Confirmed Antibody Specificity Research Articles

Abstract 3099: A new HTS ELISA for monitoring NRAS activity to expedite drug discovery

Abstract Oncogenic mutations in the KRAS, HRAS, and NRAS isoforms of the RAS gene family alter GTP binding and hydrolysis leading to errant over activation of the MAPK/ERK pathway. While the total of all such RAS mutations drives some 20-25% of all human cancers, each isoform tends to favor particular mutations that are cancer specific. Many evolving treatment strategies for these cancers target these mutant isoforms to minimize off target effects. NRAS mutations are seen in about one-fifth of cutaneous melanomas and patients with NRAS mutation melanomas have a poorer prognosis due to the high aggressiveness of RAS mutant tumors, a lack of efficient targeted therapies, or rapidly emerging resistance to existing treatments. Furthermore, there is no currently authorized targeted treatment for NRAS-mutated melanoma. As a result, there is a growing need for assays which can quantify activated NRAS. Here we describe the development of a highly specific and sensitive ELISA for determining the active NRAS protein in high throughput compatible (HTS) format. A NRAS specific recombinant antibody was developed via cloning of immunoglobulin variable regions from antigen positive B cells isolated by FACS from rabbits immunized with a NRAS peptide corresponding to a region of low homology between the RAS isoforms. Immunoblotting and a bead-based multiplex ELISA with all three RAS isoforms were used to confirm antibody specificity with recombinant HRAS, KRAS, and NRAS proteins over a wide concentration range. The new immunoassay enables in-well lysis of cells cultivated in 96-well plate. Activated NRAS in the lysate is collected on the test plate and detected using the NRAS specific antibody via chemiluminescence. We anticipate that this assay will further the discovery of novel NRAS specific therapeutics. EGF treatment of MCF7 cells demonstrated activation in cells expressing wildtype NRAS in preliminary assay validation. Evaluation of assay performance in mutant NRAS melanoma cell lines will be presented. Citation Format: Hyun Min Park, Julie Weber, Michelle Carlson, Mary Anne Jelinek. A new HTS ELISA for monitoring NRAS activity to expedite drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3099.

New insights in the renal distribution profile of TRPC3 – Of mice and men

Several reports previously investigated the Transient Receptor Potential Canonical subfamily channel 3 (TRPC3) in the kidney. However, most of the conclusions are based on animal samples or cell cultures leaving the door open for human tissue investigations. Moreover, results often disagreed among investigators. Histological description is lacking since most of these studies focused on functional aspects. Nevertheless, the same reports highlighted the potential key-role of TRPC3 in renal disorders. Hence, our interest to investigate the localization of TRPC3 in human kidneys. For this purpose, both healthy mouse and human kidney samples that were originated from tumor nephrectomies have been prepared for immunohistochemical staining using a knockout-validated antibody. A blocking peptide was used to confirm antibody specificity. A normalized weighted diaminobenzidine (DAB) area score between 0 and 3 comparable to a pixelwise H-score was established and employed for semiquantitative analysis. Altogether, our results suggest that glomeruli only express little TRPC3 compared to several segments of the tubular system. Cortical and medullary proximal tubules are stained, although intracortical differences in staining exist in mice. Intermediate tubules, however, are only weakly stained. The distal tubule was studied in three localizations and staining was marked although slightly varying throughout the different subsegments. Finally, the collecting duct was also immunolabeled in both human and mouse tissue. We therefore provide evidence that TRPC3 is expressed in various localizations of both human and mouse samples. We verify results of previous studies and propose until now undescribed localizations of TRPC3 in the mouse but especially and of greater interest in the human kidney. We thereby not only support the translational concept of the TRPC3 channel as key-player in physiology and pathophysiology of the human kidney but also present new potential targets to functional analysis.

Effect of moderate prenatal ethanol exposure on the differential expression of two histamine H3 receptor isoforms in different brain regions of adult rat offspring.

We have reported that prenatal alcohol exposure (PAE) elevates histamine H3 receptor (H3R) agonist-mediated inhibition of glutamatergic neurotransmission in the dentate gyrus. Here, we hypothesized that PAE alters the expression of two prominent H3R isoforms namely, the rH3A and rH3C isoforms, which have differing intrinsic activities for H3R agonists, in a manner that may contribute to heightened H3R function in PAE rats. In contrast to our predictions, we found different effects of sex and PAE in various brain regions with significant interactions between sex and PAE in dentate gyrus and entorhinal cortex for both isoforms. Subsequently, to confirm the PAE-and sex-induced differences on H3R isoform mRNA expression, we developed a polyclonal antibody selective for the rH3A inform. Western blots of rH3A mRNA-transfected HEK-293 cells identified a ~ 48 kDa band of binding consistent with the molecular weight of rH3A, thus confirming antibody sensitivity for rH3A protein. In parallel, we also established a pan-H3R knockout mice line to confirm antibody specificity in rodent brain membranes. Both qRT-PCR and H3R agonist-stimulated [35S]-GTPγS binding confirmed the absence of mH3A mRNA and H3 receptor-effector coupling in H3R knockout (KO) mice. Subsequent western blotting studies in both rat and mouse brain membranes were unable to detect rH3A antibody binding at ~48 kDa. Rather, the H3RA antibody bound to a ~ 55 kDa band in both rat and mouse membranes, including H3R KO mice, suggesting H3RA binding was not specific for H3Rs in rodent membranes. Subsequent LC/MS analysis of the ~55 kDa band in frontal cortical membranes identified the highly abundant beta subunit of ATPase in both WT and KO mice. Finally, LC/MS analysis of the ~48 kDa band from rH3A mRNA-transfected HEK-293 cell membranes was able to detect rH3A protein, but its presence was below the limits of quantitative reliability. We conclude that PAE alters rH3A and rH3C mRNA expression in some of the same brain regions where we have previously reported PAE-induced alterations in H3R-effector coupling. However, interpreting the functional consequences of altered H3R isoform expression was limited given the technical challenges of measuring the relatively low abundance of rH3A protein in native membrane preparations.

Expression of the Calcitonin Receptor-like Receptor (CALCRL) in Normal and Neoplastic Tissues.

Little information is available concerning protein expression of the calcitonin receptor-like receptor (CALCRL) at the protein level. Here, we developed a rabbit monoclonal antibody, 8H9L8, which is directed against human CALCRL but cross-reacts with the rat and mouse forms of the receptor. We confirmed antibody specificity via Western blot analyses and immunocytochemistry using the CALCRL-expressing neuroendocrine tumour cell line BON-1 and a CALCRL-specific small interfering RNA (siRNA). We then used the antibody for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic tissues. In nearly all tissue specimens examined, CALCRL expression was detected in the capillary endothelium, smooth muscles of the arterioles and arteries, and immune cells. Analyses of normal human, rat, and mouse tissues revealed that CALCRL was primarily present in distinct cell populations in the cerebral cortex; pituitary; dorsal root ganglia; epithelia, muscles, and glands of the larger bronchi; intestinal mucosa (particularly in enteroendocrine cells); intestinal ganglia; exocrine and endocrine pancreas; arteries, capillaries, and glomerular capillary loops in the kidneys; the adrenals; Leydig cells in the testicles; and syncytiotrophoblasts in the placenta. In the neoplastic tissues, CALCRL was predominantly expressed in thyroid carcinomas, parathyroid adenomas, small-cell lung cancers, large-cell neuroendocrine carcinomas of the lung, pancreatic neuroendocrine neoplasms, renal clear-cell carcinomas, pheochromocytomas, lymphomas, and melanomas. In these tumours with strong expression of CALCRL, the receptor may represent a useful target structure for future therapies.

Improved Efficiency of Bluetongue Viral Antigen Isolation for Successful Immunization

Bluetongue (BT) is a viral disease transmitted by Culicoides spp. The clinical presentation of BT varies widely among susceptible sheep, and in most cases results in severe illness and death in the infected animals. The mortality among susceptible sheep ranges from 2-30% but can occasionally be as high as 70%. Therefore, we investigated a new method to increase the purified BTV-antigen. BTV viral suspensions were purified using Freon-113 and ultracentrifugation through 40% sucrose. We obtained 94.5-95.8% purification of the BT-16 viral antigen. Sheep and cows were immunized with the isolated BTV antigen obtained from this method to confirm antibody specificity to BTV. The antibody activity measured by enzyme-linked immunosorbent assay (ELISA) from goat serum was 7.0log2 to 13.0log2 (on average 10.8±2.28log2) relative to that of sheep (P<0.05 to P<0.0001). We show here that this method can successfully purify BTV-16 antigen and could be used for large-scale production and other BTV serotypes.

Confirming Antibody Specificity in Xenopus.

Verifying that a new antibody recognizes its target can be difficult. In this protocol, expression of a target protein in Xenopus embryos is either knocked down using CRISPR-Cas9 technology (for zygotic proteins) or enhanced by microinjection of a synthetic mRNA (for maternal proteins). Western blotting analysis is then performed. If the antibody recognizes the target protein, the western blot will show a relatively weak band for CRISPR-injected embryos and a relatively strong band for RNA-injected embryos. This represents a straightforward, powerful strategy for confirming antibody specificity in Xenopus.

Domain-Specific Antibodies Reveal Differences in the Membrane Topologies of Apolipoprotein L1 in Serum and Podocytes.

Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.

Antibody validation for Western blot: By the user, for the user

Antibody validation for Western blot: By the user, for the user

CF-PA2Vtech: a cell-free human protein array technology for antibody validation against human proteins

Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA2Vtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein.

PO-146 Knockout validation of antibodies to components of the NF-kB signalling pathway

IntroductionNuclear factor-κB (NF-κB) plays a pivotal role in inflammation and innate immunity and aberrant regulation of NF-κB can influence cancer development and progression (Ref 1). As a result, a great deal of research is now focused on targeting this signalling pathway therapeutically. In order to accurately and reproducibly examine the NF-kB signalling cascade there is a need for highly specific antibodies. We, at Abcam, have developed knockout (KO)-validated antibodies targeted to distinct elements of the NF-κB signalling pathway to maximise experimental accuracy and reproducibility.Material and methodsOur KO validation program uses the extensive library of KO cell lines available from Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line, resulting in a frameshift and complete loss of gene expression. Target specificity is then confirmed via western blot, flow cytometry or immunocytochemistry. These KO cell lines function as true negative controls that can be used at a large scale to confirm antibody specificity to target proteins.Results and discussionsWe present antibody validation data for seven targets in the NF-κB signalling pathway. Of the 30 Abcam products tested, we demonstrate that 22 antibodies are specific. The remaining 8 have since been removed from our catalogue due to specificity concerns. We have now validated over 1000 antibodies using this KO method, including many that are relevant to immunology and immuno-oncology research.ConclusionFollowing validation with KO cell lines as a negative controls, we can recommend 22 highly specific antibodies to proteins in the NF-κB signalling pathway. KO validation is one of many strategies that we use to ensure the specificity of our antibodies. By providing robust and specific antibodies which work the first time, we hope to help researchers to improve the experimental reproducibility and accuracy of their studies.Reference1. Bastian Hoesel and Johannes A Schmid; Mol Cancer. 2013 12: 86. doi:10.1186/1476-4598-12-86

Abstract 5763: Large-scale use of knockout validation to confirm antibody specificity to key oncology targets

Abstract Antibodies are the most common tools in basic science and clinical research. Over the past several years there has been an ongoing discussion around antibody validation in light of poor experimental reproducibility between laboratories. This is in part due to the cross-reactivity between antibodies and off-target proteins and the variability between different antibody batches. As a result, experimental irreproducibility leads to wasted resources and compromises the advancement of science. In response to this, Abcam has introduced knockout (KO) validation to help produce target-specific antibodies. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line. This results in a frameshift and a complete loss of gene expression. These KO cell lines provide a true negative control for antibody validation as a lack of signal confirms that the antibody binds to its intended target. As a result, both antibody specificity and reproducibility on a large scale can be addressed. We have KO-validated over a thousand antibodies, including several that are relevant to immuno-oncology research. Examples of such targets are the ERK1/2 pathway, which regulates cell survival, proliferation and cell death. Inhibition of this pathway is an attractive target for the treatment of malignant tumors with increased ERK1/2 activity. Similarly, Ki67 expression is strongly associated with tumor cell proliferation and can be used as a marker for tumor aggressiveness. Finally, overexpression of vimentin in tumors correlates with increased growth and invasion, leading to poor prognosis. It is imperative that reproducible data can be produced on these targets to further our understanding of tumor biology and patient prognosis. Here we present the data for antibodies to these key targets that have been validated in both flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. The antibodies shown gave signal in wild-type cells but showed complete loss of signal when tested in knockout cell lines. By providing researchers with reliable and specific antibodies that work first time we hope to minimize wasted resources and improve reproducibility. Citation Format: Sam Heaton, Monica Sassi, Alejandra Solache, Bruce Hamilton, Sambhav Dave. Large-scale use of knockout validation to confirm antibody specificity to key oncology targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5763.

Large-scale use of knockout validation to confirm antibody specificity

Abstract Antibodies are among the most common tools in basic science and clinical research. However, an increasing number of studies have shown that not all antibodies are specific, leading to a growing issue of experimental irreproducibility. This is in part due to the cross-reactivity between antibodies and off-target proteins, as well as the variability between different antibody batches. These unspecific antibodies result in wasted time and resources and compromise the advancement of science. In response to this, Abcam launched a knockout (KO) validation program in 2015 as one of its antibody quality control protocols. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line, which results in a frameshift and a complete loss of gene expression. These KO cell lines function as true negative controls that can be used at a large scale to confirm antibody specificity to the protein of interest. We have KO-validated hundreds of antibodies, including several that are relevant to immunology and immuno-oncology research. Here we present the data related to key targets validated in western blot, flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. By providing researchers with reliable and specific antibodies that work first time, we hope to minimize wasted resources and improve antibody reproducibility.

Abstract 2626: Expression of human poliovirus receptor, an immune checkpoint biomarker in lung cancer

Abstract Background: Recently, immune therapy has shown notable efficacy in melanoma, non-small-cell lung cancer (NSCLC), among others, indicating that the targeting of immune checkpoints may be a promising treatment for malignant tumors. The human Poliovirus Receptor (CD155), an immune checkpoint, has been found to be overexpressed on the membranes of various tumor cells including colorectal and ovarian carcinoma. Examining PVR’s expression in NSCLC could shine light on PVR’s potential role and its possible correlation to the prognosis and outcomes for NSCLC patients. Material and Methods: Gene expression data for lung cancer cell lines and tumor tissues was derived from the Cancer Cell Line Encyclopedia (CCLE [www.broadinstitute.org/ccle]) and the TCGA database (www.cbioportal.org), respectively. Immunohistochemistry (IHC) was performed on a Ventana Benchmark XT platform using an anti-PVR antibody (Cell Signaling #D3G7H at a 1:100 dilution). IHC staining was evaluated for a NSCLC Cell Line Tissue MicroArray (TMA) and a NSCLC patient cohort TMA. The H-score system was used to generate a semi-quantitative score, ranging from 0 to 300, that evaluates the intensity and prevalence of the staining on tumor cells. Patient data was analysed to uncover correlations between outcome and PVR expression in the NSCLC TMA cohort. Western blot analysis was performed to confirm antibody specificity and PVR expression levels in three NSCLC cell lines. Results: Analysis of mRNA expression data from CCLE and TCGA databases identified that PVR is highly expressed in lung cancer. Three lung cancer cell lines were chosen based on mRNA expression for this study: NCI-H211 (high), NCI-H1650 (moderate) and NCI-H187 (negative). Cell line data revealed that PVR demonstrated higher expression levels in lung cancer than other immune checkpoints, such as PD-L1, PD-L2, and Gal-9 among others. In addition, PVR expression was not correlated with other negative immune checkpoints including PD-L1 and Gal-9. Western blot analysis confirmed that the Cell Signaling antibody was specific to the PVR protein in NSCLC cell lines. IHC staining of NSCLC cell lines revealed that non-squamous NSCLC cell lines express higher levels of PVR than do squamous cell cell lines (p<0.0001, Fisher test). The prevalence of PVR in the NSCLC patient cohort was 68% (68/100) using a H-score cutoff of ≥ 7. Analysis revealed a lower positivity of 61.7% (37/60) in squamous carcinoma as compared to 77.5% (31/40) in non-squamous NSCLC. Analysis of the patient outcome data demonstrated that higher levels of PVR protein expression is correlated with poorer outcomes in patients with both squamous lung cancer and adenocarcinoma. Conclusions: PVR protein expression could potentially serve as a new immune checkpoint biomarker in lung cancer. This study demonstrates that high PVR protein levels predict poorer outcomes in lung cancer patients; suggesting that PVR may be an important target for future immune therapy. Citation Format: Camilla Koczara, Hui Yu, Kim Ellison, Christopher Rivard, Leslie Rozeboom, Daniel Chan, Kenichi Suda, Kristine Brovsky, Rafal Dziadziuszko, Fred Hirsch. Expression of human poliovirus receptor, an immune checkpoint biomarker in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2626. doi:10.1158/1538-7445.AM2017-2626

The LRF/ZBTB7A Transcription Factor Is a BCL11A-Independent Repressor of Fetal Hemoglobin

Induction of fetal-type hemoglobin (HbF: α2γ2) is a promising means to treat hemoglobinopathies; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Such knowledge is essential to develop mechanism-based, targeted approaches to reactivate HbF production. Here, we show that Leukemia/lymphoma Related Factor (LRF), encoded by the ZBTB7A gene, is a novel and potent repressor of HbF production.To assess the effects of LRF loss on the mouse erythroid transcriptome, we performed RNA-Seq analysis using splenic erythroblasts from control and LRF conditional knockout (Zbtb7aF/F Mx1-Cre+) mice. LRF-deficient adult erythroblasts showed significant induction of Hbb-bh1, but not Hbb-y. The results were validated at the protein levels via isoelectric focusing of peripheral blood (PB) hemolysates and MALDI-TOFMS analysis. LRF loss also reactivated human fetal globin expression in vivo in LRF conditional KO mice harboring the human β-globin gene cluster as a yeast artificial chromosome transgene (β-YAC).To determine whether LRF loss could induce HbF in human erythroid cells, we employed human CD34+ hematopoietic stem and progenitor (HSPC)-derived primary erythroblasts and determined γ-globin expression levels upon shRNA-mediated LRF knockdown (KD). HbF levels in LRF KD cells (49-70%) were much greater than those seen in parental or scrambled-shRNA transduced cells. We next employed a novel human immortalized erythroid line (HUDEP-2). This line possesses an advantage over lines currently used for globin switching studies because it expresses predominantly adult hemoglobin (HbA: α2β2), with very low background HbF expression. Using CRISPR/cas9 gene modification, we knocked out ZBTB7A in HUDEP-2 cells and performed RNA-Seq analysis. As expected, γ-globin (HBG1 and HBG2) transcripts, but not those of embryonic ε-globin (HBE1), were markedly induced in ZBTB7A KO (ZBTB7AΔ/Δ) HUDEP-2 cells. ZBTB7AΔ/Δ cells exhibited HbF levels greater than 60%, while that of parental cells was less than 3%. Notably, the HbF reactivation occurred without changes in levels of transcripts encoding known HbF repressors, including BCL11A, the principal known switching factor.We next performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) with an anti-LRF antibody using HSPC-derived proerythroblasts and HUDEP-2 cells. The most enriched motif identified in either was concordant with that previously identified in vitro using CAST analysis (Maeda et. al. Nature 2005), confirming antibody specificity. Supporting a direct role of LRF at the β-globin cluster, we observed several significant enrichment of LRF-ChIP binding signals at adult (HBB), fetal (HBG1) globin loci and the upstream hypersensitivity (HS) sites within the locus control region (LCR). ATAC-Seq (for assay for transposase-accessible chromatin with high-throughput sequencing) analysis revealed strong chromatin accessibility at the γ-globin locus in ZBTB7AΔ/Δ cells. Strikingly, differential enrichment of ATAC-signals in ZBTB7AΔ/Δ cells was evident only at the γ-locus. Thus, while LRF binds to the HBB locus and HS sites as well as to the HBG1 locus, LRF depletion specifically opens chromatin at the γ-globin locus.Finally, to determine whether LRF and BCL11A suppress γ-globin expression via distinct mechanisms, we established LRF/BCL11A double knockout HUDEP-2 cells. Strikingly, HUDEP-2 lines lacking both LRF and BCL11A exhibited almost a complete switch in expression from adult- to fetal-type globin, suggesting that these two factors cumulatively represent the near entirety of γ-globin repressive activity in adult erythroid cells. Our findings reveal a novel molecular mechanism regulating γ-globin silencing and may open a new window for therapeutic targeting in the treatment of hemoglobinopathies. DisclosuresBauer:Biogen: Research Funding; Editas Medicine: Consultancy. Orkin:Editas Inc.: Consultancy.

Cross-reactivity of Antibodies Directed to the Gram-Negative Bacterium Neisseria gonorrhoeae With Heat Shock Protein 60 and ATP-Binding Protein Correlates to Reduced Mitochondrial Activity in HIBCPP Choroid Plexus Papilloma Cells

Antibacterial antibodies can cause neurologic side-effects by cross-reactivity with cellular antigens. Here we investigated interactions of antibodies to Neisseria gonorrhoeae (α-NG) - maternal infections by which increases the offspring's risk for later psychosis-with HIBCPP cells, a cell culture model of choroid plexus epithelium. Immunocytochemistry and Western blotting with α-NG, revealed organelle-like intracellular staining in HIBCPP cells, and labelling of several immunoreactive bands in cellular protein. Two-dimensional Western blotting revealed several immunopositive spots, most prominent of which were identified by mass spectrometry as mitochondrially localized proteins heat shock protein 60 (Hsp60) and ATP-binding protein β-subunit (ATPB). Similarly α-NG interacted with commercial samples of these proteins as revealed by Western blotting. Three alternative methods (JC-1, Janus green and MTT staining) revealed α-NG to cause in HIBCPP cells a significant decrease in mitochondrial activity, which could be reverted by neuroleptic drugs. Immunoreactivity of α-NG with choroid plexus epithelium in human post mortem samples suggests in vivo relevance of these findings. Finally, distinctly different staining patterns of antibodies against Neisseria meningitidis (α-NM), confirmed antibody specificity. To our knowledge this is the first report that α-NG cross-reactivity with Hsp60 and ATPB impairs mitochondrial activity in choroid plexus epithelial cells, pathogenetic relevance of which needs further clarification.

Antibodies Directed to the Gram-Negative Bacterium Neisseria gonorrhoeae Cross-React with the 60 kDa Heat Shock Protein and Lead to Impaired Neurite Outgrowth in NTera2/D1 Cells

Children of mothers with prenatal gonococcal infections are of increased risk to develop schizophrenic psychosis in later life. The present study hypothesizes an autoimmune mechanism for this, investigating interactions of a commercial rabbit antiserum directed to Neisseria gonorrhoeae (α-NG) with human NTera2/D1 cells, an established in vitro model for human neuronal differentiation. Immunocytochemistry demonstrated α-NG to label antigens on an intracellular organelle, which by Western blot analysis showed a molecular weight shortly below 72 kDa. An antiserum directed to Neisseria meningitidis (α-NM) reacts with an antigen shortly below 95 kDa, confirming antibody specificity of these interactions. Two-dimensional gel electrophoresis and partial Western transfer, allowed to localize an α-NG reactive protein spot which was identified by LC-Q-TOF MS/MS analysis as mitochondrial heat shock protein Hsp60. This was confirmed by Western blot analysis of α-NG immunoreactivity with a commercial Hsp60 protein sample, with which α-NM failed to interact. Finally, analysis of neurite outgrowth in retinoic acid-stimulated differentiating NTera2-D1 cells, demonstrates that α-NG but not α-NM treatment reduces neurite length. These results demonstrate that α-NG can interact with Hsp60 in vitro, whereas pathogenetic relevance of this interaction for psychotic symptomatology remains to be clarified.

Clinical Impact Of The Simultaneous Detection Of Anti-Erythocytic Autoantibodies In Alloimmunization

Introduction The simultaneous detection of autoantibodies and alloantibodies to red blood cells (RBC) following allogeneic transfusion is poorly understood. The incidence of autoimmune hemolysis in these patients is not well established (Young et al., Transfusion 2004; 44: 67-72). Methods Patients with a positive antibody screen, an identified alloantibody and a positive direct antiglobulin test (DAT) were selected from our hospital Blood Bank database from January 2002 to June 2013. Sex, age, main clinical diagnosis, transfusion history, alloantibody and autoantibody specificity and hemolysis parameters were analysed retrospectively in those patients with simultaneous first detection of auto and alloantibodies. In our Blood Bank, all patients to be transfused underwent a three-cell antibody screen by gel technology (Ortho Clinical Diagnosis). If the screen was positive, two microcolumn panels with eleven cells (DianaGel) and an autocontrol were performed in Liss-Coombs and with papain-treated erythrocytes for antibody identification. Test-tube testing at different temperatures with Reagent RBC (Makropanel) and a polyethyleneglycol (PEG) antiglobulin technique were carried out to confirm antibody specificity when deemed appropriate. Polyspecific and monospecific (anti-IgG and C3d) DAT (Menarini reagents) and a 6% albumin negative control were performed in all patients presenting a positive autocontrol. Acid glycine elution (Gamma ELU-KIT II) was the method of choice to prepare the eluates. Adsorption techniques were used in some cases to remove autoantibodies. Results A total of 2915 patients formed alloantibodies; 4.5% (n=132) presented both auto- and alloantibodies. The temporal relationship between the antibodies detected had the following distribution: 96.2% (n=127) simultaneous auto- and alloantibody, 2.3% alloantibody identification and on subsequent studies autoantibody and 1.5% had autoantibody previous to alloimmunization. The following results will refer to the group with simultaneous detection of auto and alloantibodies. In the selected group, 55% had a complete register of previous RBC transfusion with a median of 8.5 transfusions lifelong (range: 2 to 81) and a median of 38.5 days (range: 4 to 270 days) between last negative and first positive antibody screen. The ratio male to female was 1:1 and the median age was 67 years-old (range: 20 to 91 years-old). The most common diagnosis in these patients were: solid neoplasia (21.3%), oncohematologic disease (13.4%; mielodysplastic syndromes were included in this subgroup), chronic hepatopathy (15%; of which 58% are HVC-related), cardiovascular disease (9.5%), traumatism (7%), pregnancy (3.2%) and autoimmune disease (3.1%). The most frequent alloantibodies identified were: anti-E (39.3%) > anti-C (28.3%) > anti-K (24.5%). Most autoantibodies had no apparent specificity (67%), those that did have a specific pattern were related to Rh antigens (anti-e 7%; anti-c 1.5%; anti-D 1.5%), and the remainder 23% were crioagglutinins. A total of 16 patients (12.5%) had concomitant hemolysis at the time of antibody detection of which 43.7% (n=7) had an active cancer (57% due to oncohematologic disease), 25% (n=4) HCV hepatopathy and 12.5% autoimmune hepatitis (n=2). Conclusion Patients with de novo alloimmunization showed concomitant antierythrocytic autoantibodies in 4.5% of our population. Most cases appear to be related to previous RBC transfusion and medical conditions affecting normal immunological functioning. However, hemolysis was only found in 12.5% of these patients at the time of antibody detection and all of them had diseases with a known associated risk of developing autoimmune anemia. Disclosures: No relevant conflicts of interest to declare.

Discovery and validation of ovarian cancer biomarkers utilizing high density antibody microarrays

Here we demonstrate the utility of high-density antibody microarrays for ovarian cancer biomarker discovery. This report describes the technology and how it can be optimized for hypothesis-generating and testing experiments. Our previous results validated the high density antibody array technology platform, the current work expands on it utilizing a second generation array that we tested with a larger set of ovarian case and control serum samples. We then describe our strategies and methods for result validation, including Western immunoblots to confirm antibody specificity. By comparing and combining the current results with our previous study, we solidified the case that the markers found could be used for ovarian cancer diagnosis using this technology. These results set the stage for further validation of these potential biomarkers and the use of this technology in future biomarker discovery studies.

Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis

In this study, we investigated the hypothesis that regulatory T cells (T(reg)) are involved in the immunomodulatory effects of statins on rheumatoid arthritis (RA) patients. The 12-week study cohort consisted of 55 RA patients and 42 control subjects allocated to either a group treated with atorvastatin (AT) (20 mg/day) or a non-AT group. T(reg) numbers, suppressive function, serum inflammatory markers, and disease activity were evaluated before and after the therapy. Furthermore, the effects of AT on the frequency and suppressive function of T(reg) were determined in vitro. Our data revealed that the suppressive function of T(reg) from RA patients significantly decreased compared with that of control subjects. AT significantly reduced erythrosedimentation, C-reactive protein, and disease activity. Concomitantly, T(reg) numbers and suppressive functions were significantly improved by AT. Consistent with the in vivo experiments, AT promoted the generation of T(reg) from primary T cells and enhanced preexisting T(reg) function in vitro. Moreover, we showed that PI3K-Akt-mTOR and ERK signal pathways were involved in the induction of T(reg) by AT. In conclusion, AT significantly increased T(reg) numbers and restored their suppressive function in the RA patients, and this may be relevant in the modulation of uncontrolled inflammation in this disorder.

Évaluation des performances de quatre immunoblots commercialisés pour la confirmation de la borréliose de Lyme dans une population de patients suivis en Rhône-Alpes

Summary Objective Likewise biological diagnosis resting on the serology is fussy. Immunoblot is employed to confirm antibody specificity after positive Lyme screening. Our objective: to compare analytical performances of four immunoblots commercialized for Lyme disease in our population positive for screening of Lyme disease. Material and methods We tested 42 well-defined sera of patients at different stages of Lyme borreliosis and 33 non-confirmed diagnoses. Results by four serological confirmation tests were analysed: Europe Line ® (Virotech, InGen), Ecoblot ® (Virotech, Meridian), EU Lyme WB ® (MarDx, Trinity Biotech) and Recomblot ® (Mikrogen, DiaSorin). Reactivity of two major proteins, VlsE (IgG) and OspC (IgM) were especially studied. Technical criteria were also evaluated: automated use, simplicity of reading and cost of use. Results For the patients with Lyme disease, the IgG are founded in 85,7%, 83,3%, 76,2% and 78,6% respectively by the immunoblots (Europe Line ® , Ecoblot ® , EU Lyme WB ® and Recomblot ® ) and the IgM are founded in 69%, 54.8%, 85.7% et 73.8% des IB. For the patients with suspicion of Lyme not confirmed, the IgG immunoblots are positives in 15.2%, 21.2%, 24.2% and 18.2%, respectively. For the same patients, the IgM immunoblots are positives in 27.3%, 30.3%, 45.5% and 30.3%. Immunoblot results should be interpreted carefully, and in relation to the clinical-epidemiological and biological findings. Discrepancies in findings on identical sera indicate the need to standardise serological confirmation of Lyme borreliosis infection. A good confirmatory test would need to have proven specificity in the particular population being tested, as advised by the ISO 15189 accreditation norm.