Rat Monoclonal Antibody Research Articles

Generation of Rat Monoclonal Antibody for Mouse Nucleolin by Immunization of Ferroptosis-Induced Hepa 1-6 Cells.

Nucleolin is a multifunctional phosphoprotein that is ubiquitously distributed in the nucleus, nucleoplasm, cytoplasm, and cell membrane. The principal functions of nucleolin involve DNA and RNA metabolism, gene transcription and translation, ribosome biogenesis, and mRNA stability. Ferroptosis is a type of regulated cell death that is characterized by the iron-dependent accumulation of lipid peroxidation products. In a previous study, we produced monoclonal antibodies (mAbs) against lysates prepared from ferroptosis-induced Hepa 1-6 cells. In this study, we describe one of those rat mAbs, 4B5, which was generated against mouse nucleolin. This mAb was useful in immunofluorescence staining, immunoblotting, and immunoprecipitation experiments, and was confirmed to recognize endogenous nucleolin in mouse cell lines and tissues. We anticipate that mAb 4B5 will be useful for functional analyses of nucleolin.

Flow cytometric determination of ferroptosis using a rat monoclonal antibody raised against ferroptotic cells

Ferroptosis, a type of iron-dependent necrotic cell death, is specifically associated with increased lipid peroxidation. The dysfunction of the glutathione (GSH) production via the starvation of cysteine or the inhibition of phospholipid hydroperoxide glutathione peroxidase (GPX4) typically results in the accumulation of lipid peroxidation products and, consequently, the development of ferroptosis. We recently reported on the production of a rat monoclonal antibody, referred to as FerAb, against mouse-derived Hepa 1–6 cells that had been cultivated in cystine-deprived medium. Immunocytological analyses by means of fluorescence microscopy revealed that FerAb binds to fixed ferroptotic cells regardless of the species from which they were obtained, but not to apoptotic cells. We report herein on an in-depth characterization of the reactivity of FerAb with respect to unfixed cells by means of flow cytometry. The binding of FerAb to the cells was stimulated by incubating the cells in cystine deprived culture medium or treatment with RSL3, a GPX4 inhibitor, while treatment with staurosporine, an apoptosis inducer, had no effect on its binding to the cells. Supplementation with ferrostatin-1, a ferroptosis inhibitor, effectively suppressed the binding of FerAb to cells that had been cultivated in cystine-deprived medium or treated with RSL3, further confirming the specific binding of FerAb to ferroptotic cells. Thus, FerAb combined with a flow cytometry can be used to distinguish ferroptotic cells from living cells or apoptotic cells without the need for fixation. Applications of this combined technique will enable the quantitative evaluation of ferroptotic cells under a variety of patho-physiological conditions and will contribute to our understanding of the roles of ferroptosis in the body as well as cultured cells.

Targeting of a Conserved Epitope in Mouse and Human GPVI Differently Affects Receptor Function.

Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.

Production and Characterization of Rat Monoclonal Antibodies against the PAL Antigen of Legionella spp.

The purpose of this work was to obtain genus-specific monoclonal antibodies against the Legionella spp. recombinant PAL protein, which will subsequently allow to use them as a basis for the development of new express tests for pathogenic legionella detection. A short three-week immunization protocol for Wistar rats was used to generate rat–mouse heterohybridomas producing antibodies against PAL. Mouse myeloma cell line Sp2/0-Ag14 served as the fusion partner. Hybridization was performed using two methods: PEG-mediated fusion and electrofusion. Subsequent screening was performed by indirect solid-phase ELISA against the target protein rPAL. Specificity analysis was performed by dot-blot using a panel of lysates obtained from 39 pure cultures of different strains, which included closely related and heterologous microorganisms among others. No difference in the efficiency of stable hybridoma clones production by the two indicated cell-fusion methods was detected. Twelve clones producing specific rat monoclonal antibodies were obtained based on the screening results. The obtained rat monoclonal antibodies are highly specific towards the PAL protein of L. pneumophila of different serological groups and other pathogenic legionella and are good candidates to be used as the components of diagnostic test systems for the detection of pathogenic representatives of the Legionella genus.

Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

InstructionThe human amphoterin-induced gene and open reading frame (AMIGO) was identified as a novel cell adhesion molecule of type I transmembrane protein. AMIGO2 is one of three members of the AMIGO family (AMIGO1, 2, and 3), and the similarity between them is approximately 40% at the amino acid level. We have previously shown that AMIGO2 functions as a driver of liver metastasis. Immunohistochemical analysis of AMIGO2 expression in colorectal cancer (CRC) using a commercially available anti-AMIGO2 mouse monoclonal antibody clone sc-373699 (sc mAb) correlated with liver metastasis and poor prognosis. However, the sc mAb was found to be cross-reactive with all three molecules in the AMIGO family.MethodsWe generated a rat monoclonal antibody clone rTNK1A0012 (rTNK mAb) for human AMIGO2. The rTNK mAb was used to re-evaluate the association between AMIGO2 expression and liver metastases/clinical outcomes using the same CRC tissue samples previously reported with sc mAb.ResultsWestern blot analysis revealed that a rTNK mAb was identified as being specific for AMIGO2 protein and did not cross-react with AMIGO1 and AMIGO3. The rTNK mAb and sc mAb showed higher AMIGO2 expression, which correlates with a high frequency of liver metastases (65.3% and 47.5%, respectively), while multivariate analysis showed that AMIGO2 expression was an independent prognostic factor for liver metastases (p = 7.930E-10 and p = 1.707E-5). The Kaplan-Meier analyses showed that the rTNK mAb (p = 0.004), but not sc mAb (p = 0.107), predicted worse overall survival in patients with high AMIGO2 expression. The relationship between AMIGO2 expression and poor disease-specific survival showed a higher level of significance for rTNK mAb (p = 0.00004) compared to sc mAb (p = 0.001).ConclusionsThese results indicate that the developed rTNK1A0012 mAb is an antibody that specifically recognizes AMIGO2 by immunohistochemistry and can be a more reliable and applicable method for the diagnostic detection of liver metastases and worse prognosis in patients with high AMIGO2-expressing CRC.

Changes in mandibular CDH5, CXCL1, and PECAM1 expression following exposure to bisphosphonate and molar extraction suggest a loss of vascular endothelial cell barrier integrity plays a role in MRONJ

Objectives: Medication related osteonecrosis of the jaw (MRONJ) is a disorder characterized by loss of blood supply to the jaws and death to the bone. In our previous work, we created a rat model of MRONJ by multiple injections of 60ug/Kg zoledronic acid (ZA) via tail vein followed by extraction of a single first molar. We have previously shown in this model a decrease in the vasculature of the jaws and a delay in bone healing after 6 weeks. The current study was designed to look at mRNA expression and immunohistochemical localization of three endothelial cell markers (CDH5, CXCL1 and PECAM1) 6 weeks following ZA injection with and without molar extraction and in saline injected rats as controls. The objective of this study is to determine if the expression of these markers may serve to identify clinically significant changes in vascular endothelium barrier function associated with the onset of MRONJ. Methods: Using RT-PCR, we analyzed the expression of mRNA for angiogenic markers, six weeks after injection with either 60ug/Kg ZA or saline as a control and in ZA injected rats following first molar extraction. Routine immunohistochemical procedures with antibodies specific for rat marker proteins were used to study the immunohistochemical localization of proteins translated from the mRNA angiogenic markers with significant expression. All tissues were processed together under identical conditions. Results: We found a decrease in the mRNA expression of CDH5, and PECAM1 and an increase in CXCL1 expression six weeks after extraction and ZA injection in our rat model of MRONJ. Rat monoclonal antibodies specific for protein epitopes expressed by these genes (VE-cadherin, C-X-C motif ligand 1(CXCl1), and PECAM-1) were then used to localize proteins for these markers in tissues from ZA, saline-injected control rats, and MRONJ rats. The results show the absence of immunohistochemical localization of PECAM-1 and VE-cadherin marker proteins (-) in tissues from the MRONJ rats but strong (++++) in tissue from the saline-injected Control rats. The results also show the localization of the immunohistochemical staining for CXCl1 was high (++++) in the MRONJ rats after six weeks but absent (-) in the Controls. No mRNA or immunohistochemical localization for the three markers was seen in the rats treated with ZA without extraction. Conclusions: Our study identifies markers associated with a loss of vascular endothelial cell barrier integrity suggesting a role for this mechanism in MRONJ. This finding further suggests that CDH5, CXCL1 and PECAM1 may be useful as early markers for the changes seen in blood vessels after bisphosphonate exposure but before the frank onset of MRONJ.

Obtaining and characterization of rat monoclonal antibodies against the antigen PAL Legionella spp.

Obtaining and characterization of rat monoclonal antibodies against the antigen PAL Legionella spp.

A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG controlConclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. DisclosuresRoss: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Altered binding avidities and improved growth inhibitory effects of novel anti-HER3 mAb against human cancers in the presence of HER1-or HER2-targeted drugs

HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (KA) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The KA of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of KA, and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high KA by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI–H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high KA by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI–H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI–H1838, the reactivity and KA of Ab4 increased compared with in parent NCI–H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of KA with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116.

An Immunosensor for the Determination of Cathepsin S in Blood Plasma by Array SPRi-A Comparison of Analytical Properties of Silver-Gold and Pure Gold Chips.

The array SPR imaging (SPRi) technique is well suited to the determination of biomarkers in body fluids, called liquid biopsy. No signal enhancement or analyte preconcentration is required. With the aim of achieving signal enhancement and lowering the cost of a single determination, the replacement of gold-covered chips by silver–gold chips was investigated. The aim of this work was to investigate the analytical characteristics of a biosensor formed on a Ag/Au chip and to compare them with those of a biosensor formed on a gold chip. A biosensor for the determination of cathepsin S (Cath S) was chosen as an example. The biosensor consisted of the linker cysteamine and an immobilized rat monoclonal antibody specific for cathepsin S. Both biosensors exhibited a Langmuirian response to Cath S concentration, with linear response ranging from LOQ to 1.5 ng mL−1. The LOQ is 0.1 ng mL−1 for the biosensor formed on the Ag/Au chip, and 0.22 ng mL−1 for that formed on the gold chip. Recoveries and precision for medium and high Cath S concentrations were acceptable for both biosensors, i.e., precision better than 10% and recoveries within the range 102–105%. However, the results for the lowest Cath S concentration were better for the biosensor formed on the Ag/Au chip (9.4 and 106% for precision and recovery, respectively). Generally, no significant differences in analytical characteristics were observed between the Ag/Au and Au chips. The two biosensors were also compared in the determination of Cath S in real samples. Nine plasma samples from healthy donors and nine from patients with ovarian cancer were analyzed for Cath S concentration with the biosensors formed on Ag/Au and Au chips. The results obtained with the two biosensors were very similar and show no significant differences on the Bland–Altman plot. The Cath S concentration in the blood plasma of ovarian cancer patients was elevated by one order of magnitude as compared with the control (12.6 ± 3.6 vs. 1.6 ± 1.2 ng mL−1).

Differential regulation of the platelet GPIb‐IX complex by anti‐GPIbβ antibodies

BackgroundPlatelets' initial recognition of endothelial damage proceeds through the interaction between collagen, plasma von Willebrand factor (VWF), and the platelet glycoprotein (GP)Ib‐IX complex (CD42). The GPIb‐IX complex consists of one GPIbα, one GPIX, and two GPIbβ subunits. Once platelets are immobilized to the subendothelial matrix, shear generated by blood flow unfolds a membrane‐proximal mechanosensory domain (MSD) in GPIbα, exposing a conserved trigger sequence and activating the receptor. Currently, GPIbα appears to solely facilitate ligand‐induced activation because it contains both the MSD and the binding sites for all known ligands to GPIb‐IX. Despite being positioned directly adjacent to the MSD, the roles of GPIbβ and GPIX in signal transduction remain murky. ObjectivesTo characterize a novel rat monoclonal antibody 3G6 that binds GPIbβ. MethodsEffects of 3G6 on activation of GPIb‐IX are characterized in platelets and Chinese hamster ovary cells expressing GPIb‐IX (CHO‐Ib‐IX) and compared with those of an inhibitory anti‐GPIbβ antibody, RAM.1. ResultsBoth RAM.1 and 3G6 bind to purified GPIbβ and GPIb‐IX with high affinity. 3G6 potentiates GPIb‐IX‐associated filopodia formation in platelets or CHO‐Ib‐IX when they adhere VWF or antibodies against the ligand‐binding domain (LBD) of GPIbα. Pretreatment with 3G6 also increased anti‐LBD antibody‐induced GPIb‐IX activation. Conversely, RAM.1 inhibits nearly all GPIb‐IX‐related signaling in platelets and CHO‐Ib‐IX cells. ConclusionsThese data represent the first report of a positive modulator of GPIb‐IX activation. The divergent modulatory effects of 3G6 and RAM.1, both targeting GPIbβ, strongly suggest that changes in the conformation of GPIbβ underlie outside‐in activation via GPIb‐IX.

CD40 Agonist Monoclonal Antibody-Mediated Hepatitis in TNF-Receptor 1 Gene Knockout Mice.

Tumor necrosis factor-alpha (TNF-α) plays an important role in liver inflammation. CD40-CD40 ligand (CD40-CD40L) is a key receptor–ligand signaling pair involved in the adaptive immune response and pathogenesis of autoimmune diseases. In mice, CD40 activation leads to sickness behavior syndrome (SBS) comprising weight loss, sleep disruption and depression, which can be blocked by administration of the TNF-inhibitor etanercept. In the present study, we assessed the extent of hepatic inflammation in mice devoid of the TNF-receptor 1 (TNFR1)-mediated signaling pathway. The TNFR1-depleted (TNFR1−/−) adult mice and their wild type littermates were given a single intra-peritoneal injection of CD40 agonist monoclonal antibody (mAb) or rat IgG2a isotope control. As described previously, TNFR1−/− mice were protected from SBS upon CD40 mAb treatment. Cd40, tnf and tnfr1 mRNA and Tnf-α peptide were increased in the liver of CD40 mAb-stimulated wild type mice. Serum alanine aminotransferase was elevated in both CD40-activated wild type and TNFR1−/− mice. TNFR1−/− mice showed much less intra-parenchymal infiltrates, hepatocellular necrosis, and perivascular clusters upon CD40 mAb activation than their wild type littermates. A gene expression microarray detected increased activity of metabolic and detoxification pathways and decreased activity of inflammatory pathways. We conclude that immune activation and development of liver inflammation in CD40L interactions depend on TNFR1-mediated signaling pathways and are counteracted by alterations in metabolic pathways.

Generation of Rat Monoclonal Antibody for Human IQGAP1 by Immunization of Three-Dimensional-Cultured Cancer Cells.

The scaffold protein IQ motif containing GTPase activating protein 1 (IQGAP1) is an adherens junction component in the epithelial tissue that binds many signaling and structural molecules to regulate biological processes. It is known that IQGAP1 is overexpressed in some tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from three-dimensional (3D)-cultured DLD-1 cells to elucidate a characteristic feature of a tumor. In cancer research, 3D-cultured cancer cells are used as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. Our results showed that mAb 7E11 recognized increasing antigen in the lysate of 3D-cultured cells comparing with two-dimensional-cultured cells, and its antigen is the human IQGAP1. Furthermore, we indicated that mAb 7E11 was used in immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful in the analysis of human cancer.

Overexpression of the MSK1 Kinase in Patients With Chronic Lung Allograft Dysfunction and Its Confirmed Role in a Murine Model.

Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.

Proteomic analysis of microglia during LPS-induced acute neuroinflammation.

Abstract Dysregulation of microglia function is associated with neuroinflammation. A major limitation in understanding microglial contribution to cellular processes and their role in disease has been the lack of tools to specifically distinguish these cells from other myeloid cells. We generated and validated a rat monoclonal antibody against a selective microglia marker P2RY12. We utilized LPS administration as a model for neuroinflammation to assess alterations in P2RY12 expression, a homeostatic protein. Mice were injected with either PBS, low or high dosages of LPS daily for 4 days after which microglia were harvested from the brain. Isolated cells were co-stained with CD11b, CX3CR1, and P2RY12 as general markers for microglia. Flow cytometric quantification using live cells showed a significant increase in CD11b+ expression in both low and high dose LPS treated mice compared to the control untreated group. Low and high dose LPS injections led to a significant reduction in P2RY12 and CX3CR1 expression in these cells. These data are consistent with a decrease in P2RY12 expression under inflammatory conditions. We assessed microglial proteomic profile using BioLegend’s proprietary technology, TotalSeq™, which allows simultaneous analysis of both the transcriptome and proteome at the single-cell level. We utilized a panel of 200 antibody-oligonucleotide conjugates, including P2RY12, to analyze differentially expressed proteins under normal and LPS-induced inflammation in mice. These studies help elucidate the diversity and physiology of microglia, and potentially aid in the discovery of novel markers in response to neuroinflammation.

Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

Identification and characterization of the antigen recognized by the germ cell mAb TRA98 using a human comprehensive wet protein array.

TRA98 is a rat monoclonal antibody (mAb) which recognizes a specific antigen in the nuclei of germ cells. mAb TRA98 has been used to understand the mechanism of germ cell development and differentiation in many studies. In mice, the antigen recognized by mAb TRA98 or GCNA1 has been reported to be a GCNA gene product, but despite the demonstration of the immunoreactivity of this mAb in human testis and sperm in 1997, the antigen in humans remains unknown, as of date. To identify the human antigen recognized by mAb TRA98, a human comprehensive wet protein array was developed containing 19,446 proteins derived from human cDNAs. Using this array, it was found that the antigen of mAb TRA98 is not a GCNA gene product, but nuclear factor-κB activating protein (NKAP). In mice, mAb TRA98 recognized both the GCNA gene product and NKAP. Furthermore, conditional knockout of Nkap in mice revealed a phenotype of Sertoli cell-only syndrome. Although NKAP is a ubiquitously expressed protein, NKAP recognized by mAb TRA98 in mouse testis was SUMOylated. These results suggest that NKAP undergoes modifications, such as SUMOylation in the testis, and plays an important role in spermatogenesis.

Detection of Post-translational Modifications on MYC.

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.

Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene.

Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer‐related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference–mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1‐GFP in a GFP expression–dependent manner were selected from established hybridoma clones. Novel anti‐CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno‐histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti‐CAT1 mAbs exhibited internalization activity, antibody‐dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW‐C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs.

Two SPRi biosensors for the determination of cathepsin S in blood plasma

Cathepsin S is an emerging marker for ovarian cancer. Two ‘analytically specific’ SPRi biosensors for the determination of Cath S have been developed. The reception part of one of the biosensors consists of the rat monoclonal antibody specific for cathepsin S attached to the gold surface via covalent bonds with cysteamine linker, while the second biosensor consists of the inhibitor LY3000328 attached via hydrophobic interaction with the 1-octadecanothiol linker. Under optimized conditions, in terms of pH and receptor concentration, both biosensors have linear response ranges between LOQ (0.14 ng mL−1) and 2.5 ng mL−1, which is suitable for the determination of Cath S in blood plasma samples of ovarian cancer patients and healthy individuals, after corresponding dilution with 0.15 M PBS buffer.Precision and recoveries are quite acceptable: below 7% and 98–101% respectively for the biosensor with antibody, and below 12% and 101–103% for the biosensor with inhibitor. The biosensors were validated by the determination of Cath S in series of plasma from ovarian cancer patients and healthy volunteers using both biosensors and ELISA, giving Pearson coefficients close to 1. Plasma Cath S concentration can be used as an ovarian cancer marker, in view of the highly elevated concentrations detected.