Myelin-Associated Glycoprotein Is the Antigen for a Monoclonal IgM in Polyneuropathy

Recent studies show that IgM monoclonal antibody from patients with IgM paraproteinemia and peripheral neuropathy reacts with a protein component of human PNS myelin and an analogous component or components of human CNS myelin. We have now demonstrated that the antigen for this antibody is a specific glycoprotein component of myelin, referred to as myelin-associated glycoprotein (MAG). Human PNS and CNS myelin proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on pore-gradient slabs, and MAG was identified by the immuno-electroblot procedure with rabbit anti-MAG (rat). The identical band(s) were stained by an analogous procedure with patient serum as the first antibody. Human PNS MAG had an apparent molecular weight of 107,000. Human CNS MAG appeared as three bands: 113,000, 107,000, and 92,000. Passage of myelin proteins through a concanavalin A-Sepharose column removed the staining component. Purified patient IgM, added to a lithium diiodosalicylate extract of myelin, immunoprecipitated MAG. This antibody also cross-reacted with MAG from bovine CNS, but not from rabbit, rat, or mouse.     

Oligosaccharide-Related Epitope Specific for a Brain-Specific Glycoprotein, 1D4 Antigen

The characteristics of glycosylation of a brain-specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high-mannose and hybrid types of N-linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N-linked oligosaccharides by treatment with glycopeptidase F or removal of both N- and O-linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex-type oligosaccharide-related epitope specific for the 1D4 antigen. Results also showed that N-linked glycosylation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain-related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide-related epitope is species specific. Furthermore, results with neuraminidase-treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide-related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.     

靶向PSMA多价纳米抗体的制备及其生物学活性表征*

目的:构建原核表达系统,制备靶向前列腺特异性膜抗原(prostate-specific membrane antigen,PSMA)多价纳米抗体并初步评价其生物学活性。方法:Bglbrick法构建多价纳米抗体表达载体,转化至大肠杆菌表达并利用亲和层析法纯化。联合蛋白质电泳和Western blot验证纯化产物,BCA法检测表达量。通过免疫荧光和流式细胞术定性评估PSMA特异性亲和能力,细胞ELISA法定量检测PSMA亲和水平,流式细胞术检测内吞效率。结果:成功构建靶向PSMA单价、二价、三价和四价纳米抗体大肠杆菌表达菌株。发酵结果表明四种纳米抗体均能在摇瓶水平实现高效可溶表达,其中二价纳米抗体表达量最高[(259.14±23.56) mg/L],单价纳米抗体表达量最低[(100.58±6.27) mg/L]。亲和实验结果证实四种纳米抗体均能特异性识别并结合PSMA阳性肿瘤细胞,与单价纳米抗体相比,二价、三价和四价纳米抗体对PSMA亲和能力分别提高了3.32倍、2.29倍和2.03倍。最后的内吞实验显示四种纳米抗体均能被PSMA阳性肿瘤细胞高效摄取,30 min内的摄取率均在80%以上。结论:靶向PSMA的多价纳米抗体,尤其是二价纳米抗体,具有比单价纳米抗体更高的产量和亲和水平,且具备不亚于单价纳米抗体的内吞效率,是未来基于PSMA肿瘤诊疗试剂开发的重要候选。     

Localization of Rabies Virus Glycoprotein into the Endoplasmic Reticulum Produces Immunoprotective Antigen

Rabies virus surface glycoprotein (rabies G-protein) with (G+RS) and without (G?RS) endoplasmic reticulum retrieval signal was expressed and characterized in tobacco plants. Transgenically expressed rabies G-protein was estimated at 0.015?C0.38?% of total leaf protein. The relative migration of the rabies G-protein on SDS-PAGE was at the position, as anticipated for the viral coat protein (~66?kDa). Immunolocalization by confocal microscopy established that immunoprotective G+RS expressed in tobacco was primarily confined to ER. G+RS showed binding to Con A lectin and was susceptible to N-glycosidase F activity similar to native rabies G-protein. However, the G?RS transgenically expressed in tobacco leaves was glycosylated differently and was resitant to N-glycosidase F. Immunological studies and Rapid Fluorescent Foci Inhibition Test (RFFIT) showed that G+RS was immunogenic and immunoprotective, whereas G?RS was moderately immunogenic but non-protective against live virus challenge. Hence, plants can express the antigenic component of rabies virus with suitable glycosylation, which is important to give protection against rabies virus infection.     

Probable Identity of NILE Glycoprotein and the High-Molecular-Weight Component of L1 Antigen

We report here that anti-L1 antiserum, raised against material from embryonic brain, and anti-NILE antiserum, raised against purified NILE (nerve growth factor-inducible large external) glycoprotein of PC12 cells, immunoprecipitate from PC12 cells material of the same apparent molecular weight (230 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, each of these immune reagents has the capacity to clear from a PC12 cell extract all of the 230-kilodalton antigen recognized by the other antiserum. Finally, in immunohistochemical staining of developing cerebellum the two antisera exhibit very similar staining patterns. We suggest that the NILE glycoprotein and the high-molecular-weight component of L1 antigen are closely related molecules, and probably the same.     

Malignant Glial Neoplasms: Definition of a Humoral Host Response to Tumor-Associated Antigen(s)

There is increasing evidence that human tumors possess tumor-associated neo-antigens. The host mounts an immunological response to these antigens, as evidenced by the detection of circulating humoral antibodies in a variety of human neoplasia.An indirect immunofluorescent antibody technique was employed to detect antibodies to tumor-associated antigens in the sera of patients with malignant gliomas. Viable single cell suspensions were used to demonstrate antibodies to surface contents of tumor cells and cell preparations were snap-frozen at −160° C to demonstrate antibodies to cytoplasmic components of tumor cells. After incubation with serum, the preparations were treated with polyvalent sheep antihuman globulin conjugated to isomer-1-fluorescein isothiocyanate, washed, and examined with a Leitz incident fluorescent microscope.Of the 17 sera from histologically proven malignant glial neoplasm patients, 2 (11%) were positive for an autologous surface antibody reaction. Five (23%) of 21 were positive for an autologus cytoplasmic antibody, however, 10 (47%) of 21 of the sera gave a positive reaction for cross-reacting cytoplasmic antibodies when tested with a battery of tumor cells obtained from different patients with malignant glial tumors.No reaction was observed with normal brain tissue. Absorption studies indicated the presence of a tumor-associated antigen.This study demonstrated that certain patients with malignant gliomas possess circulating antibodies to cytoplasmic components of their own tumor cells. The fact that a number of sera cross-reacted with tumor cells obtained from different patients suggests that antigenic cross-reactivity exists between malignant glioma cells from different patients. It is suggested that with further refinement, immunofluorescent detection of antibodies could evolve as a useful diagnostic adjunct in malignant glioma.     

猕猴B病毒gC蛋白特异性抗原表位的合成和表达

目的获得B病毒gC蛋白的特异性表位抗原。方法利用长片段基因合成的方法,合成B病毒C蛋白的特性抗原表位基因,将该基因连接到pMAL-5x载体,转化到BL21受体菌进行表达,并纯化表达产物。结果成功的获得了B病毒gC蛋白的特异性抗原蛋白,该蛋白以可溶的形式表达。结论利用原核表达系统,可以产生B病毒gC蛋白的可溶性抗原,可以作为B病毒的检测抗原。     

抗原表位的研究方法及口蹄疫病毒抗原表位的研究进展

本文综述了近几年来用于B细胞表位及T细胞表位研究的常用方法及其在口蹄疫病毒抗原表位研究中的应用,并介绍了口蹄疫病毒抗原表位的研究进展.     

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A_27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8⁺ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.     

Development and Validation of Arc Nanobodies: New Tools for Probing Arc Dynamics and Function

Neurochemical Research - Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral...     

Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.     

The Potential of Nanobodies for COVID-19 Diagnostics and Therapeutics

Molecular Diagnosis & Therapy - The infectious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19). Globally, there have...     

Antigen Targeting to Dendritic Cells Allows the Identification of a CD4 T-Cell Epitope within an Immunodominant Trypanosoma cruzi Antigen

Targeting antigens to dendritic cells (DCs) by using hybrid monoclonal antibodies (mAbs) directed against DC receptors is known to improve activation and support long-lasting T cell responses. In the present work, we used the mAb αDEC205 fused to the Trypanosoma cruzi amastigote surface protein 2 (ASP-2) to identify a region of this protein recognized by specific T cells. The hybrid αDEC-ASP2 mAb was successfully generated and preserved its ability to bind the DEC205 receptor. Immunization of BALB/c mice with the recombinant mAb in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)) specifically enhanced the number of IFN-γ producing cells and CD4+ T cell proliferation when compared to mice immunized with a mAb without receptor affinity or with the non-targeted ASP-2 protein. The strong immune response induced in mice immunized with the hybrid αDEC-ASP2 mAb allowed us to identify an ASP-2-specific CD4+ T cell epitope recognized by the BALB/c MHCII haplotype. We conclude that targeting parasite antigens to DCs is a useful strategy to enhance T cell mediated immune responses facilitating the identification of new T-cell epitopes.     

新型冠状病毒抗原快速检测研发现状及展望*

新型冠状病毒肺炎疫情的全球大流行,对全球公共健康、社会和经济运转造成了重大影响。在药物研发迟滞及疫苗有效性未得到充分验证的情况下,对人群进行大规模的快速筛查,寻找潜在的感染者(尤其是轻症和无症状患者),并进行集中隔离,切断传播途径和保护易感人群是首要的任务。因此对于SARS-CoV-2感染,早期诊断尤为重要。总结现有市场上的新冠病毒抗原快速检测产品,对全球抗原快速检测市场进行分析,概述其研发的动向并展望了我国在新冠抗原检测新方法、新技术方面的自主创新能力。     

Human Cytomegalovirus Glycoprotein B Contains Autonomous Determinants for Vectorial Targeting to Apical Membranes of Polarized Epithelial Cells

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Δ717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBΔ646-906) or the TM anchor alone (gBΔ751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBΔ772-906) or the last 73 amino acids (gBΔ834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that cooperate in vectorial transport in polarized epithelial cells.     

MiR-508-5p Inhibits the Progression of Glioma by Targeting Glycoprotein Non-metastatic Melanoma B

Glioma is a severe and highly lethal brain cancer, a malignancy largely stemming from growing in a relatively restrained area of the brain. Hence, the understanding of the molecular regulation of the growth of glioma is critical for improving its treatment. MicroRNA has become a hotspot in research on diseases, especially in the initiation and progression of different types of cancer. However, the molecular function and mechanisms of miR-508-5p in gliomagenesis are still unclear. The aim of this study was to investigate miR-508-5p expression in glioma and determine its effects on proliferation. miR-508-5p expression levels, both in glioma cell lines and in tissue, were significantly lower than in a normal human astrocyte cell line or adjacent tissues. Cell growth was analyzed using a MTT assay and over-expression of miR-508-5p was found to decrease glioma cell growth. Moreover, a bioinformatic analysis was performed, showing that glycoprotein non-metastatic melanoma B (GPNMB) was a direct target for miR-508-5p in glioma cells. Furthermore, in vivo treatment with miR-508-5p reduced GPNMB protein levels in the tumor. Additionally, overexpression of GPNMB without 3′-UTR partially reversed the cell growth arrest induced by miR-508-5p over-expression in glioma cells. In conclusion, these results indicate that increased expression of miR-508-5p might be related to glioma progression, indicating a potential role of miR-508-5p for clinical therapy.     

Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies/VHH Domains

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