Antibody Detection System Research Articles

Performance verification of Siemens Atellica DL IM1600 automatic immune analyzer detection system: A-TPO and A-TG.

To study the performance of the Siemens Atellica DL IM1600 automatic immune analyzer detection system for thyroid peroxidase antibodies (A-TPO) and anti-thyroglobulin antibodies (A-TG). Perform performance verification based on CNAS and CLSI related documents, including precision, accuracy, reference interval, and linearity. In precision validation, intra-batch imprecision coefficient of variation values of A-TG and A-TPO were 3.60%, 5.40%, and 3.20%, 2.20%, inter-batch imprecision coefficient of variation values were 5.40%, 4.40%, and 5.00%, 2.50%, all within the specified range. In the accuracy verification, the A-TG comparison values were -10.94%, -1.49%, -1.14%, -7.78%, -3.28%, and the A-TPO values were 21.20%, 6.42%, 14.63%, 12.40%, and 6.55%, The absolute values were all lower than the comparison requirements. The reference interval verifies that the A-TG and A-TPO test results of health patients were within the normal reference interval of 0 to 60 U/mL. In the linear range validation, A-TG linear regression y = 1.0145x - 10.093, correlation coefficient R2 = 0.9963 > 0.95, A-TPO linear regression y = 0.9985x - 18.172, correlation coefficient R2 = 0.9954 > 0.95, indicating linear linearity. The performance verification of Siemens Atellica DL IM1600 automatic immune analyzer detection system for A-TPO and A-TG was satisfactory, suitable for clinical application.

Enzyme immunoassay system for serological diagnosis of hemorrhagic fever with renal syndrome based on inactivated purified Puumala virus (Hantaviridae: Orthohantavirus)

Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS. Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent. Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA. For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays. The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.

#2555 C4d—a novel predictor of the disease progression and treatment failure in primary focal segmental glomerulosclerosis?

Abstract Background and Aims Complement system seems to play a role in the pathogenesis of primary focal segmental glomerulosclerosis (pFSGS) where, due to unpredictable clinical course and outcome, novel prognostic factors are sought. The split product of C4 and tissue marker of complement activation, C4d, is a potential candidate whose prognostic significance was determined in this study. Method Patients >18 years with features consistent with pFSGS (full blown nephrotic syndrome, diffuse podocyte foot processes effacement and exclusion of known causes of FSGS), were recruited from our Hospital registry of kidney biopsies, from 2003 to 2021. Patients with end-stage renal disease (ESRD) defined as eGFR <15 ml/min/1.73 m2 were not included. Every renal biopsy specimen was analyzed by light, immunofluorescence and electron microscopy with additional immunochemistry for C4d performed on paraffin-embedded tissue using monoclonal rabbit antibody (detection system Ventana BenchMark Ultra). Samples with at least one non-globally sclerosed glomerulus (GSG) were examined by two renal pathologists who classified every non-GSG into C4d positive and negative. Based on ratio of C4d+ non-GGS/total N of non-GSG and C4d+ nonsclerotic glomeruli (NSG)/total N of NSG, patients were stratified into two groups: <50% and ≥50%. Primary outcome was defined as composite of ESRD and initial eGFR declining >50%. Failure to meet criteria for at least partial remission at the last follow-up visit according to KDIGO 2021 Guidelines was defined as treatment failure. Numeric variables are shown as median with interquartile range (IQR) and P value <0.05 was considered as significant. Results Relevant data of 58 included patients with renal outcomes are shown in Table. Median follow up was 90.7 (IQR 48-167) months. Patients with ≥50% of C4d+ non-GSG had higher proportion of treatment failure (Table) and worse renal survival (log-rank test, p = 0.006). In multivariate Cox regression analysis, after inclusion of age, sex, initial eGFR and 24-h proteinuria, IFTA, serum albumin and C4d category, only C4d ≥50% (HR = 4.077, 95% CI 1.101-15.09, p = 0.035) and IFTA (HR = 1.052, 95% CI 1.019-1.085, p = 0.002) remained independent predictors of progression to defined endpoint. Likewise, patients with ≥50% of C4d+ NSG/total N of NSG had higher proportion of treatment failure (chi-square, p = 0.03) and worse renal survival (log-rank, p = 0.037). Conclusion C4d deposition is an independent predictor of disease progression, treatment failure and renal survival in pFSGS. Significant influence of NSG-C4d deposition on disease progression may indicate pathophysiological role of the complement system activation in pFSGS independently of the presence of glomerular sclerosis.

Rapid quantitative detection system for measles virus-neutralizing antibodies using HiBiT-tagged virus-like particles.

Immunological testing to detect neutralizing antibodies (NAbs) is important in measles (MV) infection control. Currently, the plaque reduction neutralization testis the only credible method for measuring actual virus NAbs; however, its feasibility is hampered by drawbacks, such as long turnaround times, low throughput, and the need for laboratory biosafety equipment. To solve these problems, we developed a simple and rapid MV-NAb detection system using lentivirus-based virus-like particlesincorporated with the NanoLuc fragment peptide HiBiT comprising the MV fusion protein and hemagglutinin on their exterior surface. Overall, this simple, safe, and rapid method could be used to detect MV NAbs.

A novel lateral flow immunochromatographic assay using a recombinant VP2 antigen for total antibody detection of canine parvovirus-2.

Canine parvovirus-2 (CPV-2) is a viral disease of dogs causing acute hemorrhagic gastroenteritis and myocarditis with high morbidity and mortality rates. The infection is still widespread all over the world. Vaccines developed against infection have great importance in preventing infection. However, it is difficult to recommend a practical vaccination program without knowing the antibody level of a puppy. Despite widespread vaccination, difficulties in detecting the maternal antibodies in puppies remain the main cause of vaccination failure. The hemagglutination inhibition (HAI) test is the gold standard to determine the immune status of dogs for canine parvovirus 2, but the HAI test has several disadvantages such as the need for fresh porcine blood, well-equipped laboratory, and long incubation periods. In this study, for the first time we developed a colloidal gold-based competitive lateral flow assay (cLFA) system for the rapid detection of total antibodies in canine serum using CPV-2b-VP2 derived from field isolates. The recombinantly expressed capsid protein of CPV-2 in the prokaryotic expression system was used as a labeled molecule in cLFA. We carried out studies on our cLFA system using the standard antibody solution and the clinical samples from vaccinated puppy serum. We compared the results of the LFAs with the HAI test. Competitive lateral flow assay results showed good correlation with the gold standard method, the HAI test. In the developed platform, the limit of detection of the standard antibody was determined to be 375 ng mL-1, while the cut-off level of antibodies was observed to be 1 : 40 HAI titer in clinical samples. Our reported system will be a strong alternative for CPV-2 antibody-based detection applications.

Red Cell Alloimmunization and Antibody Specificity in Multigravida Attending Antenatal Clinic at Tertiary Care Centre from Rohilkhand Region

Introduction: Red cell alloimmunization is an immuneresponse to foreign red cell antigens which can occur due totransfusion between a donor and recipient, during pregnancybetween mother and fetus and also in transplant recipients.Such disparity may result in transfusion reactions or may beassociated with hemolytic disease of the fetus and newborn.The current study was undertaken to observe the prevalenceof red cell alloimmunization and antibody specificity inmultigravida patients with respect to epidemiological (age),obstetrical, major blood group systems and transfusion-relatedparameters.Material and Methods: This prospective cross-sectional studywas conducted over a period of 1.5 years from October 2018 toApril 2020, in the patients at risk of exposure to foreign red cellantigens in multigravida patients, in the blood bank of tertiarycare medical institute of North India.Antibody screening and identification was performed on 1055multigravida females using commercially prepared O cells(IMMUCOR, USA). Statistical evaluation by Chi-square testwas performed wherever applicable. p-value of <0.05 wasconsidered statistically significant at 95% confidence interval.Results: Prevalence of red cell alloimmunization inmultigravida females was 1.99% with anti-D being the mostcommon antibody identified. Other less commonly detectedalloantibodies were anti-E, anti-Jkb (Kidds) and anti-Kpa (Kell).A statistically significant association was found between red cellalloimmunization and increase in gravida status, presence ofbad obstetric history, age (>30 years) and Rh-D status. Whencompared with other independent variables such as parityand ABO blood group of the female, no statistical significantassociation was observed.Conclusion: Maternal alloimmunization is associated withanti-D specificity and antibodies against other clinicallysignificant minor blood group antigens such as anti-E andanti-Jkb (Kidd) and anti-Kpa (Kell). Routine antibody screeningof all antenatal females, irrespective of the Rh-D status mustbe adopted by health care system for detection of clinicallysignificant antibodies that may be associated with HDFN ordelayed hemolytic transfusion reaction.

Binding of C1q to Galectin-3 Binding Protein on Microvesicles Released by Mononuclear Cells from Patients with Systemic Lupus Erythematosus

Background: Generation of galectin-3 binding protein (G3BP)-expressing microvesicles can be induced in-vitro by Toll-like receptor 9 ligation in mononuclear cells. Microvesicles co-expressing G3BP and double-stranded DNA are associated with active lupus nephritis. However, whether the microvesicular G3BP mainly deposits from circulation or is endogenously derived is unknown. In this study, we aim to delineate the origin of G3BP on in-vitro generated microvesicles by using serum as a source of native G3BP. Methods: G3BP-expressing microvesicles, generated by stimulation of systemic lupus erythematosus patient-derived mononuclear cells with the Toll-like receptor 9-agonist ODN2395, were incubated with normal human serum, heat-inactivated human serum, recombinant human C1q or human albumin. The expression of G3BP by microvesicles was examined by flow cytometry, and the binding of soluble recombinant human C1q to recombinant human G3BP was investigated by ELISA. Results: Approximately half of the microvesicles released from mononuclear cells expressed G3BP. Surprisingly, the staining was abrogated by incubation of the microvesicles with normal human serum, while incubation with heat-inactivated human serum did not have a similar effect. Reasoning that C1q might be the heat-labile factor blocking access of our G3BP antibody detection system, we incubated microvesicles with recombinant human C1q, which on average inhibited the detectable proportion of G3BP-bearing microvesicles by 87%. Soluble recombinant human C1q bound to immobilized recombinant human G3BP in a dose-dependent manner. Conclusion: Our data suggest that soluble C1q binds to G3BP on Toll-like receptor 9-induced microvesicles released from systemic lupus erythematosus patient-derived mononuclear cells. This interaction may exarcerbate inflammation in systemic lupus erythematosus but may also serve as a general mechanism for the appropriate clearance of these potentially pathogenic factors. Keywords: microvesicles, C1q, complement, clearance, Toll-like receptor 9, peripheral blood mononuclear cells, galectin-3 binding protein, systemic lupus erythematosus

Emergence of infectious diseases and role of advanced nanomaterials in point-of-care diagnostics: a review

ABSTRACT Infectious outbreaks are the foremost global public health concern, challenging the current healthcare system, which claims millions of lives annually. The most crucial way to control an infectious outbreak is by early detection through point-of-care (POC) diagnostics. POC diagnostics are highly advantageous owing to the prompt diagnosis, which is economical, simple and highly efficient with remote access capabilities. In particular, utilization of nanomaterials to architect POC devices has enabled highly integrated and portable (compact) devices with enhanced efficiency. As such, this review will detail the factors influencing the emergence of infectious diseases and methods for fast and accurate detection, thus elucidating the underlying factors of these infections. Furthermore, it comprehensively highlights the importance of different nanomaterials in POCs to detect nucleic acid, whole pathogens, proteins and antibody detection systems. Finally, we summarize findings reported on nanomaterials based on advanced POCs such as lab-on-chip, lab-on-disc-devices, point-of-action and hospital-on-chip. To this end, we discuss the challenges, potential solutions, prospects of integrating internet-of-things, artificial intelligence, 5G communications and data clouding to achieve intelligent POCs.

A New Flow Cytometry-Based Single Platform for Universal and Differential Serodiagnosis of HTLV-1/2 Infection.

In the present work, we developed and evaluated the performance of a new flow cytometry-based single platform, referred to as “FC-Duplex IgG1 (HTLV-1/2)”, for universal and differential serodiagnosis of HTLV-1/2 infection. The proposed technology employs a system for detection of IgG1 antibodies in a single competitive immunofluorescence platform by flow cytometry using fluorescently labeled MT-2/MoT cell line mix coupled to a highly sensitive development system (Biotin/Streptavidin/Phycoerythrin). The stability of fluorescent labeling and the antigenicity of MT-2 and MoT cell lines were confirmed upon storage at −20°C for 2, 6, and 12 months. The anti-HTLV-1/2 IgG1 reactivity, expressed as percentage of positive fluorescent cells (PPFC), was evaluated for each target antigen along the titration curve of test serum samples (1:32 to 1:4,096). Upon selection of target cell line and serum dilutions with higher segregation score between groups, the performance of “FIX” and “FIX & PERM” protocols was evaluated. The “FIX” protocol presented excellent performance indices (Se = 92%/Sp = 94%/AUC = 0.96; Se = 96%/Sp = 100%/AUC = 0.99) for the universal (HTLV-1/2 vs. NI) and differential (HTLV-1 vs. HTLV-2) diagnosis of HTLV-1 infection, respectively. Optimization of the “FIX” protocol using the principle of synchronous and asynchronous pairwise analysis further improved the performance of “FC-Duplex IgG1 (HTLV-1/2)”, using the “FIX” protocol for differential diagnosis of HTLV-1 and HTLV-2 infections (Se = 100%/Sp = 100%/AUC = 1.00). In conclusion, the “FC-Duplex IgG1 (HTLV-1/2)” method represents an innovation in the biotechnology segment with the potential to compose a serological kit for differential diagnosis of HTLV-1/2 infection for reference laboratories and blood centers.

Assessing HeberFast® Line Gavac, a lateral flow immunochromatographic system for the rapid detection of anti-Bm86 antibodies in Gavac vaccinated cattle

Rhipicephalus Boophilus microplus cattle tick is a scourge for livestock production. The infestations produced by this pathogen are incompletely contained by chemical treatments, with the associated environmental pollution risks. Vaccination against cattle ticks has emerged as a feasible and environmentally friendly strategy to control tick-borne diseases. In this setting, Gavac® vaccine has proven effective in decreasing cattle tick populations through antibody responses against the tick Bm86 antigen, as part of an Integrated Control Program. However, animal vaccination programs require easy and ready-to-use screening tests to follow up the immune response in vaccinated animals under field conditions. This study reports the evaluation HeberFast® Line Gavac, a lateral flow immunochromatographic system for the rapid detection of anti Bm86 antibodies in vaccinated cattle. The system was tested on 598 serum samples taken from immunized animals, arranged in three groups according to their anti-Bm86 antibody response in ELISA (209 high, 150 medium or 239 low and 100 samples from non-immunized animals. The HeberFast® Line Gavac system was assessed for sensitivity, specificity, and concordance against the ELISA reference test. Consistency was evaluated among production batches and inter-analyst reading-independent consistency at two moments: ten minutes after completing the test and after strip drying. The system showed high sensitivity (81.6%, 82.2%, and 81%), specificity (96.7, 94.6, and 93.3%), and agreement with the ELISA reference test (75%; 74%, and 71%) for high, medium and low anti-Bm86 sera, respectively. The effectiveness of the diagnosis was 87.6; 87.1; 85.9 for high, medium, and low antibody titers, respectively. Consistency among production batches and analysts was documented, and no significant differences between evaluation times were found. These results indicate that HeberFast® Line Gavac is a valuable tool for the serological surveillance of Gavac vaccinated cattle.

Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies.

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.

Correlation of Automated Chemiluminescent Method with Enzyme-Linked Immunosorbent Assay (ELISA) Antibody Titers in Convalescent COVID-19 Plasma Samples: Development of Rapid, Cost-Effective Semi-Quantitative Diagnostic Methods.

BackgroundWe investigated the utility of an automated chemiluminescent SARS-CoV-2 IgG antibody assay platform in quantifying the amount of binding antibodies present in donated convalescent plasma.MethodsA total of 179 convalescent plasma units were analyzed for the presence of SARS-CoV-2 IgG antibodies using the Beckman-Coulter chemiluminescent immunoassay (CLIA) platform. The equipment-derived numerical values (S/Co ratio) were recorded. Aliquots from the same units were subjected to enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 S1 protein. The relationship between ELISA titers and CLIA S/Co values was analyzed using linear regression and receiver operating characteristics (ROC) curve.ResultsTwenty-one samples (11.7%) had S/Co values of less than 1.0 and were deemed negative for antibodies and convalescent plasma had S/Co values between >1.0 and 5.0 (70/179, 39.1%). Fifteen units (8.4%) had negative ELISA titer. The majority of the units (95/179. 53.1%) had titers ≥1:1024. The sensitivities of ELISA to CLIA were comparable (90.5% vs 88.3%, respectively; p=0.18). There was positive linear correlation between CLIA S/Co values and ELISA IgG titer (Rho = 0.75; Spearman’s rank = 0.82, p-value = <0.0001). The agreement between the two methods was fair, with a κ index of 0.2741. Using the ROC analysis, we identified a CLIA S/Co cutoff value of 8.2, which gives a sensitivity of 90% and a specificity of 82% in predicting a titer dilution of ≥1:1024.ConclusionThe utility of automated antibody detection systems can be extended from simply a screening method to a semi-quantitative and quantitative functional antibody analysis. CLIA S/Co values can be used to reliably estimate the ELISA antibody titer. Incorporation of chemiluminescent-based methods can provide rapid, cost-effective means of identifying anti-SARS-CoV-2 antibody titers in donated plasma for use in the treatment of COVID-19 infection.

High-Throughput Screening and Triage Assays Identify Small Molecules Targeting c-MYC in Cancer Cells.

While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells. The assay was based on a dual antibody detection system using Förster/fluorescence resonance energy transfer (FRET) and was utilized to detect endogenous c-MYC protein in the MYC amplified cancer cell lines DMS273 and Colo320 HSR. The assays were miniaturized to 1536-well plate format and utilized to screen the GlaxoSmithKline small-molecule collection of approximately 2 million compounds. In addition to the HTS assay, follow-up assays were developed and used to triage and qualify compounds. Two cellular assays used to eliminate false-positive compounds from the initially selected HTS hits were (1) a cellular toxicity assay and (2) an unstable protein reporter assay. Three positive selection assays were subsequently used to qualify compounds: (1) 384-well cell cycle flow cytometry, (2) 384-well cell growth, and (3) c-MYC gene signature reverse transcription quantitative PCR (RT-qPCR). The HTS and follow-up assays successfully identified three compounds that specifically decreased c-MYC protein levels in cancer cells and phenocopied c-MYC siRNA in terms of cell growth inhibition and gene signatures. The HTS, triage, and three compounds identified are described.

A Low Cost Antibody Signal Enhancer Improves Immunolabeling in Cell Culture, Primate Brain and Human Cancer Biopsy

The use of antibodies to identify neuronal receptors, neurotransmitters, cytoskeletal elements or pathologic protein aggregates, ion channels, adhesion molecules or other cell-type specific markers, is common practice in neuroscience. Antibody detection systems are often based on confocal, epifluorescence or brightfield microscopy. Three types of technical issues can interfere with immunolabeling: low abundance of the target protein, low specific affinity of the antibody and/or signal background sometimes related to tissue fixation. Here, giving tribute to Professor Miledi’s mentorship, we propose the application of an antibody signal enhancer (ASE) solution based on glycine, hydrogen peroxide and a detergent mix as a simple, low cost, protocol variation that significantly and specifically improves the signal to noise ratio during immunostaining experiments. We describe three new settings in which ASE improves the detection of a variety of antibodies applied on long-time stored non-human primate brain sections, cell culture monolayers and on squamous carcinomas retrieved from cervical cancer patients. The significant improvement of ASE over optimized immunohistochemical protocols used in clinical practice (i.e. cancer detection) combined with its simplicity and low cost makes it an attractive method for biomedical applications.

Interaction study of peptide-PAMAM as potential bio-nanogate for detecting anti-hepatitis B surface antigen

Bio-nanogate involves synthesized or natural molecules as a ‘gate’ towards bioreceptors and responds upon the presence of targeted analytes in nanoscale dimension. Development of bio-nanogate improves analyte selectivity and signal response across various types of biosensors. The versatility of PAMAM dendrimers to form conjugates with guest molecules, such as proteins can be utilized in forming a bio-nanogate. PAMAM interaction with peptide bioreceptor for antibody detection is of interest in this study. This study investigated the interaction of synthesized immunogenic ‘a’ determinant (aD) region of hepatitis B virus surface antigen (HBsAg) with PAMAM G4 and anti-HBsAg antibody, as a potential bio-nanogate for anti-HBsAg detection. The aD peptide fused with maltose binding protein (MBP), was confirmed with Western blotting. Nano-Differential Scanning Fluorimetry (nano-DSF) study revealed that the interaction of MBP-aD with anti-HBsAg indicated a higher thermal stability as compared to its interaction with PAMAM G4. Electrochemical impedance spectroscopy showed that a higher binding constant of MBP-aD interaction with anti-HBsAg (0.92 μM−1) was observed at maximum saturation, as compared with PAMAM G4 (0.07 μM−1). Thermodynamic parameters demonstrated that MBP-aD interacted with anti-HBsAg and PAMAM G4, through van der Waals and hydrogen bonding. These analyses suggest that the weak interaction of MBP-aD and PAMAM G4 may form a potential bio-nanogate. It is hypothesized that the presence of anti-HBsAg has a higher affinity towards MBP-aD which may displace PAMAM G4 in the anti-HBsAg detection system. This interaction study is crucial as an initial platform of using peptide-PAMAM as a bio-nanogate in an antibody detection system.

Systematic Screening Using Luminex for De Novo C3d Fixing of Class II Donor-Specific Antibodies Is Correlated With Luminex Mean Fluorescence Intensity in Renal Transplant Patients.

Chronic antibody-mediated rejection is the main cause of late kidney graft loss. The presence of donor-specific antibodies in the serum is the main criterion for this diagnosis. Single antigen Luminex assays can identify donor-specific antibodies, and semiquantitative estimates of antibodies can be assessed using mean fluorescence intensity. Recent data have shown that patients whose donor-specific antibodies fix C3d have worse clinical outcomes, implying that C3d-specific Luminex assays may provide useful prognostic data. We compared C3d donor-specific antibodies with standard immunoglobulin G donor-specific antibody mean fluorescence intensities in a cohort of patients with de novo class II donor-specific antibodies and analyzed subsequent graft survival. The included kidney graft recipients received transplants between 2005 and 2015 and had developed de novo class II donor-specific antibodies. Serum was tested using the standard single antigen Luminex technique and the C3d-fixing antibody-detection system (Immucor, Herentals, Belgium). In our patient cohort, 41/924 patients (4.4%) developed class II donor-specific antibodies, and 65 serum samples were analyzed (at baseline and follow-up). Among these samples, 43 (66%) were negative for C3d donor-specific antibodies. A mean fluorescence intensity threshold of 9000 in the single antigen Luminex assay discerned all negative (from positive) C3d donor-specific antibodies, even when all single-bead results were taken into account. Sixteen patients (39%) had poor outcomes (ie, either creatinine levels had doubled or they had lost their graft) over the median follow-up of 5 years. C3d results were significantly associated with graft survival (P = .04). We found a strong correlation between C3d-fixing antibody positivity and mean fluorescence intensity strength in the setting of de novo class II donor-specific antibodies. These results further reinforce the paradigm that the higher the mean fluorescence intensity, the more complement activation occurs. Routine C3d testing is thus unnecessary in this setting.

Development of a diagnostic system for detection of specific antibodies and antigens against Middle East respiratory syndrome coronavirus.

ABSTRACTMiddle East respiratory syndrome coronavirus (MERS‐CoV) is a single‐stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS‐CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS‐CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS‐CoV. Using a recombinant full‐length S protein, an indirect ELISA was developed and found to detect MERS‐CoV‐specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS‐CoV S protein. Neither ELISA system exhibited significant intra‐assay or inter‐assay variation, indicating good reproducibility. Moreover, the inter‐day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS‐CoV.

Technical challenges and clinical relevance of single antigen bead C1q/C3d testing and IgG subclass analysis of human leukocyte antigen antibodies.

Luminex single antigen bead assays revolutionized human leukocyte antigen (HLA) antibody detection owing to their superior sensitivity compared to conventional methods. Nevertheless, the advent of higher sensitivity came at the expense of difficulty in clinical decision-making, since not all luminex detectable antibodies are clinically relevant. Therefore, new tools such as C1q/C3d assays and IgG subclass analysis emerged with the aim to discriminate the inert antibodies from the deleterious ones. Here, we provide an overview on the technical challenges related to these different HLA antibody detection systems and briefly refer to the recent literature regarding the clinical relevance of these assays, mainly in the field of kidney transplantation.

Expression of Programmed Death Ligand 1 (PD-L1) in Posttreatment Primary Inflammatory Breast Cancers and Clinical Implications

Inflammatory breast carcinoma (IBC) is rare but is the most lethal type of breast cancer. Programmed death ligand 1 (PD-L1) expression in IBCs has been understudied. In this study, tissue microarrays of 68 IBCs were immunostained with a PD-L1 antibody using an antibody clone (28-8) and detection system approved by the US Food and Drug Administration for selecting patients with non-small cell lung cancer and melanoma for anti-PD-L1 therapy. Positive PD-L1 expression was found in 25 (36.8%) of 68 samples but was not significantly associated with the clinicopathologic variables examined. Univariate analysis of overall survival (OS) revealed that worse OS was significantly associated with positive PD-L1, negative estrogen receptor, and triple-negative status. The 5-year OS rate was 36.4% for patients with PD-L1-positive IBC and 47.3% for those with PD-L1-negative IBC. In multivariate analyses, PD-L1 status remained a statistically independent predictor of OS. These findings indicate that PD-L1 inhibitors could potentially improve the clinical outcome of patients with PD-L1-positive IBC.

Antibody response to polyomavirus primary infection: high seroprevalence of Merkel cell polyomavirus and lymphoid tissue involvement.

Human polyomaviruses (HPyVs) asymptomatically infect the human population establishing latency in the host, and their seroprevalence can reach 90% in healthy adults. Few studies have focused on the pediatric population, and there are no reports regarding the seroprevalence of all the newly isolated HPyVs among Italian children. Therefore, we investigated the frequency of serum antibodies against 12 PyVs in 182 immunocompetent children from Northeast Italy, by means of a multiplex antibody detection system. Additionally, secondary lymphoid tissues were collected to analyze the presence of HPyV DNA sequences using a specific real-time PCRs or PCRs. Almost 100% of subjects were seropositive for at least one PyV. Seropositivity ranged from 3% for antibodies against simian virus 40 (SV40) in children from 0 to 3years, to 91% for antibodies against WU polyomavirus (WUPyV) and HPyV10 in children from 8 to 17years. The mean number of PyV for which children were seropositive increased with the increasing of age: 4 standard deviations (SD) 1.8 in the 0-3-year group, 5 (SD 1.9) in the 4-7-year group, and 6 (SD 2.2) in the 8-17-year group. JC polyomavirus (JCPyV) DNA was detected in 1% of the adenoids, WUPyV in 12% of the tonsils, and 28% of the adenoids, and Merkel cell polyomavirus (MCPyV) was present in 6 and 2% of the tonsils and adenoids, respectively. Our study gives new insights on the serological evidence of exposure to PyVs during childhood, and on their possible respiratory route of transmission.