Antibody B72 Research Articles

Antiviral Activity of HIV gp120-Targeting Bispecific T Cell Engager Antibody Constructs

Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs) as well as in macrophages cocultured with autologous CD8+ T cells, the most potent being the human CD4(1 + 2) BiTE [termed CD(1 + 2) h BiTE] antibody construct and the CD4(1 + 2)L17b BiTE antibody construct. The CD4(1 + 2) h BiTE antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTE antibody constructs, as well as the CD4(1 + 2)L17b BiTE antibody construct, did not. Thus, BiTE antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential.IMPORTANCE HIV is a chronic infection well controlled with the current cART. However, we lack a cure for HIV, and the HIV pandemic goes on. Here, we showed in vitro and ex vivo that a BiTE antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE antibody constructs display efficient killing of gp120-expressing cells and inhibited replication in ex vivo HIV-infected PBMCs or macrophages. We believe that BiTE antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds which reverse latency.

Calcium Mobilization and Inhibition of Akt Reduced the Binding of PEO-1 Cells to Fibronectin.

To investigate the effects of intracellular calcium (Ca2+) mobilization, β-catenin and Akt signal pathways after the binding of metastatic ovarian cells to fibronectin. The expression levels of α4β1 and αvβ6 integrin were determined using α4, β1, αv, and β6 antibodies using flow cytometry on PEO-1 cells. The effect of [Ca2+]i on cell adhesion capacity was investigated using RTCA after stimulating PEO-1 cells using thapsigargin and tunicamycin. The binding rate of PEO-1 cells to fibronectin was also investigated in the presence of either different concentrations of cardamonin, which inhibits the accumulation of β-catenin, or different concentrations of FPA 124, which is a specific inhibitor for the PKB/Akt signal pathway, using RTCA. RTCA analysis results showed that increasing [Ca2+]i through leakage of the calcium pool was strongly effective on PEO-1 cell binding to fibronectin. Extracellular calcium influx also reduced the binding of PEO-1 cells. Cell binding to fibronectin was also inhibited with a ratio of 64% in the presence of 100 µM cardamonin compared with untreated control cells. Finally, it was found that PKB/Akt inhibition with 15 µM FPA 124 decreased the binding of PEO-1 cells to fibronectin with a ratio of 88% compared with untreated control cells. PEO-1 cell binding to fibronectin via integrins could be related to intracellular Ca2+ mobilization and Akt signaling.

The molecular mechanism of two coreceptor binding site antibodies X5 and 17b neutralizing HIV-1: Insights from molecular dynamics simulation.

The coreceptor binding site of gp120 plays an important role in HIV entry into host cell. X5 and 17b are typical coreceptor binding site antibodies with the ability to broadly neutralize HIV. Thus, here, to study the neutralizing mechanism of two antibodies and identify the source of two antibodies with different neutralizing ability, we performed molecular dynamics simulations for the complexes of X5 and 17b with gp120 and CD4. The simulation results indicate X5 and 17b mainly affects CD4 and coreceptor binding sites. Specifically, for CD4 binding site (CD4bs), the binding of antibodies has different effects on CD4bs with and without CD4. However, for coreceptor binding sites, the binding of the antibodies has consistent influence on the region adjacent to loop V3 despite of the simulated systems with or without CD4. The binding of the antibodies enhances the interactions of gp120 region adjacent to loop V3 with other region of gp120, which are unfavorable for conformational rearrangements of the region adjacent to loop V3 and further binding the coreceptor. Additionally, the interactions of loop V3 and bridging sheet with X5 lead to the close motion of loop V3 in X5 bound form, which further influences the rearrangements in gp120.

Anti-αIIb β3 immunization in Glanzmann thrombasthenia: review of literature and treatment recommendations.

Glanzmann thrombasthenia (GT)is caused by inherited defects of the αIIb β3 platelet glycoprotein. This bleeding disorder can be treated with platelet transfusion therapy, but some patients will be immunized and begin to form anti-human leucocyte antigen (HLA) and/or anti-αIIb β3 antibodies. These antibodies can bind and interfere with the function of the transfused platelets, rendering treatment ineffective. However, platelet transfusion refractoriness attributable to HLA antibodies may be managed by the selection of compatible donors, although they are not always readily available, particularly in an emergency. Thus, anti-αIIb β3 antibodies represent one of the most severe complications in GT. Both genetic and environmental factors may contribute to the risk of anti-αIIb β3 development, but the underlying pathogenic mechanisms are still unknown. Thisreviewwill summarize the current knowledge of the risk factors for development of anti-αIIb β3 antibodies in patients with GT and discuss how these findings may influence the clinical management of patients.

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations.

HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.

Hyperglycosylated hCG Drives Malignancy in Most or All Human Cancers: Tying All Research Together

Objectives: Two forms of hCG are produced, the hormone hCG binding a luteinizing hormone/hCG joint receptor and the autocrine hyperglycosylated hCG binding a TGF-ß receptor. In pregnancy, hyperglycosylated hCG drives placental cell growth and invasion in implantation of pregnancy. It also blocks apoptosis. Human cancer cells steal the hCG ß-subunit gene and use hyperglycosylated hCG and its ß-subunit to drive malignancy. Here we examine research into hyperglycosylated hCG and its ß-subunit, and show that these molecules drive malignancy in most or possibly all human cancers. Methods: Mouse monoclonal antibody B152was raised against intact hyperglycosylated hCG, batch C5. The antibody binds hyperglycosylated hCG and its ß-subunit but does not bind the hormone hCG or its subunits. Total hCG was measured using the Siemens Immulite hCG assay, hyperglycosylated hCG and its ß-subunit were measured using the antibody B152 assay. Results: Eight independent center show that the hCG ß-subunit produced by cancers promotes malignancy, enhances cancer cell growth, cancer cell invasion and blockage of apoptosis in cancers. A study of 42 choriocarcinoma cases shows that percentage hyperglycosylated hCG exactly correlates with weekly doubling rate of cancer. It is concluded that hyperglycosylated hCG drive malignancy in this cancer. In a study with 7 separate cancers it is shown that increasing concentrations of hyperglycosylated hCG enhance all cancers. Increasing concentration of monoclonal antibody B152. Hyperglycosylated hCG and its ß-subunit drives cancer growth, cancer invasion and blocks apoptosis in cancer cells. Antibody B152 suppressed cancer cell growth creating a non-malignant-like state (no growth, no invasion), with no cancer growth over a starting 70% confluency. Conclusions: Choriocarcinoma is an example of cancer driven in malignancy by hyperglycosylated hCG, cancer aggression (weekly doubling rate) exactly correlating with percent hyperglycosylated hCG. In examining cancers, antibody B152 suppresses malignancy totally halting cancer growth in 7 of 7 cancer. This confirms that only the antigens, hyperglycosylated hCG and its ß-subunit drives malignancy in cancer cases.

Crystal structure of B-cell co-receptor CD19 in complex with antibody B43 reveals an unexpected fold.

CD19 is a transmembrane protein expressed on malignant B cells, but not in other lineages or other tissues, which makes it an attractive target for monoclonal antibody-mediated immunotherapy. Anti-CD19 antibody B43 was utilized in a bispecific T-cell engager (BiTE) blinatumomab that demonstrated potency for the treatment of relapsed acute lymphoblastic leukemia. To gain insight into the mechanism of action of the antibody, the crystal structure of B43 Fab was determined in complex with CD19 and in the unbound form. The structure revealed the binding epitope, explained the lack of cross-reactivity toward non-human species, and suggested the key-and-lock mechanism of antigen recognition. Most unexpectedly, the structure revealed a unique molecular topology of CD19. Rather than a tandem of c-type immunoglobulin folds predicted from the amino acid sequence, the extracellular domain of CD19 exhibits an elongated β-sandwich formed by two immunoglobulin folds by swapping their C-terminal halves. This is the first structure of CD19, which has no sequence homologs.

Clinical significance of immunohistochemical expression of cancer/testis tumor-associated antigens (MAGE-A1, MAGE-A3/4, NY-ESO-1) in patients with non-small cell lung cancer.

This paper deals with the clinical significance of the immunohistochemical expression of MAGE-A1, MAGE-A3/4 and NY-ESO-1 antigens in patients with non-small cell lung cancer (NSCLC). The study included 80 patients with NSCLC (40 with adenocarcinoma, 40 with squamous cell carcinoma) who had undergone surgery. MAGE-A1 and MAGE-A3/4 antigen expression was determined by an immunohistochemical method using the monoclonal antibody 57B, and NY-ESO-1 antigen expression was determined with the addition of the B9.8.1.1 antibody. The expression of these antigens was compared with the clinicopathological features of the tumors and the survival of the patients. MAGE-A1, MAGE-A3/4 and NY-ESO-1 were expressed in 17.3%, 44.4% and 18.5% of NSCLC patients, respectively. A statistically higher immunohistological expression rate of MAGE-A3/4 was found in squamous cell carcinoma (P <0.001) and a significantly higher amount of tumor necrosis was observed in tumors with MAGE-3 expression (P = 0.001), but no correlation with positive lymph nodes was found. There was a statistically significant correlation between MAGE-A1 expression in adenocarcinoma and the presence of tumor necrosis (P = 0.05). Furthermore, there was a significant correlation between NY-ESO-1 expression and positive lymph nodes in adenocarcinoma, but not in squamous cell carcinoma. No statistically significant difference in patient survival was found with regard to tumor type and the observed histopathological characteristics except tumor size. Statistically significantly better survival was found in the group of patients with adenocarcinomas who had positive expression of MAGE-A3/4 (P = 0.012). This study demonstrated that the expression of MAGE-A3/4 antigen might be a valuable prognostic factor regarding survival in patients with NSCLC.

Signal peptide of HIV envelope protein impacts glycosylation and antigenicity of gp120

The HIV-1 envelope protein (Env) of early-replicating viruses encodes several distinct transmission signatures. One such signature involves a reduced number of potential N-linked glycosylation sites (PNGs). This transmission signature underscores the importance of posttranslational modifications in the fitness of early-replicating isolates. An additional signature in Env involves the overrepresentation of basic amino acid residues at a specific position in the Env signal peptide (SP). In this report, we investigated the potential impact of this SP signature on gp120 glycosylation and antigenicity. Two recombinant gp120s were constructed, one derived from an isolate that lacks this signature and a second from an early-replicating isolate that includes this signature. Chimeric gp120s were also constructed in which the two SPs were swapped between the isolates. All four gp120s were probed with glycan-, structure- and receptor- specific probes in a surface plasmon resonance binding assay. We found that the SP of Env influences qualitative aspects of Env glycosylation that in turn affect the antigenicity of Env in a major way. The SP impacts the affinity of Env for DC-SIGN, a lectin receptor expressed on dendritic cells that is believed to play a role in mucosal transmission. Additionally, affinity for the monoclonal antibodies 17b and A32, which recognize a CD4-induced, open conformation of Env is also altered. These results demonstrate that natural variation in the SP of HIV Env can significantly impact the antigenicity of mature gp120. Thus, the SP is likely subject to antibody-mediated immune pressure.

Delipidation of Plasma Has Minimal Effects on Human Butyrylcholinesterase.

Human butyrylcholinesterase (BChE) is purified in large quantities from Cohn fraction IV-4 to use for protection against the toxicity of chemical warfare agents. Small scale preliminary experiments use outdated plasma from the American Red Cross as the starting material for purifying BChE (P06276). Many of the volunteer donor plasma samples are turbid with fat, the donor having eaten fatty food before the blood draw. The turbid fat interferes with enzyme assays performed in the spectrophotometer and with column chromatography. Our goal was to find a method to remove fat from plasma without loss of BChE activity. Satisfactory delipidation was achieved by adding a solution of 10% dextran sulfate and calcium chloride to fatty plasma, followed by centrifugation, and filtration through a 0.8 μm filter. Treatment with Aerosil also delipidated fatty plasma, but was accompanied by loss of 50% of the plasma volume. BChE activity and the BChE isozyme pattern on nondenaturing gel electrophoresis were unaffected by delipidation. BChE in delipidated plasma was efficiently captured by immobilized monoclonal antibodies B2 18-5 and mAb2. The immunopurified BChE was released from antibody binding with acid and visualized as a highly enriched, denatured BChE preparation by SDS gel electrophoresis. In conclusion, delipidation with dextran sulfate/CaCl2 preserves BChE activity and the tetramer structure of BChE.

Changes in V-ATPase subunits of human urinary exosomes reflect the renal response to acute acid/alkali loading and the defects in distal renal tubular acidosis

In the kidney, final urinary acidification is achieved by V-ATPases expressed in type A intercalated cells. The B1 subunit of the V-ATPase is required for maximal urinary acidification, while the role of the homologous B2 subunit is less clear. Here we examined the effect of acute acid/alkali loading in humans on B1 and B2 subunit abundance in urinary exosomes in normal individuals and of acid loading in patients with distal renal tubular acidosis (dRTA). Specificities of B1 and B2 subunit antibodies were verified by yeast heterologously expressing human B1 and B2 subunits, and murine wild-type and B1-deleted kidney lysates. Acute ammonium chloride loading elicited systemic acidemia, a drop in urinary pH, and increased urinary ammonium excretion. Nadir urinary pH was achieved at four to five hours, and exosomal B1 abundance was significantly increased at two through six hours after ammonium chloride loading. After acute equimolar sodium bicarbonate loading, blood and urinary pH rose rapidly, with a concomitant reduction of exosomal B1 abundance within two hours, which remained lower throughout the test. In contrast, no change in exosomal B2 abundance was found following acid or alkali loading. In patients with inherited or acquired distal RTA, the urinary B1 subunit was extremely low or undetectable and did not respond to acid loading in urine, whereas no change in B2 subunit was found. Thus, both B1 and B2 subunits of the V-ATPase are detectable in human urinary exosomes, and acid and alkali loading or distal RTA cause changes in the B1 but not B2 subunit abundance in urinary exosomes.

Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike.

SUMMARYFor many enveloped viruses, binding to a receptor(s) on a host cell acts as a first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication [for review see1,2]. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. While Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env3,4, targets of broadly neutralizing antibodies (bnAbs). Thus, they are attractive immunogens for vaccine development [for review see5–8]. Here we present high-resolution cryo-electron microscopy (cryoEM) structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of ~3.7 Å and ~3.6 Å, respectively, and compare them to cryoEM reconstructions of ligand-free B41 SOSIP Env trimers or in complex with either CD4 or CD4bs antibody PGV04, at ~5.6 Å, ~5.2 Å and ~7.4 Å, respectively. Consequently, we present the most complete description and understanding of the CD4/17b-induced intermediate and provide the molecular basis of the receptor-binding induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes more buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

Abstract 223: CXCR4 Regulates B1 Cell Localization, Proliferation, Survival, and Atheroprotective IgM Production

Background: B1 cells exert protective effects in atherosclerosis through production of anti-inflammatory natural IgM antibodies that recognize oxidation-specific epitopes, such as MDA-LDL, present in diseased arteries. The bone marrow, spleen, and omental fat are known niches for B1 antibody production. However, the mechanisms underlying B1 localization to these sites and B1 antibody production are currently unclear. Methods and Results: To identify key immune mediators that may be relevant to atherosclerosis, our lab has correlated surface marker expression on peripheral blood mononuclear cells with clinical markers of atherosclerosis in a human cohort. Expression of the chemokine receptor CXCR4 on circulating B1 cells associates with decreased plaque burden in coronary arteries and increased plasma levels of anti-MDA-LDL IgM antibodies. To study the role of CXCR4 in modulating B1 cell function, we generated mice with B cell-specific loss of CXCR4 on the atherogenic ApoE -/- background (CXCR4 BKO ). Chow-fed, 8-week-old CXCR4 BKO mice demonstrate fewer B1 cells (N=6-8, p&lt;0.0001) and IgM antibody-secreting cells (N=6, p=0.004) in the bone marrow, and reduced plasma total IgM levels (N=6-8, p=0.04), relative to littermate controls (CXCR4 WT ). To determine the role of CXCR4 on the B1 cell subset specifically, we adoptively transferred CXCR4 WT or CXCR4 BKO B1 cells into lymphocyte-deficient Rag1 -/- ApoE -/- mice. After 16 weeks of Western diet feeding, recipient mice given CXCR4 BKO B1 cells demonstrate significantly reduced plasma IgM levels (n=5-7, p=0.02), and fewer donor B1 cells in the spleen, peritoneal cavity, and omental fat compared to recipients given CXCR4 WT B1 cells. Few to no donor cells were detected in the bone marrow. To determine whether CXCR4 has a role in B1 cell proliferation or survival, BrdU incorporation and expression of the death receptor FasR were assayed in CXCR4 WT and CXCR4 BKO B1 cells. B1 cells from CXCR4 BKO mice display heightened BrdU incorporation (n=4, p=0.005), yet have increased expression of FasR (n=4, p=0.01) relative to CXCR4 WT B1 cells. Conclusion: Our data demonstrate novel roles for CXCR4 in regulating several processes in B1 cells, including proliferation and survival, consequently impacting production of IgM.

Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1

ABSTRACTThe potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity. Addition of 1B7 to cultured human chondrocytes revealed the full aggrecanolytic activity of ADAMTS-5 generated by the cells. 1B7 is a useful tool to estimate the ADAMTS-5 activity and to identify its potential roles in the tissues.

HIV-1 Consensus Envelope-Induced Broadly Binding Antibodies.

Antibodies that cross-react with multiple HIV-1 envelopes (Envs) are useful reagents for characterizing Env proteins and for soluble Env capture and purification assays. We previously reported 10 murine monoclonal antibodies induced by group M consensus Env, CON-6 immunization. Each demonstrated broad cross-reactivity to recombinant Envs. Here we characterized the Env epitopes to which they bind. Seven mapped to linear epitopes in gp120, five at the Env N-terminus, and two at the Env C-terminus. One antibody, 13D7, bound at the gp120 N-terminus (aa 30-42), reacted with HIV-1-infected CD4+ T cells, and when expressed in a human IgG1 backbone, mediated antibody-dependent cellular cytotoxicity. Antibody 18F11 bound at the gp120 C-terminus (aa 445-459) and reactivity was glycan dependent. Antibodies 13D7, 3B3, and 16H3 bound to 100 percent of HIV-1 Envs tested in ELISA and sodium dodecyl sulfate/polyacrylamide gel electrophoresis/western blot analysis. These data define the epitopes of monoclonal antibody reagents for characterization of recombinant Envs, one epitope of which is also expressed on the surface of HIV-1-infected CD4+ T cells.

Development of antibody-modified chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier as a strategy for inhibiting HIV replication in astrocytes.

RNA interference (RNAi)-mediated gene silencing offers a novel treatment and prevention strategy for human immunodeficiency virus (HIV) infection. HIV was found to infect and replicate in human brain cells and can cause neuroinfections and neurological deterioration. We designed dual-antibody-modified chitosan/small interfering RNA (siRNA) nanoparticles to deliver siRNA across the blood-brain barrier (BBB) targeting HIV-infected brain astrocytes as a strategy for inhibiting HIV replication. We hypothesized that transferrin antibody and bradykinin B2 antibody could specifically bind to the transferrin receptor (TfR) and bradykinin B2 receptor (B2R), respectively, and deliver siRNA across the BBB into astrocytes as potential targeting ligands. In this study, chitosan nanoparticles (CS-NPs) were prepared by a complex coacervation method in the presence of siRNA, and antibody was chemically conjugated to the nanoparticles. The antibody-modified chitosan nanoparticles (Ab-CS-NPs) were spherical in shape, with an average particle size of 235.7±10.2nm and a zeta potential of 22.88±1.78mV. The therapeutic potential of the nanoparticles was evaluated based on their cellular uptake and gene silencing efficiency. Cellular accumulation and gene silencing efficiency of Ab-CS-NPs in astrocytes were significantly improved compared to non-modified CS-NPs and single-antibody-modified CS-NPs. These results suggest that the combination of anti-Tf antibody and anti-B2 antibody significantly increased the knockdown effect of siRNA-loaded nanoparticles. Thus, antibody-mediated dual-targeting nanoparticles are an efficient and promising delivery strategy for inhibiting HIV replication in astrocytes. Graphical abstract Graphic representation of dual-antibody-conjugated chitosan nanoparticles for the targeted delivery of siRNA across the blood-brain barrier (BBB) for inhibiting HIV replication in astrocytes. a Nanoparticle delivery to the BBB and penetration. b TfR-mediated transcytosis of nanoparticles across the epithelial cells. c B2R-mediated endocytosis of nanoparticles in astrocytes. d The molecular interactions between HIV-1 Tat protein and Cyclin T1 and Tip110 cellular proteins. e A schematic representation of chitosan nanoparticles with its components. RNAPII RNA polymerase II, TAR transactivation response RNA element, LTR long terminal repeat, Ab antibody, CS chitosan, TPP tripolyphosphate.

Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

Covalent immobilization of rabbit-antiaflatoxin-antibodies onto the poly-acrylamideacrylonitrile as well as hybrid material UREASIL and developing an optical immunosensor

Abstract The aim of this work is to describe a covalent immobilization of antibodies onto the poly- acrylamide-acrylonitrile or hybrid material UREASIL and creation of optical immunosensor for determination of aflatoxin Bl. For this purpose, mouse-anti-aflatoxin B1 antibodies with oxidized carbohydrate moieties were covalently immobilized on the membranes of polyacrylamide- polyacrylonitrile copolymer, as well as the hybrid material UREASIL. To determine the affinity&gt; binding of the immobilized antibody with afatoxin Bl was used "sandwich" method. Associated with the immobilized antibody sought ingredients interact with a surplus of secondary’ signal antibodies. The described method has been developed as a model system, which can easily be applied for the determination of aflatoxins in samples of different origin. To the best of our knowledge, this is the first study to show that in the establishment of biosensor was used hybrid material UREASIL.

Single-Molecule FRET Delineates Asymmetric Trimer Conformations during HIV-1 Entry

HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions revealed that Env trimers are dynamic, spontaneously transitioning between three distinct conformations. Binding of CD4 and coreceptor surrogate antibody 17b promotes opening of the closed Env (State 1) to stabilize an activated conformation (State 3) by way of at least one structural intermediate (State 2). Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we identify this intermediate as an asymmetric conformation where only a single CD4 molecule engages the Env trimer and individual protomers adopt distinct conformations. Binding of a second CD4 molecule is needed to completely open the trimer. Thus, our work reveals a novel asymmetric structural intermediate and gives insights into cooperativity during the opening of the HIV-1 Env trimer.

Development of a highly specific HER2 monoclonal antibody for immunohistochemistry using protein microarray chips

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.