV-1 p24 or gp41 antigen without the need of protein purification. After the addition of furimazine substrate, anti-HIV antibodies were quantitatively measured as luciferase light units. LISA showed a wide linear range of detection and was about 104-fold more sensitive than ELISA. For the detection of both anti-p24 and anti-gp41, LISA showed extraordinary sensitivity (99.5% and 100%, respectively) and equivalent specificity (100%). LISA could also monitor the change in the anti-HIV-1 antibody response over time in antiretroviral therapy (ART) treated individuals, and can sufficiently distinguish between recent and long-term HIV-1 infections. Our preliminary results indicate that LISA may provide a novel universal immunoassay platform for simultaneous HIV-1 detection, quantitative measurement of anti-HIV antibodies as well as the differentiation of HIV-1 infection stages.

Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is the most common nonpolycystic genetic kidney disease, but it remains unrecognized due to its clinical heterogeneity and lack of screening test. Moreover, the fact that the clinical feature is a poor predictor of disease outcome further highlights the need for the development of mechanistic biomarkers in ADTKD. However, low abundant urinary proteins secreted by thick ascending limb cells, where UMOD is synthesized, have posed a challenge for the detection of biomarkers in ADTKD-UMOD. In the CRISPR/Cas9-generated murine model and patients with ADTKD-UMOD, we found that immunoglobulin heavy chain-binding protein (BiP), an endoplasmic reticulum chaperone, was exclusively upregulated by mutant UMOD in the thick ascending limb and easily detected by Western blot analysis in the urine at an early stage of disease. However, even the most sensitive ELISA failed to detect urinary BiP in affected individuals. We therefore developed an ultrasensitive, plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA) to quantify urinary BiP concentration by harnessing the newly invented ultrabright fluorescent nanoconstruct, termed “plasmonic Fluor.” p-FLISA demonstrated that urinary BiP excretion was significantly elevated in patients with ADTKD-UMOD compared with unaffected controls, which may have potential utility in risk stratification, disease activity monitoring, disease progression prediction, and guidance of endoplasmic reticulum-targeted therapies in ADTKD. NEW & NOTEWORTHY Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is an underdiagnosed cause of chronic kidney disease (CKD). Lack of ultrasensitive bioanalytical tools has hindered the discovery of low abundant urinary biomarkers in ADTKD. Here, we developed an ultrasensitive plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA). p-FLISA demonstrated that secreted immunoglobulin heavy chain-binding protein is an early urinary endoplasmic reticulum stress biomarker in ADTKD-UMOD, which will be valuable in monitoring disease progression and the treatment response in ADTKD.

Objectives and background In December 2019, the first case of COVID-19 was reported in Wuhan, China. Its causative virus, is a novel strain of RNA viruses with high mortality rate. There is no definitive treatment, but among available approaches the use of recovered patients’ plasma containing specific antibodies can enhance the immune response against coronavirus. However, the dearth of eligible donors and also ABO incompatibility in plasma transfusion, have limited this therapeutic method. Therefore, it is highly desirable to introduce a simple procedure that allows efficient reduction or even removal of natural ABO antibodies. Accordingly, we aimed to evaluate a RBC-mediated adsorption technique that reduces the titer of the mentioned antibodies in plasma. Methods/materials This experimental study was conducted in Kerman University of Medical Sciences, Kerman, Iran. The pre- and post-incubation antibody titers of 168 plasma samples were determined. For incubation, each plasma sample was exposed (60 min) to different percentages of RBCs at room temperature or 4 °C. Results The results evidenced that both the concentration of RBCs and temperature had significant decreasing effects on antibody titer (P

For more than a year the whole world is suffering from the COVID-19 pandemic with no treatment option in sight. Administration of plasma from convalescent donors containing anti-SARS-CoV-2 antibodies, though promising according to case reports, failed to show a clear benefit in a greater number of trials. One reason could be varying and low antibody contents in a majority of plasma units hampering standardization and clinical efficacy. Besides, other plasma components unnecessarily transfused like coagulation factors might promote hypercoagulation seen in severe COVID-19 etiopathology. We therefore hypothesized that instead of collecting whole plasma units, convalescent donors could donate solely immunoglobulins by undergoing immunoadsorption, a mode of therapy regularly applied in autoimmune diseases. Here, we report the results of the first two antibody donations performed at the University Hospital Dusseldorf. In both cases, immunoadsorptions were very well tolerated with no side effects. Collected and neutralized eluates were concentrated using tangential flow filtration increasing the concentration of immunoglobulins 10fold as compared to peripheral blood and leading to probably eight times more neutralizing antibodies than in one plasma unit. Therefore, immunoadsorption can be used as a method of antibody donation. Whether these donated antibodies can be used as passive immunization in acutely infected patients remains to be elucidated.

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

Antibody-mediated rejection of allograft is a poorly understood problem after cardiac transplantation that complicates the postoperative course and impairs the graft function and overall survival. Although plasmapheresis and intravenous immunoglobulins have been used as standard therapies for years, there is no consensus about antibody-mediated rejection therapy and most transplantation centers have their own protocols. We describe herein a successful treatment for an acute antibody-mediated rejection of cardiac allograft combining immunoadsorption, intravenous immunoglobulins, and anti-thymocyte globulin, which manifested with polymorphic ventricular tachycardia and right ventricular dysfunction.

Enzyme immunoassays are widely used for detection of analytes within various samples. However, enzymes as labels suffer several disadvantages such as high production cost and limited stability. Catalytic nanoparticles (nanozymes) can be used as an alternative label in immunoassays overcoming the inherent disadvantages of enzymes. Prussian blue nanoparticles (PBNPs) are nanozymes composed of the Fe4[Fe(CN)6]3-based coordination polymer. They reveal peroxidase-like activity and are capable of catalyzing the oxidation of colorless 3,3',5,5'-tetramethylbenzidine in the presence of H2O2 to form intensely blue product. Here, we introduce the method for conjugation of PBNPs with antibodies and their application in nanozyme-linked immunosorbent assay (NLISA). Sandwich NLISA for detection of human serum albumin in urine was developed with limit of detection (LOD) of 1.2 ng·mL-1 and working range up to 1 μg·mL-1. Furthermore, the microbial contamination of Salmonella Typhimurium in powdered milk was detected with LOD of 6 × 103 colony-forming units (cfu)·mL-1 and working range up to 106 cfu·mL-1. In both cases, a critical comparison with the same immunoassay but using native peroxidase as label was realized. The achieved results confirmed the suitability of PBNPs for universal and robust replacement of enzyme labels.

Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating2-fold more hybridoma clones in25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.

Autoimmune encephalitis is a severe inflammatory disorder of the brain. The discovery that several non-infectious forms of encephalitis are associated with autoantibodies was a breakthrough in the care of this previously untreatable group of patients. The correlation of antibody type and titer with pattern and severity of symptoms was essential for the initiation of immunotherapies. First line therapy consists of steroids, intravenous immunoglobulins, plasma exchange or immunoadsorption. Rapid elimination of autoantibodies using selective immunoadsorption and avoiding the disadvantage of plasma substitution is a pathophysiologically guided therapeutic approach, and has been proven to be an effective therapeutic option as part of multimodal immunotherapy.

Key Points Reduction of APL antibodies by immunoadsorption may be a lifesaving therapy for the management of DAH with high titer of APL antibodies. Autologous HSCT may be a valid treatment option in patients with primary APS and no response to standard immunosuppressive therapy.

Adeno-associated virus (AAV)–based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

BACKGROUND Allogeneic adsorption (alloadsorption) involves incubating an aliquot of patient plasma with red blood cells (RBCs) of a known phenotype to facilitate removal of autoantibodies and allow for detection of remaining, potentially clinically significant alloantibodies. Alloadsorptions are routinely performed using fresh or frozen donor RBCs having particular Rh and Kidd phenotypes, and are usually enzyme or ZZAP treated before adsorptions. RBC stroma would provide a method to prepare large amounts of adsorption material that may be stored frozen and used when needed. STUDY DESIGN AND METHODS A total of 309 samples were presented to our institutions (American Red Cross, Southwest Region Texas laboratories) with serologic positive IgG autoantibodies, had allogeneic RBC stromal adsorptions performed, and underlying alloantibodies identified. After papain treatment of RBCs from group O, K- individuals who were R1 R1 , R2 R2 , and rr where at least one individual was Jk(a+b-) and one individual Jk(a-b+), stroma was prepared using digitonin digestion of the RBC membrane. Large amounts of stroma could be obtained and were aliquoted and frozen at -18°C or less for up to 2 years. RESULTS One hundred seventeen of 309 (38%) samples demonstrated alloantibodies following stromal alloadsorptions. Twenty-two different alloantibodies were identified in the stroma-adsorbed plasma, with an average of two stromal adsorptions resolving the autoantibody reactivity. The specificities of alloantibodies underlying the autoantibody included those in the Rh system (112), Kell system (24), Duffy system (14), Kidd system (30), MNS system (19), Lutheran system (2), and Diego system (2). CONCLUSION Successful removal of autoantibody using RBC stromal adsorption was obtained in 308 samples and allowed for the identification of underlying alloantibodies in 117 samples. Preparation and use of RBC stroma should significantly benefit immunohematology reference laboratories in their antibody investigations.

Human astroviruses are an important cause of viral gastroenteritis globally, yet few studies have investigated the serostatus of adults to establish rates of previous infection. Here, we applied biolayer interferometry immunosorbent assay (BLI-ISA), a recently developed serosurveillance technique, to measure the presence of blood plasma IgG antibodies directed towards the human astrovirus capsid spikes from serotypes 1–8 in a cross-sectional sample of a United States adult population. The seroprevalence rates of IgG antibodies were 73% for human astrovirus serotype 1, 62% for serotype 3, 52% for serotype 4, 29% for serotype 5, 27% for serotype 8, 22% for serotype 2, 8% for serotype 6, and 8% for serotype 7. Notably, seroprevalence rates for capsid spike antigens correlate with neutralizing antibody rates determined previously. This work is the first seroprevalence study evaluating all eight classical human astrovirus serotypes.

Improving the performance of conventional enzyme-linked immunosorbent assay (ELISA) is of great importance to meet the demand of early clinical diagnosis of various diseases. Herein, we report a feasible enzyme-free immunosorbent assay (EFISA) system using antibody conjugated Au-Pt/SiO2 nanocomposites (APS NCs) as labels. In this system, Au-Pt/SiO2 nanospheres (APS NPs) were first synthesized by wet chemical method and exhibited intrinsic peroxidase and catalase-like activity with excellent water-solubility. Then APS NCs were utilized as labels to replace HRP conjugated antibody, and Fe3O4 magnetic beads (MBs) to entrap the analyte. To discuss the performance of EFISA system, Human IgG was served as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples. The system boosted the detection limit of HIgG to 75pgmL(-1) with a RSD below 5%, a 264-fold improvement as compared with conventional ELISA. This is the first time that APS NCs have been used and successfully optimized for the sensitive dilution detection of PCV2 antibody (5:10(7)) in ELISA. Besides, APS NCs have advantages related to low cost, easy preparation, good stability and tunable catalytic activity, which make them a potent enzyme mimetic candidate and may find potential applications in bioassays and clinical diagnostics.

Liver disease, in the form of hepatocellular carcinoma (HCC) accounts for > 700,000 deaths worldwide. A major reason for this is late diagnosis of HCC. The currently used biomarker, serum alpha-fetoprotein (AFP) is elevated in 40–60% of those with HCC and other markers that can either compliment or replace AFP are desired. Our previous work has identified a number of proteins that contain altered glycans in HCC. Specifically, these altered glycans were increased levels of core and outer arm fucosylation. To determine the clinical usefulness of those identified glycoproteins, a plate based assay was developed that allowed for the detection of fucosylated glycoforms. While this method was applicable to a number of independent patient sets, it was unable to specifically detect fucosylated glycoforms in many patient samples. That is, some material was present in serum that led to non-specific signal in the lectin- fluorescence -linked immunosorbent assay (lectin-FLISA). To address this issue, a systematic process was undertaken to identify the material. This material was found to be increased levels of lectin reactive IgM. Removal of both IgG and IgM using a multi-step protein A/G incubation and filtration step removed the contaminating signal and allowed for the analysis of specific protein glycoforms. This assay was subsequently used on two sample sets, one that was shown previously to be unable to be tested via a lectin FLISA and in a larger independent sample set. The clinical usefulness of this assay in the early detection of HCC is discussed.

BackgroundThese last months, dozens of SARS-CoV-2 serological tests have become available with varying performances. A major effort was completed to compare 17 serological tests.MethodsIn a preliminary phase, we compared 17 IgG, IgM, IgA and pan Ig serological tests including ELISA, LFA, CLIA and ECLIA on a panel of 182 sera, comprising 113 sera from hospitalized patients with a positive RT-PCR, and 69 sampled before 1stNovember 2019, expected to give a positive and negative results, respectively. In a second phase, the five best performing and most available tests were further evaluated on a total of 582 sera (178 and 404 expected positive and negative, respectively), allowing the assessment of 20 possible cross-reactions with other virus.ResultsIn the preliminary phase, among eight IgG/pan-Ig ELISA or CLIA/ECLIA tests, four had a sensitivity and specificity above 90% and 98% respectively, and on six IgM/IgA tests, only one was acceptable. Only one LFA test on three showed good performances for both IgG and IgM. For all the tests IgM and IgG aroused concomitantly. In the second phase, no tests showed particular cross-reaction. We observed an important heterogeneity in the development of the antibody response, and that anti-nucleocapside (anti-N) antibodies appeared earlier than the anti-spike (anti-S) proteins.ConclusionsThe identified SARS-CoV-2 serology tests may be used for the diagnostic of CoviD-19 for negative RT-PCR patients presenting severe to mild suggestive symptoms or particular clinical presentation. Detection of both anti-N and anti-S could be complementary to increase the sensitivity of the analysis.

Autoimmune neurological diseases are often treated by immunoadsorption using a conventional plasma separator and tryptophan-immobilized column (IA). However, there is only one case report on treatment with immunoadsorption using a selective plasma separator and tryptophan-immobilized column (SeIA) in clinical practice. This study aimed to investigate the removal characteristics of antibodies against acetylcholine receptors (AChRAb), immunoglobulin G, fibrinogen, and factor XIII (FXIII) in IA and SeIA in four patients with myasthenia gravis. A total of 19 sessions of immunoadsorption were performed (five sessions of IA and 14 sessions of SeIA) when the processed plasma volume was 2 L. The corresponding reductions were 52.5% ± 6.2% for AChRAb, 58.8% ± 4.2% for fibrinogen, and 36.9% ± 5.5% for FXIII after one session of IA. The corresponding reductions were 45.2% ± 9.9% for AChRAb, 3.5% ± 6.9% for fibrinogen, and -4.6% ± 11.1% for FXIII after one session of SeIA. The removal rates for AChRAb, fibrinogen, and FXIII in IA were significantly higher than those in SeIA. IA could effectively remove AChRAb, and SeIA could retain fibrinogen and FXIII. IA can be combined with SeIA, resulting in both IgG autoantibodies removal by IA and retention of coagulation factors by SeIA.

Background Patients in China with juvenile-onset myasthenia gravis present early, with a high prevalence of purely ocular symptoms, spontaneous remission rates, and low antibody seropositivity. Antibody detection using a cell-based assay has been reported to increase the diagnostic sensitivity in adult-onset myasthenia gravis. However, this method in patients with juvenile-onset myasthenia gravis has not been investigated. Methods Patients with juvenile-onset myasthenia gravis who had not received prednisone or immunosuppressive therapy were recruited between June 2015 and April 2018 at the Huashan Hospital. Clinical information was collected. Serum anti-acetylcholine receptor antibodies were detected via cell-based assay with HEK293T cells expressing acetylcholine receptor subunits and rapsyn. Additionally, the IgG antibody subclass was identified. Results Eighty-two patients with juvenile-onset myasthenia gravis were enrolled in the current study. Among them, 48 patients were anti-acetylcholine receptor positive (58.5%) and 34 were seronegative (41.5%), as assessed via enzyme-linked immunosorbent assay. Cell-based assay yielded 63 positive subjects (76.8%) and 19 seronegative subjects (23.2%). All the enzyme-linked immunosorbent assay-positive samples showed robust immunofluorescence in the cell-based assay, whereas 15 of 34 enzyme-linked immunosorbent assay-negative patients (44.1%) were found to have low-affinity acetylcholine receptor antibodies. Among all the cell-based assay-positive patients, 41 were positive for both adult and fetal acetylcholine receptor antibodies (50.0%), 18 were found positive for only adult acetylcholine receptor antibodies (21.9%), and four were found to possess only fetal acetylcholine receptor antibodies (4.9%). Fifteen antibody-positive samples underwent subclassification and were confirmed to be IgG1 subclass predominant (n = 15, including eight adult and fetal acetylcholine receptor antibody positive, five only adult acetylcholine receptor antibody positive, and two only fetal acetylcholine receptor antibody positive). There were no significant differences in clinical features among patients with different antibody profiles. Conclusions The cell-based assay showed increased sensitivity in acetylcholine receptor antibody detection in Chinese patients with juvenile-onset myasthenia gravis, and most cases of Chinese juvenile-onset myasthenia gravis are still acetylcholine receptor autoantibody mediated. Furthermore, the antibodies detected are predominately of the IgG1 subclass.

Zika virus (ZIKV) has emerged as a major global public health concern due to its link as a causative agent of human birth defects. Laboratory diagnosis of suspected ZIKV infections by serological testing of specimens collected a week or more after symptom onset primarily relies on detection of anti-ZIKV-specific IgM antibodies by enzyme-linked immunosorbent assay coupled with detection of ZIKV-specific neutralizing antibody by neutralization tests. A definitive diagnosis based on serological assays is possible during primary ZIKV infections; however, due to the cross-reactivity of antibodies elicited during flaviviral infections, a definitive diagnosis is not always possible, especially among individuals who have previously been exposed to closely related flaviviruses, such as dengue virus (DENV). Here, we investigated the neutralizing IgM antibody profiles of 33 diagnostic specimens collected from individuals with suspected primary and secondary flaviviral infections acquired when visiting areas experiencing active ZIKV transmission in 2015 and 2016. Specimens collected between 1 day and 3 months postexposure were tested for ZIKV and dengue virus type 1 (DENV1) and type 2 (DENV2) by the plaque reduction neutralization test (PRNT) before and after IgG depletion. We found that IgG depletion prior to neutralization testing had little effect in differentiating samples from individuals with secondary infections taken less than 3 weeks postexposure; however, IgG depletion significantly reduced the cross-reactive neutralizing antibody titers and increased the percentage of cases discernible by PRNT from 15.4% (95% confidence interval [CI], 4.3 to 42.2%) to 76.9% (95% CI, 49.7 to 91.8%) for samples collected between roughly 3 and 12 weeks postexposure. These results highlight the potential of IgG depletion to improve the specificity of PRNT for better confirmation and differential diagnosis of flavivirus infections.

The latest therapeutic approaches to Alzheimer disease are using intravenous immunoglobulin (IVIG) products. Therefore, the detailed characterization of target-specific antibodies naturally occurring in IVIG products is beneficial. We have focused on characterization of antibodies isolated against tau protein, a biomarker of Alzheimer's disease, from Flebogamma IVIG product. The analysis of IgG subclass distribution indicated skewing toward IgG3 in anti-tau-enriched IgG fraction. The evaluation of their reactivity and avidity with several recombinant tau forms was performed by ELISA and blotting techniques. Truncated non-phosphorylated tau protein (amino acids 155-421) demonstrated the highest reactivity and avidity index. We provide the first detailed insight into the reactivity of isolated natural antibodies against tau protein.

Background To date, there have been no studies evaluating the effect of isolated lipoprotein(a) (Lp(a)) lowering therapy on carotid atherosclerosis progression. Methods We enrolled 30 patients who had coronary heart disease (CHD) verified by angiography, Lp(a) level ≥50 mg/dL, and low density lipoprotein cholesterol (LDL-C) level ≤2.6 mmol/L (100 mg/dL) on chronic statin therapy. Subjects were allocated in a 1:1 ratio to receive apheresis treatment on a weekly basis with immunoadsorption columns ("Lp(a) Lipopak" ® , POCARD Ltd., Russia) added to atorvastatin, or atorvastatin monotherapy. The primary efficacy end-point was the change from baseline in the mean intima-media thickness (IMT) of the common carotid arteries. Results After one month run-in period with stable atorvastatin dose, LDL-C level was 2.3 ± 0.3 mmol/L and Lp(a) – 105 ± 37 mg/dL. As a result of acute effect of specific Lp(a) apheresis procedures, Lp(a) level decreased by an average of 73 ± 12% to a mean of 29 ± 16 mg/dL, and mean LDL-C decreased by 17 ± 3% to a mean of 1.8 ± 0.2 mmol/L. In the apheresis group, changes in carotid IMT at 9 and 18 months from baseline were −0.03 ± 0.09 mm (p = 0.05) and −0.07 ± 0.15 mm (p = 0.01), respectively. In the atorvastatin group no significant changes in lipid and lipoprotein parameters as well as in carotid IMT were received over 18-month period. Two years after study termination carotid IMT increased by an average of 0.02 ± 0.08 mm in apheresis group and by 0.06 ± 0.10 mm in the control group (p = 0.033). Conclusion Isolated extracorporeal Lp(a) elimination over an 18 months period produced regression of carotid intima–media thickness in stable CHD patients with high Lp(a) levels. This effect was maintained for two years after the end of study. Trial Registration: Clinicaltrials.gov (NCT02133807).

One major hallmark of atopic dermatitis (AD) is the elevated level of total serum IgE, which has been reported to be partly of the autoreactive type in a subset of patients. Immunoadsorption (IA) has been successfully applied in various classical autoantibody-mediated diseases such as pemphigus. Recent reports proposed the use of IA also for patients with severe AD and high total serum IgE levels. In this mini-review, we summarize the current knowledge about this novel treatment approach for AD and briefly discuss the so far incompletely known role of autoreactive IgE as potential target of IA therapy in this common inflammatory skin disorder.

Glial fibrillary acidic protein (GFAP) is expressed in the central nervous system and the level of GFAP normally rises with brain injury and astroglial tumors. So, serum GFAP is used as a marker for diagnosing various types of brain damage and astroglial tumors. In this study, a new sensor based on carbon dots (CDs) linked with antibodies to specifically detect GFAP in human serum was developed. Anti-GFAP (Ab1) linked with protein A/G agarose resin (PA/G) as a capture antibody (PA/G-Ab1) and anti-GFAP (Ab2) labeled with CDs as a detection antibody (CDs-Ab2) were prepared firstly. Then the CD-linked antibody immunosorbent assay (CLAISA) method was constructed based on the sandwich conjunction reaction among PA/G-Ab1, GFAP, and CDs-Ab2. CLAISA, using the fluorescence of PA/G-Ab1-GFAP-Ab2-CDs as the direct signal, enabled the proposed immunosensor to detect GFAP sensitively with a linear range of 0.10-8.00 ng ml-1 and a detection limit of 25 pg ml-1. This method was applied to the determination of GFAP in human serum by the standard addition method, and the results showed high accuracy and precision. Considering the easy synthetic process and excellent performance of CLAISA, this method has great potential to be used to monitor GFAP in the clinic.

We retrospectively evaluated the ability of protein-A immunoadsorption (IA) as compared to that of conventional plasma exchanges (PE) in reducing the mean fluorescence intensity (MFI) of anti-HLA antibodies assessed by the single antigen assay in sensitized renal transplant recipients. Change in MFI of 441 anti-HLA antibodies was measured after 1 single session of IA or after 3 consecutive daily PE sessions. While both strategies were able to significantly lower the amount of anti-HLA antibodies, the relative reduction in MFI was higher after IA as compared to PE (-69 vs. -58%, respectively, p = 0.003). This better efficacy of IA was observed despite a lower total volume of treated plasma (105 ± 6 vs. 160 ± 16 ml/kg after IA and after PE, respectively). Our data suggest a higher efficiency of IA over conventional PE sessions to remove anti-HLA antibodies and call for a larger evaluation of IA to confirm its potential added value in desensitization protocols.

Introduction Immunoadsorption (IA) is increasingly used instead of plasma exchange due to lower risk of side effects and a higher selectivity. As a consequence of the reduction of immunoglobulins (Ig), the rate of infectious complications might increase in those patients. We therefore aimed to investigate the infection rate following IA without intravenous IG (IVIG) substitution in our apheresis center, where patients do not receive IVIG on a regular basis. Material and methods We conducted a retrospective analysis of the IA treatments performed between 2010 and 2015 without IVIG substitution and collected data on patient age, diagnosis, number of IA treatments, serum levels of Ig, total protein, albumin, C-reactive protein (CRP) and infectious complications that occurred within 2 months after the IA treatment cycle. Results A total number of 52 patients (27 females) received at least 5 IA sessions using the following adsorbers: TheraSorb™-Ig (n = 3), TheraSorb™-Ig flex (n = 44), TheraSorb™ Ig pro (n = 1) and TheraSorb™-IgE (n = 5). The median number of treatment sessions was 8.8 [range 5–16], the median IgG reduction was 82 [11–99] %. Serum albumin was decreased by 8%. The median CRP levels remained normal until the end of therapy and within 2 months after that (3.10 and 4.30 mg/L respectively). Only 4 patients had infections (7.7%). Three of them received additional immunosuppressive therapy. Conclusions Immunoadsorption leads to a significant reduction of IgG. CRP as inflammatory marker is not affected. Even without substitution of IVIG the complication rate directly linked with IA is low and questionable.

Atopic dermatitis (AD) is a common disease affecting up to 10-20% of the population with the largest disease burden in childhood. Treatment options include basic emollient treatment, topical as well as systemic immunosuppressants. The pathogenesis is complex and among various triggers, genetic predisposition and immunological alterations contribute to development of disease. Atopy is common in patients with AD and many patients have high levels of Immunoglobulin E (IgE), some of which recognizes exogenous or auto/self-allergens. Treatment options targeting IgE such as specific immunotherapy against e.g. house dust mites or using anti-IgE antibodies (omalizumab) showed variable results that were not convincing. We now review recent data on the application of unspecific and IgE-selective immunoadsorption (IA) in AD. All in all, 53 patients have been treated with non-specific pan Ig IA and 28 patients with IgE-selective IA. Side effects were rarely seen. The efficacy of IgE depletion was generally high (

The human antibody repertoire is a unique repository of information regarding infection, inflammation, and autoimmunity of the past, present, and future. However, antibodies can span vast ranges of concentrations with varying affinities and the repertoire is often heavily polarized by a few species. These complexities lead to difficulties detecting and characterizing low abundance antibody species that may be relevant to disease. We therefore developed a method to selectively remove antibodies from a sample in proportion to the titer of the species prior to analysis, referred to as high-titer depletion (HTD). Peptides from a large random peptide display library were enriched towards binding high-titer antibody species and utilized as binding reagents to deplete the corresponding species from the specimen. HTD enabled the discovery of antibody binding specificities using random peptide library screening with reduced cross-reactivity and background signal and improved coverage of low abundance species. With HTD, three monoclonal antibody species were detected at concentrations at least an order of magnitude lower than without HTD. Additionally, 92 serum antibody specificities were readily discovered from an individual specimen using HTD compared to only 25 specificities without HTD. Parameters affecting the extent of depletion such as the concentration of depleted serum were also adjusted to reproducibly improve the coverage of antibody specificities. These results demonstrate that HTD could be employed for the discovery of rare specificities related to disease and enable extensive characterization of the antibody repertoire. Moreover, the strategy of depletion in proportion to titer could be extended to other applications with complex biological samples to improve discovery.

Background. Recipient desensitization using blood group (BG)-specific immunoadsorption (ABO-IA) has proven to enable successful kidney transplantation across major ABO barriers. In this context, the efficiency of non-antigen-specific (semiselective) IA adsorbers has not yet been established. The objective of our study was to quantify anti-A/B antibody depletion by protein A-, peptide ligand- and anti-human immunoglobulin-based semiselective IA in comparison to ABO-IA. Methods. Eight ABO-IA-treated transplant candidates and 39 patients subjected to semiselective IA for a variety of different indications outside the context of ABO-incompatible transplantation were included. Antibody patterns (IgG, IgG1-4 subclasses, IgM, C4d-fixing reactivities) were analysed applying conventional agglutination testing and flow cytometry. Results. As assessed by sensitive flow cytometric antibody detection, ABO-IA-based desensitization led to a profound even though often incomplete reduction of anti-A/B reactivities. Persistent complement- or non-complement-fixing reactivities, however, were not associated with transplant rejection or capillary C4d deposition. Single sessions of semiselective IA turned out to be more effective than ABO-IA in decreasing levels of anti-A/B IgG [median reduction to 28 versus 59% (ABO-IA) of baseline values, P < 0.001). In contrast, BG-specific IgM (74 versus 30%, P < 0.001) and IgG3 (72 versus 42%, P < 0.05) were reduced to a lesser extent, without differences between tested adsorber types. Analysis of four consecutive IA sessions revealed that inferior efficiency could not be overcome by serial treatment. Conclusion. Our observation of limited adsorption capacities regarding distinct BG-specific Ig (sub)classes suggests caution in applying semiselective IA techniques in ABO-incompatible kidney transplantation

Immunoadsorption with subsequent immunoglobulin substitution (IA/IgG) represents a therapeutic approach for patients with dilated cardiomyopathy (DCM). Here, we studied which molecular cardiac alterations are initiated after this treatment. Transcription profiling of endomyocardial biopsies with Affymetrix whole genome arrays was performed on 33 paired samples of DCM patients collected before and 6 months after IA/IgG. Therapy-related effects on myocardial protein levels were analysed by label-free proteome profiling for a subset of 23 DCM patients. Data were analysed regarding therapy-associated differences in gene expression and protein levels by comparing responders (defined by improvement of left ventricular ejection fraction ≥20 % relative and ≥5 % absolute) and non-responders. Responders to IA/IgG showed a decrease in serum N-terminal proBNP levels in comparison with baseline which was accompanied by a decreased expression of heart failure markers, such as angiotensin converting enzyme 2 or periostin. However, despite clinical improvement even in responders, IA/IgG did not trigger general inversion of DCM-associated molecular alterations in myocardial tissue. Transcriptome profiling revealed reduced gene expression for connective tissue growth factor, fibronectin, and collagen type I in responders. In contrast, in non-responders after IA/IgG, fibrosis-associated genes and proteins showed elevated levels, whereas values were reduced or maintained in responders. Thus, improvement of LV function after IA/IgG seems to be related to a reduced gene expression of heart failure markers and pro-fibrotic molecules as well as reduced fibrosis progression. Electronic supplementary material The online version of this article (doi:10.1007/s00395-016-0569-y) contains supplementary material, which is available to authorized users.

The detrimental impact of preformed anti-HLA donor-specific antibodies (DSA) is well defined, contrarily to non-donor-specific antibodies (NDSA). We sought to evaluate their clinical impact in a cohort of 724 kidney graft recipients in whom anti-HLA antibodies were thoroughly screened and identified in pre-transplant sera by solid-phase assays. NDSA or DSA were detected in 100 (13.8%) and 47 (6.5%) recipients respectively, while 577 (79.7%) were non-allosensitized (NaS). Incidence of antibody-mediated rejection at 1-year was 0.7%, 4.0% and 25.5% in NaS, NDSA and DSA patients, respectively (NaS vs. NDSA P=0.004; NaS vs. DSA P0.001; NDSA vs. DSA P0.001). Graft survival was lowest in DSA (78.7%), followed by NDSA (88.0%) and NaS (93.8%) recipients (NaS vs. NDSA P=0.015; NaS vs. DSA P0.001; NDSA vs. DSA P=0.378). Multivariable competing risk analysis confirmed both NDSA (sHR=2.19; P=0.025) and DSA (sHR=2.87; P=0.012) as significant predictors of graft failure. The negative effect of NDSA and DSA on graft survival was significant in patients receiving no induction (P=0.019) or an anti-IL-2 receptor antibody (P0.001), but not in those receiving anti-thymocyte globulin (P=0.852). The recognition of the immunological risk associated with preformed DSA but also NDSA have important implications in patients' risk stratification, and may impact clinical decisions at transplant.

Allergen-specific IgE measurements and the clinical history are the cornerstones of allergy diagnosis. During the past decades, both characterization and standardization of allergen extracts and assay technology have improved. Here we discuss the uses, advantages, misinterpretations, and limitations of ImmunoCAP IgE assays (Thermo Fisher Scientific/Phadia, Uppsala, Sweden) in the field of allergology. They can be performed as singleplex (ImmunoCAP) and, for the last decade, as multiplex (Immuno Solid-phase Allergen Chip [ISAC]). The major benefit of ImmunoCAP is the obtained quantified allergen-specific IgE antibody level and the lack of interference from allergen-specific IgG antibodies. However, ImmunoCAP allergen extracts are limited to the composition of the extract. The introduction of allergen molecules has had a major effect on analytic specificity and allergy diagnosis. They are used in both singleplex ImmunoCAP and multiplex ImmunoCAP ISAC assays. The major advantage of ISAC is the comprehensive IgE pattern obtained with a minute amount of serum. The shortcomings are its semiquantitative measurements, lower linear range, and cost per assay. With respect to assay performance, ImmunoCAP allergen extracts are good screening tools, but allergen molecules dissect the IgE response on a molecular level and put allergy research on the map of precision medicine.

Antibodies against MuSK seem to be the pathogenic factor in approximately 5–8% of myasthenia gravis (MG) patients. We aim to develop an antigen-specific therapy in which only MuSK antibodies will be removed from patients' plasma using MuSK extracellular domain (MuSK-ECD) as immunoadsorbent. We showed that two different immunoadsorbents, very efficiently and selectively depleted the MuSK antibodies from all tested sera, were stable during the procedure and were reusable. Furthermore, animal experiments showed that the treatment has no toxic effects to the animals. We conclude that the MuSK-ECD-mediated immunoadsorption can be used as an efficient antigen-specific therapy for MuSK-MG.

Myasthenia gravis is an autoimmune disease usually caused by autoantibodies against the muscle nicotinic acetylcholine receptor (nAChR). Current treatments are not specific, and thus often cause side effects. Here, we elaborate on our previous findings on antigen specific immunoadsorption towards scaling up the method as well as testing whole blood apheresis. The average percent of plasma or whole blood immunoadsorption was up to 79.5%±2.9. Moreover, neither pyrogens were co-administered nor did complement activation occur after immunoadsorption. Thus, antigen-specific apheresis of anti-AChR autoantibodies seems a safe and effective treatment for myasthenia gravis that can be scaled up for clinical testing.

Immunoassays are commonly used for clinical diagnosis, although interferences have been well documented. The streptavidin-biotin interaction provides an efficient and convenient method to manipulate assay components and is currently used in several immunoassay platforms. To date, there has been no report in the literature of interference from endogenous anti-streptavidin antibodies; however, such antibodies would potentially affect multiple diagnostic platforms. We report results from a patient being treated for thyroid dysfunction who demonstrated a T-uptake result of less than 0.2 and a nonlinear thyroid stimulating hormone dilution that suggested an immunoassay interference. Protein-A sepharose pretreatment corrected the nonlinear dilution and revealed an interference trend of falsely decreased results, as measured by sandwich assay, and falsely elevated results, as measured by competitive assay. The results of streptavidin-agarose adsorption were comparable to adsorption with protein-A sepharose. To our knowledge, this is the first published description of an endogenous anti-streptavidin antibody interfering with clinical laboratory assays.

Intravenous immunoglobulin (IVIG) products are being investigated as possible therapeutic agents for mild cognitive impairment and Alzheimer's disease (AD). Anti-Aβ antibodies have been measured by ELISA in unfractionated IVIG products and in affinity-purified antibodies from these products, but it is unclear if similar results are obtained with these two approaches. Measurements of anti-Aβ antibodies in unfractionated IVIG may be confounded by the presence of polyvalent antibodies which can bind to multiple antigens, including those on ELISA plates; whether this is an issue when measuring anti-Aβ antibodies in purified antibody eluates from IVIG is also unknown. The objective of this study was to clarify these issues. The concentrations of specific antibodies to Aβ1-42 monomer and the percentages of specific binding to it were compared via ELISA between three unfractionated IVIG products (Gamunex [Talecris], Gammagard [Baxter], and Flebogamma [Grifols]) and their affinity-purified anti-Aβ antibodies. The concentrations of anti-Aβ antibodies in unfractionated IVIG products were higher than in their respective purified anti-Aβ eluates, and the rank order of the IVIG products with respect to their anti-Aβ concentrations differed between the two types of samples. The percentages of specific binding to Aβ were lower for unfractionated IVIG than for purified anti-Aβ eluates. These findings indicate that ELISA measurements of specific anti-Aβ antibodies and percentages of specific binding to Aβ produce different results depending upon whether these measurements are made in unfractionated IVIG products or their purified anti-Aβ antibodies. Polyvalent binding occurs even with purified anti-Aβ antibodies eluated from IVIG products, but it is less extensive than with unfractionated IVIG.

We report on a novel, high-dimensional method to detect autoantibodies that are complexed with their natural autoantigens. Specifically, autoantibody-autoantigen complexes in serum or plasma are directly incubated onto a high-density antibody microarray. Detection of the bound autoantibody-antigen complex is made via fluorescently labeled anti-human immunoglobulin G or other immunoglobulin isotype secondary antibodies and quantification in a microarray scanner. Uncomplexed antibodies do not interfere with this assay. The whole process is very rapid and applicable for high-throughput screening without the need for production of proteins or immunoglobulin purification from the samples. Using these methods, we found that plasma from healthy individuals contains hundreds of autoantibodies complexed with cellular proteins. Thus, this highly sensitive, multiplex method is capable of discovering new autoantibody-antigen or circulating immune complexes many of which will likely be useful for disease detection and characterization.

Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are the key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/anti-B quantification and specificity determination or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered Chinese hamster ovary (CHO) cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains. Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) (PSGL-1/mIgG(2b)) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or α1,2Fuc-T2 and the A or B gene-encoded α1,3GalNAcT or α1,3Gal-T, respectively. Selected clones with the correct glycophenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography-mass spectrometry was used to characterize the released O-glycans. Clones producing PSGL-1/mIgG(2b) carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps. The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in enzyme-linked immunosorbent assay or as affinity matrices in IA columns is explored.

For establishing cells that secrete antibodies most efficiently (e.g., hybridomas, CHO (Chinese hamster ovary) cells), the screening and subsequent breeding of promising cells have been performed at the single-colony level, which requires several weeks to propagate a substantial number of cells by forming colonies from single cells for evaluation by the conventional assays. However, this screening process lacks high-throughput performance in time and colony numbers. Therefore, development of novel methods is expected to identify single cells secreting higher amounts of antibodies in real-time and in a nondestructive manner without colony formation. In this study, we prepared lipid-labeled antimouse IgG Fc antibodies (capture molecules) that were uniformly displayed on the surface of candidate cells. Secreted nascent antibodies were subsequently sandwiched between capture molecules and fluorescence-labeled antimouse IgG F(ab')(2) F(ab')(2) (detection molecules). This newly developed method is hereinafter referred to as a cell surface-fluorescence immunosorbent assay (CS-FIA). The fluorescence intensity of each cell was found to correlate well with the amount of sandwiched antibodies (from 6.25 fg/cell to 6.40 pg/cell). When about 4 × 10(3) cells of mouse hybridomas were subjected to CS-FIA, we isolated 28 hybridomas showing the highest fluorescence intensity within a day. Furthermore, after propagation of single cells to about 10(5) cells (after 2 weeks), 20 hybridomas were still able to secrete higher amounts (up to 7-fold) of antibodies than parental hybridomas. Our results demonstrate that CS-FIA is a powerful method for the single-cell-based establishment of cells that secrete most efficiently not only antibodies but also various biomolecules.

Applications of trastuzumab are limited to breast cancer patients with high Her2-expressing tumors. We developed a T-cell receptor mimic (TCRm) monoclonal antibody (hereafter called RL1B) that targets the Her2-E75 peptide (residues 369-377)-HLA-A2 complex and examined its effects in Her2-expressing cancer cells.RL1B binding affinity was determined by surface plasmon resonance and specificity was demonstrated using Her2 antigen-positive and negative tumor cell lines. Immunohistochemistry was used to assess binding to frozen sections of human carcinomas (n = 3). Antitumor activity mediated by RL1B and trastuzumab against Her2(+) tumor cell lines was evaluated using the WST-1 cell viability assay and caspase-3 and poly(ADP-ribose) polymerase cleavage assays. A xenograft mouse model (n = 6 per group) was used to assess RL1B antitumor activity. Mechanisms of RL1B-mediated cytotoxicity were evaluated with confocal microscopy, flow cytometry, and histology. All statistical tests were two-sided.RL1B bound with high specificity and affinity to the E75 peptide-HLA-A2 complex in all Her2(+) and HLA-A2(+) cancer cell lines and human carcinomas. Compared with control antibody, RL1B suppressed growth of low Her2-expressing breast tumors in mice (mean volume, RL1B vs control = 241 mm(3) vs 1531 mm(3); P = .0109) and statistically significantly increased mouse survival (P = .0098). It reduced viability compared to control monoclonal antibody-treated cells and statistically significantly increased caspase 3 activation of all Her2(+) carcinoma cell lines tested, whereas trastuzumab induced apoptosis only in high Her2-expressing cancer cells. Mechanisms of RL1B cytotoxicity were associated with antibody internalization and intracellular signaling.The TCRm RL1B could be a new approach to immunotherapy of Her2-expressing malignancies.

A patient with thymoma-associated neuromyotonia and voltage-gated potassium channel (Kv1.2 and Kv1.6) antibodies by immunoprecipitation and rat brain immunolabeling was treated successfully with immunoadsorption and cyclophosphamide. Curiously, glutamic acid decarboxylase antibodies, absent at onset, appeared later. Stiff-person syndrome was absent, but fast blink reflex recovery suggested enhanced brainstem excitability. The range of antibodies produced in thymoma-associated neuromyotonia is richer, and the timing of antibody appearance more complex, than previously suspected.

Item does not contain fulltext AIMS: Immunoadsorption with subsequent immunoglobulin G substitution (IA/IgG) represents a novel therapeutic approach in the treatment of dilated cardiomyopathy (DCM) which leads to the improvement of left ventricular ejection fraction (LVEF). However, response to this therapeutic intervention shows wide inter-individual variability. In this pilot study, we tested the value of clinical, biochemical, and molecular parameters for the prediction of the response of patients with DCM to IA/IgG. METHODS AND RESULTS: Forty DCM patients underwent endomyocardial biopsies (EMBs) before IA/IgG. In eight patients with normal LVEF (controls), EMBs were obtained for clinical reasons. Clinical parameters, negative inotropic activity (NIA) of antibodies on isolated rat cardiomyocytes, and gene expression profiles of EMBs were analysed. Dilated cardiomyopathy patients displaying improvement of LVEF (>/=20 relative and >/=5% absolute) 6 months after IA/IgG were considered responders. Compared with non-responders (n = 16), responders (n = 24) displayed shorter disease duration (P = 0.006), smaller LV internal diameter in diastole (P = 0.019), and stronger NIA of antibodies. Antibodies obtained from controls were devoid of NIA. Myocardial gene expression patterns were different in responders and non-responders for genes of oxidative phosphorylation, mitochondrial dysfunction, hypertrophy, and ubiquitin-proteasome pathway. The integration of scores of NIA and expression levels of four genes allowed robust discrimination of responders from non-responders at baseline (BL) [sensitivity of 100% (95% CI 85.8-100%); specificity up to 100% (95% CI 79.4-100%); cut-off value: -0.28] and was superior to scores derived from antibodies, gene expression, or clinical parameters only. CONCLUSION: Combined assessment of NIA of antibodies and gene expression patterns of DCM patients at BL predicts response to IA/IgG therapy and may enable appropriate selection of patients who benefit from this therapeutic intervention.

Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we developed an immunoaffinity purification and liquid chromatography-mass spectrometry (LC-MS) strategy for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. Using a purified BChE protein, we initially identified the exact phosphorylation site on the serine residue (S198) with a 108 Da modification by both MS/MS and accurately measured parent ion masses and quantified the extent of phosphorylation on S198 following paraoxon treatment to be >99.9%. Then, the phosphorylated BChE peptide in paraoxon-treated human plasma following immunoaffinity purification was successfully identified based on the accurate measured mass and retention time information initially obtained from the purified BChE protein. Thus, immunoaffinity purification combined with LC-MS represents a viable approach for the detection and quantification of phosphorylated BChE as an exposure biomarker of organophosphates and nerve agents.

Intrathecal synthesis of the antibodies specific to neurotrofic viruses: measles (M), rubella (R), Varicella-Zoster (Z), and/or H. simplex (H), known as “MRZH-reaction” plays important diagnostic role in multiple sclerosis (MS). Whereas the analysis of the oligoclonal IgG bands provides high sensitivity, the MRZH-reaction shows high specificity, and hence these methods complement each other. For the first time we applied multiplexing bead-based technology to simultaneously analyze cerebrospinal fluid (CSF) and serum concentrations of antibodies against these viruses, and to calculate the antibody specific indices (ASI’s). The method shows reasonable precision: intra-assay, 2.9–6.7%, and inter-assay, 2.0–3.2%. The results are comparable with these obtained with other methods (ELISAs), including two runs of the certified external quality control schemes. Eighty-one percent of the MS cases ( n = 27) and none of the sex- and age-matched controls ( n = 14), except one subject with “borderline” anti-measles ASI of 1.5, showed intrathecal synthesis of IgG against at least one of the viruses discussed. The ratios of the MRZH-positive cases in the MS group were: 12/22 for M, 12/19 for R, 13/26 for Z, and 7/26 for H. We conclude that the multiplexing technology can be applied as a tool to study the intrathecal immune response in the diagnosis of MS.

Treatment of pemphigus patients is still challenging and, in some cases, conventional therapy with systemic corticosteroids in combination with adjuvant corticosteroid-sparing immunosuppressive drugs is not sufficient to induce clinical remission. More recently, high-dose intravenous immunoglobulins, immunoadsorption, and the monoclonal anti-CD20 antibody, rituximab, have been established as additional successful therapeutic options. This contribution covers both conventional therapies and most current treatment strategies for pemphigus.

The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble.

It has been known for 40 years that cytotoxic human leukocyte antigen (HLA) antibodies are associated with graft rejection. However, the complement-dependent cytotoxicity assay (CDC) used to define these clinically deleterious antibodies suffers from a lack of sensitivity and specificity. Recently, methods exploiting immunoglobulin G (IgG) antibody binding to HLA single antigen beads (SAB) have overcome sensitivity and specificity drawbacks but introduced a new dilemma: which of the much broader set of antibodies defined by these methods are clinically relevant. To address this, we developed a complement-fixing C1q assay on the HLA SAB that combines sensitivity, specificity, and functional potential into one assay. We compared the CDC, IgG, and C1q assays on 96 sera having 2,118 defined antibodies and determined that CDC detects only 19% of complement-fixing antibodies detected by C1q, whereas C1q detects only 47% of antibodies detected by IgG. In the same patient, there is no predictability by IgG mean fluorescence intensity (MFI) as to which of the antibodies will bind C1q because fixation is independent of MFI values. In 3 clinical studies, C1q + antibodies appear to be more highly correlated than those detected by IgG alone for antibody-mediated rejection in hearts as well as for kidney transplant glomerulopathy and graft failure.

In the transplant recipient patients receive immunosuppressive therapy, the possibility of reactivation of the old infection or acquisition of infection from a donor's tissue increases. In this study, IgM and IgG anti-Toxoplasma immunoglobulins seroconversion in renal transplant recipients (RTRs) have been evaluated before and after transplantation. This is a prospective cohort study on a total of 102 RTRs. Two serum samples were obtained from each patient. The first was taken before administration of any immunosuppressive drugs such as corticosteroids and the second was taken 3 months after transplantation. The IgM and IgG anti-Toxoplasma antibodies were assayed by enzyme-linked flourescence assay (ELFA) and enzyme-linked immunosorbent assay (ELISA) techniques. IgM/immunosorbent agglutination assay (ISAGA) method has also been used. All RTRs were tested for toxoplasmosis before and after transplantation. ELFA identified 65 (63·7%) pre-transplantation samples as IgG+ and did not detect any positive IgM samples. However, IgM was detected in three (2·9%) post-transplantation samples by this method. Forty-nine (48%) pre-transplantation samples were reported IgG+ by ELISA and no IgM positive sample was identified by this method. ELISA has detected two (1·9%) IgM-positive reactions in post-transplantation samples. By IgM/ISAGA method, we have detected no IgM positive reactions in pre-transplantation samples, whereas 3 months later (second sampling) IgM antibody was detected in 3 (2·9%) cases. Secondary toxoplasmosis infection was observed in 30 cases per 1000 RTRs, which indicates that screening for toxoplasmosis infection should be performed in developed countries for these patients. On the other hand, as the risk of re-active toxoplasmosis infection exists in developing nations, they should consider the necessary preventive measures to control this condition.