Monoclonal Antibodies.

ERIC Educational Resources Information Center

Killington, R. A.; Powell, K. L.

1984-01-01

Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

Development of an analytical method to assess the occupational health risk of therapeutic monoclonal antibodies using LC-HRMS.

PubMed

Reinders, Lars M H; Klassen, Martin D; Jaeger, Martin; Teutenberg, Thorsten; Tuerk, Jochen

2018-04-01

Monoclonal antibodies are a group of commonly used therapeutics, whose occupational health risk is still discussed controversially. The long-term low-dose exposure side effects are insufficiently evaluated; hence, discussions are often based on a theoretical level or extrapolating side effects from therapeutic dosages. While some research groups recommend applying the precautionary principle for monoclonal antibodies, others consider the exposure risk too low for measures taken towards occupational health and safety. However, both groups agree that airborne monoclonal antibodies have the biggest risk potential. Therefore, we developed a peptide-based analytical method for occupational exposure monitoring of airborne monoclonal antibodies. The method will allow collecting data about the occupational exposure to monoclonal antibodies. Thus, the mean daily intake for personnel in pharmacies and the pharmaceutical industry can be determined for the first time and will help to substantiate the risk assessment by relevant data. The introduced monitoring method includes air sampling, sample preparation and detection by liquid chromatography coupled with high-resolution mass spectrometry of individual monoclonal antibodies as well as sum parameter. For method development and validation, a chimeric (rituximab), humanised (trastuzumab) and a fully humanised (daratumumab) monoclonal antibody are used. A limit of detection between 1 μg per sample for daratumumab and 25 μg per sample for the collective peptide is achieved. Graphical abstract Demonstration of the analytical workflow, from the release of monoclonal antibodies to the detection as single substances as well as sum parameter.

[International classification of various types of monoclonal antibodies].

PubMed

Scheen, A J

2009-01-01

Significant advances in the development of monoclonal antibodies ("mabs") have been acknowledged during the last two decades. Successive developments led to the marketing of murine antibodies ("o-mab" first, followed by chimeric antibodies ("xi-mab"), humanised antibodies ("zu-mab") and, finally, human monoclonal antibodies ("u-mab"). In order to facilitate the distinction between the various monoclonal antibodies used in clinical practice, an international nomenclature has been proposed with the use of a specific suffix corresponding to the origine/source of "mabs" preceded by an infix referring to the medicine's target. The efforts in developing new types of monoclonal antibodies aimed at improving their pharmacokinetics (longer half-life), pharmacodynamics (better efficacy because of stronger affinity to human receptor), and safety profile (less antigenic and immunogenic reactions). These progresses could be obtained thanks to the remarkable development of molecular biotechnology.

Selectivity verification of cardiac troponin monoclonal antibodies for cardiac troponin detection by using conventional ELISA

NASA Astrophysics Data System (ADS)

Fathil, M. F. M.; Arshad, M. K. Md; Gopinath, Subash C. B.; Adzhri, R.; Ruslinda, A. R.; Hashim, U.

2017-03-01

This paper presents preparation and characterization of conventional enzyme-linked immunosorbent assay (ELISA) for cardiac troponin detection to determine the selectivity of the cardiac troponin monoclonal antibodies. Monoclonal antibodies, used to capture and bind the targets in this experiment, are cTnI monoclonal antibody (MAb-cTnI) and cTnT monoclonal antibody (MAb-cTnT), while both cardiac troponin I (cTnI) and T (cTnT) are used as targets. ELISA is performed inside two microtiter plates for MAb-cTnI and MAb-cTnT. For each plate, monoclonal antibodies are tested by various concentrations of cTnI and cTnT ranging from 0-6400 µg/l. The binding selectivity and level of detection between monoclonal antibodies and antigen are determined through visual observation based on the color change inside each well on the plate. ELISA reader is further used to quantitatively measured the optical density of the color changes, thus produced more accurate reading. The results from this experiment are utilized to justify the use of these monoclonal antibodies as bio-receptors for cardiac troponin detection by using field-effect transistor (FET)-based biosensors coupled with substrate-gate in the future.

Antibodies and Selection of Monoclonal Antibodies.

PubMed

Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

NASA Astrophysics Data System (ADS)

Massey, Richard J.; Schochetman, Gerald

1981-07-01

The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

PubMed

vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

2011-07-01

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

Humanized Antibodies for Antiviral Therapy

NASA Astrophysics Data System (ADS)

Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

1991-04-01

Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: implication for diagnosis of Kala-azar

PubMed Central

Nourizadeh, Ezzat; Zargar, Seyed Jalal; Alimohammadian, Mohammad Hossein; Ajdary, Soheila; Mahdavi, Mahdi

2018-01-01

Objective(s): Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran. Materials and Methods: First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c smice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting. Results: Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones. Conclusion: This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar.

Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

PubMed

Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

2010-03-01

The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.

PubMed Central

Nato, F; Mazie, J C; Fournier, J M; Slizewicz, B; Sagot, N; Guibourdenche, M; Postic, D; Riou, J Y

1991-01-01

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents. PMID:1909346

An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies.

PubMed

Sanders, Melanie; Guo, Yirong; Iyer, Abhishek; García, Yara Ruiz; Galvita, Anastasia; Heyerick, Arne; Deforce, Dieter; Risseeuw, Martijn D P; Van Calenbergh, Serge; Bracke, Marc; Eremin, Sergei; Madder, Annemieke; De Saeger, Sarah

2014-01-01

An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml⁻¹. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml⁻¹, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml⁻¹. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.

Monoclonal antibodies against the rat liver glucocorticoid receptor.

PubMed Central

Okret, S; Wikström, A C; Wrange, O; Andersson, B; Gustafsson, J A

1984-01-01

Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR. Images PMID:6200880

Identification of a vesicular-arbuscular mycorrhizal fungus by using monoclonal antibodies in an enzyme-linked immunosorbent assay.

PubMed

Wright, S F; Morton, J B; Sworobuk, J E

1987-09-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

PubMed

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Synergistic protection of mice against plague with monoclonal antibodies specific for the F1 and V antigens of Yersinia pestis.

PubMed

Hill, Jim; Copse, Catherine; Leary, Sophie; Stagg, Anthony J; Williamson, E Diane; Titball, Richard W

2003-04-01

Monoclonal antibodies specific for Yersinia pestis V antigen and F1 antigen, administered singly or in combination, protected mice in models of bubonic and pneumonic plague. Antibodies showed synergy when administered prophylactically and as a therapy 48 h postinfection. Monoclonal antibodies therefore have potential as a treatment for plague.

Using Monoclonal Antibodies to Prevent Mucosal Transmission of Epidemic Infectious Diseases

PubMed Central

Zeitlin, Larry; Cone, Richard A.

1999-01-01

Passive immunization with antibodies has been shown to prevent a wide variety of diseases. Recent advances in monoclonal antibody technology are enabling the development of new methods for passive immunization of mucosal surfaces. Human monoclonal antibodies, produced rapidly, inexpensively, and in large quantities, may help prevent respiratory, diarrheal, and sexually transmitted diseases on a public health scale. PMID:10081672

Therapeutic Recombinant Monoclonal Antibodies

ERIC Educational Resources Information Center

Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

The therapeutic monoclonal antibody market

PubMed Central

Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

2015-01-01

Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion. PMID:25529996

Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

PubMed

Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

2015-08-01

Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies

PubMed Central

Hutchinson, Alistair P.; Nicklin, Stephen

2015-01-01

Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti-PSMA Monoclonal Antibody J591 in Patients With High-Risk Castrate Biochemically Relapsed Prostate Cancer

DTIC Science & Technology

2015-09-01

Award Number: W81XWH-09-1-0596 TITLE: A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti- PSMA Monoclonal Antibody J591 in Patients With High...1-0596 A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti- PSMA Monoclonal Antibody J591 in Patients With High-Risk Castrat Biochemically Relapsed...in December 2014 with approval to proceed without modifications. 15. SUBJECT TERMS Prostate cancer, PSA, PSMA , monoclonal antibody

Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assay †

PubMed Central

Wright, Sara F.; Morton, Joseph B.; Sworobuk, Janis E.

1987-01-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains. PMID:16347441

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

PubMed Central

Sineh sepehr, Koushan; Baradaran, Behzad; Majidi, Jafar; Abdolalizadeh, Jalal; Aghebati, leili; Zare Shahneh, Fatemeh

2013-01-01

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells. PMID:24312821

[Design of next generation antibody drug conjugates].

PubMed

Zhu, Gui-Dong; Fu, Yang-Xin

2013-07-01

Chemotherapy remains one of the major tools, along with surgery, radiotherapy, and more recently targeted therapy, in the war against cancer. There have appeared a plethora of highly potent cytotoxic drugs but the poor discriminability between cancerous and healthy cells of these agents limits their broader application in clinical settings. Therapeutic antibodies have emerged as an important class of biological anticancer agents, thanks to their ability in specific binding to tumor-associated antigens. While this important class of biologics can be used as single agents for the treatment of cancer through antibody-dependent cell cytotoxicity (ADCC), their therapeutical efficacy is often limited. Antitumor antibody drug conjugates (ADCs) combine the target-specificity of monoclonal antibody (mAb) and the highly active cell-killing drugs, taking advantages of the best characteristics out of both components. Thus, insufficiency of most naked mAbs in cancer therapy has been circumvented by arming the immunoglobulin with cytotoxic drugs. Here mAbs are used as vehicles to transport potent payloads to tumor cells. ADCs contain three main components: antibody, linker and cytotoxics (also frequently referred as payload). Antibodies can recognize and specifically bind to the tumor-specific antigens, leading to an antibody-assisted internalization, and payload release. While ADC has demonstrated tremendous success, a number of practical challenges limit the broader applications of this new class of anticancer therapy, including inefficient cellular uptake, low cytotoxicity, and off-target effects. This review article aims to cover recent advances in optimizing linkers with increased stability in circulation while allowing efficient payload release within tumor cells. We also attempt to provide some practical strategies in resolving the current challenges in this attractive research area, particularly to those new to the field.

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus).

PubMed

Wellehan, James F X; Green, Linda G; Duke, Diane G; Bootorabi, Shadi; Heard, Darryl J; Klein, Paul A; Jacobson, Elliott R

2009-09-01

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate.

PubMed

Marcoux, Julien; Champion, Thierry; Colas, Olivier; Wagner-Rousset, Elsa; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianférani, Sarah

2015-08-01

Antibody-drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla(®)). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin(®)), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1. © 2015 The Protein Society.

Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

NASA Astrophysics Data System (ADS)

Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

1987-05-01

A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

Monoclonal antibodies and method for detecting dioxins and dibenzofurans

DOEpatents

Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

1989-01-01

Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

From rabbit antibody repertoires to rabbit monoclonal antibodies.

PubMed

Weber, Justus; Peng, Haiyong; Rader, Christoph

2017-03-24

In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

PubMed

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

A survey of manufacturing and handling practices for monoclonal antibodies by pharmacy, nursing and medical personnel.

PubMed

Alexander, M; King, J; Lingaratnam, S; Byrne, J; MacMillan, K; Mollo, A; Kirsa, S; Green, M

2016-04-01

There is a paucity of data available to assess the occupational health and safety risk associated with exposure to monoclonal antibodies. Industry standards and published guidelines are conflicting or outdated. Guidelines offer contrary recommendations based on an array of methodological approaches. This survey aimed to describe current practices, beliefs and attitudes relating to the handling of monoclonal antibodies by Australian medical, nursing and pharmacy clinicians. An electronic survey was distributed between June and September 2013. Respondents were surveyed on three focus areas: institutional guideline availability and content, current practices and attitudes. Demographic data relating to respondent and primary place of practice were also collected. A total of 222 clinicians completed the survey, with representation from all targeted professional groups and from a variety of geographic locations. 92% of respondents reported that their institution prepared or administered monoclonal antibodies, with 87% specifically handling anti-cancer monoclonal antibodies. Monoclonal antibodies were mostly prepared onsite (84-90%) and mostly within pharmacy clean-rooms (75%) and using cytotoxic cabinets (61%). 43% of respondents reported access to institutional monoclonal antibody handling guidelines with risk reduction strategies including training and education (71%), spill and waste management (71%), procedures for transportation (57%) and restricted handling (50%). Nurses had a stronger preference towards pharmacy manufacturing than both doctors and pharmacists for a range of clinical scenarios. 95% of all respondents identified that professional or regulatory body guidelines are an important resource when considering handling practices. Monoclonal antibodies are most commonly handled according to cytotoxic drug standards and often in the absence of formal guidelines. © The Author(s) 2014.

Neutralizing anti-interleukin-1β antibodies modulate fetal blood-brain barrier function after ischemia.

PubMed

Chen, Xiaodi; Sadowska, Grazyna B; Zhang, Jiyong; Kim, Jeong-Eun; Cummings, Erin E; Bodge, Courtney A; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Threlkeld, Steven W; Banks, William A; Stonestreet, Barbara S

2015-01-01

We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1β antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1β monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin-1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1β monoclonal antibody levels were higher (P<0.03), and interleukin-1β protein concentrations (P<0.03) and protein expressions (P<0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (P<0.04), and Ki showed an inverse linear correlation (r= -0.65, P<0.02) with anti-interleukin-1β monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1β monoclonal antibody infusions after ischemia result in brain anti-interleukin-1β antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1β protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1β, contributes to impaired blood-brain barrier function after ischemia in the fetus. Copyright © 2014 Elsevier Inc. All rights reserved.

Polyclonal and monoclonal antibodies in clinic.

PubMed

Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

2014-01-01

Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

Indium 111 ZCE-025 immunoscintigraphy in occult recurrent colorectal cancer with elevated carcinoembryonic antigen level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doerr, R.J.; Abdel-Nabi, H.; Merchant, B.

1990-02-01

We investigated the utility of scanning with indium 111 labeled to monoclonal antibody in 13 patients after curative resection of colorectal cancer who had elevated carcinoembryonic antigen levels and negative results of clinical workup. Each patient received 1 mg of anti-carcinoembryonic antigen monoclonal antibody type ZCE 025 labeled with 5.5 mCi of {sup 111}In, plus 9 to 39 mg of the same antibody unlabeled. Patients underwent scanning 3 to 7 days after infusion by planar and emission computed tomography. ZCE-025 monoclonal antibody imaging detected tumor recurrence or metastasis in 11 of 13 patients. In one patient the monoclonal antibody scanmore » gave a true-negative result, and in one patient the monoclonal antibody scan failed to disclose a metachronous cecal primary. Tumor sites identified were the pelvis (2 patients), abdominal wall (2), retroperitoneum (1), lymph nodes (3); liver (2), bone (2), and lung (1). The accurate localization of colorectal carcinoma recurrences by means of {sup 111}In ZCE-025 monoclonal antibody demonstrates the usefulness of this diagnostic agent in the setting of elevated carcinoembryonic antigen level and negative results of clinical and radiologic workup.« less

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

PubMed

Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

1985-01-29

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

PubMed

Worobec, E A; Shastry, P; Smart, W; Bradley, R; Singh, B; Paranchych, W

1983-09-01

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

PubMed Central

Worobec, E A; Shastry, P; Smart, W; Bradley, R; Singh, B; Paranchych, W

1983-01-01

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types. Images PMID:6136463

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

PubMed

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Rajendran, Madhusudan; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R; García-Sastre, Adolfo; Basler, Christopher F; Munoz-Fontela, Cesar; Krammer, Florian

2017-08-15

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 -/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro , suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics. Copyright © 2017 American Society for Microbiology.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

PubMed Central

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R.; García-Sastre, Adolfo; Basler, Christopher F.

2017-01-01

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2−/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics. PMID:28592526

Structure of solid tumors and their vasculature: Implications for therapy with monoclonal antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dvorak, H.F.; Nagy, J.A.; Dvorak, A.M.

Delivery of monoclonal antibodies to solid tumors is a vexing problem that must be solved if these antibodies are to realize their promise in therapy. Such success as has been achieved with monoclonal antibodies is attributable to the local hyperpermeability of the tumor vasculature, a property that favors antibody extravasation at tumor sites and that is mediated by a tumor-secreted vascular permeability factor. However, leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and by other tumor cells that together represent significant barriers to penetration by extravasated monoclonal antibodies. For this reason, alternativemore » approaches may be attractive. These include the use of antibody-linked cytotoxins, which are able to kill tumor cells without immediate contact, and direction of antibodies against nontumor cell targets, for example, antigens unique to the tumor vascular endothelium or to tumor stroma. 50 refs.« less

Construction of Rabbit Immune Antibody Libraries.

PubMed

Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

2018-01-01

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.

PubMed

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.

Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

PubMed Central

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

DOEpatents

Meagher, Richard B.; Laterza, Vince

2006-12-12

The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

Generation of Gene-Engineered Chimeric DNA Molecules for Specific Therapy of Autoimmune Diseases

PubMed Central

Gesheva, Vera; Szekeres, Zsuzsanna; Mihaylova, Nikolina; Dimitrova, Iliyana; Nikolova, Maria; Erdei, Anna; Prechl, Jozsef

2012-01-01

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of self-reactive B and T cells and autoantibody production. In particular, double-stranded DNA-specific B cells play an important role in lupus progression, and their selective elimination is a reasonable approach for effective therapy of SLE. DNA-based vaccines aim at the induction of immune response against the vector-encoded antigen. Here, we are exploring, as a new DNA-based therapy of SLE, a chimeric DNA molecule encoding a DNA-mimotope peptide, and the Fv but not the immunogenic Fc fragment of an FcγRIIb-specific monoclonal antibody. This DNA construct was inserted in the expression vector pNut and used as a naked DNA vaccine in a mouse model of lupus. The chimeric DNA molecule can be expressed in eukaryotic cells and cross-links cell surface receptors on DNA-specific B cells, delivering an inhibitory intracellular signal. Intramuscular administration of the recombinant DNA molecule to lupus-prone MRL/lpr mice prevented increase in IgG anti-DNA antibodies and was associated with a low degree of proteinuria, modulation of cytokine profile, and suppression of lupus nephritis. PMID:23075110

[A New Simple Technique for Producing Labeled Monoclonal Antibodies for Antibody Pair Screening in Sandwich-ELISA].

PubMed

Zaripov, M M; Afanasieva, G V; Glukhova, X A; Trizna, Y A; Glukhov, A S; Beletsky, I P; Prusakova, O V

2015-01-01

A simple and fast method for obtaining biotin-labeled monoclonal antibodies was developed usingcontent of hybridoma culture supernatant sufficient to select antibody pairs in sandwich ELISA. The method consists in chemical biotinylation of antigen-bound antibodies in a well of ELISA plate. Using as an example target Vaccinia virus A27L protein it was shown that the yield of biotinylated reactant is enough to set comprehensive sandwich ELISA for a moderate size panel of up to 25 monoclonal antibodies with an aim to determine candidate pairs. The technique is a cheap and effective solution since it avoids obtaining preparative amounts of antibodies.

Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

PubMed

Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

2015-12-01

Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

Development, characterization, and use of monoclonal and polyclonal antibodies against the myxosporean, Ceratomyxa shasta

USGS Publications Warehouse

Bartholomew, J.L.; Rohovec, J.S.; Fryer, J.L.

1989-01-01

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.

Monoclonal antibodies and recombinant immunoglobulins for the treatment of multiple sclerosis.

PubMed

Gensicke, Henrik; Leppert, David; Yaldizli, Özgür; Lindberg, Raija L P; Mehling, Matthias; Kappos, Ludwig; Kuhle, Jens

2012-01-01

Multiple sclerosis (MS) is an inflammatory and degenerative disease leading to demyelination and axonal damage in the CNS. Autoimmunity plays a central role in MS pathogenesis. Per definition, monoclonal antibodies are recombinant biological compounds with a well defined target, thus carrying the promise of targeting pathogenic cells or molecules with high specificity, avoiding undesired off-target effects. Natalizumab was the first monoclonal antibody to be approved for the treatment of MS. Several other monoclonal antibodies are in development and have demonstrated promising efficacy in phase II studies. They can be categorized according to their mode of action into compounds targeting (i) leukocyte migration into the CNS (natalizumab); (ii) cytolytic antibodies (rituximab, ocrelizumab, ofatumumab, alemtuzumab); or (iii) antibodies and recombinant proteins targeting cytokines and chemokines and their receptors (daclizumab, ustekinumab, atacicept, tabalumab [Ly-2127399], secukinumab [AIN457]). In this review, we discuss the specific molecular targets, clinical efficacy and safety of these compounds and discuss criteria to anticipate the position of monoclonal antibodies in the diversifying armamentarium of MS therapy in the coming years.

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

PubMed

Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

2016-12-01

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na + -K + -ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na + -K + -ATPase activity was detected by SensoLyte R FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na + -K + -ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na + -K + -ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na + -K + -ATPase α1 subunit is prepared.

The two novel DLL4-targeting antibody-drug conjugates MvM03 and MGD03 show potent anti-tumour activity in breast cancer xenograft models.

PubMed

Wang, Shijing; Zhou, Rihong; Sun, Fumou; Li, Renjie; Wang, Min; Wu, Min

2017-11-28

The anti-human Delta-like 4 (DLL4) monoclonal antibody MMGZ01 has a high affinity to hrDLL4 and arrests the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype, promotes immature vessels, and effectively reduces breast cancer cell growth in vivo. To develop a much more effective therapy, we conjugated MMGZ01 with two small-molecule cytotoxic agents, i.e., monomethyl auristatin E (MMAE) and doxorubicin (DOX), with different linkers to generate antibody-drug conjugates (ADCs), i.e., MMGZ01-vc-MMAE (named MvM03) and MMGZ01-GMBS-DOX (named MGD03), that are more potent therapeutic agents than naked antibody therapeutic agents. The produced anti-DLL4 ADCs can be effectively directed against DLL4 and internalized. Then, the release of MMAE or DOX into the cytosol can induce G2/M or G0/G1 phase growth arrest and cell death through the induction of apoptosis. In vitro, MvM03 was highly potent and selective against DLL4 cell lines. The anti-DLL4 ADCs, particularly MvM03, showed more potent anti-tumour activity than Docetaxel, which is an inhibitor of the depolymerisation of microtubules, in two xenograft breast cancer tumour models. Our findings indicate that anti-DLL4 ADCs have promising potential as an effective therapy for breast cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

DOEpatents

Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

1994-01-01

Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

Identification of a novel group of Serpulina hyodysenteriae isolates by using a lipopolysaccharide-specific monoclonal antibody.

PubMed Central

Alderton, M R; Smith, S C; Coloe, P J

1993-01-01

A monoclonal antibody to Serpulina hyodysenteriae 8930 was produced and was used to probe pronase-treated cell lysates of S. hyodysenteriae isolates in immunblots. The results showed that the monoclonal antibody was specific for only five closely related S. hyodysenteriae isolates: 8930, 5380, 70A, RMIT 88, and RMIT 97. Images PMID:8501237

[Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

PubMed

Dzagurova, T K; Solopova, O N; Sveshnikov, P G; Korotina, N A; Balovneva, M V; Leonovich, O A; Varlamov, N E; Malkin, G A; Sotskova, S E; Tkachenko, E A

2013-01-01

The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

PubMed

Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

2010-07-01

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

PubMed

Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

2014-01-25

One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

Generation of Murine Monoclonal Antibodies by Hybridoma Technology.

PubMed

Holzlöhner, Pamela; Hanack, Katja

2017-01-02

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Development of a colloidal gold immunochromatographic test strip for detection of lymphocystis disease virus in fish.

PubMed

Sheng, X Z; Song, J L; Zhan, W B

2012-10-01

To develop a gold immunochromatographic test strip for on-site rapid detection of lymphocystis disease virus (LCDV). Monodispersional colloidal gold and gold-labelled anti-LCDV monoclonal antibody (McAb) 2D11 were prepared and characterized by UV-visible spectroscopy and transmission electron microscopy. Gold-labelled probe was used as the detection antibody, and goat anti-mouse IgG at the control line and anti-LCDV McAb 1A8 at the test line of the test strip served as the capture antibody. The positive results could be easily judged by the presence of a red test line with naked eye within 10 min. The test strip, in good agreement with enzyme-linked immunosorbent assay and dot-blotting in sensitivity and LCDV detection, gave a detection limit of 1 μg ml(-1) of LCDV and was stable for 6 months at room temperature and 12 months at 4°C. The test strip was specific, simple and convenient for rapid detection of LCDV presenting good stability and reproducibility. This ready-to-use test strip allows on-site rapid detection of LCDV in fish without the requirement of specialized equipments and professional personnel, which could augment the practical application for diagnosis of LCDV even in disadvantage areas. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

DOEpatents

Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

1994-08-02

Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

Monoclonal Antibodies Passively Protect BALB/c Mice Against Burkholderia mallei Aerosol Challenge

DTIC Science & Technology

2006-03-01

November 2005 Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei , the... Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei , the etiologic... Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice. Vaccine 23:1986–1992. 17. Vyshelesskii, S. N. 1974. Glanders (Equinia). Tr

Scintigraphy of normal mouse ovaries with monoclonal antibodies to ZP-2, the major zona pellucida protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

East, I.J.; Keenan, A.M.; Larson, S.M.

1984-08-31

The zona pellucida is an extracellular glycocalyx, made of three sulfated glycoproteins, that surrounds mammalian oocytes. Parenterally administered monoclonal antibodies specific for ZP-2, the most abundant zona protein, localize in the zona pellucida. When labeled with iodine-125, these monoclonal antibodies demonstrate a remarkably high target-to-nontarget tissue ratio and provide clear external radioimaging of ovarian tissue.

Study of blood group B antigen with a specific monoclonal antibody (anti-B, b-183).

PubMed Central

Rouger, P; Edelman, L; Doinel, C; Reviron, J; Salmon, C; Bach, J F

1983-01-01

A murine anti-B monoclonal antibody was obtained by the hybridoma technique. This antibody called anti-B (b-183) is of IgM nature; it is capable of agglutinating normal B, B3, Bx, cis AB and some acquired B red cells. Its association constant is 1.1 X 10(8) l/mol, and appears high compared to those of the monoclonal anti-A. This monoclonal anti-B was used to determine the number of B sites on B3 and Bx red cells. PMID:6840810

Monoclonal Antibodies against the Drosophila Nervous System

NASA Astrophysics Data System (ADS)

Fujita, Shinobu C.; Zipursky, Stephen L.; Benzer, Seymour; Ferrus, Alberto; Shotwell, Sandra L.

1982-12-01

A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.

The Case for Adjunctive Monoclonal Antibody Immunotherapy in Schizophrenia.

PubMed

Miller, Brian J; Buckley, Peter F

2016-06-01

This article presents the case in favor of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted. Then key evidence for immune dysfunction in patients with schizophrenia is considered. Next, previous trials of adjunctive anti-inflammatory or other immunotherapy in schizophrenia are discussed. Then evidence for psychosis as a side effect of immunotherapy for other disorders is discussed. Also presented is preliminary evidence for adjunctive monoclonal antibody immunotherapy in psychiatric disorders. Finally, important considerations in the design and implementation of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Systemic Front Line Therapy of Follicular Lymphoma: When, to Whom and How.

PubMed

Pavanello, Francesca; Steffanoni, Sara; Ghielmini, Michele; Zucca, Emanuele

2016-01-01

The natural history of follicular lymphoma is usually characterized by an indolent course with a high response rate to the first line therapy followed by recurrent relapses, with a time to next treatment becoming shorter after each subsequent treatment line. More than 80% of patients have advanced stage disease at diagnosis. The time of initiation and the nature of the treatment is mainly conditioned by symptoms, tumor burden, lymphoma grading, co-morbidities and patients preference. A number of clinical and biological factors have been determined to be prognostic in this disease, but the majority of them could not show to be predictive of response to treatment, and therefore can't be used to guide the treatment choice. CD20 expression is the only predictive factor recognized in the treatment of FL and justifies the use of "naked" or "conjugated" anti-CD20 monoclonal antibodies as a single agent or in combination with chemo- or targeted therapy. Nevertheless, as this marker is almost universally found in FL, it has little role in the choice of treatment. The outcome of patients with FL improved significantly in the last years, mainly due to the widespread use of rituximab, autologous and allogeneic transplantation in young and fit relapsed patients, the introduction of new drugs and the improvement in diagnostic accuracy and management of side effects. Agents as new monoclonal antibodies, immuno-modulating drugs, and target therapy have recently been developed and approved for the relapsed setting, while studies to evaluate their role in first line treatment are still ongoing. Here we report our considerations on first line treatment approach and on the potential factors which could help in the choice of therapy.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

PubMed

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein.

PubMed

Smith, D S; Kitts, D D

1994-03-01

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

Varied distribution of RhD epitopes in the Indian population.

PubMed

Kulkarni, S S; Gupte, S C; Vasantha, K; Mohanty, D; Ghosh, K

2007-01-01

Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

In vivo T-cell activation by a monoclonal αCD3ε antibody induces preterm labor and birth

PubMed Central

Gomez-Lopez, Nardhy; Romero, Roberto; Arenas-Hernandez, Marcia; Ahn, Hyunyoung; Panaitescu, Bogdan; Vadillo-Ortega, Felipe; Sanchez-Torres, Carmen; Salisbury, Katherine S; Hassan, Sonia S.

2016-01-01

PROBLEM Activated/effector T cells seem to play a role in the pathological inflammation associated with preterm labor. The aim of this study was to determine whether in vivo T-cell activation by a monoclonal αCD3ε antibody induces preterm labor and birth. METHOD OF STUDY Pregnant B6 mice were intraperitoneally injected with a monoclonal αCD3ε antibody or its isotype control. The gestational age and the rates of preterm birth and pup mortality at birth, as well as the fetal heart rate and umbilical artery pulsatility index, were determined. RESULTS Injection of a monoclonal αCD3ε antibody led to preterm labor/birth [αCD3ε 83 ± 16.97% (10/12) vs. isotype 0% (0/8)], and increased the rate of pup mortality at birth [αCD3ε 87.30 ± 8.95% (77/85) vs. isotype 4.91 ± 4.34% (3/59)]. In addition, injection of a monoclonal αCD3ε antibody decreased the fetal heart rate and increased the umbilical artery pulsatility index when compared to isotype controls. CONCLUSION In vivo T-cell activation by a monoclonal αCD3ε antibody in late gestation induces preterm labor and birth. PMID:27658719

Production and characterization of murine monoclonal antibody against synthetic peptide of CD34.

PubMed

Maleki, Leili Aghebati; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Akbari, Aliakbar Movassaghpour

2013-01-01

The treatment of hematologic malignancies and immunodeficiency diseases are offered by hematopoietic stem cells (HSCs) as a unique self-renewal and differentiation source which most commonly is selected by CD34 surface marker for HSC. The purpose of this study was to develop and characterize monoclonal antibody against CD34 antigen for detection of hematopoietic stem cells. Balb/c mice were immunized with two synthetic peptides of CD34 and Spleen cells were fused with SP2/0.Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution. Large scale of monoclonal antibodies was produced by mouse ascites production of mAb (in vivo) method. Monoclonal antibody was purified by chromatography. Then reactivity of these antibodies was evaluated in different immunological assays including ELISA, immunofluorescence (IF), western blot (WB) and flowcytometry. In this study, between five positive clone wells, two clones were chosen for limiting dilution. Limiting dilution product was one monoclone (3-D5 monoclone) with absorbance about 2. Isotype of this mAb was identified as IgG1 class with Kappa (κ) light chain. This antibody is highly specific and functional in biomedical applications such as ELISA, flowcytometry, immunofluorescence, and western blot assays.

Monoclonal Antibodies to Ferric Pseudobactin, the Siderophore of Plant Growth-Promoting Pseudomonas putida B10

PubMed Central

Buyer, Jeffrey S.; Sikora, Lawrence J.; Kratzke, Marian G.

1990-01-01

Monoclonal antibodies to ferric pseudobactin, the siderophore (microbial iron transport agent) of plant growth-promoting Pseudomonas putida B10, have been developed. Three immunoglobulin G subclass 1-type monoclonal antibodies have been characterized. Each antibody appears to be unique on the basis of their reactions with ferric pseudobactin and with culture supernatants from other pseudomonads. None of the three cross-reacts with ferric pseudobactin-type siderophores produced by seven other pseudomonads. However, P. aeruginosa ATCC 15692 and P. fluorescens ATCC 17400 produced relatively high-molecular-mass compounds (mass greater than approximately 30,000 daltons) that did react with the antibodies. The compound from P. aeruginosa was not iron regulated, while the compound from P. fluorescens was produced only under iron-limiting conditions. A competitive assay using these antibodies has a detection limit of 5 × 10−12 mol of ferric pseudobactin. This is, to our knowledge, the first report of monoclonal antibodies reactive with siderophores. PMID:16348116

NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, the National Cancer Institute (NCI) Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

Single-domain monoclonal antibodies for the treatment of hepatocellular carcinoma | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute seeks parties to license human monoclonal antibodies and immunoconjugates and co-develop, evaluate, and/or commercialize large-scale antibody production and hepatocellular carcinoma (HCC) xenograft mouse models.

Monoclonal Antibody Therapy for Advanced Neuroblastoma

Cancer.gov

NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

NASA Astrophysics Data System (ADS)

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

2017-05-01

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Monoclonal antibodies for chronic pain: A practical review of mechanisms and clinical applications

PubMed Central

Yeh, Ju-Fen; Akinci, Aysen; Al Shaker, Mohammed; Chang, Ming Hong; Danilov, Andrei; Guillen, Rocio; Johnson, Kirk W; Kim, Yong-Chul; Skljarevski, Vladimir; Dueñas, Héctor J; Tassanawipas, Warat

2017-01-01

Context Monoclonal antibodies are being investigated for chronic pain to overcome the shortcomings of current treatment options. Objective To provide a practical overview of monoclonal antibodies in clinical development for use in chronic pain conditions, with a focus on mechanisms of action and relevance to specific classes. Methods Qualitative review using a systematic strategy to search for randomized controlled trials, systematic and nonsystematic (narrative) reviews, observational studies, nonclinical studies, and case reports for inclusion. Studies were identified via relevant search terms using an electronic search of MEDLINE via PubMed (1990 to June 2017) in addition to hand-searching reference lists of retrieved systematic and nonsystematic reviews. Results Monoclonal antibodies targeting nerve growth factor, calcitonin gene-related peptide pathways, various ion channels, tumor necrosis factor-α, and epidermal growth factor receptor are in different stages of development. Mechanisms of action are dependent on specific signaling pathways, which commonly involve those related to peripheral neurogenic inflammation. In clinical studies, there has been a mixed response to different monoclonal antibodies in several chronic pain conditions, including migraine, neuropathic pain conditions (e.g., diabetic peripheral neuropathy), osteoarthritis, chronic back pain, ankylosing spondylitis, and cancer. Adverse events observed to date have generally been mild, although further studies are needed to ensure safety of monoclonal antibodies in early stages of development, especially where there is an overlap with non-pain-related pathways. High acquisition cost remains another treatment limitation. Conclusion Monoclonal antibodies for chronic pain have the potential to overcome the limitations of current treatment options, but strategies to ensure their appropriate use need to be determined. PMID:29056066

Altered antigenicity of human monoclonal antibodies derived from human-mouse heterohybridomas.

PubMed

Kan-Mitchell, J; Andrews, K L; Gallardo, D; Mitchell, M S

1987-04-01

We have generated milligram quantities of human monoclonal antibodies (Hu-MAbs) in the ascites of pristane-primed nude mice injected with human-mouse heterohybridomas. After contaminating mouse immunoglobulins were removed by affinity chromatography, an enzyme immunosorbent assay (EIA) was used to measure the concentrations of human immunoglobulins. Ten different partially purified preparations were tested. The titration curves with all 5 IgG Hu-MAbs were unusual, reaching a plateau at a very low apparent maximum concentration of antibody. In contrast, the EIA yielded more usual titration curves and thus apparently more reliable estimates of the concentrations of 4 IgM and 1 IgA monoclonal antibodies. An analogous EIA for the quantitation of mouse IgG monoclonal antibodies also gave accurate estimates. To understand the nature of the discrepancy with human IgG, 5 Hu-MAbs of the 3 classes (2 IgG, 2 pentameric IgM and 1 IgA) were purified to homogeneity for a more detailed analysis. The inability to quantitate the human IgG monoclonal antibodies by EIA was not due to defective molecules, as shown by SDS polyacrylamide gel electrophoresis. The human IgG monoclonal antibodies were found to consist of intact heavy and light chains, as were the IgM and IgA antibodies. The possibility that the human IgG monoclonal antibodies differed antigenically from polyclonal IgG was explored by comparing the concentrations by EIA with the protein concentrations determined by absorbance at 280 nm. This analysis permitted a comparison of the detectability of antigenic determinants on Hu-MAbs with those on polyclonal Ig with goat antibodies to Ig or Ig subclass. The IgG monoclonal antibodies differed from polyclonal IgG in both their heavy and light chains. Goat antiserum monospecific for the gamma chain in fact underestimated the concentration by as much as one hundred-fold. IgM and IgA monoclonal antibodies were less antigenically distinct from their polyclonal counterparts even though their light chains were also underestimated, because goat monospecific antibodies were more efficient at recognizing their heavy chains. The molecular basis for the observed difference in antigenicity is not yet known. These findings have important implications for the analysis of the binding of IgG Hu-MAbs. A direct binding assay with the label directly conjugated to the Hu-MAb should be used in preference to an indirect assay with a labeled detecting antibody to maximize the sensitivity of the assay. The altered antigenicity of IgG Hu-MAbs may also imply decreased immunogenicity when they are given in vivo as carriers for radionuclides or cytotoxic antitumor materials.

Antibody immunoprophylaxis and immunotherapy for influenza virus infection: Utilization of monoclonal or polyclonal antibodies?

PubMed

Berry, Cassandra M

2018-03-04

Control programs for emerging influenza are in urgent need of novel therapeutic strategies to mitigate potentially devastating threats from pathogenic strains with pandemic potential. Current vaccines and antivirals have inherent limitations in efficacy, especially with rapid evolutionary changes of influenza viruses. Antibody-based antiviral protection harnesses the natural power of the immune system. Antibodies present prophylactic and therapeutic intervention options for prevention and control of influenza, especially for at-risk populations. Specific monoclonal antibodies are well defined in purity and initial efficacy but polyclonal antibodies are easier to scale-up and cost-effective with long-term efficacy, using batches with broadly neutralizing properties against influenza variants. This review presents the pros and cons of monoclonal versus polyclonal antibody therapy for influenza.

Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta.

PubMed

Willott, E; Trenczek, T; Thrower, L W; Kanost, M R

1994-12-01

We have made 140 monoclonal antibodies to hemocytes (insect blood cells) from Manduca sexta. Four of these antibodies, when used in immunofluorescent microscopy of fixed hemocytes, distinguish the four main morphologically distinct hemocyte types. Plasmatocytes, granular cells, and oenocytoids are each recognized by a unique antibody specific to that type; spherulocytes are recognized by an antibody that also binds to plasmatocytes. When used in flow cytometry with nonfixed hemocytes, three of the four antibodies bind their respective cells; the oenocytoid marker failed to bind to any hemocytes. This set of four monoclonal antibodies may be useful for labeling individual cell types and for separating the different hemocyte types for further study of hemocyte functions.

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

PubMed

Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Development of an Antigen Capture Enzyme-Linked Immunosorbent Assay for Virus Detection Based on Porcine Epidemic Diarrhea Virus Monoclonal Antibodies

PubMed Central

Wang, Zanyu; Jiyuan, Yin; Su, Chen; Xinyuan, Qiao

2015-01-01

Abstract Porcine epidemic diarrhea virus (PEDV), a coronavirus, can cause acute diarrhea and dehydration in pigs. In the current study, two positive monoclonal cell lines (5D7 and 3H4) specific for PEDV were established, and the immunoreactivity of the monoclonal antibodies was confirmed by immunofluorescence and dot-immunobinding assays. A method, termed antigen capture enzyme-linked immunosorbent assay (AC-ELISA), which used the monoclonal antibody 5D7 as the detecting antibody and rabbit antiserum of PEDV protein S as the capture antibody, was developed. Compared with the reverse transcription polymerase chain reaction method of detecting PEDV in fecal samples, AC-ELISA showed similar sensitivity and specificity. These results suggested that AC-ELISA would be useful for the diagnosis and epidemiological studies of PEDV. PMID:25658793

Guardians at the gate

PubMed Central

2009-01-01

Patents provide one of the few protections companies can avail themselves of to help protect their therapeutic monoclonal antibody products. Just as the therapeutic monoclonal antibody field is constantly evolving, so too is the legal environment surrounding these inventions. In a series of articles, the general state of the law surrounding therapeutic antibodies will be explained, and important challenges to this technology area will be discussed. Much is at stake when companies market therapeutic monoclonal antibodies; therefore, a firm understanding of this important form of protection is critically important for anyone developing such products. PMID:20068400

Palladium-109 labeled anti-melanoma monoclonal antibodies

DOEpatents

Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

1984-04-30

The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratcliffe, D.R.

This study on passive immunity dealt principally with the following topics: pathogenesis of the pneumonia produced by influenza virus (PR8) in CF-1 mice; the distribution and retention of monoclonal antibody instilled intratracheally (IT) into the lung; and prophylaxis and therapy of influenza pneumonia using specific monoclonal antibody (IgG 2a/k anti-HA). The fate of a single 50 ul bolus of antibody instilled IT was determined by monitoring the activity of /sup 125/I-labelled monoclonal IgG in the lungs and by lavage recovery of functional antibody.Antibody was demonstrated in high concentrations for the first 3 days and was present in the lungs formore » a period of 7 days. For prophylaxis several trials indicated that monoclonal antibody provided significant protection from lethal effects of the virus. Antibody given to clinically ill mice on day 3 produced a highly significant reduction in mortality (P < 0.001) when compared to control mice. The treatment reversed the weight loss and apparently arrested the development of lesions in most of the mice within 2 days following antibody administration.« less

Fine mapping of HIV-1 Nef-epitopes by monoclonal antibodies.

PubMed

Siakkou, H; Jahn, S; Kienzle, N; Ulrich, R; Grötzinger, C; Schneider, T; Kohleisen, B; Pauli, G; Spohn, R; Jung, G

1993-01-01

A panel of newly isolated murine monoclonal antibodies is described which are specific for the Nef protein of the human immunodeficiency virus type 1 (HIV-1). Epitope mapping using recombinant Nef-related proteins, synthetic peptides and lipopeptides showed 3 independent antigenic determinants located within the regions of amino acids 83-93, 175-190 and 86-166 of the Nef protein. None of the monoclonal antibodies reacted with recombinant Nef proteins of HIV-2.

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

PubMed

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd < or = 1 x 10(-8) M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

NCI Requests Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through July 9, 2012.

Immunoscintigraphy and radioimmunotherapy in Cuba: experiences with labeled monoclonal antibodies for cancer diagnosis and treatment (1993-2013).

PubMed

Peña, Yamilé; Perera, Alejandro; Batista, Juan F

2014-01-01

INTRODUCTION The availability of monoclonal antibodies in Cuba has facilitated development and application of innovative techniques (immunoscintigraphy and radioimmunotherapy) for cancer diagnosis and treatment. Objective Review immunoscintigraphy and radioimmunotherapy techniques and analyze their use in Cuba, based on the published literature. In this context, we describe the experience of Havana's Clinical Research Center with labeled monoclonal antibodies for cancer diagnosis and treatment during the period 1993-2013. EVIDENCE ACQUISITION Basic concepts concerning cancer and monoclonal antibodies were reviewed, as well as relevant international and Cuban data. Forty-nine documents were reviewed, among them 2 textbooks, 34 articles by Cuban authors and 13 by international authors. All works published by the Clinical Research Center from 1993 through 2013 were included. Bibliography was obtained from the library of the Clinical Research Center and Infomed, Cuba's national health telematics network, using the following keywords: monoclonal antibodies, immunoscintigraphy and radioimmunotherapy. RESULTS Labeling the antibodies (ior t3, ior t1, ior cea 1, ior egf/r3, ior c5, h-R3, 14F7 and rituximab) with radioactive isotopes was a basic line of research in Cuba and has fostered their use as diagnostic and therapeutic tools. The studies conducted demonstrated the good sensitivity and diagnostic precision of immunoscintigraphy for detecting various types of tumors (head and neck, ovarian, colon, breast, lymphoma, brain). Obtaining different radioimmune conjugates with radioactive isotopes such as 99mTc and 188Re made it possible to administer radioimmunotherapy to patients with several types of cancer (brain, lymphoma, breast). The objective of 60% of the clinical trials was to determine pharmacokinetics, internal dosimetry and adverse effects of monoclonal antibodies, as well as tumor response; there were few adverse effects, no damage to vital organs, and a positive tumor response in a substantial percentage of patients. CONCLUSIONS Cuba has experience with production and radiolabeling of monoclonal antibodies, which facilitates use of these agents. Studies in Cuba conducted by the Clinical Research Center over the past 20 years have yielded satisfactory results. Evidence obtained suggests promising potential of monoclonal antibodies and nuclear medicine, with immunoscintigraphy and radioimmunotherapy techniques providing alternatives for cancer diagnosis and treatment in Cuba.

A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

NASA Astrophysics Data System (ADS)

Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

1981-05-01

Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

PubMed

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

Coarse grained modeling of transport properties in monoclonal antibody solution

NASA Astrophysics Data System (ADS)

Swan, James; Wang, Gang

Monoclonal antibodies and their derivatives represent the fastest growing segment of the bio pharmaceutical industry. For many applications such as novel cancer therapies, high concentration, sub-cutaneous injections of these protein solutions are desired. However, depending on the peptide sequence within the antibody, such high concentration formulations can be too viscous to inject via human derived force alone. Understanding how heterogenous charge distribution and hydrophobicity within the antibodies leads to high viscosities is crucial to their future application. In this talk, we explore a coarse grained computational model of therapeutically relevant monoclonal antibodies that accounts for electrostatic, dispersion and hydrodynamic interactions between suspended antibodies to predict assembly and transport properties in concentrated antibody solutions. We explain the high viscosities observed in many experimental studies of the same biologics.

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

PubMed

Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

2015-01-01

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

[Diagnostic and therapeutic use of human anti-D (Rho) monoclonal antibodies. Evaluation and perspectives].

PubMed

Rouger, P; Goossens, D; Champomier, F; Tsikas, G; Liberge, G; Leblanc, J; Richard, C; Bailleul, C; Salmon, C

1985-12-01

Human monoclonal antibodies will be essential in medicine. They are valuable tools for biological diagnosis and therapeutics. Our model, human monoclonal antibodies directed against the Rhesus D antigen can be used for the determination of the Rhesus D phenotype and for the suppression of Rh(D) immunisation in women. These new products require new procedures of preparation, new regulations for the quality controls, which will be discussed in this paper.

Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Ferguson, Carly N.; Gucinski-Ruth, Ashley C.

2016-05-01

Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

NCI Requests Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through February 5, 2016.

NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through July 11, 2014.

NCI Requests Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through July 12, 2013.

Monoclonal antibodies passively protect BALB/c mice against Burkholderia mallei aerosol challenge.

PubMed

Treviño, Sylvia R; Permenter, Amy R; England, Marilyn J; Parthasarathy, Narayanan; Gibbs, Paul H; Waag, David M; Chanh, Tran C

2006-03-01

Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei, the etiologic agent of glanders, and were shown to be effective in passively protecting mice against a lethal aerosol challenge. The antibodies appeared to target lipopolysaccharide. Humoral antibodies may be important for immune protection against B. mallei infection.

Interferon-γ : The Major Mediator of Resistance against Toxoplasma gondii

NASA Astrophysics Data System (ADS)

Suzuki, Yasuhiro; Orellana, Manuel A.; Schreiber, Robert D.; Remington, Jack S.

1988-04-01

Mice were injected with a monoclonal antibody to interferon-γ to examine the importance of endogenous production of this lymphokine in resistance against infection with the sporozoan parasite Toxoplasma gondii. Mice with intraperitoneal infections of T. gondii that received no antibody survived and developed chronic T. gondii infection, whereas the infected mice that received the monoclonal antibody died of toxoplasmosis. The activation of macrophages, which kill T. gondii in vivo, was inhibited by administration of the monoclonal antibody, but the production of antibodies to T. gondii was not suppressed. The fact that an antibody to interferon-γ can eliminate resistance to acute Toxoplasma infection in mice suggests that this lymphokine is an important mediator of host resistance to this parasite.

A novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da

2009-08-01

The pursuit of efficient and highly targeting-selective transporters is an active topic in cancer-targeting therapy. In this study, a novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes (SWCNTs) was developed to investigate cancer cell targeting in vitro. SWCNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). PL-PEG functionalized SWCNTs were then conjugated with fluorescein isothiocyanate (FITC) labeled integrin αvβ3 monoclonal antibody to construct SWCNT-integrin αvβ3 monoclonal antibody system (denoted as SWCNT-PEG-mAb). In vitro study revealed that the system had a high efficiency in cancer cell targeting in integrin αvβ3 positive U87MG cells. Moreover, the SWCNT-PEG-mAb is stable in physiological media, and can be readily transported into U87MG cells via integrin αvβ3-mediated endocytosis in cell. In summary, the integrin αvβ3 monoclonal antibody labeled SWCNT is a potential carrier-candidate for cancer-imaging and drug-delivering in cancer-targeting therapy.

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

PubMed Central

1988-01-01

We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

Monoclonal antibodies specific for African swine fever virus proteins.

PubMed Central

Sanz, A; García-Barreno, B; Nogal, M L; Viñuela, E; Enjuanes, L

1985-01-01

We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles. Images PMID:3882998

Radioimmunotherapy of non-Hodgkin's lymphoma: molecular targeting and novel agents.

PubMed

Pauwels, Ernest K J; Erba, Paola

2007-03-01

In recent years monoclonal antibodies have played an important role in cancer therapy. This successful track is grosso modo based upon developments in the production of desired antibody molecules, the identification of suitable tumor antigens and the construction of chimeric and fully humanized antibodies. Especially in hematologic disorders, notably in non-Hodgkin's disease, the monoclonal antibody rituximab has proven to be of value in relapsed or refractory disease. Yet, to overcome the nonoptimal properties of this drug, especially in relation to the time to next therapy, radiolabeled immunoconjugates have been synthesized. For this purpose, the radionuclide yttrium-90 has been linked to the monoclonal antibody ibritumomab via the chelator tiuxetan. The most recent clinical results of this radiolabeled agent versus the nonradioactive drug treatment are reviewed in this paper. Furthermore, attention is paid to the monoclonal antibody tositumomab labeled with iodine-131, of which the first clinical results have become available most recently. This overview also mentions possibilities to increase the therapeutic efficacy of radionuclide immunoconjugates. This can be achieved by enhancing the targeting characteristics of the antibody and the use of alpha radiation-emitting radionuclides.

Rapid preparative separation of monoclonal antibody charge variants using laterally-fed membrane chromatography.

PubMed

Sadavarte, Rahul; Madadkar, Pedram; Filipe, Carlos Dm; Ghosh, Raja

2018-01-15

Monoclonal antibodies undergo various forms of chemical transformation which have been shown to cause loss in efficacy and alteration in pharmacokinetic properties of these molecules. Such modified antibody molecules are known as variants. They also display physical properties such as charge that are different from intact antibody molecules. However, the difference in charge is very subtle and separation based on it is quite challenging. Charge variants are usually separated using ion-exchange column chromatography or isoelectric focusing. In this paper, we report a rapid and scalable method for fractionating monoclonal antibody charge variants, based on the use of cation exchange laterally-fed membrane chromatography (LFMC). Starting with a sample of monoclonal antibody hIgG1-CD4, three well-resolved fractions were obtained using either pH or salt gradient. These fractions were identified as acidic, neutral and basic variants. Each of these fractions contained intact heavy and light chains and so antibody fragmentation had no role in variant generation. The separation was comparable to that using column chromatography but was an order of magnitude faster. Copyright © 2017 Elsevier B.V. All rights reserved.

Gaining insights into the consequences of target-mediated drug disposition of monoclonal antibodies using quasi-steady-state approximations.

PubMed

Grimm, Hans Peter

2009-10-01

Target-mediated drug disposition (TMDD) is frequently reported for therapeutic monoclonal antibodies and is linked to the high affinity and high specificity of antibody molecules for their target. Understanding TMDD of a monoclonal antibody should go beyond the empirical description of its non-linear PK since valuable insights on the antibody-target interaction itself can be gained. This makes its mechanistic understanding precious for the drug development process, in particular for the optimization of new antibody molecules, for the design and interpretation of pharmacokinetic studies, and possibly even for the evaluation of efficacy and dose selection of drug candidates. Using the observation that the molecular (microscopic) processes are usually much more rapid than the pharmacokinetic (macroscopic) processes, a series of quasi-steady-state conditions on the microscopic level is proposed to bridge the gap between simple empirical and complex mechanistic descriptions of TMDD. These considerations show the impact of parameters such as target turnover, target expression, and target accessibility on the pharmacokinetics and pharmacodynamics of monoclonal antibodies.

Idiotypic Cascades in Cancer Patients Treated with Monoclonal Antibody CO17-1A

NASA Astrophysics Data System (ADS)

Wettendorff, Martine; Iliopoulos, Dimitrios; Tempero, Margaret; Kay, David; Defreitas, Elaine; Koprowski, Hilary; Herlyn, Dorothee

1989-05-01

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.

Common idiotypes expressed on human, monoclonal, abnormal immunoglobulins and cryoglobulins with polyreactive autoantibody activities.

PubMed Central

Barbouche, M R; Guilbert, B; Makni, S; Gorgi, Y; Ayed, K; Avrameas, S

1993-01-01

Several human monoclonal immunoglobulins with the same autoantibody activity have been shown to have cross-reactive idiotypes (CRI). In this study, using polyclonal anti-idiotypic antibodies, we found that 28% of human monoclonal immunoglobulins with polyreactive autoantibody activity from myeloma, Waldenström's macroglobulinaemia and cryoglobulinaemia patients shared common idiotype(s). Furthermore, the latter were expressed on human and murine natural MoAbs (respectively in 12% and 22% of the clones tested) and on human IgG preparations used for therapeutic intravenous injections (IVIg) and which contain natural antibodies. These findings suggest that monoclonal immunoglobulins could arise from the proliferation of a clone that normally produces a natural antibody. The existence of common idiotype(s) between monoclonal immunoglobulins and IVIg could be relevant to the improvement noted after treatment with IVIg in patients suffering from peripheral neuropathies associated with monoclonal gammopathy. PMID:8428386

The generation of monoclonal antibodies and their use in rapid diagnostic tests

USDA-ARS?s Scientific Manuscript database

Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

Development of monoclonal antibodies that recognize Treponema pallidum.

PubMed Central

Saunders, J M; Folds, J D

1983-01-01

We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay. PMID:6347899

Human Monoclonal Antibodies Targeting Glypican-2 in Neuroblastoma | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute’s Laboratory of Molecular Biology (NCI LMB) have developed and isolated several single domain monoclonal human antibodies against GPC2. NCI seeks parties interested in licensing or co-developing GPC2 antibodies and/or conjugates.

Myosin: A Link between Streptococci and Heart

NASA Astrophysics Data System (ADS)

Krisher, Karen; Cunningham, Madeleine W.

1985-01-01

Murine monoclonal antibodies to Streptococcus pyogenes reacted with skeletal muscle myosin. High molecular weight proteins in extracts of human heart tissue that reacted with an antibody to S. pyogenes also reacted with a monoclonal antibody to ventricular myosin. Adsorption of the antibody to streptococci with S. pyogenes simultaneously removed reactivity of the antibody for either S. pyogenes or myosin. These results indicate that myosin shares immunodeterminants with a component of S. pyogenes.

Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

NASA Astrophysics Data System (ADS)

Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

2016-10-01

Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

Antibody therapeutics for treating prostate cancer: where are we now and what comes next?

PubMed

Vlachostergios, Panagiotis J; Galletti, Giuseppe; Palmer, Jessica; Lam, Linda; Karir, Beerinder S; Tagawa, Scott T

2017-02-01

Progress in the understanding of molecular events of carcinogenesis and cancer evolution as well as the identification of tumor antigens has led to the development of different targeted therapeutic approaches, including the use of monoclonal antibodies (mAbs). Prostate cancer (PC) is highly amenable to mAb targeting given the existence of prostate-specific targets and the natural history and localization of metastatic disease. Areas covered: Several aspects of the PC phenotype, including growth factors, angiogenesis mediators, bone microenvironment signals, and immune evasion pathways, have become areas of ongoing investigation in terms of mAb targeting. These are reviewed. The greatest success so far has been the development of mAbs against prostate-specific tumor antigen (PSMA), which opened an opportunity to improve diagnostic accuracy and simultaneously target metastatic disease. Expert opinion: As mAb use in PC continues to evolve, more accurate imaging of the extent of disease and more effective mAb therapies (naked or conjugated with drugs, toxins or radioactive molecules) are emerging. In addition, the combination of mAbs with other treatment modalities is expected to further improve responses and overall survival. Identification of validated biomarkers is necessary for better recognition of patient subgroups who will derive the greatest benefit from mAb therapy.

[The electron microscopic observation of the effect of monoclonal antibody on the form and structure of mutans streptococci OMZ176].

PubMed

Wen, L; Yue, S

1996-01-01

The effect of monoclonal antibody on the form and structure of Mutans Streptococci OMZ176 was studied. The result showed that a great number of Mutans Streptococci OMZ176 was agglutianated after treating with monoclonal antibody prepared by a cell wall protein antigen (molecular weight 220 kd) of Mutans Streptococci OMZ176. Bacterial cells were swollen obviously. The gap between cell wall and cytoplasmic was widened. The electronic density of cell plasm was greatly decreased.

Determination of a Unique Epitope Binding Site for a Complement-Lysis- Enhancing Monoclonal Antibody, 3D12, on the Galactose Adherence Lectin of Entamoeba histolytica, Using BIAcore.

DTIC Science & Technology

1992-05-01

COMPLEMENT-LYSIS-ENHANCING MONOCLONAL ANTIBODY, 3D12, ON THE GALACTOSE ADHERENCE LECTIN OF ENTAMOEBA HISTOLYTICA, USING BIAcore Sheila J. Wood...Binding 5. FUNDING NUMBERS Site for a Complement-Lysis-Enhancing Monoclonal Antibody, 3D12, on the Galactose Adherence Lectin of Entamoeba Hiiutolitica...Mechani sms of pathogenicity used by Entamoeba histolytica to invade the bloodstream and cause liver abscess, include complement mediated lysis

Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis.

PubMed

Wootla, Bharath; Watzlawik, Jens O; Stavropoulos, Nikolaos; Wittenberg, Nathan J; Dasari, Harika; Abdelrahim, Murtada A; Henley, John R; Oh, Sang-Hyun; Warrington, Arthur E; Rodriguez, Moses

2016-06-01

Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside.

Immunohistochemical detection of chlamydiae in formalin-fixed tissue sections: comparison of a monoclonal antibody with yolk derived antibodies (IgY).

PubMed

Kunz, U S; Pospischil, A; Paccaud, M F

1991-06-01

Immunohistological detection of chlamydiae in formalin-fixed and paraffin-embedded sections of various organs from several species is described. In a retrospective study, two antisera, a commercially available monoclonal murine antibody (IgMur) and vitelline immunoglobulins (IgY), extracted from the egg yolk of immunized hens, were compared and tested for their applicability under routine condition. Both antisera were applied to tissues from which chlamydiae had been isolated or in which the presence of chlamydiae had been suspected in specially stained sections. Antigen labelling was optimal with the monoclonal antibody. Vitelline immunoglobulins produced some unspecific reactions, especially in lung tissue sections. Because of the antigenic relationship between the vitelline antibodies and tissues of birds, IgY are not suitable for the detection of psittacosis on avian substrates, when using an indirect immunological method. Staining in other tissues e.g. intestine or placenta was of equal quality as that attained with monoclonal antibodies. Depending on the advantages and disadvantages in every individual case, one of the two antibodies may be chosen for further studies. Vitelline antibodies should be preferred with respect to animal welfare.

Fusion antibody for Alzheimer's disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation.

PubMed

Boado, Ruben J; Zhang, Yufeng; Zhang, Yun; Xia, Chun-Fang; Pardridge, William M

2007-01-01

Delivery of monoclonal antibody therapeutics across the blood-brain barrier is an obstacle to the diagnosis or therapy of CNS disease with antibody drugs. The immune therapy of Alzheimer's disease attempts to disaggregate the amyloid plaque of Alzheimer's disease with an anti-Abeta monoclonal antibody. The present work is based on a three-step model of immune therapy of Alzheimer's disease: (1) influx of the anti-Abeta monoclonal antibody across the blood-brain barrier in the blood to brain direction, (2) binding and disaggregation of Abeta fibrils in brain, and (3) efflux of the anti-Abeta monoclonal antibody across the blood-brain barrier in the brain to blood direction. This is accomplished with the genetic engineering of a trifunctional fusion antibody that binds (1) the human insulin receptor, which mediates the influx from blood to brain across the blood-brain barrier, (2) the Abeta fibril to disaggregate amyloid plaque, and (3) the Fc receptor, which mediates the efflux from brain to blood across the blood-brain barrier. This fusion protein is a new antibody-based therapeutic for Alzheimer's disease that is specifically engineered to cross the human blood-brain barrier in both directions.

Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis

PubMed Central

Stavropoulos, Nikolaos; Wittenberg, Nathan J.; Dasari, Harika; Abdelrahim, Murtada A.; Henley, John R.; Oh, Sang-Hyun; Warrington, Arthur E.; Rodriguez, Moses

2016-01-01

Introduction Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Areas Covered Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Expert Opinion Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside. PMID:26914737

Discovery of an antibody for pan-ebolavirus therapy.

PubMed

Furuyama, Wakako; Marzi, Andrea; Nanbo, Asuka; Haddock, Elaine; Maruyama, Junki; Miyamoto, Hiroko; Igarashi, Manabu; Yoshida, Reiko; Noyori, Osamu; Feldmann, Heinz; Takada, Ayato

2016-02-10

During the latest outbreak of Ebola virus disease in West Africa, monoclonal antibody therapy (e.g., ZMapp) was utilized to treat patients. However, due to the antigenic differences among the five ebolavirus species, the current therapeutic monoclonal antibodies are only effective against viruses of the species Zaire ebolavirus. Although this particular species has indeed caused the majority of human infections in Central and, recently, West Africa, other ebolavirus species (e.g., Sudan ebolavirus and Bundibugyo ebolavirus) have also repeatedly caused outbreaks in Central Africa and thus should not be neglected in the development of countermeasures against ebolaviruses. Here we report the generation of an ebolavirus glycoprotein-specific monoclonal antibody that effectively inhibits cellular entry of representative isolates of all known ebolavirus species in vitro and show its protective efficacy in mouse models of ebolavirus infections. This novel neutralizing monoclonal antibody targets a highly conserved internal fusion loop in the glycoprotein molecule and prevents membrane fusion of the viral envelope with cellular membranes. The discovery of this highly cross-neutralizing antibody provides a promising option for broad-acting ebolavirus antibody therapy and will accelerate the design of improved vaccines that can selectively elicit cross-neutralizing antibodies against multiple species of ebolaviruses.

Antibodies directed against receptor tyrosine kinases

PubMed Central

FAUVEL, Bénédicte; Yasri, Aziz

2014-01-01

Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy. PMID:24859229

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

PubMed

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Gold nanoparticle based Tuberculosis immunochromatographic assay: the quantitative ESE Quanti analysis of the intensity of test and control lines.

PubMed

Mdluli, Phumlani; Tetyana, Phumlani; Sosibo, Ndabenhle; van der Walt, Hendriëtte; Mlambo, Mbuso; Skepu, Amanda; Tshikhudo, Robert

2014-04-15

A rapid dual channel lateral flow assay for the detection of Mycobacterium Tuberculosis antibodies (MTB 38 kDa monoclonal antibody) in human blood was developed. The MTB 6-14-38 kDa fusion antigen and anti-Protein A were used as the capture proteins for test and control lines respectively. Protein A labeled 40 nm gold nanoparticles were used as the detection conjugate. Whole blood and serum were spiked with MTB 38 kDa monoclonal antibody to make a positive sample model. The developed lateral flow was used to test MTB 38 kDa monoclonal antibody, and a detection limit of 5 ng/ml was used as a cut-off concentration of the analytes. The effect of the analyte concentration on the MTB lateral flow assay was studied using the variation of the intensity obtained from a ESE Quanti reader. There was a direct correlation between the analyte (MTB 38 kDa monoclonal antibody) concentration and the intensity of the test line. The intensity increased with an increase in the concentration of MTB 38 kDa monoclonal antibody, while in contrast, an increase in analyte concentration decreased the intensity of the control line. © 2013 Published by Elsevier B.V.

Stages of Adult Non-Hodgkin Lymphoma

MedlinePlus

... radiolabeled monoclonal antibody. Monoclonal antibodies are given by infusion . Proteasome inhibitor therapy blocks the action of proteasomes ... and given back to the patient through an infusion. These reinfused stem cells grow into (and restore) ...

Treatment Option Overview (Adult Non-Hodgkin Lymphoma)

MedlinePlus

... radiolabeled monoclonal antibody. Monoclonal antibodies are given by infusion . Proteasome inhibitor therapy blocks the action of proteasomes ... and given back to the patient through an infusion. These reinfused stem cells grow into (and restore) ...

Treatment Options for Non-Hodgkin Lymphoma

MedlinePlus

... radiolabeled monoclonal antibody. Monoclonal antibodies are given by infusion . Proteasome inhibitor therapy blocks the action of proteasomes ... and given back to the patient through an infusion. These reinfused stem cells grow into (and restore) ...

The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line

NASA Technical Reports Server (NTRS)

Smiley, S. A.; Gillock, E. T.; Black, M. C.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1997-01-01

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

PubMed

Waldmann, Herman

2014-01-01

One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein.

PubMed

Hempel, Franziska; Maurer, Michael; Brockmann, Björn; Mayer, Christian; Biedenkopf, Nadine; Kelterbaum, Anne; Becker, Stephan; Maier, Uwe G

2017-07-27

The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014-2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle. In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized. This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle.

Reagent Target Request for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

NCI's Antibody Characterization Program provides reagents and other critical resources to support protein/peptide measurements and analysis. In an effort to produce and distribute well-characterized monoclonal antibodies to the scientific community, the program is seeking cancer related protein targets for antibody production and characterization for distribution to the research community. Submission Period: May 20, 2011 - July 1, 2011.

Beta-Catenin Stability in Breast Cancer

DTIC Science & Technology

1999-09-01

monoclonal anti-ß-catenin antibody (Clone#14) and the anti-FLAG™ antibody were purchased from Transduction Labs and Kodak, respectively. Affinity purified...anti-HA (BabCo) and anti-ß-catenin antibodies (Transduction labs ). To test the hypothesis that the tumor suppressor adenomatous polyposis coli (APC...ß-catenin was detected first using 1:50 dilution of a mouse monoclonal antibody (Transduction Labs ) followed by Texas Red-conjugated goat anti

Reminder: NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to improve rigor and reproducibility, the National Cancer Institute (NCI) Antibody Characterization Program requests cancer-related protein targets for monoclonal antibody production and distribution to the scientific community. The program from The Office of Cancer Clinical Proteomics Research provides well-characterized

Investigation of antigen-antibody interactions of sulfonamides with a monoclonal antibody in a fluorescence polarization immunoassay using 3D-QSAR models

USDA-ARS?s Scientific Manuscript database

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MAbSMR) produced against sulfamerazine was carried out by Distance Comparison (DISCOtech), comparative molecular field analysis (CoMFA), and comparative molecular si...

A Rabbit Anti-pT1989 ATR Monoclonal Antibody for Use in Immunoassays | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) have developed a monoclonal antibody against ataxia telangiectasia-mutated and Rad3-related (ATR) kinase phosphorylated at threonine 1989. The antibody can be used for pharmacodynamic assays to quantify drug action on the ATR target.

A novel micro-emulsion and micelle assembling method to prepare DEC205 monoclonal antibody coupled cationic nanoliposomes for simulating exosomes to target dendritic cells.

PubMed

Li, Kexin; Chang, Shasha; Wang, Zhongyan; Zhao, Xiuli; Chen, Dawei

2015-08-01

Cationic biomimetic exosomes were prepared using a novel micro-emulsion and micelle assembling method by introducing DEC205 monoclonal antibody as specific ligand to target dendritic cells (DCs). The Box-Behnken experimental design was applied for optimization of nanoliposomes (NLip) and DEC205 monoclonal antibody was then conjugated on the surface of NLip (DEC205-NLip). NLip and DEC205-NLip respectively had an average size of 62.7 ± 6.33 nm and 81.64 ± 4.25 nm, zeta potential of +30.5 ± 2.3 mV and +19.8 ± 1.8 mV and encapsulation efficiency of 91.02 ± 3.1% and 93.10 ± 2.2%. In addition, the toxicity studies confirmed DEC205 monoclonal antibody could significantly reduce the cytotoxicity of the cationic lipid against DCs. And the cellular uptake experiment evaluated the significant targeting effect of the DEC205 monoclonal antibody on DC cells. In conclusion, the novel method presented here to prepare biomimetic exosomes was an efficient approach to develop antigen carriers for specific DCs targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of Tryptophan Fluorescence Bandwidth-Maximum Plot in Analysis of Monoclonal Antibody Structure.

PubMed

Huang, Cheng-Yen; Hsieh, Ming-Ching; Zhou, Qinwei

2017-04-01

Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.

Correlation of monoclonal and polyclonal somatostatin receptor 5 antibodies in pancreatic neuroendocrine tumors

PubMed Central

Kaemmerer, Daniel; Lupp, Amelie; Peter, Luisa; Fischer, Elke; Schulz, Stefan; Klöppel, Günter; Hommann, Merten

2013-01-01

Aims: To evaluate the frequency of somatostatin-receptor 5 (SSTR 5) in pancreatic neuroendocrine tumors by using monoclonal and polyclonal antibodies. Material and Method: we analyzed 66 proven pancreatic neuroendocrine tumors immunohistochemically with monoclonal (clone UMB-4) and polyclonal SSTR 5-antibodies. Immunoreactive score (IRS) and DAKO-score Her2/neu were evaluated. Results: Immunohistochemistry analysis demonstrated for the IRS a significant higher staining of all specimen using the monoclonal antibodies ( IRS SSTR5 poly vs IRS SSTR 5 mono; 20.0% vs 30.3% p < 0.001) by a correlation of 0.21; p = 0.04. For the HER2 score there was also a significant higher staining in the monoclonal group (Her2 SSTR 5 poly vs Her2 SSTR 5 mono; 21.5% vs 28.8% p < 0.001) by a correlation of 0.20; p = 0.08. Conclusion: Both antibodies are useful in staining of SSTR, although UMB-4 demonstrated a 10% higher SSTR 5 staining. Due to the previous underestimated expression rate of SSTR 5, current standards in diagnostics and therapy should be reconsidered. The increasing usage of long-acting pansomatostatin receptor analogues will rise the adverse effects connected to SSTR5 binding. PMID:23236542

Intermolecular Interactions and the Viscosity of Highly Concentrated Monoclonal Antibody Solutions.

PubMed

Binabaji, Elaheh; Ma, Junfen; Zydney, Andrew L

2015-09-01

The large increase in viscosity of highly concentrated monoclonal antibody solutions can be challenging for downstream processing, drug formulation, and delivery steps. The objective of this work was to examine the viscosity of highly concentrated solutions of a high purity IgG1 monoclonal antibody over a wide range of protein concentrations, solution pH, ionic strength, and in the presence / absence of different excipients. Experiments were performed with an IgG1 monoclonal antibody provided by Amgen. The steady-state viscosity was evaluated using a Rheometrics strain-controlled rotational rheometer with a concentric cylinder geometry. The viscosity data were well-described by the Mooney equation. The data were analyzed in terms of the antibody virial coefficients obtained from osmotic pressure data evaluated under the same conditions. The viscosity coefficient in the absence of excipients was well correlated with the third osmotic virial coefficient, which has a negative value (corresponding to short range attractive interactions) at the pH and ionic strength examined in this work. These results provide important insights into the effects of intermolecular protein-protein interactions on the behavior of highly concentrated antibody solutions.

Stable long-term cultures of self-renewing B cells and their applications.

PubMed

Kwakkenbos, Mark J; van Helden, Pauline M; Beaumont, Tim; Spits, Hergen

2016-03-01

Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen-presenting cells for the discovery of tumor neoantigens. © 2016 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

Immunoactive two-dimensional self-assembly of monoclonal antibodies in aqueous solution revealed by atomic force microscopy

NASA Astrophysics Data System (ADS)

Ido, Shinichiro; Kimiya, Hirokazu; Kobayashi, Kei; Kominami, Hiroaki; Matsushige, Kazumi; Yamada, Hirofumi

2014-03-01

The conformational flexibility of antibodies in solution directly affects their immune function. Namely, the flexible hinge regions of immunoglobulin G (IgG) antibodies are essential in epitope-specific antigen recognition and biological effector function. The antibody structure, which is strongly related to its functions, has been partially revealed by electron microscopy and X-ray crystallography, but only under non-physiological conditions. Here we observed monoclonal IgG antibodies in aqueous solution by high-resolution frequency modulation atomic force microscopy (FM-AFM). We found that monoclonal antibodies self-assemble into hexamers, which form two-dimensional crystals in aqueous solution. Furthermore, by directly observing antibody-antigen interactions using FM-AFM, we revealed that IgG molecules in the crystal retain immunoactivity. As the self-assembled monolayer crystal of antibodies retains immunoactivity at a neutral pH and is functionally stable at a wide range of pH and temperature, the antibody crystal is applicable to new biotechnological platforms for biosensors or bioassays.

Monoclonal antibody form and function: manufacturing the right antibodies for treating drug abuse.

PubMed

Peterson, Eric; Owens, S Michael; Henry, Ralph L

2006-05-26

Drug abuse continues to be a major national and worldwide problem, and effective treatment strategies are badly needed. Antibodies are promising therapies for the treatment of medical problems caused by drug abuse, with several candidates in preclinical and early clinical trials. Monoclonal antibodies can be designed that have customized affinity and specificity against drugs of abuse, and because antibodies can be designed in various forms, in vivo pharmacokinetic characteristics can be tailored to suit specific clinical applications (eg, long-acting for relapse prevention, or short-acting for overdose). Passive immunization with antibodies against drugs of abuse has several advantages over active immunization, but because large doses of monoclonal antibodies may be needed for each patient, efficient antibody production technology is essential. In this minireview we discuss some of the antibody forms that may be effective clinical treatments for drug abuse, as well as several current and emerging production systems that could bridge the gap from discovery to patient use.

Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions

PubMed Central

Geng, Steven B.; Cheung, Jason K.; Narasimhan, Chakravarthy; Shameem, Mohammed; Tessier, Peter M.

2014-01-01

A limitation of using monoclonal antibodies as therapeutic molecules is their propensity to associate with themselves and/or with other molecules via non-affinity (colloidal) interactions. This can lead to a variety of problems ranging from low solubility and high viscosity to off-target binding and fast antibody clearance. Measuring such colloidal interactions is challenging given that they are weak and potentially involve diverse target molecules. Nevertheless, assessing these weak interactions – especially during early antibody discovery and lead candidate optimization – is critical to preventing problems that can arise later in the development process. Here we review advances in developing and implementing sensitive methods for measuring antibody colloidal interactions as well as using these measurements for guiding antibody selection and engineering. These systematic efforts to minimize non-affinity interactions are expected to yield more effective and stable monoclonal antibodies for diverse therapeutic applications. PMID:25209466

Determination of trace amount of cyanobacterial toxin in water by microchip based enzyme-linked immunosorbent assay.

PubMed

Pyo, Dongjin; Hahn, Jong Hoon

2009-01-01

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a microchip based enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin(KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. Since the ELISA test was highly sensitive, the newly developed microchip based ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 0.025 and 0.3 ng/mL.

[Monoclonal antibodies ICO-02 to blast cell antigens in patients with chronic myeloleukemia in blast crisis].

PubMed

Baryshnikov, A Iu

1984-01-01

Mice were immunized with blood cells of a patient with chronic granulocytic leukemia, and their cells were subsequently used for the preparation of hybridoma ICO-02. This hybridoma is continuously producing monoclonal antibodies which reacted with cells in 4 out of 13 patients with blastic crisis of chronic granulocytic leukemia and in 6 out of 38 patients with acute lymphoblastic leukemia. Antibodies reacted with blast cells in 2 out of 3 patients with undifferentiated blastic crisis of chronic myelocytic leukemia and in 2 out of 5 patients with lymphoid variant of blastic crisis of chronic granulocytic leukemia. Cells of 6 patients with acute lymphoblastic leukemia which reacted with the monoclonal antibodies had immunological markers of T lymphocytes bone-marrow precursors. Monoclonal antibodies did not react with cells of blood and bone marrow from healthy people and from patients with chronic lymphocytic leukemia, acute myeloblastic leukemia, acute myelomonocytic leukemia, acute monoblastic leukemia and lymphosarcoma.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ristow, Sandra S.; Arnzen, Jeanene M.; Leong, JoAnn Ching

Seventeen strains of infectious hematopoietic necrosis virus (IHNV) from different geographical regions and from different fish stocks were typed by polyacrylamide gel electrophoresis, indirect fluorescence with 27 monoclonal antibodies against both the G and N proteins of the virus, and by serum neutralization with six monoclonal anti-glycoprotein antibodies. In addition, many other IHNV isolates have been examined. Studying the isolates with the antibodies has shown that a greater amount of variation exists between isolates than was first predicted by the application of the polyacrylamide technique. Isolates within electrophoretic types I-V may be further classified according to their reactions with themore » monoclonal antibodies in indirect fluorescence. Serum neutralization with selected anti-glycoprotein antibodies in conjunction with fluorescence analysis confirms one of the original findings of Hsu et al. (1986) that two different species in a single facility can be infected with the same isolate. Variation among isolates as measured by reactivity with the monoclonal library appears to be greater within the G protein than within the N protein sequence. 9 refs., 7 figs., 6 tabs.« less

DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST FATHEAD MINNOW (PIMEPHALES PROMELAS) VITELLOGENIN

EPA Science Inventory

We have obtained a panel of monoclonal antibodies directed against fathead minnow vitellogenin (Vtg) for use in sensitive ELISAs to quantify the response of exposure in vivo to estrogen or estrogen mimics.

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors1

PubMed Central

Bruckheimer, Elizabeth M; Fazenbaker, Christine A; Gallagher, Sandra; Mulgrew, Kathy; Fuhrmann, Stacy; Coffman, Karen T; Walsh, William; Ready, Shannon; Cook, Kim; Damschroder, Melissa; Kinch, Michael; Kiener, Peter A; Woods, Rob; Gao, Changshou; Dall'Acqua, William; Wu, Herren; Coats, Steven

2009-01-01

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells. PMID:19484140

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

PubMed

Winkler, Mark T; Bushey, Ryan T; Gottlin, Elizabeth B; Campa, Michael J; Guadalupe, Eross S; Volkheimer, Alicia D; Weinberg, J Brice; Patz, Edward F

2017-01-01

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Influence of pH on heat-induced aggregation and degradation of therapeutic monoclonal antibodies.

PubMed

Ishikawa, Tomoyoshi; Ito, Takahiko; Endo, Ryosuke; Nakagawa, Keiko; Sawa, Eiji; Wakamatsu, Kaori

2010-01-01

Monoclonal antibodies are widely used for the treatment of various diseases, and because therapeutic monoclonal antibodies are stored in an aqueous solution or in a lyophilized state, the preparation of a stabilizing formulation that prevents their deterioration (degradation and aggregation) is crucial. Given the structural similarities of the immunoglobulin G (IgG) framework regions and a diversity of only four subclasses, we aimed to find common conditions that stabilize many different antibodies. In this study, we analyzed the effect of pH (the most critical factor in establishing a stable formulation) on human monoclonal antibodies from subclasses IgG1, IgG2, and IgG4, all of which have been utilized in antibody therapeutics. We found that human IgGs are stable with minimal heat-induced degradation and aggregation at pH 5.0-5.5 irrespective of their subclass. We also found that IgG1 is more susceptible to fragmentation, whereas IgG4 is more susceptible to aggregation. This basic information emphasizing the influence of pH on IgG stability should facilitate the optimization of formulation conditions tailored to individual antibodies for specific uses.

Enzyme-linked immunosorbent assay with monoclonal and single-chain variable fragment antibodies selective to coplanar polychlorinated biphenyls.

PubMed

Inui, Hideyuki; Takeuchi, Tetsuya; Uesugi, Akari; Doi, Fumito; Takai, Mikio; Nishi, Kosuke; Miyake, Shiro; Ohkawa, Hideo

2012-02-22

Coplanar polychlorinated biphenyls (Co-PCBs) consisting of non-ortho and mono-ortho-chlorinated PCBs are dioxin-like compounds and cause wide contamination in the environment. To monitor Co-PCB residues, it was attempted to establish an enzyme-linked immunosorbent assay (ELISA) with monoclonal and recombinant antibodies selective to Co-PCBs. When 3,3',5,5'-tetrachlorobiphenoxybutyric acid (PCBH)-keyhole limpet hemocyanin conjugate was immunized into mice, two monoclonal antibodies, Mab-0217 and Mab-4444, were obtained. 3,3',5,5'-Tetrachlorobiphenyl (PCB80) was determined with an IC(50) value of 2.6 and 0.46 ng mL(-1) in ELISA based on Mab-0217 and Mab-4444, respectively. Mab-4444 cross-reacted with Co-PCB congeners, except for PCB77 and PCB81. Mab-0217 reacted with PCB80 and cross-reacted with PCB111. A single-chain variable fragment (scFv) antibody derived from Mab-4444 was produced in recombinant Escherichia coli cells. The scFv antibody showed nearly the same sensitivity toward PCBH as the parent monoclonal antibody in ELISA. These results clearly suggested that Mab-4444 and its scFv antibodies were suitable for monitoring the representative congeners of Co-PCBs.

ERBB oncogene proteins as targets for monoclonal antibodies.

PubMed

Polanovski, O L; Lebedenko, E N; Deyev, S M

2012-03-01

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

In-situ Detection of Squalane in Sedimentary Organic Matter Using Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Bailey, J. V.; Corsetti, F. A.; Moldowan, J. M.; Fago, F.; Caron, D.

2008-12-01

Sedimentary geolipids can serve as powerful tools for reconstructing ancient ecosystems, but only if investigators can demonstrate that the hydrocarbons are indigenous to their host rocks. The association of molecules with primary sedimentary fabrics could indicate a syngenetic relationship. However, traditional biomarker analyses require extraction from large quantities of powdered rock, confounding detailed spatial correlations. Biological studies commonly use antibodies as extremely sensitive molecular probes. When coupled with fluorescent labels, antibodies allow for the visual localization of molecules. Here we show that monoclonal antibodies that bind specifically to geolipid compounds can be used for in situ detection and labeling of such compounds in mineral-bound organic macerals. Monoclonal antibodies to squalene, produced for human health studies, also react with the geolipid, squalane. We show that squalene antibodies do not react with other common sedimentary hydrocarbons. We also show that squalane antibodies bind specifically to isolated organic-rich lamina in Eocene-age, squalane-containing rocks. These results suggest that squalane is confined to discrete organo-sedimentary fabrics within those rocks, providing evidence for its syngeneity. The chemical similarity of squalane to other sedimentary hydrocarbons hints at the potential for developing monoclonal antibodies to a variety of biomarkers that could then be localized in rocks, sediments, and extant cells.

Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

PubMed Central

Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

2015-01-01

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586

A Mini-Review for Cancer Immunotherapy: Molecular Understanding of PD-1/PD-L1 Pathway & Translational Blockade of Immune Checkpoints

PubMed Central

Li, Yongshu; Li, Fangfei; Jiang, Feng; Lv, Xiaoqing; Zhang, Rongjiang; Lu, Aiping; Zhang, Ge

2016-01-01

Interference of the binding of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has become a new inspiring immunotherapy for resisting cancers. To date, the FDA has approved two PD-1 monoclonal antibody drugs against cancer as well as a monoclonal antibody for PD-L1. More PD-1 and PD-L1 monoclonal antibody drugs are on their way in clinical trials. In this review, we focused on the mechanism of the PD-1/PD-L1 signaling pathway and the monoclonal antibodies (mAbs) against PD-1 and PD-L1, which were approved by the FDA or are still in clinical trials. And also presented is the prospect of the PD-1/PD-L1 immune checkpoint blockade in the next generation of immunotherapy. PMID:27438833

Survey of Latin American Neuroimmunologists on the Treatment of Multiple Sclerosis with Monoclonal Antibodies.

PubMed

Fragoso, Yara Dadalti

2015-01-01

Natalizumab and alemtuzumab are monoclonal antibodies approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). A third monoclonal antibody, daclizumab, should soon become another alternative for RRMS therapy. A group of 26 doctors working at specific MS Units in seven different Latin American countries participated in the present study. All 26 neurologists had experience with natalizumab for the treatment of MS and were willing to discuss strategies for improving this treatment. Most neurologists had no confidence in starting a patient on natalizumab and alemtuzumab, which are new and efficient drugs approved by North American, European and most Latin American health agencies. The Latin American specialists felt they were not properly informed on daclizumab. Specific pharmacovigilance programs for each of these monoclonal antibodies were considered very important by the neurologists, who were also willing to discuss these therapeutic options with peers from other countries.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural smaples

USDA-ARS?s Scientific Manuscript database

A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

Therapeutic monoclonal antibodies for respiratory diseases: Current challenges and perspectives, March 31 - April 1, 2016, Tours, France.

PubMed

Desoubeaux, Guillaume; Reichert, Janice M; Sleeman, Matthew; Reckamp, Karen L; Ryffel, Bernhard; Adamczewski, Jörg P; Sweeney, Theresa D; Vanbever, Rita; Diot, Patrice; Owen, Caroline A; Page, Clive; Lerondel, Stéphanie; Le Pape, Alain; Heuze-Vourc'h, Nathalie

2016-01-01

Monoclonal antibody (mAb) therapeutics have tremendous potential to benefit patients with lung diseases, for which there remains substantial unmet medical need. To capture the current state of mAb research and development in the area of respiratory diseases, the Research Center of Respiratory Diseases (CEPR-INSERM U1100), the Laboratory of Excellence "MAbImprove," the GDR 3260 "Antibodies and therapeutic targeting," and the Grant Research program ARD2020 "Biotherapeutics" invited speakers from industry, academic and government organizations to present their recent research results at the Therapeutic Monoclonal Antibodies for Respiratory Diseases: Current challenges and perspectives congress held March 31 - April 1, 2016 in Tours, France.

A monoclonal IgM smooth muscle antibody reactive with fibroblast stress fibres produced by immunization with Treponema pallidum.

PubMed Central

Strugnell, R A; Underwood, J R; Clarke, F M; Pedersen, J S; Chalmers, P J; Faine, S; Toh, B H

1983-01-01

A monoclonal IgM smooth muscle antibody secreted by a hybrid (MMI-1) of mouse plasmacytoma NS-1 with spleen cells from mouse immunized with Treponema pallidum was detected by indirect immunofluorescence tests on frozen tissue sections and on acetone fixed monolayers of rat and human fibroblasts. The antibody did not react with acetone fixed smears of T. pallidum but reacted with smooth muscle fibres and with striations of skeletal and cardiac muscle. In non-muscle cells, the antibody stained liver in a 'polygonal' pattern, thymus with accentuated staining of the thymic medulla, renal glomeruli and the brush border and peritubular fibrils of renal tubules. In fibroblast monolayers, the antibody stained stress fibres in an interrupted pattern. Immunoblotting with muscle proteins and the antibody showed labelling of a 100K molecule. The cellular distribution of the mouse monoclonal antibody is similar to that obtained with anti-actin antibody suggesting that the corresponding antigen may be an actin binding protein. Images Fig. 3 PMID:6347470

[Antibody therapy for Alzheimer's disease].

PubMed

Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

2011-11-01

In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

Immunogenicity of an interferon-beta1a product.

PubMed

Kauffman, M A; Sterin-Prync, A; Papouchado, M; González, E; Vidal, A J; Grossberg, S E; Chuppa, S; Odoriz, B; Vrech, C; Diez, R A; Ferro, H H

2011-01-01

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Novel monoclonal antibodies to study tissue regeneration in planarians.

PubMed

Ross, Kelly G; Omuro, Kerilyn C; Taylor, Matthew R; Munday, Roma K; Hubert, Amy; King, Ryan S; Zayas, Ricardo M

2015-01-21

Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments. In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

The analysis with monoclonal antibodies of the heterogeneity of Ia glycoproteins on chronic lymphocytic leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Addis, J.B.; Tisch, R.; Falk, J.A.

The accessible Ia molecules on the surface of chronic lymphocytic leukemia (CLL) cells were quantitated in the cellular radioimmunoassay with saturating concentrations of monoclonal antibodies. Monoclonal antibody 21w4, like DA/2 antibody, recognizes monomorphic determinants of human Ia antigens.The amount of 21w4 or DA/2 bound to CLL cells derived from eight patients (varying from 2.6 to 13.9 x 10/sup 5/ molecules/cell) appears to be the maximum observed with the antibodies studied. Two other antibodies, 18d5 and 21r5, although also directed at nonpolymorphic Ia determinants, bind differentially to CLL cells, with the ratios of 21r5/21w4 and 18d5/21w4 varying from 0.08 to 0.90.more » Sequential immunoprecipitation studies have established that the four epitopes 18d5, 21r5, 21w4, and DA/2 were present on the same molecules. All Ia molecules express 21w4 and DA/2 epitopes, whereas only certain subsets of Ia molecules carry accessible 21r5 or 18d5 epitopes. Competitive binding studies showed that the epitopes recognized by the four monoclonal antibodies were different. Monoclonal antibodies 21r5 and 21w4 did not inhibit each other's binding. Furthermore, binding of 21w4 to CLL cells potentiated the binding of /sup 125/I-21r5 IgG to the same cells, suggesting that binding of 21w4 antibody induces a conformational change in the molecule that renders 21r5 epitopes more accessible.« less

Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys.

PubMed

Oswald, Wendelien B; Geisbert, Thomas W; Davis, Kelly J; Geisbert, Joan B; Sullivan, Nancy J; Jahrling, Peter B; Parren, Paul W H I; Burton, Dennis R

2007-01-01

Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.

Indium-111 labeled anti-melanoma monoclonal antibodies

DOEpatents

Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

1984-04-30

A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

Hybridoma technology

USDA-ARS?s Scientific Manuscript database

The generation of hybridoma cell lines by the fusion of splenocytes from immunized mice with immortal myeloma cells is a well established method for the production of monoclonal antibodies. Although other methods have emerged as an effective alternative for the generation of monoclonal antibodies t...

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum (Didelphis virginiana) serum.

PubMed

Sánchez, E E; García, C; Pérez, J C; De La Zerda, S J

1998-10-01

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occurring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra (Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five-step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.

Antibody-Directed Cytotoxic Agents: Use of Monoclonal Antibody to Direct the Action of Toxin A Chains to Colorectal Carcinoma Cells

NASA Astrophysics Data System (ADS)

Gilliland, D. Gary; Steplewski, Zenon; Collier, R. John; Mitchell, Kenneth F.; Chang, Tong H.; Koprowski, Hilary

1980-08-01

We have constructed cell-specific cytotoxic agents by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-1A was modified by attachment of 3-(2-pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linked conjugates that retained the original binding specificity of the antibody moiety. The conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.

Blockade of the antigen-antibody reaction using benzil condensation with the guanidyl residue of arginine.

PubMed

Montero, C; Segura, D I; Gutierrez, M

1991-03-01

Benzil blockade of the guanidyl group of arginine was tried on sections of paraffin-embedded tissue fixed in two different fixatives, in an attempt to evaluate the relevance of this amino acid to the reaction of several proteins with their corresponding antibodies. The two fixatives were 10% formaldehyde, and Bouin's fluid without acetic acid. Both polyclonal and monoclonal antibodies against proteins or peptides (lysozyme, adrenocorticotropic hormone, growth hormone, placental lactogen, and prolactin) were used on human biopsies or material from autopsies. The blockade was effective when monoclonal antibodies were used, whereas no effect or only a small decrease of the intensity of the reaction was observed with polyclonal antibodies. The least definitive result was obtained with prolactin, where a complete blockade was never achieved with monoclonal antibodies. Calcitonin, a peptide that does not contain arginine, was used as a control not susceptible to benzil blockade; no blockade of immunostaining was observed.

Monoclonal antibody technologies and rapid detection assays

USDA-ARS?s Scientific Manuscript database

Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

Functional single-walled carbon nanotubes based on an integrin αvβ3 monoclonal antibody for highly efficient cancer cell targeting

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da; Zhou, Feifan; Wang, Huiying; Tang, Yonghong

2009-03-01

The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on an integrin αvβ3 monoclonal antibody was developed and was used for cancer cell targeting in vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). The PL-PEG functionalized SWNTs were then conjugated with protein A. A SWNT-integrin αvβ3 monoclonal antibody system (SWNT-PEG-mAb) was thus constructed by conjugating protein A with the fluorescein labeled integrin αvβ3 monoclonal antibody. In vitro study revealed that SWNT-PEG-mAb presented a high targeting efficiency on integrin αvβ3-positive U87MG cells with low cellular toxicity, while for integrin αvβ3-negative MCF-7 cells, the system had a low targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific integrin targeting of the monoclonal antibody. In conclusion, SWNT-PEG-mAb developed in this research is a potential candidate for cancer imaging and drug delivery in cancer targeting therapy.

Requests Cancer Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, the National Cancer Institute (NCI) Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. The program from The Office of Cancer Clinical Proteomics Research provides reagents and other critical resources that support protein and/or peptide measurements and analysis.

Monoclonal Antibodies Against Fusicoccin with Binding Characteristics Similar to the Putative Fusicoccin Receptor of Higher Plants 1

PubMed Central

Feyerabend, Martin; Weiler, Elmar W.

1987-01-01

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [3H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 1014Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10−9 molar and 1.85 × 10−9 molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10−9 molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [3H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. Images Fig. 2 PMID:16665786

[Biotechnological advances in monoclonal antibody therapy: the RANK ligand inhibitor antibody].

PubMed

Kiss, Emese; Kuluncsics, Zénó; Kiss, Zoltán; Poór, Gyula

2010-12-26

Biological drugs have been used since the middle of the last century in medicine. Nowadays we are witnesses of the intensive development and wider administration of these drugs in clinical practice. Around 250 biological drugs are available and more than 350 million patients have been treated since their marketed authorization. Among the biologics there are protein based macromolecules, which mass production can be performed with the help of biotechnology. This term referring to the use of living organisms for production of molecules, was introduced by the Hungarian engineer, Károly Ereky. The present review focuses on the research, production and development of monoclonal antibodies manufactured by biotechnology. Some steps of this development have changed our immunological knowledge and the outcome of several diseases. The development of antibodies was highly recognized by two Nobel prizes. Authors detail the structure and functions of immunoglobulins, and their development, including fully human monoclonal antibodies. The RANKL inhibitor denosumab, a fully human IgG2 monoclonal antibody belongs to this latter group and it is available for treatment of osteoporosis. Authors also summarize the basic process of bone metabolism and the benefits of RANK ligand inhibition.

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bidart, J.M.; Troalen, F.; Salesse, R.

1987-06-25

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptidesmore » spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.« less

Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

PubMed

Sierralta, W D; Thole, H H

1996-05-01

The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform.

PubMed

Foltz, Ian N; Gunasekaran, Kannan; King, Chadwick T

2016-03-01

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Establishment and characterization of monoclonal and polyclonal antibodies against human intestinal fatty acid-binding protein (I-FABP) using synthetic regional peptides and recombinant I-FABP.

PubMed

Kajiura, Satoshi; Yashiki, Tetsuya; Funaoka, Hiroyuki; Ohkaru, Yasuhiko; Nishikura, Ken; Kanda, Tatsuo; Ajioka, Yoichi; Igarashi, Michihiro; Hatakeyama, Katsuyoshi; Fujii, Hiroshi

2008-01-01

We have succeeded in raising highly specific anti-human intestinal fatty acid-binding protein (I-FABP) monoclonal antibodies by immunizing animals with three synthetic regional peptides, i.e., the amino terminal (RP-1: N-acetylated 1-19-cysteine), middle portion (RP-2: cysteinyl-91-107) and carboxylic terminal (RP-3: cysteinyl-121-131) regions of human I-FABP, and the whole I-FABP molecule as antigens. We also raised a polyclonal antibody by immunizing with a recombinant (r) I-FABP. To ascertain the specificity of these antibodies for human I-FABP, the immunological reactivity of each was examined by a binding assay using rI-FABP, partially purified native I-FABP and related proteins such as liver-type (L)-FABP, heart-type (H)-FABP, as well as the regional peptides as reactants, and by Western blot analysis. In addition, the expression and distribution of I-FABP in the human gastrointestinal tract were investigated by an immunohistochemical technique using a carboxylic terminal region-specific monoclonal antibody, 8F9, and a polyclonal antibody, DN-R2. Our results indicated that both the monoclonal and polyclonal antibodies established in this study were highly specific for I-FABP, but not for L-FABP and H-FABP. Especially, the monoclonal antibodies raised against the regional peptides, showed regional specificity for the I-FABP molecule. Immunoreactivity of I-FABP was demonstrated in the mucosal epithelium of the jejunum and ileum by immunohistochemical staining, and the immunoreactivity was based on the presence of the whole I-FABP molecule but not the presence of any precursors or degradation products containing a carboxylic terminal fragment. It is concluded that some of these monoclonal and polyclonal antibodies, such as 8F9, 4205, and DN-R2, will be suitable for use in research on the immunochemistry and clinical chemistry of I-FABP because those antibodies can recognize both types of native and denatured I-FABP. In order to detect I-FABP in blood samples, it is essential to use this type of antibody, reactive to native type of I-FABP. It is anticipated that, in the near future, such a method for measuring I-FABP will be developed as a useful tool for diagnosing intestinal ischemia by using some of these antibodies.

Anti-EGFR-Conjugated Hollow Gold Nanospheres Enhance Radiocytotoxic Targeting of Cervical Cancer at Megavoltage Radiation Energies

NASA Astrophysics Data System (ADS)

Liu, Jiao; Liang, Ying; Liu, Ting; Li, Dengke; Yang, Xingsheng

2015-05-01

The study aimed to confirm that anti-epidermal growth factor receptor (EGFR) monoclonal antibody-conjugated hollow gold nanospheres (anti-EGFR/HGNs) can be selectively uptaken by cervical cancer cells and induce its apoptosis when combined with radiotherapy, as a result enhancing radiosensitivity of cervical cancer cells. HGNs with a mean diameter of 54.6 ± 7.11 nm and wall thickness of 5.01 ± 2.23 nm were viewed by transmission electron microscopy (TEM). Cell uptake was assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The cytotoxicity on HeLa cells, which were used in our experiment, was assessed by CCK-8 assay. Cell cycle and apoptosis were examined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry (FCM). The expression of several critical apoptosis-related proteins, including Bcl-2, Bax, Bad, and active caspase 3, was tested by western blot analysis. Cells treated by anti-EGFR/HGNs showed an obvious increase in nanoparticle uptake compared to naked HGNs. Anti-EGFR/HGNs combined with radiation resulted in a significant growth inhibition, compared with radiation combined with naked HGNs. Anti-EGFR/HGNs remarkably increased the ratio of HeLa cells in the G2/M phase and induced more apoptosis by an obvious deregulation of Bcl-2 and upregulation of Bax, Bad, and caspase 3 when combined with radiation. Therefore, anti-EGFR/HGNs can increase the targeted uptake of HGNs by HeLa cells and enhance radiocytotoxic targeting of cervical cancer at megavoltage radiation energies.

Monoclonal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms.

PubMed

Rael, E D; Salo, R J; Zepeda, H

1986-01-01

Hybridomas secreting monoclonal antibodies against Mojave toxin were established. The antibodies were used for identifying cross-reacting proteins in individual C. s. scutulatus and other Crotalus venoms and to isolate Mojave toxin. The antibodies recognized five bands with a pI range from 5.1 to 6.1 in immunoblots of electrofocused crude venom and Mojave toxin purified by immunoaffinity chromatography. The specificity of the antibodies was for the basic subunit of the toxin, which resolved into four bands of pI between 9.3 and 9.6. Individual C. s. scutulatus venoms of snakes from Texas and southern Arizona had multiple bands with pI's ranging from 4.9 to 6.3. Cross-reacting proteins were also recognized by the antibodies in the electrophoresed venoms of C. basiliscus, C. d. durissus, C. d. terrificus, C. h. horridus and C. v. concolor, and may be isolated by immunoaffinity chromatography with the monoclonal antibodies.

Modern affinity reagents: Recombinant antibodies and aptamers.

PubMed

Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

2015-12-01

Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

1988-01-01

The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

Monoclonal antibodies bind identically to both spores and hyphae of Aspergillus fumigatus.

PubMed

Reijula, K E; Kurup, V P; Kumar, A; Fink, J N

1992-05-01

Immunoelectron microscopy (IEM) was used to determine the binding of six monoclonal antibodies (MoAbs) produced against Aspergillus fumigatus antigens present on or within the conidia and hyphae of the fungus. Antigen-antibody complexes were demonstrated in EM using labelled colloidal gold particles (15 nm). Three out of 6 MoAbs (C9, F12 and H10) reacted only with the cytoplasmic components of A. fumigatus while the remaining three (B12, F6G5 and D6E6) showed reactivity to both cytoplasm and cell wall of the conidia and hyphae. The results indicate that IEM is of considerable value in determining and selecting monoclonal antibodies having specific reactivity with diverse antigenic components.

Therapeutic monoclonal antibodies for respiratory diseases: Current challenges and perspectives, March 31 – April 1, 2016, Tours, France

PubMed Central

Desoubeaux, Guillaume; Reichert, Janice M.; Sleeman, Matthew; Reckamp, Karen L.; Ryffel, Bernhard; Adamczewski, Jörg P.; Sweeney, Theresa D.; Vanbever, Rita; Diot, Patrice; Owen, Caroline A.; Page, Clive; Lerondel, Stéphanie; Le Pape, Alain; Heuze-Vourc'h, Nathalie

2016-01-01

ABSTRACT Monoclonal antibody (mAb) therapeutics have tremendous potential to benefit patients with lung diseases, for which there remains substantial unmet medical need. To capture the current state of mAb research and development in the area of respiratory diseases, the Research Center of Respiratory Diseases (CEPR-INSERM U1100), the Laboratory of Excellence “MAbImprove,” the GDR 3260 “Antibodies and therapeutic targeting,” and the Grant Research program ARD2020 “Biotherapeutics” invited speakers from industry, academic and government organizations to present their recent research results at the Therapeutic Monoclonal Antibodies for Respiratory Diseases: Current challenges and perspectives congress held March 31 – April 1, 2016 in Tours, France. PMID:27266390

Production of large numbers of hybridomas producing monoclonal antibodies against rat IgE using mast cell-deficient w/wv and sl/sld strains of mice.

PubMed

Rup, B J

1989-08-15

A number of different mouse strains and immunization protocols were used to attempt to make monoclonal antibodies against rat IgE for use in studies of the structure, biological activities and regulation of this class of antibody. Successful production of large numbers of monoclonal antibodies was achieved when mast cell deficient (w/wv and sl/sld) but not conventional (BALB/c, CAF1 or SJL) mice were used. These results suggest that the poor response of conventional strains of mice to rat IgE may be due to the presence of mast cells bearing high affinity receptors for IgE in these mice.

Fully-human Monoclonal Antibodies Against Human EphrinB2 and EphB4 | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute's Cancer and Inflammation Program is seeking statements of capability or interest from parties interested in licensing fully-human monoclonal antibodies against human EphrinB2 and EphB4.

Development and evaluation of monoclonal antibodies for paxilline

USDA-ARS?s Scientific Manuscript database

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrati...

Class-specific effector functions of therapeutic antibodies.

PubMed

Pascal, Virginie; Laffleur, Brice; Cogné, Michel

2012-01-01

Physiology usually combines polyclonal antibodies of multiple classes in a single humoral response. Beyond their common ability to bind antigens, these various classes of human immunoglobulins carry specific functions which can each serve specific goals. In many cases, the function of a monoclonal therapeutic antibody may thus be modulated according to the class of its constant domains. Depending on the immunoglobulin class, different functional assays will be used in order to evaluate the functional activity of a monoclonal antibody.

The Biological and Clinical Significance of Androgen Receptor Variants

DTIC Science & Technology

2014-04-01

immunohistochemistry. We tested two antibodies purportedly specific for AR-V7: 1) a mouse monoclonal antibody available from A&G Precision Antibody...a gift from Dr. Luo and tested on AR-V7- high and -low cases from the mixed-grade cohort in our lab. We used a monoclonal antibody against AR as a...fold, as compared with conventional avidin- biotinylated enzyme complex (ABC) procedures. We tested this protocol on four cases of the mixed-grade

Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies.

PubMed

Shahmohammadi-Farid, Sima; Ghods, Roya; Jeddi-Tehrani, Mahmood; Bayat, Ali-Ahmad; Mojtabavi, Nazanin; Razavi, Alireza; Zarnani, Amir-Hassan

2017-12-01

Siglec-F (SF) is a surface glycoprotein expressed by mouse eosinophils and induces caspase- and mitochondria-dependent apoptosis after engagement with its cognate ligand or specific antibodies. This targeting eosinophils by monoclonal antibodies may help diverse diseases associated with increased frequency of eosinophils including allergy and asthma. In this paper, production of murine and rat monoclonal antibodies (mAbs) against Siglec-F has been addressed. Balb/c mice were immunized with siglec-F1 (SF1) and siglec-F2 (SF2) synthetic peptides conjugated to a carrier protein. Rats were immunized with Chinese hamster ovary CHO cells overexpressing Siglec-F (CHO-SF) or with Siglec-F-human immunoglobulin FC fusion protein (CHO-SF-Ig). Hybridomas were produced by standard protocol and screened for their reactivity by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and flow cytometry. In parallel, polyclonal antibodies were generated in New Zealand White rabbits immunized with SF1 and SF2 peptides. Three mouse and three rat mAbs were generated against synthetic peptides and SF-Ig, respectively. All mouse monoclonal and rabbit polyclonal antibodies reacted well with immunizing molecules in ELISA and detected specific band of Siglec-F in WB. However, they failed to detect native molecule in flow cytometry analysis. Quite the contrary, rat mAbs did not reacted with the denatured protein in WB, instead exhibited significant reactivity with CHO-SF cells in flow cytometry. Based on the heavily glycosylated nature of Siglec-F, it seems that generation of anti-SF antibodies able to detect native protein needs a properly folded molecule for immunization. Monoclonal antibodies reported here are invaluable tools for studying linear and conformation epitopes of SF and tracing mouse eosinophils.

Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

PubMed Central

Winkelmann, D. A.; Bourdieu, L.; Kinose, F.; Libchaber, A.

1995-01-01

We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement. PMID:7787107

Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies.

PubMed

Aboklaish, Ali F; Ahmed, Shatha; McAllister, Douglas; Cassell, Gail; Zheng, Xiaotian T; Spiller, Owen B

2016-08-01

Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel mAb against a human CD34 peptide reacts with the native protein on CD34+ cells.

PubMed

Shams, Mahmood; Jeddi-Tehrani, Mahmood; Notash Haghighat, Farzaneh; Bayat, Ali Ahmad; Mahmoudian, Jafar; Rezvani, Mohammad Reza

2013-12-01

‎Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem ‎cells (HSCs) and the small-‎vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a ‎marker for diagnosis ‎and classification of leukemia. Anti CD34 antibodies are used for isolation and ‎purification ‎of HSCs from bone marrow, peripheral blood and cord blood. To characterize a newly produced monoclonal antibody against a human CD34 peptide. Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line. ‎ELISA experiment revealed that the antibody recognized CD34 peptide. Western ‎blot analysis on TF1 ‎cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.‎ ‎Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

PubMed Central

Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

2014-01-01

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research

Cancer.gov

Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

New Stx2e monoclonal antibodies for immunological detection and distinction of Stx2 subtypes

USDA-ARS?s Scientific Manuscript database

Background Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, most antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype. Methods and Findings Four monoclonal antibodie...

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

PubMed

Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Using Monoclonal Antibody to Determine Lead Ions with a Localized Surface Plasmon Resonance Fiber-optic Biosensor

PubMed Central

Lin, Tsao-Jen; Chung, Mon-Fu

2008-01-01

A novel reflection-based localized surface plasmon resonance (LSPR) fiber­optic probe has been developed to determine the heavy metal lead ion concentration. Monoclonal antibody as the detecting probe containing massive amino groups to capture Pb(II)-chelate complexes was immobilized onto gold nanoparticle-modified optical fiber (NMAuOF). The optimal immobilizing conditions of monoclonal antibody on to the NMAuOF are 189 μg/mL in pH7.4 PBS for 2 h at 25°C. The absorbability of the functionalized NMAuOF sensor increases to 12.2 % upon changing the Pb(II)-EDTA level from 10 to 100 ppb with a detection limit of 0.27 ppb. The sensor retains 92.7 % of its original activity and gives reproducible results after storage in 5% D-(+)-Trehalose dehydrate solution at 4°C for 35 days. In conclusion, the monoclonal antibody-functionalized NMAuOF sensor shows a promising result for determining the concentration of Pb(II) with high sensitivity. PMID:27879723

Anti-EGFR monoclonal antibody in cancer treatment: in vitro and in vivo evidence

PubMed

Quatrale, Anna Elisa; Petriella, Daniela; Porcelli, Letizia; Tommasi, Stefania; Silvestris, Nicola; Colucci, Giuseppe; Angelo, Angelo; Azzariti, Amalia

2011-01-01

The complexity of EGFR signaling network suggests that the receptor could be promising targets for new personalised therapy. In clinical practice two strategies targeting the receptor are available; they utilise monoclonal antibodies, directed towards the extracellular domain of EGFR, and small molecule tyrosine kinase inhibitors, which bind the catalytic kinase domain of the receptor. In this review, we summarise currently known pre-clinical data on the antitumor effects of monoclonal antibodies, which bind to EGFR in its inactive configuration, competing for ligand binding and thereby blocking ligand-induced EGFR tyrosine kinase activation. As a consequence of treatment, key EGFR-dependent intracellular signals in cancer cells are affected. Data explaining the mechanisms of action of anti-EGFR monoclonal antibodies, currently used in clinical setting and under development for the treatment of solid tumors, are revised with the aim to provide an overview of the most important preclinical studies showing the impact of this class of EGFR targeted agents on tumor biology.

A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica.

PubMed Central

Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Jaramillo, C; Castañon, G; Hall, A; Guhl, F; Ruiz-Palacios, G; Warhurst, D C

1992-01-01

Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively. Images PMID:1452651

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments.

PubMed

Greunke, Kerstin; Spillner, Edzard; Braren, Ingke; Seismann, Henning; Kainz, Sabine; Hahn, Ulrich; Grunwald, Thomas; Bredehorst, Reinhard

2006-07-13

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.

The main immunogenic region of acetylcholine receptors does not provoke the formation of antibodies of a predominant idiotype.

PubMed

Killen, J A; Hochschwender, S M; Lindstrom, J M

1985-08-01

Anti-idiotype antibodies were induced in rats by immunization with rat monoclonal antibodies to the main immunogenic region of acetylcholine receptors. These anti-idiotype antibodies showed very little crossreaction with other rat monoclonal antibodies which bind to the same region of the receptor. When the rats producing these anti-idiotype antibodies were immunized with receptor, they showed no net decrease in anti-receptor antibody production. These data indicate that, although more than half of the antibodies produced by rats immunized with receptor are directed at a small region, many anti-receptor idiotypes are involved in this response and anti-idiotype therapy is not beneficial.

Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

PubMed Central

Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

2013-01-01

The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

Monoclonal antibody specific for IDH1 R132H mutation.

PubMed

Capper, David; Zentgraf, Hanswalter; Balss, Jörg; Hartmann, Christian; von Deimling, Andreas

2009-11-01

IDH1 R132H mutations occur in approximately 70% of astrocytomas and oligodendroglial tumors. We developed a mouse monoclonal antibody targeting the IDH1 R132H mutation. Here, we show the high specificity and sensitivity of this antibody on Western blots and tissue sections from formalin fixed paraffin embedded tumor specimens. This antibody is highly useful for tumor classification, in detecting single infiltrating tumor cells and for the characterization of the cellular role of mutant IDH1 protein.

THE ECSTACY OF GOLD Patent Expirations for Trastuzumab, Bevacizumab, Rituximab, and Cetuximab.

PubMed

Serna-Gallegos, Tasha R; LaFargue, Christopher J; Tewari, Krishnansu S

2017-11-22

Fully humanized monoclonal antibodies have revolutionized the treatment of many solid tumors, including breast, lung, colorectal, and ovarian cancer. Among the most widely used monoclonal antibodies in clinical oncology are cetuximab, trastuzumab, rituximab, and bevacizumab. This article will review these four notable monoclonal antibodies, their role in clinical oncology, and the drug patents that are nearing expiration. They are used in both first and second line treatment regimens for multiple common malignancies. With recent patent expirations, pharmaceutical companies involved in biosimilar manufacture are looking to establish ownership over these financial monopolies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Biotechnology and genetic engineering in the new drug development. Part II. Monoclonal antibodies, modern vaccines and gene therapy.

PubMed

Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

2013-01-01

Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.

Therapeutic monoclonal antibodies in human breast milk: a case study.

PubMed

Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A

2014-04-01

Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

The form variation of the capsular polysaccharide K1 is not a critical virulence factor of Escherichia coli in a neonatal mouse model of infection.

PubMed

Colino, J; Outschoorn, I

1999-10-01

Escherichia coli K1 is a prevalent cause of Gram-negative neonatal bacteraemia and meningitis in humans. Its capsular polysaccharide K1 (CpsK1) has been identified as an important virulence factor. Nevertheless, the biological and pathogenic implications of its O-acetylated and non-O-acetylated forms are poorly understood. In an attempt to address this, we monitored the expression of both CpsK1 form variants in a neonatal mouse infection model. In the absence of anti-CpsK1 antibodies, no CpsK1 form variant selection was observed during the course of infection. The administration of monoclonal antibodies specific for CpsK1 provided a high level of protection. The monoclonal antibodies that recognized both CpsK1 forms (MGB12) provided protection from up to 850 LD(50). By contrast, the administration of the monoclonal antibodies (MGB15) specific for non-O-acetylated CpsK1 cleared only bacteria expressing this CpsK1 form; a few mouse pups remained bacteraemic, and the bacteria in the blood had O-acetylated CpsK1. In those pups, the infection progressed in a similar fashion to that in mice not treated with monoclonal antibody. Moreover, when the number of bacteria expressing the O-acetylated CpsK1 in the inoculated dose is considered independently, the LD(50)was similar to that for the original strain in pups that had not been treated with monoclonal antibodies (35 CFU). These results suggest that whereas variation in acetylation form per se does not reinforce virulence, it could enable E. coli to avoid immune defenses. This highlights the importance of using highly specific monoclonal antibodies in immunotherapeutic approaches to E. coli K1 neonatal meningitis. Copyright 1999 Academic Press.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

1984-11-29

The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

Evaluation of a newly developed quantitative heart-type fatty acid binding protein assay based on fluorescence immunochromatography using specific monoclonal antibodies.

PubMed

Kang, Keren; Wu, Peidian; Li, Wenmei; Tang, Shixing; Wang, Jihua; Luo, Xiaochun; Xie, Mingquan

2015-01-01

To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.

Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody

PubMed Central

Wacker, Daniel; Kapoor, Mili; Zhang, Ai; Han, Gye Won; Basu, Shibom; Patel, Nilkanth; Messerschmidt, Marc; Weierstall, Uwe; Liu, Wei; Katritch, Vsevolod; Roth, Bryan L.; Stevens, Raymond C.

2017-01-01

Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. PMID:28716900

In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

NASA Astrophysics Data System (ADS)

Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

1983-05-01

A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

PubMed

Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

2012-05-01

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

PubMed Central

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

2014-01-01

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

PubMed

Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

2013-08-01

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Britton, W.J.; Hellqvist, L.; Basten, A.

1985-12-01

Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bandsmore » of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.« less

Tumour dormancy and cell signalling--III: Role of hypercrosslinking of IgM and CD40 on the induction of cell cycle arrest and apoptosis in B lymphoma cells.

PubMed

Marches, R; Racila, E; Tucker, T F; Picker, L; Mongini, P; Hsueh, R; Vitetta, E S; Scheuermann, R H; Uhr, J W

1995-06-01

Polyclonal anti-IgM antibodies were more effective than monoclonal antibodies in inducing dormancy in SCID mice bearing a murine B lymphoma (BCL1). Under saturating conditions, both polyclonal and monoclonal anti-Ig antibodies induced cell cycle arrest (CCA) in both BCL1 cells and human B lymphoma cells (Daudi) but polyclonal antibodies were far more effective at inducing apoptosis. A mixture of several monoclonal antibodies specific for noncrossreactive epitopes on C mu mimicked the effects of a polyclonal anti-mu. Hypercrosslinking mIgM by a polyclonal antibody against the primary monoclonal anti-mu markedly increased apoptosis and CCA. Hence, the extent of crosslinking of IgM and the resultant singnalling may be a major factor in inducing and maintaining dormancy and in determining whether lymphoma cells respond by apoptosis or CCA. In contrast to mIgM, another B cell receptor, CD40, which induces CCA when crosslinked did not induce apoptosis after hypercrosslinking. The results are consistent with the hypothesis that aspects of the CCA and apoptotic pathways are independent. When anti-CD40 was added with anti-mu to Daudi cells, the proportion of cells undergoing apoptosis was increased.

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

NASA Astrophysics Data System (ADS)

Du, Yi; May, Kimberly; Xu, Wei; Liu, Hongcheng

2012-07-01

The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed

Papac, D I; Hoyes, J; Tomer, K B

1994-09-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed Central

Papac, D. I.; Hoyes, J.; Tomer, K. B.

1994-01-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP. PMID:7530543

A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia

PubMed Central

Varshney, Avanish K.; Sunley, Kevin M.; Bowling, Rodney A.; Kwan, Tzu-Yu; Mays, Heather R.; Rambhadran, Anu; Zhang, Yanfeng; Martin, Rebecca L.; Cavalier, Michael C.; Simard, John

2018-01-01

Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia. PMID:29364906

Optical Imaging of Mammaglobin Expression of Breast Cancer

DTIC Science & Technology

2003-05-01

monoclonal anti-MMG antibodies with a near infrared fluorescent probe for optical imaging and 64Cu -DOTA for positron emission tomography (mPET...Preliminary results indicate that the mPET imaging with 64Cu -DOTA-anti MMG monoclonal antibodies showed predominant liver uptake in mice. In contrast, the

INITIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE FUNGAL HEMOLYSIN STACHYLYSIN FROM STACHYBOTRYS CHARTARUM

EPA Science Inventory

Stachybotrys chartarum is known to produce the hemolysin stachylysin and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the dev...

Acute treatment with XMetA activates hepatic insulin receptors and lowers blood glucose in normal mice

USDA-ARS?s Scientific Manuscript database

It has been proposed that monoclonal antibodies may become therapeutics for metabolic diseases such as diabetes mellitus. We have previously characterized an allosteric monoclonal antibody to the human insulin receptor (IR), XMetA, that activated metabolic signaling leading to enhanced glucose tran...

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

PubMed

Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

2012-12-01

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

PubMed

Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

2012-01-01

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

A human recombinant monoclonal antibody to cocaine: Preparation, characterization and behavioral studies

PubMed Central

Eubanks, Lisa M.; Ellis, Beverly A.; Cai, Xiaoqing; Schlosburg, Joel E.; Janda, Kim D.

2014-01-01

Cocaine abuse remains prevalent worldwide and continues to be a major health concern; nonetheless, there is no effective therapy. Immunopharmacothery has emerged as a promising treatment strategy by which anti-cocaine antibodies bind to the drug blunting its effects. Previous passive immunization studies using our human monoclonal antibody, GNCgzk, resulted in protection against cocaine overdose and acute toxicity. To further realize the clinical potential of this antibody, a recombinant IgG form of the antibody has been produced in mammalian cells. This antibody displayed a high binding affinity for cocaine (low nanomolar) in line with the superior attributes of the GNCgzk antibody and reduced cocaine-induced ataxia in a cocaine overdose model. PMID:25205191

Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin Neutralization Assays and Their Synergistic Action

PubMed Central

Ngundi, Miriam M.; Meade, Bruce D.; Little, Stephen F.; Quinn, Conrad P.; Corbett, Cindi R.; Brady, Rebecca A.

2012-01-01

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax. PMID:22441391

Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A

NASA Astrophysics Data System (ADS)

Leserman, Lee D.; Barbet, Jacques; Kourilsky, François; Weinstein, John N.

1980-12-01

Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets1-3. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human β2-microglobulin [antibody B1.1G6 (IgG2a, κ) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

Cross-reactivity of a human IgG1 anticitrullinated fibrinogen monoclonal antibody to a citrullinated profilaggrin peptide

PubMed Central

Trier, Nicole Hartwig; Leth, Maria Louise; Hansen, Paul Robert; Houen, Gunnar

2012-01-01

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It is characterized by persistent joint inflammation, resulting in loss of joint function, morbidity and premature mortality. The presence of antibodies against citrullinated proteins is a characteristic feature of RA and up to 70% of RA patients are anticitrullinated protein antibody (ACPA) positive. ACPA responses have been widely studied and are suggested to be heterogeneous, favoring antibody cross-reactivity to citrullinated proteins. In this study, we examined factors that may influence cross-reactivity between a commercial human anticitrullinated fibrinogen monoclonal antibody and a citrullinated peptide. Using a citrullinated profilaggrin sequence (HQCHQEST- Cit-GRSRGRCGRSGS) as template, cyclic and linear truncated peptide versions were tested for reactivity to the monoclonal antibody. Factors such as structure, peptide length and flanking amino acids were found to have a notable impact on antibody cross-reactivity. The results achieved contribute to the understanding of the interactions between citrullinated peptides and ACPA, which may aid in the development of improved diagnostics of ACPA. PMID:23076998

Monoclonal IgA Antibodies for Aflatoxin Immunoassays

PubMed Central

Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

2016-01-01

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

Dog Erythrocyte Antigen 1 (DEA 1): Mode of Inheritance and Initial Characterization

PubMed Central

Polak, Klaudia; Acierno, Michelle; Raj, Karthik; Mizukami, Keijiro; Siegel, Don L.; Giger, Urs

2015-01-01

Background The Dog Erythrocyte Antigen (DEA) 1 blood group system remains poorly defined. Objectives The purpose of the study was to determine the DEA 1 mode of inheritance and to characterize the DEA 1 antigen and alloantibodies. Animals Canine research colony families, clinic canine patients, and DEA 1.2+ blood bank dogs were studied. Methods Canine blood was typed by flow cytometry and immunochromatographic strips using anti-DEA 1 monoclonal antibodies. Gel column experiments with polyclonal and immunoblotting with monoclonal anti-DEA 1 antibodies were performed to analyze select samples. Cross-reactivity of human typing reagents against canine RBCs and one monoclonal anti-DEA 1 antibody against human RBC panels was assessed. Results Typing of 12 families comprising 144 dogs indicated an autosomal dominant inheritance with ≥4 alleles: DEA 1− (0) and DEA 1+ weak (1+), intermediate (2+) and strong (3+ and 4+). Samples from 6 dogs previously typed as DEA 1.2+ were typed as DEA 1+ or DEA 1− using monoclonal antibodies. Human typing reagents produced varied reactions in tube agglutination experiments against DEA 1+ and DEA 1− RBCs. Polypeptide bands were not detected on immunoblots using a monoclonal anti-DEA 1 antibody, therefore the anti-DEA 1 antibody may be specific for conformational epitopes lost during denaturation. Conclusions The autosomal dominant inheritance of DEA 1 with ≥4 alleles indicates a complex blood group system; the antigenicity of each DEA 1+ type will need to be determined. The biochemical nature of the DEA 1 antigen(s) appears different from human blood group systems tested. PMID:26291052

Influence of Snail on Integrin Beta l Expression/Activity in Breast Carcinoma

DTIC Science & Technology

2008-09-01

sensitive for this purpose (data not shown). Next, we tested the ability of a monoclonal Snail-1 antibody from our collaborator, Dr. Antonio Garcia de...monoclonal antibody from our collaborator, Dr. Antonio Garcia de Herreros (commercially- available antibodies were not of sufficient sensitivity...expression/function in breast tumor cells. Breast Cancer Res. Treat. In Press. 2. Wanami, L.S., Chen, H., Peiro, S., Garcia de Herreros, A., and

Influence of Snail on Integrin Beta 1 Expression/Activity in Breast Carcinoma

DTIC Science & Technology

2008-09-01

sensitive for this purpose (data not shown). Next, we tested the ability of a monoclonal Snail-1 antibody from our collaborator, Dr. Antonio Garcia de...monoclonal antibody from our collaborator, Dr. Antonio Garcia de Herreros (commercially- available antibodies were not of sufficient sensitivity...expression/function in breast tumor cells. Breast Cancer Res. Treat. In Press. 2. Wanami, L.S., Chen, H., Peiro, S., Garcia de Herreros, A., and

Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein.

PubMed

Kaba, Hani E J; Maier, Natalia; Schliebe-Ohler, Nicole; Mayer, Yvonne; Müller, Peter P; van den Heuvel, Joop; Schuchhardt, Johannes; Hanack, Katja; Bilitewski, Ursula

2015-01-01

We selected the immunogenic cell wall ß-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Breast Mucin Tumor-Specific Epitopes for Cancer Immunotherapy

DTIC Science & Technology

1998-09-01

reactivity with tumor-specific monoclonal antibodies show that antigenicity is maximized with the 40 amino acid MUC1-mtr2. By contrast, the MUC1-mtr3...associated mucins (7). The presence of tumor-specific epitopes is evidenced by the development of many monoclonal antibodies (mAb) that recognize...P1-P5 in the tandem repeat sequence (7). This epitope was identified by competition of antibody binding to tumor- specific mucin by synthetic

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

1991-10-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

1991-01-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

Uses of monoclonial antibody 8H9

DOEpatents

Cheung, Nai-Kong V.

2015-06-23

This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

A model system for the evaluation of radioimmunoimaging of tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koizumi, M.; Endo, K.; Sakahara, H.

1985-05-01

The authors have developed a simple model system that can be used to evaluate methods of radioimmunoimaging of tumors, using human chorionic gonadropin (hCG) as a model antigen, and a monoclonal antibody against hCG ..beta..-subunit as a model antibody. HCG was coated on a polystylene spherical bead with a quarter inch in diameter, and coated beads were washed extensively with phosphate buffered saline, and glycine acid buffer to remove the easily dissociable antigen. HCG-coated beads were put into the subcutaneous tissue on the back of mice. At 24 hr after the transplantation, when serum hCG was not detectable by themore » conventional RIA, radiolabeled antibodies were injected and its bio-distribution monitored. The %ID/g for the hCG coated beads increased to a maximum of 48 hr after the injection of radioiodinad antibody, whereas the %ID/g for most organs decreased with time. As a nonspecific antigen, beads coated with bovine serum albumin were transplanted and its uptake was as low as about one 50th of hCG-coated ones. The %ID/g of radioiodinated monoclonal antibody against human thyroglobulin (a nonspecific antibody) for hCG-coated beads was also negligible. Thus, the localization index (%ID of specific antibody / %ID of nonspecific antibody) reached to 15.0 at 24 hr, 35.5 at 48 hr and 57.8 at 96 hr after the injection. The biodistribution of In-111 labeled specific monoclonal antibody, prepared through the chelation with DTPA, demonstrated similar results with radioiodinated ones. This mouse model system that did not involve the use of tumors, yielded high localization index and reproducibilities and could be used to evaluate different methods for radiolabelng monoclonal antibodies.« less

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

PubMed Central

Boyd, Mary A.; Wang, Jin Y.; Tulapurkar, Mohan E.; Pasetti, Marcela F.; Levine, Myron M.; Simon, Raphael

2016-01-01

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity. PMID:26998925

Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

PubMed Central

Cohen, Matthew R.; Huynh, Kevin W.; Cawley, Daniel; Moiseenkova-Bell, Vera Y.

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function. PMID:24392006

Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies.

PubMed

Cohen, Matthew R; Huynh, Kevin W; Cawley, Daniel; Moiseenkova-Bell, Vera Y

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca(2+)-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function.

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

NASA Astrophysics Data System (ADS)

Liu, Hongcheng; Gaza-Bulseco, Georgeen; Chumsae, Chris

2009-12-01

Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly.

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

Monoclonal Antibody and an Antibody-Toxin Conjugate to a Cell Surface Proteoglycan of Melanoma Cells Suppress in vivo Tumor Growth

NASA Astrophysics Data System (ADS)

Bumol, T. F.; Wang, Q. C.; Reisfeld, R. A.; Kaplan, N. O.

1983-01-01

A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

PubMed

McClain, Mark S; Cover, Timothy L

2007-04-01

The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.

Identification of monoclonal antibodies cross-reactive with bottlenose dolphin orthologues of the major histocompatibility complex and leukocyte differentiation molecules

USDA-ARS?s Scientific Manuscript database

The slow progress in understanding immunotoxic effects of environmental contaminants and their influence on disease susceptibility and immunopathogenesis of the bottlenose dolphin (Tursiops truncates) and other species of cetaceans, is largely due to lack of monoclonal antibody (mAb) reagents. As re...

Development and Characterization of Mouse Monoclonal Antibodies Reactive with Chicken CD83

USDA-ARS?s Scientific Manuscript database

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein con...

Survey of citrus tristeza virus populations in Central California that react with MCA13 monoclonal antibody

USDA-ARS?s Scientific Manuscript database

The Citrus Pest Detection Program (CPDP) of the Central California Tristeza Eradication Agency monitors Citrus tristeza virus (CTV) in Central California. MCA13 is a severe strain discriminating monoclonal antibody used to screen for potentially virulent CTV isolates. MCA13-reactive CTV isolates are...

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-1ß

USDA-ARS?s Scientific Manuscript database

Two mouse monoclonal antibodies (mAbs) specific for chicken interleukin-1ß (chIL-1ß) were produced and characterized. Both mAbs identified a 66.0 kDa recombinant protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant fusion ...

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-8

USDA-ARS?s Scientific Manuscript database

Interleukin-8 (IL-8) is a key mediator in neutrophil-mediated acute proinflammatory response and activates multiple signaling pathways downstream of two receptors (CXCR1 and CXCR2). However, there have not been any monoclonal antibodies which specifically detect chicken IL8 (chIL8) and define its b...

Development and characterization of mouse monoclonal antibodies reactive with chicken IL1 Beta

USDA-ARS?s Scientific Manuscript database

Two mouse monoclonal antibodies (mAbs) specific for chicken interleukin-1 Beta (chIL-1 Beta) were produced and characterized. Both mAbs identified a 66.0 kDa recombinant protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant...

Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

USDA-ARS?s Scientific Manuscript database

One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3...

Serological diversity demonstrable by a set of monoclonal antibodies to eight serotypes of the mutans streptococci.

PubMed

Ota, F; Ota, M; Mahmud, Z H; Mohammad, A; Yamato, M; Kassu, A; Kato, Y; Tomotake, H; Batoni, G; Campa, M

2006-01-01

A set of monoclonal antibodies were prepared by the conventional cell fusion of myeloma cells (SP2/0-Ag14) with spleen cells from BALB/c mice immunised with whole cells of a strain of mutans streptococci. Their specificities were examined against 35 reference strains of mutans streptococci, 34 reference strains of other oral streptococci and 8 reference strains of other microorganisms often inhabiting the oral cavity. Specificity was examined by enzyme immunoassay using whole cells. A total of 52 strains, consisting of 19 strains isolated in Japan, 19 strains isolated in Italy and 14 strains isolated in England, were characterised by conventional physiological and biochemical tests and then serotyped by the use of 8 monoclonal antibodies with different specificities. They were also confirmed by guanine-plus-cytosine contents of their nucleic acid and DNA-DNA hybridisation test. The results indicated that all monoclonal antibodies are useful for identification of 8 serotypes of the mutans streptococci responsible for dental caries. They also suggest the existence of more serological varieties among mutans species.

[New method for analyzing pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies].

PubMed

Zhao, Yan; Qu, Hui-Hua; Wang, Qing-Guo

2013-09-01

Study on pharmacodynamic material basis of traditional Chinese medicines is one of the key issues for the modernization of traditional Chinese medicine. Having introduced the monoclonal antibody technology into the study on pharmacodynamic material basis of traditional Chinese medicines, the author prepared the immunoaffinity chromatography column by using monoclonal antibodies in active components of traditional Chinese medicines, so as to selectively knock out the component from herbs or traditional Chinese medicine compounds, while preserving all of the other components and keeping their amount and ratio unchanged. A comparative study on pharmacokinetics and pharmacodynamics was made to explicitly reveal the correlation between the component and the main purpose of traditional Chinese medicines and compounds. The analysis on pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies is a new method for study pharmacodynamic material basis in line with the characteristics of traditional Chinese medicines. Its results can not only help study material basis from a new perspective, but also help find the modern scientific significance in single herb or among compounds of traditional Chinese medicines.

Pathogenesis and morbidity of autoantibody syndromes in Waldenstrom's macroglobulinemia.

PubMed

Stone, Marvin J

2011-02-01

A number of autoantibody syndromes have been identified that are mediated by monoclonal macroglobulins. Chronic cold agglutinin disease (CAD), mixed cryoglobulinemia (MC), and various polyneuropathies produce symptoms and signs such as cold sensitivity and sensorimotor neuropathies that bring the patients to attention ("the patients tell you"). Many of these patients would be classified as having monoclonal gammopathy of undetermined significance (MGUS) were it not for the consequences of antigen-antibody interaction. Other patients with autoantibody syndromes have overt Waldenström's macroglobulinemia (WM). These individuals present at an earlier stage than WM patients who do not have evident antibody activity. Thus the presence of autoreactive antibodies influences clinical presentation and natural history of the disorder. The frequency of monoclonal IgMs with functional antibody activity is unknown. The principal problem is detection. New methods have been described which may more precisely identify the antigens reacting with M-proteins. Some "autoimmune" disorders may represent cross-reactions to exogenous antigens. Elucidation of antigens reacting with monoclonal IgM proteins may provide important insights into the pathogenesis of WM.

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

PubMed Central

Raybould, T J; Crouch, C F; Acres, S D

1987-01-01

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves. PMID:2880866

Production of monoclonal antibody against clonazepam for immunoassay of benzodiazepine drugs in swine tissues.

PubMed

Shan, Wen C; Cui, Ya L; He, Xin; Zhang, Lei; Liu, Jing; Wang, Jian P

2015-01-01

The objective of the present study was to produce a generic monoclonal antibody for immunoassay of residues of benzodiazepine drugs in swine tissues. Clonazepam was used to synthesize a hapten that was coupled to bovine serum albumin as an immunogen for the production of monoclonal antibody. Results showed that the obtained monoclonal antibody was able to recognize five benzodiazepine drugs simultaneously (clonazepam, flunitrazepam nitrazepam, diazepam, and oxazepam). The cross-reactivities were in the range of 24-100% and the limits of detection were in the range of 0.2-1.5 ng mL(-1) depending on the drug. Then a competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of five benzodiazepines in swine tissues (muscle, liver and kidney). The recoveries of five analytes from the fortified blank samples were in the range of 74.5-96.5% with coefficients of variation lower than 16.7%. Therefore, this immunoassay could be used as a rapid and simple method for the screening of residues of five benzodiazepine drugs in animal-derived foods.

Antibodies to the Glycoprotein GP2 Subunit Cross-React between Old and New World Arenaviruses.

PubMed

Amanat, Fatima; Duehr, James; Oestereich, Lisa; Hastie, Kathryn M; Ollmann Saphire, Erica; Krammer, Florian

2018-01-01

Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays. IMPORTANCE Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective vaccine or therapy is urgently needed. Here, we describe efforts to produce cross-reactive monoclonal antibodies that bind to both New and Old World arenaviruses. All of our MAbs seem to be nonneutralizing and nonprotective and target subunit 2 of the glycoprotein. Due to the lack of reagents such as recombinant glycoproteins and antibodies for rapid detection assays, our MAbs could be beneficial as analytic and diagnostic tools. Copyright © 2018 Amanat et al.

Preparation and characterization of monoclonal antibodies recognizing unique epitopes on sexually differentiated rat liver cytochrome P-450 isozymes.

PubMed

Morgan, E T; Rönnholm, M; Gustafsson, J A

1987-07-14

Cytochrome P-450 isozymes P-450(16 alpha), P-450(15 beta), and P-450DEa are immunochemically related, as indicated by mutual cross-reactivity with polyclonal antibody preparations. We have isolated five monoclonal antibodies to P-450(15 beta) and one antibody to P-450(16 alpha) that show selectivity for the respective antigens. High frequencies of cross-reactivity were observed, indicating a high degree of homology among P-450(16 alpha), P-450(15 beta), and P-450DEa. All of the P-450(15 beta-specific antibodies bound to the same epitope, or closely grouped epitopes, supporting this conclusion. The specificity of each monoclonal antibody was characterized by enzyme-linked immunosorbent assay. Western immunoblotting, and antibody-Sepharose immunoadsorption of solubilized rat liver microsomes. Antibodies F22 and F23, which were apparently identical, were specific for P-450(15 beta) by these criteria. However, the apparent specificities of antibodies F3 and F20 for P-450(15 beta), and of M16 for P-450(16 alpha), were highly dependent on the analytical technique used. The five anti-P-450(15 beta) antibodies all inhibited the catalytic activity of microsomal P-450(15 beta), by a maximum of 70%. However, they also produced a similar inhibition of microsomal P-450(16 alpha-specific antibody M16 and F23 have a low-affinity interaction with an epitope on P-450(16 alpha). The P-450(16 alpha)-specific antibody M16 was not inhibitory. The results indicate that the apparent specificity of a monoclonal antibody for an antigen determined by, e.g., Western blotting does not allow the conclusive identification of a protein in another system, e.g., immunoprecipitation of in vitro translation reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical and biological properties of a mouse monoclonal antibody reactive to prunus necrotic ringspot ilarvirus.

PubMed

Aebig, J A; Jordan, R L; Lawson, R H; Hsu, H T

1987-01-01

A monoclonal antibody reacting with prunus necrotic ringspot ilarvirus was tested in immunochemical studies, neutralization of infectivity assays, and by immuno-electron microscopy. The antibody was able to detect the 27,000 Mr coat protein of prunus necrotic ringspot ilarvirus in western blots and also detected all polypeptide fragments generated after incubation of whole virus with proteolytic enzymes. In neutralization of infectivity studies, the antibody blocked virus infectivity, although it did not precipitate the antigen in agar gel Ouchterlony double diffusion tests. Immuno-electron microscopy confirmed that the antibody coats virions but does not cause clumping. The antibody may be a useful tool for investigating coat protein-dependent initiation of ilarvirus infection.

Monitoring multiple myeloma patients treated with daratumumab: teasing out monoclonal antibody interference.

PubMed

McCudden, Christopher; Axel, Amy E; Slaets, Dominique; Dejoie, Thomas; Clemens, Pamela L; Frans, Sandy; Bald, Jaime; Plesner, Torben; Jacobs, Joannes F M; van de Donk, Niels W C J; Moreau, Philippe; Schecter, Jordan M; Ahmadi, Tahamtan; Sasser, A Kate

2016-06-01

Monoclonal antibodies are promising anti-myeloma treatments. As immunoglobulins, monoclonal antibodies have the potential to be identified by serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). Therapeutic antibody interference with standard clinical SPE and IFE can confound the use of these tests for response assessment in clinical trials and disease monitoring. To discriminate between endogenous myeloma protein and daratumumab, a daratumumab-specific immunofixation electrophoresis reflex assay (DIRA) was developed using a mouse anti-daratumumab antibody. To evaluate whether anti-daratumumab bound to and shifted the migration pattern of daratumumab, it was spiked into daratumumab-containing serum and resolved by IFE/SPE. The presence (DIRA positive) or absence (DIRA negative) of residual M-protein in daratumumab-treated patient samples was evaluated using predetermined assessment criteria. DIRA was evaluated for specificity, limit of sensitivity, and reproducibility. In all of the tested samples, DIRA distinguished between daratumumab and residual M-protein in commercial serum samples spiked with daratumumab and in daratumumab-treated patient samples. The DIRA limit of sensitivity was 0.2 g/L daratumumab, using spiking experiments. Results from DIRA were reproducible over multiple days, operators, and assays. The anti-daratumumab antibody was highly specific for daratumumab and did not shift endogenous M-protein. As the treatment of myeloma evolves to incorporate novel monoclonal antibodies, additional solutions will be needed for clinical monitoring of patient responses to therapeutic regimens. In the interim, assays such as DIRA can inform clinical outcomes by distinguishing daratumumab from endogenous M-protein by IFE.

[Avidity of polyreactive immunoglobulins].

PubMed

Bobrovnik, S A

2014-01-01

An analysis of the mechanism of interaction between polyreactive immunoglobulins (PRIG) and antigen was conducted and it was shown that most of the traditional methods of antibody affinity evaluation are not applicable for PRIG affinity. The comparative assessment of the mouse and human PRIG avidity against ovalbumin and horse myoglobin and the avidity of specific monoclonal antibodies against ovalbumin have shown that the avidity of PRIG not only is much less than the avidity of monoclonal antibodies but even exceeds it.

Tumor-specific novel taxoid-monoclonal antibody conjugates.

PubMed

Ojima, Iwao; Geng, Xudong; Wu, Xinyuan; Qu, Chuanxing; Borella, Christopher P; Xie, Hongsheng; Wilhelm, Sharon D; Leece, Barbara A; Bartle, Laura M; Goldmacher, Victor S; Chari, Ravi V J

2002-12-19

Taxoids bearing methyldisulfanyl(alkanoyl) groups for taxoid-antibody immunoconjugates were designed, synthesized and their activities evaluated. A highly cytotoxic C-10 methyldisulfanylpropanoyl taxoid was conjugated to monoclonal antibodies recognizing the epidermal growth factor receptor (EGFR) expressed in human squamous cancers. These conjugates were shown to possess remarkable target-specific antitumor activity in vivo against EGFR-expressing A431 tumor xenografts in severe combined immune deficiency mice, resulting in complete inhibition of tumor growth in all the treated mice.

Microbials for the production of monoclonal antibodies and antibody fragments.

PubMed

Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

2014-01-01

Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

NASA Astrophysics Data System (ADS)

Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

PubMed

Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

2017-02-01

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

Prediction and Reduction of the Aggregation of Monoclonal Antibodies.

PubMed

van der Kant, Rob; Karow-Zwick, Anne R; Van Durme, Joost; Blech, Michaela; Gallardo, Rodrigo; Seeliger, Daniel; Aßfalg, Kerstin; Baatsen, Pieter; Compernolle, Griet; Gils, Ann; Studts, Joey M; Schulz, Patrick; Garidel, Patrick; Schymkowitz, Joost; Rousseau, Frederic

2017-04-21

Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

The protective role of immunoglobulins in fungal infections and inflammation.

PubMed

Elluru, Sri Ramulu; Kaveri, Srini V; Bayry, Jagadeesh

2015-03-01

Increased incidence of fungal infections in the immunocompromised individuals and fungi-mediated allergy and inflammatory conditions in immunocompetent individuals is a cause of concern. Consequently, there is a need for efficient therapeutic alternatives to treat fungal infections and inflammation. Several studies have demonstrated that antibodies or immunoglobulins have a role in restricting the fungal burden and their clearance. However, based on the data from monoclonal antibodies, it is now evident that the efficacy of antibodies in fungal infections is dependent on epitope specificity, abundance of protective antibodies, and their isotype. Antibodies confer protection against fungal infections by multiple mechanisms that include direct neutralization of fungi and their antigens, inhibition of growth of fungi, modification of gene expression, signaling and lipid metabolism, causing iron starvation, inhibition of polysaccharide release, and biofilm formation. Antibodies promote opsonization of fungi and their phagocytosis, complement activation, and antibody-dependent cell toxicity. Passive administration of specific protective monoclonal antibodies could also prove to be beneficial in drug resistance cases, to reduce the dosage and associated toxic symptoms of anti-fungal drugs. The longer half-life of the antibodies and flexibilities to modify their structure/forms are additional advantages. The clinical data obtained with two monoclonal antibodies should incite interests in translating pre-clinical success into the clinics. The anti-inflammatory and immunoregulatory role of antibodies in fungal inflammation could be exploited by intravenous immunoglobulin or IVIg.

A Simple Method to Avoid Nonspecific Signal When Using Monoclonal Anti-Tau Antibodies in Western Blotting of Mouse Brain Proteins.

PubMed

Petry, Franck R; Nicholls, Samantha B; Hébert, Sébastien S; Planel, Emmanuel

2017-01-01

In Alzheimer's disease and other tauopathies, tau displays several abnormal post-translation modifications such as hyperphosphorylation, truncation, conformation, and oligomerization. Mouse monoclonal antibodies have been raised against such tau modifications for research, diagnostic, and therapeutic purposes. However, many of these primary antibodies are at risk of giving nonspecific signals in common Western blotting procedures. Not because they are unspecific, but because the secondary antibodies used to detect them will also detect the heavy chain of endogenous mouse immunoglobulins (Igs), and give a nonspecific signal at the same molecular weight than tau protein (around 50 kDa). Here, we propose the use of anti-light chain secondary antibodies as a simple and efficient technique to prevent nonspecific Igs signals at around 50 kDa. We demonstrate the efficacy of this method by removing artifactual signals when using monoclonal antibodies directed at tau phosphorylation (AT100, 12E8, AT270), tau truncation (TauC3), tau oligomerization (TOMA), or tau abnormal conformation (Alz50), in wild-type, 3×Tg-AD, and tau knockout mice.

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label.

PubMed

Malakaneh, M; Rasaee, M J; Rahbarizadeh, F; Madani, R; Forozandeh, M M; Khabiri, K; Alimohammadian, M H

2001-04-01

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.

Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Weibin; Chen, Aizhong; Miao, Yi

Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarilymore » targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.« less

The Use of Monoclonal Antibodies in Human Prion Disease

NASA Astrophysics Data System (ADS)

Bodemer, Walter

Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations.

PubMed

Petit Cocault, Laurence; Fleury, Maud; Clay, Denis; Larghero, Jérôme; Vanneaux, Valérie; Souyri, Michèle

2016-04-01

Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Development of a nanogold-based immunochromatographic assay for detection of morphine in urine using the Amor-HK16 monoclonal antibody.

PubMed

Dehghannezhad, Ardeshir; Paknejad, Maliheh; Rasaee, Mohammad Javad; Omidfar, Kobra; Seyyed Ebrahimi, Shadi Sadat; Ghahremani, Hossein

2012-12-01

A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line. Goat anti-mouse antibody was used as a binder in the control line in the detection zone of the strip. Colloidal gold particles of diameter approximately 20 nm were prepared and conjugated to the monoclonal antibody. The detection limit of the test strip was found to be 2000 ng/mL of morphine in urine samples. Reliability was determined by performing the ICG test on 103 urine samples and comparing the results with those obtained by thin-layer chromatography. The sensitivity of the test was 100%, and the analysis time for the assay was approximately 5 min. The new ICG method was adequately sensitive and accurate for the rapid screening of morphine in urine.

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.; Van Emon, Jeanette M.; Bigbee, Carolyn L.

1992-01-01

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.; Van Emon, J.M.; Bigbee, C.L.

1992-04-28

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples. 6 figs.

Characterization and use of new monoclonal antibodies to CD11c,CD14, and CD163 to analyze the phenotypic complexity of ruminantmonocyte subsets

USDA-ARS?s Scientific Manuscript database

The sequencing of the bovine genome and development of mass spectrometry, in conjunction with flow cytometry (FC), have afforded an opportunity to complete the characterization of the specificity of monoclonal antibodies (mAbs), only partially characterized during previous international workshops fo...

Agonistic Human Monoclonal Antibodies against Death Receptor 4 (DR4) | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute is seeking parties interested in licensing human monoclonal antibodies (mAbs) that bind to death receptor 4 ("DR4"). The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been recognized as promising targets for cancer treatment.

Development of an enzyme-linked immunosorbent assay method specific for the detection of G-group aflatoxins

USDA-ARS?s Scientific Manuscript database

To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G1 and G2. Of the final three positive and stable hybridomas obtained, hybridoma 2G6 produced a monoclonal antibody that did not cross-react wi...

Identification of HLA-DP polymorphism with DP alpha and DP beta probes and monoclonal antibodies: correlation with primed lymphocyte typing.

PubMed Central

Bodmer, J; Bodmer, W; Heyes, J; So, A; Tonks, S; Trowsdale, J; Young, J

1987-01-01

Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP beta probes and a DP alpha probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of DPw1, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defined and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies. Images PMID:2885841

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

PubMed

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Dashboard systems: Pharmacokinetic/pharmacodynamic mediated dose optimization for monoclonal antibodies.

PubMed

Mould, Diane R; Dubinsky, Marla C

2015-03-01

Many marketed drugs exhibit high variability in exposure and response. While these drugs are efficacious in their approved indications, finding appropriate dose regimens for individual patients is not straightforward. Similar dose adjustment problems are also seen with drugs that have a complex relationship between exposure and response and/or a narrow therapeutic window. This is particularly true for monoclonal antibodies, where prolonged dosing at a sub-therapeutic dose can also elicit anti-drug antibodies which will further compromise safety and efficacy. Thus, finding appropriate doses quickly would represent a substantial improvement in healthcare. Dashboard systems, which are decision-support tools, offer an improved, convenient means of tailoring treatment for individual patients. This article reviews the clinical need for this approach, particularly with monoclonal antibodies, the design, development, and testing of such systems, and the likely benefits of dashboard systems in clinical practice. We focus on infliximab for reference. © 2015, The American College of Clinical Pharmacology.

Reshaping Human Antibodies: Grafting an Antilysozyme Activity

NASA Astrophysics Data System (ADS)

Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

1988-03-01

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Monoclonal Idiotope Vaccine against Streptococcus pneumoniae Infection

NASA Astrophysics Data System (ADS)

McNamara, Mary K.; Ward, Ronald E.; Kohler, Heinz

1984-12-01

A monoclonal anti-idiotope antibody coupled to a carrier protein was used to immunize BALB/c mice against a lethal Streptococcus pneumoniae infection. Vaccinated mice developed a high titer of antibody to phosphorylcholine, which is known to protect against infection with Streptococcus pneumoniae. Measurement of the median lethal dose of the bacteria indicated that anti-idiotope immunization significantly increased the resistance of BALB/c mice to the bacterial challenge. Antibody to an idiotope can thus be used as an antigen substitute for the induction of protective immunity.

Multiple functions of caprylic acid-induced impurity precipitation for process intensification in monoclonal antibody purification.

PubMed

Trapp, Anja; Faude, Alexander; Hörold, Natalie; Schubert, Sven; Faust, Sabine; Grob, Thilo; Schmidt, Stefan

2018-05-02

New emerging technologies delivering benefits in terms of process robustness and economy are an inevitable prerequisite for monoclonal antibody purification processes intensification. Caprylic acid was proven as an effective precipitating agent enabling efficient precipitaton of product- and process-related impurities while leaving the antibody in solution. This purification step at mild acidic pH was therefore introduced in generic antibody platform approaches after Protein A capture and evaluated for its impact regarding process robustness and antibody stability. Comparison of 13 different monoclonal antibodies showed significant differences in antibody recovery between 65-95% during caprylic acid-induced impurity precipitation. Among six compared physicochemical properties, isoelectric point of the antibody domains was figured out to correlate with yield. Antibodies with mild acidic pI of the light chain were significantly susceptible to caprylic acid-induced precipitation resulting in lower yields. Virus clearance studies revealed that caprylic acid provided complete virus inactivation of an enveloped virus. Multiple process relevant factors such as pH range, caprylic acid concentration and antibody stability were investigated in this study to enable an intensified purification process including caprylic acid precipitation for HCP removal of up to 2 log 10 reduction values at mAb yields >90% while also contributing to the virus safety of the process. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid micromotor-based naked-eye immunoassay.

PubMed

de Ávila, Berta Esteban-Fernández; Zhao, Mingjiao; Campuzano, Susana; Ricci, Francesco; Pingarrón, José M; Mascini, Marcello; Wang, Joseph

2017-05-15

A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H 2 O 2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables "on the move" specific detection of the target cortisol down to 0.1μgmL -1 in just 2min, using ultrasmall (50µL) sample volumes. Copyright © 2017 Elsevier B.V. All rights reserved.

Phase Separation in Solutions of Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

2012-02-01

We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

Immuno Nanosensor for the Ultrasensitive Naked Eye Detection of Tuberculosis.

PubMed

Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Jaafar; Wasoh, Helmi; Md Noor, Siti Suraiya; Ahmad Raston, Nurul Hanun; Mohammad, Faruq

2018-06-14

In the present study, a beneficial approach for the ultrasensitive and affordable naked eye detection and diagnosis of tuberculosis (TB) by utilizing plasmonic enzyme-linked immunosorbent assay (ELISA) via antibody-antigen interaction was studied. Here, the biocatalytic cycle of the intracellular enzymes links to the formation and successive growth of the gold nanoparticles (GNPs) for ultrasensitive detection. The formation of different colored solutions by the plasmonic nanoparticles in the presence of enzyme labels links directly to the existence or non-existence of the TB analytes in the sample solutions. For disease detection, the adapted protocol is based mainly on the conventional ELISA procedure that involves catalase-labeled antibodies, i.e., the enzymes consume hydrogen peroxide and further produce GNPs with the addition of gold (III) chloride. The amount of hydrogen peroxide remaining in the solution determines whether the GNPs solution is to be formed in the color blue or the color red, as it serves as a confirmation for the naked eye detection of TB analytes. However, the conventional ELISA method only shows tonal colors that need a high concentration of analyte to achieve high confidence levels for naked eye detection. Also, in this research, we proposed the incorporation of protein biomarker, Mycobacterium tuberculosis ESAT-6-like protein esxB (CFP-10), as a means of TB detection using plasmonic ELISA. With the use of this technique, the CFP-10 detection limit can be lowered to 0.01 µg/mL by the naked eye. Further, our developed technique was successfully tested and confirmed with sputum samples from patients diagnosed with positive TB, thereby providing enough evidence for the utilization of our technique in the early diagnosis of TB disease.

Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

PubMed Central

Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

1991-01-01

The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

PubMed

Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

1991-11-01

The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients.

Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies.

PubMed

Serroni, Anna; Magistrali, Chiara Francesca; Pezzotti, Giovanni; Bano, Luca; Pellegrini, Martina; Severi, Giulio; Di Pancrazio, Chiara; Luciani, Mirella; Tittarelli, Manuela; Tofani, Silvia; De Giuseppe, Antonio

2017-05-25

Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2 Δ1-25 -His 6 ) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2 Δ1-25 -His 6 was obtained after purification by Ni 2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2 Δ1-25 -His 6 . Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

PubMed

Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh

2017-02-01

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately used for the diagnosis of fascioliasis.

The evaluation of renal ischaemic damage: the value of CD10 monoclonal antibody staining and of biochemical assessments of tissue viability

PubMed Central

Tagboto, S; Griffiths, A Paul

2007-01-01

Background It is well recognised that there is often a disparity between the structural changes observed in the kidney following renal injury and the function of the organ. For this reason, we carried out studies to explore possible means of studying and quantifying the severity of renal ischaemic damage using a laboratory model. Methods To do this, freshly isolated rabbit kidney tissue was subjected to warm (37°C) or cold (1°C) ischaemia for 20 hours. Following this, the tissue was stained using Haematoxylin and Eosin (H+E), Periodic Schiff reagent (PAS) and the novel monoclonal antibody CD10 stain. Additionally, ischaemic damage to the kidneys was assessed by biochemical tests of tissue viability using formazan-based colorimetry. Results CD 10 antibody intensely stained the brush border of control kidney tissue with mild or no cytoplasmic staining. Cell injury was accompanied by a redistribution of CD10 into the lumen and cell cytoplasm. There was good correlation between a score of histological damage using the CD 10 monoclonal antibody stain and the biochemical assessment of viability. Similarly, a score of histological damage using traditional PAS staining correlated well with that using the CD10 antibody stain. In particular, the biochemical assay and the monoclonal antibody staining techniques were able to demonstrate the efficacy of Soltran (this solution is used cold to preserve freshly isolated human kidneys prior to transplantation) in preserving renal tissue at cold temperatures compared to other randomly selected solutions. Conclusion We conclude that the techniques described using the CD10 monoclonal antibody stain may be helpful in the diagnosis and assessment of ischaemic renal damage. In addition, biochemical tests of viability may have an important role in routine histopathological work by giving additional information about cellular viability which may have implications on the function of the organ. PMID:17531101

Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

NASA Technical Reports Server (NTRS)

Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Verification of the Cross Immunoreactivity of A60, a Mouse Monoclonal Antibody against Neuronal Nuclear Protein.

PubMed

Mao, Shanping; Xiong, Guoxiang; Zhang, Lei; Dong, Huimin; Liu, Baohui; Cohen, Noam A; Cohen, Akiva S

2016-01-01

A60, the mouse monoclonal antibody against the neuronal nuclear protein (NeuN), is the most widely used neuronal marker in neuroscience research and neuropathological assays. Previous studies identified fragments of A60-immunoprecipitated protein as Synapsin I (Syn I), suggesting the antibody will demonstrate cross immunoreactivity. However, the likelihood of cross reactivity has never been verified by immunohistochemical techniques. Using our established tissue processing and immunofluorescent staining protocols, we found that A60 consistently labeled mossy fiber terminals in hippocampal area CA3. These A60-positive mossy fiber terminals could also be labeled by Syn I antibody. After treating brain slices with saponin in order to better preserve various membrane and/or vesicular proteins for immunostaining, we observed that A60 could also label additional synapses in various brain areas. Therefore, we used A60 together with a rabbit monoclonal NeuN antibody to confirm the existence of this cross reactivity. We showed that the putative band positive for A60 and Syn I could not be detected by the rabbit anti-NeuN in Western blotting. As efficient as Millipore A60 to recognize neuronal nuclei, the rabbit NeuN antibody demonstrated no labeling of synaptic structures in immunofluorescent staining. The present study successfully verified the cross reactivity present in immunohistochemistry, cautioning that A60 may not be the ideal biomarker to verify neuronal identity due to its cross immunoreactivity. In contrast, the rabbit monoclonal NeuN antibody used in this study may be a better candidate to substitute for A60.

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

PubMed

Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

2016-01-01

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

PubMed Central

Rickert, Keith W.; Grinberg, Luba; Woods, Robert M.; Wilson, Susan; Bowen, Michael A.; Baca, Manuel

2016-01-01

ABSTRACT The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3–5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

Use of Monoclonal Antibodies to Lipopolysaccharide for Antigenic Analysis of Coxiella burnetii

PubMed Central

Hotta, Akitoyo; Kawamura, Midori; To, Ho; Andoh, Masako; Yamaguchi, Tsuyoshi; Fukushi, Hideto; Amano, Ken-Ichi; Hirai, Katsuya

2003-01-01

Antigenic differences among Coxiella burnetii strains were analyzed. The monoclonal antibodies against the lipopolysaccharide outer core did not react with the strains containing a QpRS plasmid or with plasmidless strains, whereas they reacted with strains containing a QpH1 or QpDV plasmid. C. burnetii isolates could be divided into two groups immunologically. PMID:12682176

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

USDA-ARS?s Scientific Manuscript database

A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster

Cancer.gov

A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared

Conserved and Variant Epitopes of Plasmodium vivax Duffy Binding Protein as Targets of Inhibitory Monoclonal Antibodies

PubMed Central

Ntumngia, Francis B.; Schloegel, Jesse; Barnes, Samantha J.; McHenry, Amy M.; Singh, Sanjay; King, Christopher L.

2012-01-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains. PMID:22215740

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies.

PubMed

Ntumngia, Francis B; Schloegel, Jesse; Barnes, Samantha J; McHenry, Amy M; Singh, Sanjay; King, Christopher L; Adams, John H

2012-03-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.

Impact of IgG Fc-Oligosaccharides on Recombinant Monoclonal Antibody Structure, Stability, Safety, and Efficacy.

PubMed

Liu, Hongcheng; Nowak, Christine; Andrien, Bruce; Shao, Mei; Ponniah, Gomathinayagam; Neill, Alyssa

2017-09-01

Glycosylation of the conserved asparagine residue in the CH2 domain is the most common posttranslational modification of recombinant monoclonal antibodies. Ideally, a consistent oligosaccharide profile should be maintained from early clinical material to commercial material for the development of recombinant monoclonal therapeutics, though variation in the profile is a typical result of process changes. The risk of oligosaccharide variation posed to further development is required to be thoroughly evaluated based on its impact on antibody structure, stability, efficacy and safety. The variation should be controlled within a range so that there is no detrimental impact on safety and efficacy and thus allowing the use of early phase safety and efficacy data to support project advancement to later phase. This review article focuses on the current scientific understanding of the commonly observed oligosaccharides found in recombinant monoclonal antibodies and their impact on structure, stability and biological functions, which are the basis to evaluate safety and efficacy. It also provides a brief discussion on critical quality attribute (CQA) assessment with regard to oligosaccharides based on the mechanism of action (MOA). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1173-1181, 2017. © 2017 American Institute of Chemical Engineers.

Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

NASA Astrophysics Data System (ADS)

Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

1993-03-01

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

Development of an enzyme-linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti-glycyrrhizic acid monoclonal antibody.

PubMed

Zhang, Yue; Qu, Huihua; Zeng, Wenhao; Zhao, Yan; Shan, Wenchao; Wang, Xueqian; Wang, Qingguo; Zhao, Yan

2015-07-01

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti-glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line (Sp2/0-Ag14). Subsequently, an indirect, competitive enzyme-linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12-2500 ng/mL. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies.

PubMed

Girkontaite, I; Frischholz, S; Lammi, P; Wagner, K; Swoboda, B; Aigner, T; Von der Mark, K

1996-09-01

For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

Relationship between natural and heme-mediated antibody polyreactivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadzhieva, Maya; Vassilev, Tchavdar; Bayry, Jagadeesh

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivitymore » of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.« less

Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

PubMed

Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

1984-09-01

In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the therapeutic use of IgG1 monoclonal antibodies.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

PubMed Central

2012-01-01

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib. PMID:23234355

Development and characterization of a TAPIR-like mouse monoclonal antibody to amyloid-beta.

PubMed

Wang, Jun; Hara, Hideo; Makifuchi, Takao; Tabira, Takeshi

2008-06-01

Tissue amyloid plaque immuno-reactive (TAPIR) antibody was better related to the effect of immunotherapy in Alzheimer's disease (AD) than ELISA antibody. Here we used a hybridoma technique to develop a TAPIR-like anti-human amyloid-beta (Abeta) mouse monoclonal antibody. The obtained monoclonal antibody, 3.4A10, was an IgG2b isotype and recognized N-terminal portion of Abeta1-42 without binding denatured or native amyloid-beta protein precursor. It had higher affinity to Abeta1-42 than to Abeta1-40 by Biacore affinity analysis and stained preferably the peripheral part of senile plaques and recognized the plaque core less than 4G8. It inhibited the Abeta1-42 fibril formation as well as degraded pre-aggregated Abeta1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. The in vivo studies showed that 3.4A10 treatment decreased amyloid burden compared to the control group and significantly reduced Abeta42 levels rather than Abeta40 levels in brain lysates as well as the Abeta*56 oligomer (12mer) in TBS fraction of the brain lysates. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage. We conclude that 3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody, and has a potential of therapeutic application for AD.

Next generation and biosimilar monoclonal antibodies

PubMed Central

2011-01-01

The Next Generation and Biosimilar Monoclonal Antibodies: Essential Considerations Towards Regulatory Acceptance in Europe workshop, organized by the European Centre of Regulatory Affairs Freiburg (EUCRAF), was held February 3–4, 2011 in Freiburg, Germany. The workshop attracted over 100 attendees from 15 countries, including regulators from 11 agencies, who interacted over the course of two days. The speakers presented their authoritative views on monoclonal antibodies (mAbs) as attractive targets for development, the experience to date with the regulatory process for biosimilar medicinal products, the European Medicines Agency draft guideline on biosimilar mAbs, as well as key elements in the development of mAbs. Participants engaged in many lively discussions, and much speculation on the nature of the quality, non-clinical and clinical requirements for authorization of biosimilar mAbs. PMID:21487235

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

A silicon dioxide modified magnetic nanoparticles-labeled lateral flow strips for HBs antigen.

PubMed

Zhang, Xueqing; Jiang, Lin; Zhang, Chunlei; Li, Ding; Wang, Can; Gao, Feng; Cui, Daxiang

2011-12-01

Herein we reported a new type of silicon dioxide wrapped magnetic nanoparticles-labeled lateral flow strip for detection of HBs antigen in sera. The SiO2 wrapped Fe3O4 nanocomposites were prepared and characterized by HR-TEM, FTIR and magnetometer. As-prepared nanocomposites were used to label anti-HBV surface monoclonal antibody, the lateral flow strips were constructed, and 100 specimens of sera were collected and tested. Results showed that the prepared SiO2 wrapped Fe3O4 nanocomposites were shell/core structure, well dispersed, with the size of 25 nm in diameter, the thickness of the shell was about 3 nm, their magnetic saturation intensity was 44.3 meu g(-1). Clinical sera specimens test results showed that the prepared lateral flow strips were with the detection limitation of 5 pg/mL by naked eye observation, and 0.1 pg/mL by CCD reader or MAR Analyzer, specificity was 100%. In conclusion, one kind of silicon dioxide wrapped magnetic nanoparticles-labeled lateral flow strip for ultrasensitive detection of HBs antigen was successfully developed, its ease of use, sensitiveness and low-cost make it well-suited for population-based on-the-site hepatitis B screening.

Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery

NASA Astrophysics Data System (ADS)

Sen, K. Ilker; Tang, Wilfred H.; Nayak, Shruti; Kil, Yong J.; Bern, Marshall; Ozoglu, Berk; Ueberheide, Beatrix; Davis, Darryl; Becker, Christopher

2017-05-01

Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests.

Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

PubMed Central

Wang, Ying; Lomakin, Aleksey; Latypov, Ramil F.; Benedek, George B.

2011-01-01

We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective interprotein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable proetin condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia. PMID:21921237

Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

PubMed Central

2015-01-01

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called “acidic species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity. PMID:25136741

Production of anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen.

PubMed

Maleki, Leili Aghebati; Shanehbandi, Dariush; Majidi, Jafar; Yusefi, Mehdi; Abdolalizadeh, Jalal; Orangi, Mona; Baradaran, Behzad

2013-01-01

CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14. The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes.

Vectored Intracerebral Immunization with the Anti-Tau Monoclonal Antibody PHF1 Markedly Reduces Tau Pathology in Mutant Tau Transgenic Mice.

PubMed

Liu, Wencheng; Zhao, Lingzhi; Blackman, Brittany; Parmar, Mayur; Wong, Man Ying; Woo, Thomas; Yu, Fangmin; Chiuchiolo, Maria J; Sondhi, Dolan; Kaminsky, Stephen M; Crystal, Ronald G; Paul, Steven M

2016-12-07

Passive immunization with anti-tau monoclonal antibodies has been shown by several laboratories to reduce age-dependent tau pathology and neurodegeneration in mutant tau transgenic mice. These studies have used repeated high weekly doses of various tau antibodies administered systemically for several months and have reported reduced tau pathology of ∼40-50% in various brain regions. Here we show that direct intrahippocampal administration of the adeno-associated virus (AAV)-vectored anti-phospho-tau antibody PHF1 to P301S tau transgenic mice results in high and durable antibody expression, primarily in neurons. Hippocampal antibody levels achieved after AAV delivery were ∼50-fold more than those reported following repeated systemic administration. In contrast to systemic passive immunization, we observed markedly reduced (≥80-90%) hippocampal insoluble pathological tau species and neurofibrillary tangles following a single dose of AAV-vectored PHF1 compared with mice treated with an AAV-IgG control vector. Moreover, the hippocampal atrophy observed in untreated P301S mice was fully rescued by treatment with the AAV-vectored PHF1 antibody. Vectored passive immunotherapy with an anti-tau monoclonal antibody may represent a viable therapeutic strategy for treating or preventing such tauopathies as frontotemporal dementia, progressive supranuclear palsy, or Alzheimer's disease. We have used an adeno-associated viral (AAV) vector to deliver the genes encoding an anti-phospho-tau monoclonal antibody, PHF1, directly to the brain of mice that develop neurodegeneration due to a tau mutation that causes frontotemporal dementia (FTD). When administered systemically, PHF1 has been shown to modestly reduce tau pathology and neurodegeneration. Since such antibodies do not readily cross the blood-brain barrier, we used an AAV vector to deliver antibody directly to the hippocampus and observed much higher antibody levels and a much greater reduction in tau pathology. Using AAV vectors to deliver antibodies like PHF1 directly to brain may constitute a novel approach to treating various neurodegenerative disorders, such as FTD and Alzheimer's disease. Copyright © 2016 the authors 0270-6474/16/3612425-11$15.00/0.

Human antibody technology and the development of antibodies against cytomegalovirus.

PubMed

Ohlin, Mats; Söderberg-Nauclér, Cecilia

2015-10-01

Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Very large scale monoclonal antibody purification: the case for conventional unit operations.

PubMed

Kelley, Brian

2007-01-01

Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using today's commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the world's largest single-plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non-conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.

Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

NASA Astrophysics Data System (ADS)

Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

1993-05-01

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

Naked DNA Immunization for Prevention of Prostate Cancer in a Dunning Rat Prostate Tumor Model

DTIC Science & Technology

2005-06-01

immunized with H PSA-T or H PSMA-T developed antibodies against the target antigen. In contrast, immunization with the "secreted" vaccines, HPSMA-S or...HPSA-S resulted in production of antibodies against the target antigen. The antibodies were of mixed (Thl and Th2) type (IgGl and IgG2a). When priming...was performed with the "truncated" version of the vaccines (H PSMA-T or H PSA-T), however and boosting with the "secreted" ones, the antibodies were

Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

PubMed

Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

2017-12-01

Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

Partners | Office of Cancer Clinical Proteomics Research

Cancer.gov

Developmental Studies Hybridoma Bank at the University of Iowa NCI’s OCCPR works closely with The University of Iowa's Developmental Studies Hybridoma Bank (DSHB) that distributes all hybridomas and monoclonal antibodies from NCI's Clinical Proteomic Technologies for Cancer initiative (CPTC). DSHB supplies researchers with monoclonal antibodies, which may be ordered as tissue culture supernatants, ascites, or concentrate; selected hybridomas are also available as frozen or growing cells.

Production of Murine Monoclonal Antibodies using Traditional and Novel Technology

DTIC Science & Technology

2010-03-01

1 17. LIMITATION OF ABSTRACT UL 18. NUMBER OF PAGES 17 19a. NAME OF RESPONSIBLE PERSON Sandra J . Johnson 19b. TELEPHONE NUMBER (include...users should direct such requests to the National Technical Information Service. Acknowledgments The author would like to acknowledge Dr. Bonnie J ...Monoclonal Antibodies: Principles and Practice; Academic Press: London, 1996. Goyache, Joaquin; Orden, Jose A.; Blanco , Jose L.; Hernandez , Javier

Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.

PubMed Central

Brûlet, P; Babinet, C; Kemler, R; Jacob, F

1980-01-01

Two-dimensional gel electrophoresis has allowed the detection of proteins characteristic of inner cell mass and trophectoderm in mouse blastocyst. Certain of the proteins characterizing trophectoderm copurify with intermediate filaments from trophectoderm and a trophoblastoma cell line. A monoclonal antibody prepared against proteins of these intermediate filaments labels a filament network in trophectoderm but not in inner cell mass cells. Images PMID:6933460

Comparative analysis of monoclonal antibody N-glycosylation using stable isotope labelling and UPLC-fluorescence-MS.

PubMed

Millán Martín, Silvia; Delporte, Cédric; Farrell, Amy; Navas Iglesias, Natalia; McLoughlin, Niaobh; Bones, Jonathan

2015-03-07

A twoplex method using (12)C6 and (13)C6 stable isotope analogues (Δmass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection.

Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

USDA-ARS?s Scientific Manuscript database

Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopus japonicus. In this study, four monoclonal antibodies (MAbs) (3C1, 3D9, 2F2, 2A8) against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no ...

The combination of anti-KIR monoclonal antibodies with anti-PD-1/PD-L1 monoclonal antibodies could be a critical breakthrough in overcoming tumor immune escape in NSCLC.

PubMed

He, Yayi; Liu, Sangtian; Mattei, Jane; Bunn, Paul A; Zhou, Caicun; Chan, Daniel

2018-01-01

The anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) monoclonal antibody has a good effect in the treatment of non-small cell lung cancer (NSCLC), but not all PD-1/PD-L1 positive patients can get benefit from it. Compensatory expression of other immune checkpoints may be correlated with the poor efficacy of anti-PD-1/PD-L1 monoclonal antibodies. The inhibitory human leukocyte antigen (HLA)/killer cell Ig-like receptor (KIR) can effectively block the killing effect of natural killer (NK) cells on tumors. Our previous studies have confirmed that high expression of KIR was correlated with poor prognosis of NSCLC. Inhibitory KIR expression was positively correlated with the expression of PD-1. The expressions of KIR 2D (L1, L3, L4, S4) (BC032422/ADQ31987/NP_002246/NP_036446, Abcam) and PD-1 (NAT 105, Cell marque) proteins was assessed by immunohistochemistry. The expression of inhibitory KIR in tumor cells or tumor infiltrating lymphocytes (TILs) is associated with PD-1 expression. Among PD-1 positive patients, 76.3% were KIR 2D (L1, L3, L4, S4) positive on tumor cells, and 74.6% were KIR 2D (L1, L3, L4, S4) positive on TILs. We compared the expression of inhibitory KIR before and after treatment with nivolumab in 11 patients with NSCLC. We found that five (45.5%) patients had positive expression of inhibitory KIR in tumor tissue after being treated with anti-PD-1 monoclonal antibodies, two of whom exhibited a significant increase in expression of inhibitory KIR, and three showed no change. PD-1 expression was correlated with KIR 2D (L1, L3, L4, S4) on tumor cells or TILs. The resistance to anti-PD-1 monoclonal antibody treatment might be related to KIR. The inhibitory HLA/KIR could combine with the PD-1/PD-L1 signaling pathway negatively regulating NSCLC tumor immunity.

Recovery and purification process development for monoclonal antibody production

PubMed Central

Ma, Junfen; Winter, Charles; Bayer, Robert

2010-01-01

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

PubMed

Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

1988-07-01

The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested.

Immune Checkpoint PD-1/PD-L1: Is There Life Beyond Antibodies?

PubMed

Konstantinidou, Markella; Zarganes-Tzitzikas, Tryfon; Magiera-Mularz, Katarzyna; Holak, Tad A; Dömling, Alexander

2018-04-23

The PD-1/PD-L1 interaction has emerged as a significant target in cancer immunotherapy. Current medications include monoclonal antibodies, which have shown impressive clinical results in the treatment of several types of tumors. The cocrystal structure of human PD-1 and PD-L1 is expected to be a valuable starting point for the design of novel inhibitors, along with the recent crystal structures with monoclonal antibodies, small molecules, and macrocycles. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of lymphopoietic stem cells with a monoclonal antibody to the rat transferrin receptor.

PubMed Central

Jefferies, W A; Brandon, M R; Williams, A F; Hunt, S V

1985-01-01

A mouse monoclonal IgG2a antibody, designated MRC OX-26, is shown to be specific for the rat transferrin receptor, but does not block transferrin binding. The antibody labelled a myeloma, three leukaemia cell lines and normal dividing cells of various types, but also bound to a number of nondividing normal tissues. No labelling of lymphopoietic stem cells could be detected, even though approximately 25% of bone marrow and over 95% of fetal liver cells were clearly labelled. Images Figure 1 Figure 3 PMID:2981766

[Batch release of immunoglobulin and monoclonal antibody products].

PubMed

Gross, S

2014-10-01

The Paul-Ehrlich Institute (PEI) is an independent institution of the Federal Republic of Germany responsible for performing official experimental batch testing of sera. The institute decides about the release of each batch and performs experimental research in the field. The experimental quality control ensures the potency of the product and also the absence of harmful impurities. For release of an immunoglobulin batch the marketing authorization holder has to submit the documentation of the manufacture and the results of quality control measures together with samples of the batch to the PEI. Experimental testing is performed according to the approved specifications regarding the efficacy and safety. Since implementation of the 15th German drug law amendment, the source of antibody is not defined anymore. According to § 32 German drug law, all batches of sera need to be released by an official control laboratory. Sera are medicinal products, which contain antibodies, antibody fragments or fusion proteins with a functional antibody portion. Therefore, all batches of monoclonal antibodies and derivatives must also be released by the PEI and the marketing authorization holder has to submit a batch release application. Under certain circumstances a waiver for certain products can be issued with regard to batch release. The conditions for such a waiver apply to the majority of monoclonal antibodies.

Radioimmunotherapy of nude mice with intraperitoneally growing ovarian cancer xenograft utilizing 211At-labelled monoclonal antibody MOv18.

PubMed

Andersson, H; Lindegren, S; Back, T; Jacobsson, L; Leser, G; Horvath, G

2000-01-01

The aim of this study was to investigate the therapeutic efficacy of 211At-labelled monoclonal antibody given intraperitoneally to nude mice with intraperitoneal growth of a human ovarian cancer cell line. Female nude mice were inoculated intraperitoneally with 1 x 10(7) cells of the human ovarian cancer cell line NIH:OVCAR 3. After about two weeks they were injected with the 211At-labelled specific monoclonal antibody MOv18 intraperitoneally. For comparison, other groups of mice were given the same labelled antibody intravenously, 211At-labelled unspecific antibody C242 intraperitoneally or unalbelled MOv18 intraperitoneally. Six weeks later the animals were sacrificed and the occurrence of tumour and ascites was determined. When the mice were treated with 211At-labelled MOv18 intraperitoneally 9 out of 10 were apparently free of both ascites and tumour compared to none of the mice given unlabelled antibody. 211At-labelled MOv18 given intravenously or 211At-labelled unspecific antibody given intraperitoneally were less effective. Regional radioimmunotherapy with the alpa-emitter 211Astatine seems to be an effective treatment of nude mice with intraperitoneally growing human ovarian cancer. Hopefully this treatment can be given in an adjuvant setting to women with minimal residual ovarian cancer in the future.

Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

PubMed

Patel, Rekha; Andrien, Bruce A

2010-01-01

Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.

Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

PubMed

Geylis, Valeria; Steinitz, Michael

2006-01-01

The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen.

PubMed

Caldas, Cristina; Coelho, Verônica; Kalil, Jorge; Moro, Ana Maria; Maranhão, Andrea Q; Brígido, Marcelo M

2003-05-01

Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

PubMed

Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

2014-08-01

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of koi herpesvirus (KHV) using a monoclonal antibody against Cyprinus carpio IgM.

PubMed

Li, Yingying; Zheng, Shucheng; Wang, Qing; Bergmann, Sven M; Zeng, Weiwei; Wang, Yingying; Liu, Chun; Shi, Cunbin

2017-08-01

Koi herpesvirus disease (KHVD) is associated with high mortality in both common carp and koi carp (Cyprinus carpio L.) worldwide. The indirect detection of fish viruses based on the identification of antibodies has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against carp IgM. By using hybridoma-monoclonal antibody technology, one hybridoma cell line secreting MAbs against IgM from carp was established. In western blot analysis, the secreted MAb from cell line A5-E10 recognized the heavy chain of IgM from common carp or koi but did not react with immunoglobulins from three different fish species: grass carp (Ctenopharyngodon idella), tilapia (Oreochromis mossambicus) and Mandarin fish (Siniperca chuatsi). These results demonstrated that this MAb is highly specific for the IgM of carp and suggested that it can be used for monitoring the immunity level of carp, for example for indirect KHV diagnosis by antibody ELISA. We therefore established an indirect ELISA, which was tested using 200 serum samples from koi from three farms. The final results showed that 147 (73.5%) samples were confirmed to be KHV antibody negative and 53 (26.5%) were definitely positive, containing antibodies against KHV.

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

PubMed

Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

2013-11-13

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Specific antibody for pesticide residue determination produced by antibody-pesticide complex

USDA-ARS?s Scientific Manuscript database

A new method for specific antibody production was developed using antibody (Ab)-pesticide complex as a unique immunogen. Parathion (PA) was the targeted pesticide, and rabbit polyclonal antibody (Pab) and mouse monoclonal antibody (Mab) were used as carrier proteins. The Ab-PA complexes were genera...

Staphylococcal enterotoxins bind H-2Db molecules on macrophages

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1995-01-01

We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

Characterization of a monoclonal antibody against P57, the C3/C3b-cleaving proteinase expressed in human erythrocyte membranes.

PubMed

Fiandino-Tirel, A; Barel, M; Lyamani, F; Gauffre, A; Hermann, J; Frade, R

1991-08-01

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection.

PubMed

Mauko, Lea; Nordborg, Anna; Hutchinson, Joseph P; Lacher, Nathan A; Hilder, Emily F; Haddad, Paul R

2011-01-15

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) column coupled with electrospray ionization mass spectrometry (ESI-MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC-HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC-HILIC column and ESI-MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC-HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies. Copyright © 2010 Elsevier Inc. All rights reserved.

Label-free detection and characterization of the binding of hemagglutinin protein and broadly neutralizing monoclonal antibodies using terahertz spectroscopy

NASA Astrophysics Data System (ADS)

Sun, Yiwen; Zhong, Junlan; Zhang, Cunlin; Zuo, Jian; Pickwell-MacPherson, Emma

2015-03-01

Hemagglutinin (HA) is the main surface glycoprotein of the influenza A virus. The H9N2 subtype influenza A virus is recognized as the most possible pandemic strain as it has crossed the species barrier, infecting swine and humans. We use terahertz spectroscopy to study the hydration shell formation around H9 subtype influenza A virus's HA protein (H9 HA) as well as the detection of antigen binding of H9 HA with the broadly neutralizing monoclonal antibody. We observe a remarkable concentration dependent nonlinear response of the H9 HA, which reveals the formation process of the hydration shell around H9 HA molecules. Furthermore, we show that terahertz dielectric properties of the H9 HA are strongly affected by the presence of the monoclonal antibody F10 and that the terahertz dielectric loss tangent can be used to detect the antibody binding at lower concentrations than the standard ELISA test.

Monoclonal antibody binding to the macrophage-specific receptor sialoadhesin alters the phagocytic properties of human and mouse macrophages.

PubMed

De Schryver, Marjorie; Cappoen, Davie; Elewaut, Dirk; Nauwynck, Hans J; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

2017-02-01

Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab') 2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background. Copyright © 2016 Elsevier Inc. All rights reserved.

Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

PubMed

Caballero, Armando R; Foletti, Davide L; Bierdeman, Michael A; Tang, Aihua; Arana, Angela M; Hasa-Moreno, Adela; Sangalang, Emma Ruth B; O'Callaghan, Richard J

2015-08-01

To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis.

PubMed

King, Cory; Patel, Rekha; Ponniah, Gomathinayagam; Nowak, Christine; Neill, Alyssa; Gu, Zhenyu; Liu, Hongcheng

2018-05-15

In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen. Copyright © 2018 Elsevier B.V. All rights reserved.

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

PubMed Central

Auerbach, Marcy R.; Yan, Donghong; Vij, Rajesh; Hongo, Jo-Anne; Nakamura, Gerald; Vernes, Jean-Michel; Meng, Y. Gloria; Lein, Samantha; Chan, Pamela; Ross, Jed; Carano, Richard; Deng, Rong; Lewin-Koh, Nicholas; Xu, Min; Feierbach, Becket

2014-01-01

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans. PMID:24722349

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques.

PubMed

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A; Veazey, Ronald S

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross-react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define "memory" T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in approximately 44% of rhesus macaques (Macaca mulatta) of Indian but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques.

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques

PubMed Central

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A.; Veazey, Ronald S.

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define “memory” T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in ~44% of rhesus macaques (Macaca mulatta) of Indian, but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques. PMID:18304631

Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody.

PubMed

Sai, Jiayang; Zhao, Yan; Shan, Wenchao; Qu, Baoping; Zhang, Yue; Cheng, Jinjun; Qu, Huihua; Wang, Qingguo

2016-03-01

This work developed a novel immunochemical approach for the quality control of saikosaponin d using an enzyme-linked immunosorbent assay. Splenocytes from mice immunized with the saikosaponin d-bovine serum albumin conjugate were fused with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line, and a hybridoma secreting monoclonal antibody against saikosaponin d was successfully obtained. The prepared anti-saikosaponin d monoclonal antibody 1E7F3 has a novel characteristic, showing weak reactivity with compounds that are structurally related to saikosaponin d. Using monoclonal antibody 1E7F3, a specific and reliable enzyme-linked immunosorbent assay was developed to detect saikosaponin d. The system shows a full measurement range from 156.25 to 5000.00 ng × mL(-1). Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10.00%. The recovery rates ranged from 92.36% to 101.00%, meeting the requirements for biological samples. There was a good correlation between the enzyme-linked immunosorbent assay and high-performance liquid chromatography analyses of saikosaponin d, and the saikosaponin d levels in formulated Chinese medicines were successfully determined. Furthermore, immunoaffinity column chromatography was established using this anti-saikosaponin d monoclonal antibody, and the elution profile of saikosaponin d was detected by a Bio-Rad QuadTec UV/Vis detector at 203 nm. The results demonstrate that we generated a reliable and more efficient assay system for measuring saikosaponin d and provide a potential approach for purifying and separating saikosaponin d. Georg Thieme Verlag KG Stuttgart · New York.

Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein

PubMed Central

Kearney, Moira R.; Siddique, Abdullah; Ali, Ibne K.; Gilchrist, Carol A.; Arju, Tuhinur; Hoffstrom, Benjamin; Nguyen, Felicia K.; Petri, William A.; Haque, Rashidul; Cangelosi, Gerard A.

2016-01-01

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection. PMID:27152855

Monoclonal antibodies reactive with secreted-excreted products from the amphids and the cuticle surface of Globodera pallida affect nematode movement and delay invasion of potato roots.

PubMed

Fioretti, L; Porter, A; Haydock, P J; Curtis, R

2002-12-19

This paper describes Excreted-secreted proteins (ES) proteins that were immunolocalised in the cuticle, amphids and subventral glands of second stage juveniles of the two species of potato cyst nematodes (Globodera pallida and Globodera rostochiensis). Monoclonal antibodies reactive with these ES proteins were used in a bioassay to investigate their effect on nematode movement and on their ability to invade potato roots. Antibodies recognising the nematode cuticle surface and the amphids affected nematode movement and delayed nematode penetration of roots. These effects were temporary, since the nematodes were able to recover and infect potato roots. Movement of second stage juveniles treated with the antibodies was impaired for the first 30 min after inoculation: the juveniles remained close to the point of introduction and moved slowly and abnormally. They recovered normal movement after 1-2 h, possibly because the turnover rate of the secreted proteins meant that they were no longer blocked by the monoclonal antibodies. No effect was observed on second stage juveniles treated with an antibody reactive with secretions from the oesophageal glands. Nematodes treated with antibodies reactive with the nematode cuticle surface were notably more affected than those treated with other antibodies; nematodes failed to recover movement when in continuous contact with the antibodies. It is possible that the physical presence of the antibodies on the nematode surface affected their motility. Nematodes treated with antibodies reactive with secretions from the amphids were temporarily unable to move towards potato roots and their exploratory behaviour was greatly affected by the antibody treatment. Whether these antibodies were able to inhibit temporarily the function of the amphids or this effect was due to physical presence of the antibodies blocking the amphidial pore remains to be determined.

[Monoclonal antibodies in diagnosis of acute leukemias].

PubMed

Krawczyńska, A; Robak, T

1996-01-01

Immunophenotyping has become an essential component for the study of acute myeloblastic (AML) and lymphoblastic (ALL) leukaemias. The recent development of highly specific monoclonal antibodies (Mc Ab) to differentiation antigens (CD) of haematopoetic cells have made it readily available to clinical laboratories in most major hospitals. Immunophenotyping complements standard morphology by providing information on lineage, stage of differentiation and clonality. In addition some of the flow cytometry findings have independent prognostic significance. Monoclonal antibodies useful in defining lineage (B-cell versus T-cell) and stages of differentiation of ALL. It can be also used in identifying characteristic feature of AML and aiding in lineage determination in acute leukaemias that are morphologically undifferentiated. Surface immunophenotyping is especially helpful for recognizing mixed lineage acute leukaemia and diagnosing certain rare entities such as erythroleukaemia (M6), acute megakaryocytic leukaemia (M7) and minimally differentiation acute myeloid leukaemia.

Radiolabeled monoclonal antibodies (for the radiology administrator).

PubMed

Wahl, R

1992-11-01

Cheaper, faster, safer, these are not the attributes of 1993 automobiles, but criteria for new diagnostic tests in medicine. To achieve these characteristics, medicine is increasingly looking to biotechnology for answers. And the mother of all biotechnology is monoclonal antibody research. In past issues, Administrative Radiology published articles discussing the role of biotechnology in the development of radiopharmaceuticals used in nuclear medicine. In this issue, Richard Wahl, M.D., reviews, in plain talk, the current status and prospects for diagnostic imaging with radiolabeled monoclonal antibodies. Are there any such procedures of value today? Are there any that are FDA approved? Will there ever be such agents that are either useful or approved? If so, will any insurance carrier pay for them? For the answers to these and other "hot" questions, the reader is encouraged to continue on and read this month's Technology Review section.

Surface plasmon resonance biosensing: Approaches for screening and characterising antibodies for food diagnostics.

PubMed

Yakes, B J; Buijs, J; Elliott, C T; Campbell, K

2016-08-15

Research in biosensing approaches as alternative techniques for food diagnostics for the detection of chemical contaminants and foodborne pathogens has increased over the last twenty years. The key component of such tests is the biorecognition element whereby polyclonal or monoclonal antibodies still dominate the market. Traditionally the screening of sera or cell culture media for the selection of polyclonal or monoclonal candidate antibodies respectively has been performed by enzyme immunoassays. For niche toxin compounds, enzyme immunoassays can be expensive and/or prohibitive methodologies for antibody production due to limitations in toxin supply for conjugate production. Automated, self-regenerating, chip-based biosensors proven in food diagnostics may be utilised as rapid screening tools for antibody candidate selection. This work describes the use of both single channel and multi-channel surface plasmon resonance (SPR) biosensors for the selection and characterisation of antibodies, and their evaluation in shellfish tissue as standard techniques for the detection of domoic acid, as a model toxin compound. The key advantages in the use of these biosensor techniques for screening hybridomas in monoclonal antibody production were the real time observation of molecular interaction and rapid turnaround time in analysis compared to enzyme immunoassays. The multichannel prototype instrument was superior with 96 analyses completed in 2h compared to 12h for the single channel and over 24h for the ELISA immunoassay. Antibodies of high sensitivity, IC50's ranging from 4.8 to 6.9ng/mL for monoclonal and 2.3-6.0ng/mL for polyclonal, for the detection of domoic acid in a 1min analysis time were selected. Although there is a progression for biosensor technology towards low cost, multiplexed portable diagnostics for the food industry, there remains a place for laboratory-based SPR instrumentation for antibody development for food diagnostics as shown herein. Copyright © 2016 Elsevier B.V. All rights reserved.

A gold nanoparticle-based semi-quantitative and quantitative ultrasensitive paper sensor for the detection of twenty mycotoxins

NASA Astrophysics Data System (ADS)

Kong, Dezhao; Liu, Liqiang; Song, Shanshan; Suryoprabowo, Steven; Li, Aike; Kuang, Hua; Wang, Libing; Xu, Chuanlai

2016-02-01

A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan reader, with the calculated limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.04-0.17, 0.06-49, 0.15-0.22, 0.056-0.49 and 0.53-1.05 μg kg-1, respectively. The analytical results of spiked samples were in accordance with the accurate content in the simultaneous detection analysis. This newly developed ICA strip assay is suitable for the on-site detection and rapid initial screening of mycotoxins in cereal samples, facilitating both semi-quantitative and quantitative determination.A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan reader, with the calculated limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.04-0.17, 0.06-49, 0.15-0.22, 0.056-0.49 and 0.53-1.05 μg kg-1, respectively. The analytical results of spiked samples were in accordance with the accurate content in the simultaneous detection analysis. This newly developed ICA strip assay is suitable for the on-site detection and rapid initial screening of mycotoxins in cereal samples, facilitating both semi-quantitative and quantitative determination. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09171c

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.

PubMed

Peng, Yanfen; Gelder, Victor Van; Amaladoss, Anburaj; Patel, Kadamb Haribhai

2016-10-21

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

PubMed

Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

2015-05-01

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0.05) decreased relative to the amount of PA remained in the solution after passing through unmodified as well as protein A modified poly(AAm-AGE) cryogel columns, indicates efficient PA removal from spiked PBS over 60 min of circulation. The high adsorption capacity towards anthrax toxin PA of the cryogel adsorbents indicated potential application of these materials for treatment of Bacillus anthracis infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Art of Making Antibodies.

ERIC Educational Resources Information Center

Headon, Denis R.

1986-01-01

Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)

Immunochemical detection of food-derived isothiocyanate as a lysine conjugate.

PubMed

Nakamura, Toshiyuki; Kitamoto, Noritoshi; Osawa, Toshihiko; Kato, Yoji

2010-01-01

In a previous study we prepared monoclonal antibody against allyl isothiocyanate (AITC)-modified lysine (Lys), and found that AITC reacted with Lys under physiological conditions in vitro (T. Nakamura et al., Chem. Res. Toxicol., 22, 536-542 (2009)). In the present study, antibodies against benzyl isothiocyanate (ITC), 6-methylsulfinylhexyl ITC and phenethyl ITC modified protein were prepared, and the respective monoclonal antibodies, B6C9, 6MS3D10, and PE3A10 were obtained. These antibodies were applied to ITC detection in food using shredded Wasabia japonica (wasabi) and ground Carica papaya (papaya) seed by trapping ITC with biotin-labeled bovine serum albumin. ITC formation from the wasabi and papaya seed samples was confirmed using the antibodies in a dose-dependent manner. These antibodies might be applicable in identifying food-derived ITC.

Progress with the use of monoclonal antibodies for the treatment of systemic lupus erythematosus.

PubMed

Jordan, Natasha; Lutalo, Pamela Mk; D'Cruz, David P

2015-01-01

In recent years, significant progress has been made in the use of monoclonal antibodies in the treatment of systemic lupus erythematosus (SLE). Advances in our understanding of the complexity of SLE immunopathogenesis have led to the testing of several biologic agents in clinical trials. Monoclonal therapies currently emerging or under development include B-cell depletion therapies, agents targeting B-cell survival factors, blockade of T-cell co-stimulation and anticytokine therapies. Issues remain, however, regarding clinical trial design and outcome measures in SLE which need to be addressed to optimize translation of these promising therapies into clinical practice.

Comparison of carbohydrate and peptide biotinylation on the immunological activity of IgG1 murine monoclonal antibodies.

PubMed

Miralles, F; Takeda, Y; Escribano, M J

1991-07-05

When the classical amino acid esterification procedure was used for the biotinylation of the IgG1 monoclonal antibody J28 it resulted in a loss of immunological activity. This antibody recognizes the fetoacinar pancreatic (FAP) antigen and the decrease in reactivity was directly proportional to the molar biotin/antibody ratio indicating substitutions at or near the antibody combining site. This effect was specific to J28 since the IgG1 Mab F22 which recognises the same antigen was not damaged by this procedure. Active Mab J28 conjugates were obtained using biotinylation via oligosaccharide moieties. The biotinylation efficiency using this method was dependent on the previous degree of antibody periodate oxidation and the maximal substitution was 3 mol biotin per mol of antibody. Using these conditions the sensitivity of the biotinylated J28 for the FAP antigen was similar to that obtained when using non-substituted antibody in the two antibodies technique.

Mechanisms of Action of Therapeutic Antibodies for Cancer

PubMed Central

Redman, JM; Hill, EM; AlDeghaither, D; Weiner, LM

2015-01-01

The therapeutic utility of antibodies and their derivatives is achieved by various means. The FDA has approved several targeted antibodies that disrupt signaling of various growth factor receptors for the treatment of a number of cancers. Rituximab, and other anti-CD20 monoclonal antibodies are active in B cell malignancies. As more experience has been gained with anti-CD20 monoclonal antibodies, the multifactorial nature of their anti-tumor mechanisms has emerged. Other targeted antibodies function to dampen inhibitory checkpoints. These checkpoint inhibitors have recently achieved dramatic results in several cancers, including melanoma. These and related antibodies continue to be investigated in the clinical and pre-clinical settings. Novel antibody structures that target two or more antigens have also made their way into clinical use. Tumor targeted antibodies can also be conjugated to chemo- or radiotherapeutic agents, or catalytic toxins, as a means to deliver toxic payloads to cancer cells. Here we provide a review of these mechanisms and a discussion of their relevance to current and future clinical applications. PMID:25911943

Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

1988-01-01

eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

NASA Technical Reports Server (NTRS)

Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

1990-01-01

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

Mechanistic Investigation on Grinding-Induced Subvisible Particle Formation during Mixing and Filling of Monoclonal Antibody Formulations.

PubMed

Gikanga, Benson; Hui, Ada; Maa, Yuh-Fun

2018-01-01

Processing equipment involving grinding of two solid surfaces has been demonstrated to induce subvisible particle formation in monoclonal antibody drug product manufacturing processes. This study elucidated potential stress types associated with grinding action to identify the stress mechanism responsible for subvisible particle formation. Several potential stress types can be associated with the grinding action, including interfacial stresses (air-liquid and liquid-solid), hydraulic/mechanical shear stress, cavitation, nucleation of stressed protein molecules, and localized thermal stress. More than one stress type can synergically affect monoclonal antibody product quality, making it challenging to determine the primary mode of stress. Our strategy was to assess and rule out some stress types through platform knowledge, rational judgments, or via small-scale models, for example, rheometer/rotator-stator homogenizer for hydraulic/mechanical shear stress, sonicator for cavitation, etc. These models may not provide direct evidence but can offer rational correlations. Cavitation, as demonstrated by sonication, proved to be quite detrimental to monoclonal antibody molecules in forming not just subvisible particles but also soluble high-molecular-weight species as well as low-molecular-weight species. This outcome was not consistent with that of grinding monoclonal antibodies between the impeller and the drive unit of a bottom-mounted mixer or between the piston and the housing of a rotary piston pump, both of which formed only subvisible particles without obvious high-molecular-weight species and low-molecular-weight species. In addition, a p -nitrophenol model suggested that cavitation in the bottom-mounted mixer is barely detectable. We attributed the grinding-induced, localized thermal effect to be the primary stress to subvisible particle formation based on a high-temperature, spray-drying model. The heat effect of spray drying also caused subvisible particles, in the absence of significant high-molecular-weight species and low-molecular-weight species, in spray-dried monoclonal antibody powders. This investigation provides a mechanistic understanding of the underlying stress mechanism leading to monoclonal antibody subvisible particle formation as the result of drug product processing involving grinding of solid surfaces. LAY ABSTRACT: Subvisible particles present in therapeutic protein formulations could adversely affect drug product safety and efficacy. We previously illustrated that grinding action of the solid surfaces in some bottom-mounted mixers and piston pump is responsible for subvisible particle formation of monoclonal antibody formulations. In this study, we delved into mechanistic understanding of the stress types associated with solid surface grinding. The approach was to employ several scale-down stress models with known stress types. Protein formulations stressed in these models were analytically characterized for subvisible particles and other degradants. Some commonly known stress types-such as air-liquid interface, mechanical stress, cavitation, nucleation, and thermal effect-were assessed in this study. The stress model yielding a degradation profile matching that of bottom-mounted mixers and piston pump warranted further assessment. Localized, thermal stress proved to be the most feasible mechanism. This study, along with previously published results, may further advance our understanding of these particular drug product manufacturing processes and benefit scientists and engineers in overcoming these development challenges. © PDA, Inc. 2018.

Noninvasive in vivo multispectral optoacoustic imaging of apoptosis in triple negative breast cancer using indocyanine green conjugated phosphatidylserine monoclonal antibody

NASA Astrophysics Data System (ADS)

Kannadorai, Ravi Kumar; Udumala, Sunil Kumar; Sidney, Yu Wing Kwong

2016-12-01

Noninvasive and nonradioactive imaging modality to track and image apoptosis during chemotherapy of triple negative breast cancer is much needed for an effective treatment plan. Phosphatidylserine (PS) is a biomarker transiently exposed on the outer surface of the cells during apoptosis. Its externalization occurs within a few hours of an apoptotic stimulus by a chemotherapy drug and leads to presentation of millions of phospholipid molecules per apoptotic cell on the cell surface. This makes PS an abundant and accessible target for apoptosis imaging. In the current work, we show that PS monoclonal antibody tagged with indocyanine green (ICG) can help to track and image apoptosis using multispectral optoacoustic tomography in vivo. When compared to saline control, the doxorubicin treated group showed a significant increase in uptake of ICG-PS monoclonal antibody in triple negative breast tumor xenografted in NCr nude female mice. Day 5 posttreatment had the highest optoacoustic signal in the tumor region, indicating maximum apoptosis and the tumor subsequently shrank. Since multispectral optoacoustic imaging does not involve the use of radioactivity, the longer the circulatory time of the PS antibody can be exploited to monitor apoptosis over a period of time without multiple injections of commonly used imaging probes such as Tc-99m Annexin V or F-18 ML10. The proposed apoptosis imaging technique involving multispectral optoacoustic tomography, monoclonal antibody, and near-infrared absorbing fluorescent marker can be an effective tool for imaging apoptosis and treatment planning.

The effect of monoclonal antibodies to the human transferrin receptor on transferrin and iron uptake by rat and rabbit reticulocytes.

PubMed

McArdle, H J; Morgan, E H

1984-02-10

The effect of monoclonal antibodies to the human transferrin receptor on transferrin and iron uptake by rat and rabbit reticulocytes has been examined. The antibodies used were as follows: T58/1.4, B3/25.4, 42/6.3, T56/14.3.1, and 43/31. The effects were the same, irrespective of the antibody. Transferrin and iron uptake were stimulated in both rat and rabbit reticulocytes. The stimulation was not due to an increase in the number or affinity of the receptors, but rather to an increase in the rate of turnover of the receptors. Electron microscopy suggested that the antibody acted by facilitating the formation of coated pits containing the transferrin-receptor complex.

Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

PubMed Central

Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

1988-01-01

The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested. PMID:3262464

Analysis of the antigen recognition sites of anti-methamphetamine monoclonal antibodies (II): unique feature of MA-3 antibody.

PubMed

Ishimaru, M; Morikawa, K; Hifumi, E; Itoh, T; Uda, T

2000-01-01

A monoclonal antibody against methamphetamine (MA-3 mAb) was found to be strongly bound to ephedrine. This feature was quite different from that of other fourteen mAbs against MA. Analyses of cDNA sequence and steric conformation by molecular modeling revealed that one hydrophilic pocket was generated in the heavy chain of MA-3 mAb involving CDRH-1 and CDRH-2. Asn33, Asn35, Asn50 and Asp52 were the main components of the unique pocket capable of binding to the hydroxyl group of ephedrine.

Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys.

PubMed

Desallais, Lucille; Bouchez, Caroline; Mouhsine, Hadley; Moreau, Gabriel; Ratsimandresy, Rojo; Montes, Matthieu; Do, Hervé; Quintin-Colonna, Françoise; Zagury, Jean-François

2016-01-19

Interleukin-6 (IL-6) overproduction has been involved in the pathogenesis of several chronic inflammatory diseases and the administration of an anti-IL-6 receptor monoclonal antibody has been proven clinically efficient to treat them. However, the drawbacks of monoclonal antibodies have led our group to develop an innovative anti-IL-6 strategy using a peptide-based active immunization. This approach has previously shown its efficacy in a mouse model of systemic sclerosis. Here the safety, immunogenicity, and efficacy of this strategy was assessed in non human primates. No unscheduled death and clinical signs of toxicity was observed during the study. Furthermore, the cynomolgus monkeys immunized against the IL-6 peptide produced high levels of anti-IL-6 antibodies as well as neutralizing antibodies compared to control groups. They also showed an important decrease of the cumulative inflammatory score following a delayed-type hypersensitivity reaction induced by the Tetanus vaccine compared to control groups (minus 57,9%, P = 0.014). These findings are highly significant because the immunizing IL-6 peptide used in this study is identical in humans and in monkeys and this novel anti-IL-6 strategy could thus represent a promising alternative to monoclonal antibodies.

Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumeta, Masahiro, E-mail: kumeta@lif.kyoto-u.ac.jp; Hirai, Yuya; Yoshimura, Shige H.

2013-12-10

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do notmore » take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.« less

Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis.

PubMed

De Benedictis, Paola; Minola, Andrea; Rota Nodari, Elena; Aiello, Roberta; Zecchin, Barbara; Salomoni, Angela; Foglierini, Mathilde; Agatic, Gloria; Vanzetta, Fabrizia; Lavenir, Rachel; Lepelletier, Anthony; Bentley, Emma; Weiss, Robin; Cattoli, Giovanni; Capua, Ilaria; Sallusto, Federica; Wright, Edward; Lanzavecchia, Antonio; Bourhy, Hervé; Corti, Davide

2016-04-01

Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20-RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. © 2016 Humabs BioMed SA Published under the terms of the CC BY 4.0 license.

Characterization of two anti-dengue human monoclonal antibodies prepared from PBMCs of patients with dengue illness in Thailand.

PubMed

Li, Z-Y; Yamashita, A; Kawashita, N; Sasaki, T; Pan, Y; Ono, K-I; Ikuta, K; Li, Y-G

2016-06-01

The global spread of the four dengue virus (DENV) serotypes (dengue-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, two monoclonal antibodies prepared by using peripheral blood mononuclear cells (PBMCs) from patients with dengue fever were characterized. Epitope mapping revealed that amino acid residues 254-278 in domain II of the viral envelope protein E were the target region of these antibodies. A database search revealed that certain sequences in this epitope region showed high conservation among the four serotypes of DENV. These two human monoclonal antibodies could neutralize DENV-2,-4 more effectively than DENV-1,-3. The amino acid sequences could not explain this difference in neutralizing activity. However, the 3D structure results showed that amino acid 274 could be the critical residue for the difference in neutralization. These results may provide basic information for the development of a dengue vaccine.

[Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

PubMed

Yu, Min; Tan, De-Yong; Qian, Wei; Lai, Jian-Hua; Sun, Gui-Lin

2004-05-01

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong response in serum recovered cells. The antibody No.2 had the distinctive response to the serum recovered cells in different incubation time (15min, 30min, 1h, 2h, 4h, 8h, 12h and 48h) after serum starvation. The results showed that No.2 antibody would be useful to research the factors of cell cycle regulation and apply to tumor diagnosis.

Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

PubMed

Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

2016-07-01

Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhancement of immunogenic response and protection in model rats by CSTM nanoparticles anticaries DNA vaccine.

PubMed

Li, Hongjiao; Lu, Yiming; Xiang, Jingjie; Jiang, Hailong; Zhong, Yanqiang; Lu, Ying

2016-06-01

To construct anticaries DNA vaccine and evaluate its ability to elicit mucosal and systemic immune responses in rats. wapA fragment was cloned into pVAX1 plasmid to generate pVAX1-wapA. The pVAX1-wapA/trimethyl chitosan nanoparticles were prepared by complex coacervation method. Significantly higher specific IgG antibody titers were observed in rats immunized with nanoparticles compared with rats immunized with naked pVAX1-wapA. Anti-WapA IgA and IgG antibody levels after intranasal immunization were significantly higher than those following intramuscular delivery of nanoparticles or naked pVAX1-wapA. Furthermore, fewer enamel, slight dentin and dentin moderate lesions were observed in rats immunized with nanoparticles. The results implicate WapA as an excellent candidate for anticaries vaccine development and nanoparticles as an effective delivery system.

Combination of immunoglobulins and natural killer cells in the context of CMV and EBV infection.

PubMed

Frenzel, K; Lehmann, J; Krüger, D H; Martin-Parras, L; Uharek, L; Hofmann, J

2014-04-01

Cytomegalovirus (CMV)-specific hyperimmunoglobulin (CMV-HIG) is used to treat and prevent CMV infection in immunocompromised patients, and anti-CD20 monoclonal antibody is successfully used in the treatment for post-transplant lymphoproliferative disease caused by Epstein-Barr virus (EBV). Two immunological approaches have been suggested to further improve the control of viral reproduction in patients with active disease: first, the use of monoclonal antibodies with specificity against viral epitopes and second, coadministration of cells with the capacity to promote antibody-dependent cell-mediated cytotoxicity. Here, we have evaluated the effectiveness of these strategies in vitro (alone and in combination) with neutralization and cytotoxicity assays. Our results indicate that monoclonal antibodies (in particular SM5-1) can be as effective as CMV-HIG in neutralizing-cell-free CMV. Moreover, our data indicate that antibody-mediated elimination (either by moAb or by HIG) of EBV-infected cells can be significantly enhanced by NK cells. Using human NK cells that have been purified, cultured and expanded under GMP conditions, we were able to demonstrate that the combination of NK cells and antibodies could represent a feasible and highly effective clinical approach to achieve control of EBV infections. Especially in leukopenic patients with low numbers of ADCC-promoting cells, the combination of adoptively transferred NK cells and antiviral antibodies offers a promising strategy that should be tested in clinical trials.

Monoclonal Antibodies against the MET/HGF Receptor and Its Ligand: Multitask Tools with Applications from Basic Research to Therapy

PubMed Central

Prat, Maria; Oltolina, Francesca; Basilico, Cristina

2014-01-01

Monoclonal antibodies can be seen as valuable tools for many aspects of basic as well as applied sciences. In the case of MET/HGFR, they allowed the identification of truncated isoforms of the receptor, as well as the dissection of different epitopes, establishing structure–function relationships. Antibodies directed against MET extracellular domain were found to be full or partial receptor agonists or antagonists. The agonists can mimic the effects of the different isoforms of the natural ligand, but with the advantage of being more stable than the latter. Thus, some agonist antibodies promote all the biological responses triggered by MET activation, including motility, proliferation, morphogenesis, and protection from apoptosis, while others can induce only a migratory response. On the other hand, antagonists can inhibit MET-driven biological functions either by competing with the ligand or by removing the receptor from the cell surface. Since MET/HGFR is often over-expressed and/or aberrantly activated in tumors, monoclonal antibodies can be used as probes for MET detection or as “bullets” to target MET-expressing tumor cells, thus pointing to their use in diagnosis and therapy. PMID:28548076

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

PubMed

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

Analysis of the synthetic pyrethroids, permethrin and 1(R)-phenothrin, in grain using a monoclonal antibody-based test

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skerritt, J.H.; Hill, A.S.; McAdam, D.P.

1992-07-01

A monoclonal antibody generated to the synthetic pyrethroid-related hapten, (3-phenoxybenzyl)-2,2-dimethylcyclopropane-1, 3-dicarboxylate-protein conjugate, was used to develop assays for determinations of permethrin and 1(R)-phenothrin in wheat grain and flour milling fractions. The earlier 3-h assay was simplified using two approaches. The antibody was directly conjugated to the enzyme horseradish peroxidase (HRP), which removes a separate incubation and washing step from the assay. Also, an assay has been developed using microwell-bound monoclonal antibody and a HRP-labeled 3-phenoxybenzoic acid derivative. These assay formats have advantages in increased sensitivity and, in the case of the latter assay, accuracy with grain and flour samples. Themore » most sensitive assay format could detect 1.5 ng/mL permethrin; 50% inhibition of antibody binding occurred at 10 ng/mL. These values corresponded to 75 and 500 ppb, respectively, in the original wheat sample. Methanol was the most effective pyrethroid extractant. Use of a simple cleanup procedure for ground grain extracts improved ELISA accuracy but could by omitted for screening purposes.« less

Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever.

PubMed

Mire, Chad E; Cross, Robert W; Geisbert, Joan B; Borisevich, Viktoriya; Agans, Krystle N; Deer, Daniel J; Heinrich, Megan L; Rowland, Megan M; Goba, Augustine; Momoh, Mambu; Boisen, Mathew L; Grant, Donald S; Fullah, Mohamed; Khan, Sheik Humarr; Fenton, Karla A; Robinson, James E; Branco, Luis M; Garry, Robert F; Geisbert, Thomas W

2017-10-01

There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

Spotlight on Anti‐CGRP Monoclonal Antibodies in Migraine: The Clinical Evidence to Date

PubMed Central

Guerzoni, Simona; Pini, Luigi Alberto

2017-01-01

Abstract Migraine, a common neurovascular brain disorder, represents a severe and widespread health problem; along with medication‐induced (medication‐overuse) headache, it is the third‐leading cause of disability worldwide. Currently, its therapeutic management remains unsatisfactory for several reasons; up to 40% of migraineurs are eligible for prophylactic treatment, but there are issues of efficacy, safety, and adherence. In recent years the evidence on the role of calcitonin gene‐related peptide (CGRP) in migraine pathophysiology has been consolidated, so new and promising treatments for migraine pain and its possible prevention have been developed. The following review reports the results of the clinical trials conducted so far with each of the new monoclonal antibodies targeting CGRP or its receptor, with particular reference to safety, tolerance, and efficacy in migraine prevention. Moreover, the pharmacological characterization and further developments of each monoclonal antibody are reported, based on current knowledge. PMID:28409893

Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody.

PubMed

Zhao, Yinli; Li, Guoxi

2016-01-01

A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1-128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93-97.5%, and the coefficient of variation [CV (%)] were from 5.55-8.38%. For interassay reproducibility, recoveries were from 89.5-95.1%, the coefficient of variation [CV (%)] were from 5.26-9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

Radioimmune localization of occult carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duda, R.B.; Zimmer, A.M.; Rosen, S.T.

1990-07-01

Patients with a rising serum carcinoembryonic antigen level and no clinical or roentgenographic evidence of recurrent or metastatic cancer present a treatment dilemma. Eleven such patients, 10 with a previously treated colorectal carcinoma and 1 with a previously treated breast carcinoma, received an injection of the anticarcinoembryonic antigen monoclonal antibody ZCE-025 labeled with the radioisotope indium 111. Nuclear scintigraphy was performed on days 3 and 5 through 7 to detect potential sites of tumor recurrence. The monoclonal antibody scan accurately predicted the presence or absence of occult malignancy in 7 (64%) patients. Second-look laparotomy confirmed the monoclonal antibody scan resultsmore » in the patients with colorectal cancer, and magnetic resonance imaging confirmed metastatic breast cancer. This study demonstrates that In-ZCE-025 can localize occult carcinoma and may assist the surgeon in facilitating the operative exploration. In-ZCE-025 assisted in the initiation of adjuvant therapy for the patient with breast cancer.« less

Introduction to the application of QbD principles for the development of monoclonal antibodies.

PubMed

Finkler, Christof; Krummen, Lynne

2016-09-01

Quality by Design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter introduces a publication series on the application of Quality by Design for biopharmaceuticals, with a focus on the development of recombinant monoclonal antibodies. The development of and overview on the QbD concept applied by Roche and Genentech is described and essential QbD elements are presented. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Developmental toxicity testing of monoclonal antibodies: an enhanced pre- and postnatal study design option.

PubMed

Stewart, Jane

2009-09-01

For many monoclonal antibodies (mAb), the preferred species for general and reproductive safety testing is often the cynomolgus monkey. This article describes the rationale for combining the traditional "segmented" embryofetal development study with the pre- and postnatal development (PPND) study into a single "enhanced" PPND study design in the cynomolgus monkey where a single cohort of animals is exposed throughout gestation and allowed to give birth naturally. The proposed "enhanced" PPND study design evaluates all the stages of the traditional two study design using fewer animals. It also assesses the functional consequences of mid to late gestational exposure. This is of particular relevance to the risk assessment of monoclonal antibodies where fetal exposure to maternal IgG increases as pregnancy progresses and where morphologic examination of a pre-term fetus may not be adequate to reveal the presence of adverse effects on functional development of key target organs.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice.

PubMed

Brunn, Nicholas D; Mauze, Smita; Gu, Danling; Wiswell, Derek; Ueda, Roanna; Hodges, Douglas; Beebe, Amy M; Zhang, Shuli; Escandón, Enrique

2016-03-01

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

PubMed Central

Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

2013-01-01

Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

Monoclonal antibodies with group specificity toward sulfonamides: Selection of hapten and antibody selectivity

USDA-ARS?s Scientific Manuscript database

Although many antibodies to sulfonamides have been generated, immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have properties dependent on the immunizing hapten structure and have always suffered from high selectivity for individual sulfonamides....

A 1H NMR method for the analysis of antigen-antibody interactions: binding of a peptide fragment of lysozyme to anti-lysozyme monoclonal antibody.

PubMed

Ito, W; Nishimura, M; Sakato, N; Fujio, H; Arata, Y

1987-09-01

A proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

PubMed Central

Calow, Jenny; Bockau, Ulrike; Struwe, Weston B.; Nowaczyk, Marc M.; Loser, Karin; Crispin, Max

2016-01-01

ABSTRACT Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity. PMID:27594301

Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies.

PubMed Central

Greenberg, H; McAuliffe, V; Valdesuso, J; Wyatt, R; Flores, J; Kalica, A; Hoshino, Y; Singh, N

1983-01-01

Ten monoclones directed to the 42,000-dalton inner structural protein of rotavirus were analyzed. Eight monoclones reacted broadly with antigenic domains common to virtually all mammalian rotaviruses. Two monoclones had specificities similar or identical to previously characterized subgroup specificities. These subgroup monoclones were more efficient in detecting subgroup antigen than either hyperimmune or postinfection antisera. Using the subgroup monoclones, we determined that some animal as well as human rotavirus strains carry subgroup 2 specificity and that epizootic diarrhea of infant mice virus and turkey rotavirus are antigenically distinct from other mammalian rotavirus strains. Images PMID:6185436

Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

PubMed

Igawa, Tomoyuki

2017-01-01

Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

Challenges and opportunities for monoclonal antibody therapy in veterinary oncology.

PubMed

Beirão, Breno C B; Raposo, Teresa; Jain, Saurabh; Hupp, Ted; Argyle, David J

2016-12-01

Monoclonal antibodies (mAbs) have come to dominate the biologics market in human cancer therapy. Nevertheless, in veterinary medicine, very few clinical trials have been initiated using this form of therapy. Some of the advantages of mAb therapeutics over conventional drugs are high specificity, precise mode of action and long half-life, which favour infrequent dosing of the antibody. Further advancement in the field of biomedical sciences has led to the production of different forms of antibodies, such as single chain antibody fragment, Fab, bi-specific antibodies and drug conjugates for use in diagnostic and therapeutic purposes. This review describes the potential for mAbs in veterinary oncology in supporting both diagnosis and therapy of cancer. The technical and financial hurdles to facilitate clinical acceptance of mAbs are explored and insights into novel technologies and targets that could support more rapid clinical development are offered. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab)

PubMed Central

Wanczyk, Heather; Barker, Tolga; Rood, Debra; Zapata, Daniel I.; Howell, Amy R.; Richardson, Stewart K.; Zinckgraf, John; Marusov, Gregory P.; Lynes, Michael A.; Silbart, Lawrence K.

2013-01-01

Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers. PMID:23518474

Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

NASA Astrophysics Data System (ADS)

Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

1992-09-01

The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies.

PubMed

Corrêa, Arthur Luiz; Senna, José Procópio Moreno; de Sousa, Álvaro Paiva Braga

2016-05-01

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.

Host virus and pneumococcus-specific immune responses in high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia: implications for disease progression

PubMed Central

Criado, Ignacio; Muñoz-Criado, Santiago; Rodríguez-Caballero, Arancha; Nieto, Wendy G.; Romero, Alfonso; Fernández-Navarro, Paulino; Alcoceba, Miguel; Contreras, Teresa; González, Marcos; Orfao, Alberto; Almeida, Julia

2017-01-01

Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal B-cell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins. These findings probably reflect asymptomatic chronic reactivation of humoral immune responses against host viruses associated with expanded virus-specific antibody levels and progressively decreased protection against other micro-organisms, denoting a severe humoral immunodeficiency state not reflected by the overall plasma immunoglobulin levels. Alternatively, these results could reflect a potential role of ubiquitous viruses in the pathogenesis of the disease. Further analyses are necessary to establish the relevance of such asymptomatic humoral immune responses against host viruses in the expansion of the tumor B-cell clone and progression from monoclonal B-cell lymphocytosis to CLL. PMID:28385786

Emerging monoclonal antibodies against Clostridium difficile infection.

PubMed

Péchiné, Séverine; Janoir, Claire; Collignon, Anne

2017-04-01

Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

Evaluation of oriented lysozyme immobilized with monoclonal antibody

NASA Astrophysics Data System (ADS)

Aoyagi, Satoka; Okada, Keigo; Shigyo, Ayako; Man, Naoki; Karen, Akiya

2008-12-01

The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.

An immuno-biosensor system based on quartz crystal microbalance for avian influenza virus detection

NASA Astrophysics Data System (ADS)

Liu, Shengping; Chen, Guoming; Zhou, Qi; Wei, Yunlong

2007-12-01

For the quick detection of Avian Influenza Virus (AIV), a biosensor based on Quartz Crystal Microbalance (QCM) was fabricated according to the specific bonding principle between antibody and antigen. Staphylococcal Protein A (SPA) was extracted from Staphylococcus and purified. Then SPA was coated on the surface of QCM for immobilizing AIV monoclonal antibodies. The use of AIV monoclonal antibody could enhance the specificity of the immuno-biosensor. A multi-channel piezoelectricity detection system for the immuno-biosensor was developed. The system can work for the quick detection of AIV antigen in the case of the entirely aqueous status owe to one special oscillating circuit designed in this work. The optimum conditions of SPA coating and AIV monoclonal antibody immobilization were investigated utilizing the multi-channel detection system. The preliminary application of the immuno-biosensor system for detection of AIV was evaluated. Results indicate that the immuno-biosensor system can detect the AIV antigens with a linear range of 3-200ng/ml. The system can accomplish the detection of AIV antigens around 40 minutes.

Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients.

PubMed

Thong, Kwai-Lin; Tang, Swee-Seong; Tan, Wen-Siang; Devi, Shamala

2007-01-01

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.

Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins.

PubMed

Liu, Jing; Zhang, Hui C; Duan, Chang F; Dong, Jun; Zhao, Guo X; Wang, Jian P; Li, Nan; Liu, Jin Z; Li, Yu W

2016-11-01

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18-107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody-drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.

Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

PubMed

Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I

2017-01-01

The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products

NASA Technical Reports Server (NTRS)

Chen, F.; Haber, E.; Matsueda, G. R.

1992-01-01

The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype.

PubMed

Tatarewicz, Suzanna M; Juan, Gloria; Swanson, Steven J; Moxness, Michael S

2012-08-31

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation. ATAs that developed post-treatment were specific for the F(ab')2, whereas, pre-existing ATAs were specific to the Fc region. Further characterization of the pre-existing ATAs identified the specific epitope to be the G1m1 allotype determinant in the Fc of the therapeutic. A novel competitive bridging assay was developed to verify that serum IgG1 from subjects with pre-existing anti-G1m1 antibodies was homozygous for the antithetical allotype (G1m3). The endogenous G1m allotype of all subjects was assessed and correlation to ATA incidence and adverse events was evaluated. Interestingly, the pre-existing anti-allotype antibody in subjects persisted but was not augmented after dosing, indicating the lack of a secondary immune response to this epitope. These studies indicate the relationship of the therapeutic allotype and the corresponding allotype of subjects is an important component to further understand the impact of immunogenicity on the safety and efficacy of therapeutic antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palomo, Concepción; Mas, Vicente; Vázquez, Mónica

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to exploremore » the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.« less

Pathogenic and Nonpathogenic Strains of Entamoeba histolytica can be Differentiated by Monoclonal Antibodies to the Galactose-Specific Adherence Lectin

DTIC Science & Technology

1991-04-01

AD- A235 913 DEVELOPMENT Ei ENGINEERING CENTER CRDEC-TR-268 PATHOGENIC AND NONPATHOGENIC STRAINS OF ENTAMOEBA HISTOLYTICA CAN BE DIFFERENTIATED BY...Pathogenic and Nonpathogenic Strains of Entamoeba Histolytica can be Differentiated by Monoclonal PR-IFJlX2XXRPEW Antibodies to the Galactose-Specific...galactose lectin produced by Entamoeba histolytica provide the basis for development of a model system for the environmental detection of adherence and

Ganciclovir prophylaxis decreases frequency and severity of cytomegalovirus disease in seropositive liver transplant recipients treated with OKT3 monoclonal antibodies.

PubMed Central

Lumbreras, C; Otero, J R; Herrero, J A; Gomez, R; Lizasoain, M; Aguado, J M; Colina, F; Garcia, I; Moreno, E; Noriega, A R

1993-01-01

The efficacy of ganciclovir, given prophylactically, to prevent cytomegalovirus-related disease was evaluated in liver transplant recipients, mostly seropositive, under treatment with OKT3 monoclonal antibodies. The incidence of cytomegalovirus disease and visceral involvement was reduced, respectively, from 52 and 36% in the control group to 12 and 8% in the ganciclovir-treated patients. Leukopenia was a frequent (32%) side effect of ganciclovir administration. PMID:8285641

A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti-PSMA Monoclonal Antibody J591in Patients with High-Risk Castrate, Biochemically Relapsed Prostate Cancer

DTIC Science & Technology

2012-09-01

micrometastases that may be targeted with radioimmunotherapy. Prostate specific membrane antigen (PSMA) is the single, most well-established, highly restricted...Radiolabeled anti- prostate specific membrane antigen (PSMA) monoclonal antibody J591 (177Lu-J591) for nonmetastatic castration-resistant prostate cancer...rationale for systemic salvage targeted anti- prostate specific membrane antigen radioimmunotherapy. Adv Urol 2012, Article ID 921674, doi:10.1155

Pathogenesis of systemic inflammatory diseases in childhood: "Lessons from clinical trials of anti-cytokine monoclonal antibodies for Kawasaki disease, systemic onset juvenile idiopathic arthritis, and cryopyrin-associated periodic fever syndrome".

PubMed

Yokota, Shumpei; Kikuchi, Masako; Nozawa, Tomo; Kanetaka, Taichi; Sato, Tomomi; Yamazaki, Kazuko; Sakurai, Nodoka; Hara, Ryoki; Mori, Masaaki

2015-01-01

Inflammation has often been considered to be a nonspecific response and to play a bridging role in the activation of adaptive immunity. However, it is now accepted that inflammation is the product of an independent innate immune system closely linked to the adaptive immune system. The key mediators of inflammation are inflammatory cytokines, as determined by multiple lines of evidence both in vitro and in vivo. Due to the crucial role of inflammatory cytokines in the pathogenesis of autoimmune disorders, anti-cytokine treatment has been developed as a therapy for rheumatoid arthritis, juvenile idiopathic arthritis (JIA), and inflammatory bowel diseases. We recently completed several clinical trials of anti-cytokine treatment for children with systemic inflammatory diseases: anti-IL-6 receptor monoclonal antibody (tocilizumab) for children with two subtypes of JIA (poly-JIA and systemic JIA), anti-TNF-α monoclonal antibody (infliximab) for children with Kawasaki disease, and anti-IL-1-β monoclonal antibody (canakinumab) for children with cryopyrin-associated periodic syndrome. This review summarizes the basis of inflammation in terms of innate immunity and adaptive immunity in these systemic inflammatory diseases, clinical efficacy, and tolerability of these biologic agents, and attempts to determine the roles of individual inflammatory cytokines in disease pathogenesis.

Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

PubMed Central

Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae

2015-01-01

Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

Effect of ambient light on monoclonal antibody product quality during small-scale mammalian cell culture process in clear glass bioreactors.

PubMed

Mallaney, Mary; Wang, Szu-Han; Sreedhara, Alavattam

2014-01-01

During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production. © 2014 American Institute of Chemical Engineers.

De novo sequencing and resurrection of a human astrovirus-neutralizing antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bogdanoff, Walter A.; Morgenstern, David; Bern, Marshall

Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parentmore » monoclonal antibody to bind to the astrovirus capsid protein. Furthermore, this antibody can now potentially be developed as a therapeutic and diagnostic agent.« less

[Monoclonal antibodies against inflammatory mediators for the treatment of patients with sepsis].

PubMed

Matsubara, Tomoyo

2002-03-01

Sepsis is a common cause of morbidity and mortality, particularly in immunocompromised and critically ill patients. Recently, a new designation, systemic inflammatory response syndrome(SIRS), has been studied. When an abnormal generalized inflammatory reaction is due to an infection, the terms sepsis and SIRS are synonymous. The systemic response to infection is mediated via the macrophage-derived cytokines that target end organ receptors in response to injury or infection. One strategy used to perturb the septic cascade is to block a particular inflammatory molecule. Results have been published on clinical trials in sepsis patients treated with several monoclonal antibodies, such as antiendotoxin antibodies, anti-tumor necrosis factor antibodies, and anti CD14 antibodies. In this chapter, the results of clinical trials in patients and in vivo data from animal models of sepsis are summarized.

De novo sequencing and resurrection of a human astrovirus-neutralizing antibody

DOE PAGES

Bogdanoff, Walter A.; Morgenstern, David; Bern, Marshall; ...

2016-03-14

Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parentmore » monoclonal antibody to bind to the astrovirus capsid protein. Furthermore, this antibody can now potentially be developed as a therapeutic and diagnostic agent.« less

Antibody Characterization Lab | Office of Cancer Clinical Proteomics Research

Cancer.gov

The Antibody Characterization Lab (ACL), an intramural reference laboratory located at the Frederick National Laboratory for Cancer Research in Frederick, Maryland, thoroughly characterizes monoclonal antibodies or other renewable affinity binding reagents for use in cancer related research.

Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

One target, different effects: a comparison of distinct therapeutic antibodies against the same targets.

PubMed

Shim, Hyunbo

2011-10-31

To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-α, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-α have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-α, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.

Conservation of myeloid surface antigens on primate granulocytes.

PubMed

Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

1983-02-01

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

131-I coupled to monoclonal antibodies as therapeutic agents for neuroectodermally derived tumors: fact or fiction?

PubMed

Kemshead, J T; Jones, D H; Lashford, L; Prichard, J; Gordon, I; Breatnach, F; Coakham, H B

1986-01-01

It has been suggested that monoclonal antibodies may be useful in targeting cytotoxic compounds to tumor cells. We have explored their use in targeting 131-I to highly radiosensitive primitive neural tumors such as neuroblastoma and pineoblastomas. Two routes of administration have been employed, intravenous and intrathecal. Our current experience in using radiolabelled antibodies is described, indicating toxicities seen and any therapeutic benefit observed. The results of the study suggest that if targeted radiation has a role in the treatment of these malignancies, it will be restricted to the eradication of small tumor masses from the body.

DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

NASA Astrophysics Data System (ADS)

Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

1984-08-01

A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

A monoclonal antibody that recognizes B cells and B cell precursors in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coffman, R.L.; Weissman, I.L.

1981-02-01

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

In vitro production of monoclonal antibodies under serum-free conditions using a compact and inexpensive hollow fibre cell culture unit.

PubMed

Klerx, J P; Jansen Verplanke, C; Blonk, C G; Twaalfhoven, L C

1988-07-22

A compact and easily portable hollow fibre cell culture system using commercially available components is described. The construction is relatively cheap and simple. As the hollow fibre cell culture cartridge we chose an inexpensive haemodialyser. Though not specially developed for this purpose this performed excellently in our system. Using a serum-free medium supplemented with ethanolamine, selenium and transferrin, an average antibody production of 30-200 mg per cartridge per day could be achieved, depending on the cell line. Because a serum-free medium was used, monoclonal antibodies could readily be purified on a large scale.

Structure of the extracellular domain of matrix protein 2 of influenza A virus in complex with a protective monoclonal antibody.

PubMed

Cho, Ki Joon; Schepens, Bert; Seok, Jong Hyeon; Kim, Sella; Roose, Kenny; Lee, Ji-Hye; Gallardo, Rodrigo; Van Hamme, Evelien; Schymkowitz, Joost; Rousseau, Frederic; Fiers, Walter; Saelens, Xavier; Kim, Kyung Hyun

2015-04-01

The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e. M2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Rapid Multiplex Assay for Serotyping Pneumococci with Monoclonal and Polyclonal Antibodies

PubMed Central

Yu, Jigui; Lin, Jisheng; Benjamin, William H.; Waites, Ken B.; Lee, Che-hung; Nahm, Moon H.

2005-01-01

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy. PMID:15634965

[Anti-PD-1 antibody: basics and clinical application].

PubMed

Tanaka, Yoshimasa; Okamura, Haruki

2013-09-01

Although the treatment of cancer with monoclonal antibodies has long been pursued, T cell-directed immunotherapy has met with limited success. Recently, much attention has been devoted to the blockade of PD-1 signaling to activate an immune response to cancer. PD-1, a protein expressed on T cells, is a member of the CD28 superfamily, and it transmits coinhibitory signals upon engagement with its ligands PD-L1 and PD-L2. Accumulating evidence suggests that the PD-1 system plays pivotal roles in the regulation of autoimmunity, transplantation immunity, infectious immunity, and tumor immunity. Because the interaction of PD-1 with its ligands occurs in the effector phase of killer T cell responses in peripheral blood, anti-PD-1 and anti-PD-L1 monoclonal antibodies are ideal as specific agents to augment T cell responses to tumors with fewer adverse events than with the inhibition of CTLA-4, because the interaction of CTLA-4 with its ligands occurs in the priming phase of T cell responses within lymph nodes. In recent phase I clinical trials, objective responses were observed in patients with melanoma, renal cell carcinoma, and non-small cell lung cancer who underwent immunotherapy with an anti-PD-1 monoclonal antibody. In addition, the antitumor activity of an anti-PD-L1 monoclonal antibody was observed in patients with melanoma, renal cell carcinoma, non-small cell lung cancer, and ovarian cancer. The next frontier of immunotherapy targeting the PD-1 axis is to define patient selection criteria and explore combination therapy with other therapeutic manipulations such as adoptive immunotherapies.

Human hybrid hybridoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tiebout, R.F.; van Boxtel-Oosterhof, F.; Stricker, E.A.M.

1987-11-15

Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. The authors have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. They fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1kappa antibody directed against tetanus toxiod and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture mediummore » containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassay. The results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies. Bispecific antibodies activity was measured by means of two radioimmunoassays.« less

Suppression of allo-human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg) versus a monoclonal anti-HLA-E IgG that mimics HLA-I reactivities of IVIg.

PubMed

Zhu, D; Ravindranath, M H; Terasaki, P I; Miyazaki, T; Pham, T; Jucaud, V

2014-08-01

B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other β2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo. © 2014 British Society for Immunology.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

PubMed

Perruchini, Claire; Pecorari, Frederic; Bourgeois, Jean-Pierre; Duyckaerts, Charles; Rougeon, François; Lafaye, Pierre

2009-11-01

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Detection of cystatin C biomarker for clinical measurement of renal disease by developed ELISA diagnostic kits.

PubMed

Jiang, Renren; Xu, Chao; Zhou, Xiaoli; Wang, Tianhao; Yao, Gang

2014-09-12

Human cystatin C (HCC) is a potential biomarker for tubular damage and impaired renal function. It is difficult to obtain efficient paired monoclonal antibodies against HCC with low molecular to meet the requirements for clinical application The present study was to establish a stable and repeatable measurement for HCC with self-made monoclonal antibodies (McAbs) and Variable domain of heavy chain of heavy-chain antibody (VHHs) increase the sensitivity. With hybridoma technology and phage display technology: R-HCC as a screening antigen and N-HCC as the detector for antigens to obtain the specific antibody and established an enzyme-linked immunosorbent assay for human cystatin C using self-made McAbs and VHHs. We have successfully obtained three McAbs; 5 F2, 4E4, 1E11 and four VHHs; 3-2, 3-24, 3-33 and 4-5 which were specific for HCC. The measurement of HCC was established with the self-made monoclonal antibodies and VHHs with a high sensitivity the lower limit of detection at 0.5 ng/ml and the detection range at 0.5 ~ 31.3 ng/ml. Our data provides a new approach for paired antibody screening and testing of the small molecular biomarker with a single dominant epitope, with the important biological and clinical significance.

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography

PubMed Central

Dutta, Amit K.; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A.; Zhang, Ada W.; Tustian, Andrew D.; Zydney, Andrew L.; Shinkazh, Oleg

2015-01-01

Recent studies using simple model systems have demonstrated that Continuous Countercurrent Tangential Chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an “after binder” to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (~0.67 g/L) and one with high titer (~6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to that obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. PMID:25747172

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

PubMed

Dutta, Amit K; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A; Zhang, Ada W; Tustian, Andrew D; Zydney, Andrew L; Shinkazh, Oleg

2015-11-10

Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. Copyright © 2015 Elsevier B.V. All rights reserved.

Functional Analysis of the Anti-adalimumab Response Using Patient-derived Monoclonal Antibodies♦

PubMed Central

van Schouwenburg, Pauline A.; Kruithof, Simone; Votsmeier, Christian; van Schie, Karin; Hart, Margreet H.; de Jong, Rob N.; van Buren, Esther E. L.; van Ham, Marieke; Aarden, Lucien; Wolbink, Gertjan; Wouters, Diana; Rispens, Theo

2014-01-01

The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (Kd between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity. PMID:25326381

Drug-protein conjugates: haptenation of 1-methyl-10 alpha-methoxydihydrolysergol and 5-bromonicotinic acid to albumin for the production of epitope-specific monoclonal antibodies against nicergoline.

PubMed

Gabor, F; Hamilton, G; Pittner, F

1995-09-01

Two types of monoclonal antibodies were used for the determination of nicergoline in biological matrices. The antibodies were prepared with the hydrolysis products 5-bromonicotinic acid and 1-methyl-10 alpha-methoxydihydrolysergol after hemisuccinoylation to haptens. The current amide bond-generating methods (mixed anhydride-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, and dicyclohexylcarbodiimide/N-hydroxysuccinimide methods) were used in bovine serum albumin (BSA)-coupling techniques and yielded conjugates that were haptenated to varying extents. The conjugates exhibiting 23 mol of 1-methyl-10 alpha-methoxydihydrolysergol (MMD) or 41 mol of 5-bromonicotinic acid (BNA) per mole of BSA were used for both immunization of mice and for coating the wells of the microtiter plates to select hybridomas and investigate specificity of the obtained antibodies. The results of hapten-inhibition ELISA using antigen-coated wells indicate that the supernatant of MMD-specific hybridoma exhibited 50% inhibition of antibody binding at 17 +/- 2 micrograms of MMD and at 24.5 +/- 2 micrograms of nicergoline, and the BNA-specific hybridoma exhibited similar inhibition at 147 +/- 6 micrograms of BNA and 500 +/- 30 micrograms of nicergoline. A main requirement for analytical purposes is that two different types of monoclonal antibodies recognize two different epitopes on nicergoline and its main metabolite, as shown by hapten-inhibition ELISA.

Fc gamma receptor 3a genotype predicts overall survival in follicular lymphoma patients treated on SWOG trials with combined monoclonal antibody plus chemotherapy but not chemotherapy alone

PubMed Central

Persky, Daniel O.; Dornan, David; Goldman, Bryan H.; Braziel, Rita M.; Fisher, Richard I.; LeBlanc, Michael; Maloney, David G.; Press, Oliver W.; Miller, Thomas P.; Rimsza, Lisa M.

2012-01-01

Background Fc gamma receptor polymorphisms were linked to outcome in follicular lymphoma patients treated with single-agent rituximab, an anti-CD20 monoclonal antibody. In particular, 158F/F genotype of Fc gamma receptor 3A and 131R/R genotype of Fc gamma receptor 2A correlated with worse outcome compared to high-affinity 158V/V and 131H/H, respectively. We examined this association in the context of anti-CD20 monoclonal antibody combined with chemotherapy, as compared to chemotherapy alone, in follicular lymphoma patients treated on SWOG clinical trials. Design and Methods Tissue from 142 SWOG patients treated with chemotherapy alone (protocol S8809, n=70) or combined chemotherapy and anti-CD20 monoclonal antibody (rituximab and Iodine I-131 tositumomab on protocols S9800 and S9911, n=30 and 42, respectively) was analyzed. DNA was extracted and assayed for Fc gamma receptor 3A V158F and 2A R131H polymorphisms using a TaqMan SNP assay. Stratified Cox’s regression was used to assess association with overall survival. Results For Fc gamma receptor 3A, there was an association with overall survival in the combination therapy trials but not in the chemotherapy-only trial. Having at least one Fc gamma receptor 3A V allele was associated with improved overall survival versus F/F (HR=0.33, 95% CI, 0.11, 0.96, P=0.042). For overall survival, there was evidence of a statistical interaction between the use of mAb and the number of V alleles (0, 1, or 2) (P=0.006). There was no such association for Fc gamma receptor 2A. Conclusions Fc gamma receptor 3A polymorphism status may be predictive of survival in follicular lymphoma patients receiving treatments containing an anti-CD20 antibody but not treatment with chemotherapy alone. Thus, Fc gamma receptor 3A polymorphisms may be important to consider in designing new follicular lymphoma trials and new anti-CD20 monoclonal antibodies. PMID:22271896

Measurement of Lp(a) with a two-step monoclonal competitive sandwich ELISA method.

PubMed

Morikawa, W; Iki, R; Terano, T; Funatsu, A; Sugiuchi, H; Uji, Y; Okabe, H

1995-06-01

To evaluate the results of Lipoprotein (a)[Lp(a)] measurements by a competitive two-step monoclonal enzyme-linked immuno sorbent assay method comparing them with those by a conventional ELISA. Serum having various isoforms of Lp(a) and purified Lp(a) were assayed using the method described here and commercially available kits. The reference range was determined with the use of 324 normal subjects by means of calculation from Lp(a) results of logarithmic transformation. Our method takes advantage of a competitive reaction between fixed antibody and free antibody to Lp(a), having the detection range up to 1000 mg/L with the lowest detection limit of 2 mg/L. The anti-Lp(a) monoclonal antibody employed in the assay system reacts uniformly with all phenotypes of Lp(a) but showing very low cross-reactivity for plasminogen and LDL. Within-run and between-run precisions were excellent, giving CVs of 2.9 and 4.0% with mean values of 145 and 635 mg/L, respectively. In comparison of the results by our method with those by a polyclonal method (Biopool) or a monoclonal antibody method (Terumo), they correlated well; Y (our method) = 0.99 x (polyclonal method, Biopool) - 1.9, r = 0.994 (n = 60), and Y = 0.94 X(monoclonal method, Terumo) -9.8, r = 0.97 (n = 60), respectively. The reference range was 105.9 +/- 25.4 mg/L, the difference between the sexes was not significant. Our method has proven highly accurate and specific. It is applicable with auto analyzer because it does not require such a pre-dilution step as is necessary for Lp(a) determination by conventional ELISA assay. Accordingly, we can conclude that our test method is workable for both clinical laboratories and mass screening.

Monoclonal antibodies as cancer therapeutics.

PubMed

Elloumi, Jihene; Jellali, Karim; Jemel, Ikram; Aifa, Sami

2012-04-01

Three main targets were subjected for the most approved monoclonal antibodies (mAbs) in cancer therapy: EGFR in solid cancer, the clusters of differentiation in blood cancer and VEGF in angiogenesis. Meanwhile side effects, the elevated costs and resistance problems are limiting the efficiency of mAbs as targeted therapy. The combinatory therapy with chemo or radiotherapy has improved the efficiency of mAbs. The present review aims to shed more light on the immunotherapy and the related patents that were developed for cancer treatment.

Monoclonal antibodies directed against surface molecules of multicell spheroids

NASA Technical Reports Server (NTRS)

Martinez, Andrew O.

1993-01-01

The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

Use of monoclonal antibodies as an effective strategy for treatment of ciguatera poisoning.

PubMed

Inoue, Masayuki; Lee, Nayoung; Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

2009-06-01

Ciguatera is a global food poisoning caused by the consumption of fish that have accumulated sodium channel activator toxins, ciguatoxins. At present, most diagnosed cases of ciguatera are treated with symptomatic and supportive remedies, and no specific therapy has been devised. Here we report that ciguatoxin CTX3C can be effectively neutralized in vitro and in vivo by simultaneous use of two anti-ciguatoxin monoclonal antibodies, providing the first rational approach toward directly preventing and treating ciguatera.

[Production of the monoclonal antibodies to the rabies virus nucleoprotein].

PubMed

Gribencha, S V; Kozlov, A Iu; Kostina, L V; Elakov, A L; Losich, M A; Tsibezov, V V; Zaberezhnyĭ, A D; Aliper, T I

2013-01-01

Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate.

Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

PubMed

Smith, Kenneth; Muther, Jennifer J; Duke, Angie L; McKee, Emily; Zheng, Nai-Ying; Wilson, Patrick C; James, Judith A

2013-05-01

B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23. Copyright © 2012 Elsevier GmbH. All rights reserved.

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

PubMed

Hill, A S; Giersch, T M; Loh, C S; Skerritt, J H

1999-10-01

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

Human Monoclonal Antibody-Based Therapy in the Treatment of Invasive Candidiasis

PubMed Central

Bugli, Francesca; Cacaci, Margherita; Martini, Cecilia; Torelli, Riccardo; Posteraro, Brunella; Sanguinetti, Maurizio; Paroni Sterbini, Francesco

2013-01-01

Invasive candidiasis (IC) represents the leading fungal infection of humans causing life-threatening disease in immunosuppressed and neutropenic individuals including also the intensive care unit patients. Despite progress in recent years in drugs development for the treatment of IC, morbidity and mortality rates still remain very high. Historically, cell-mediated immunity and innate immunity are considered to be the most important lines of defense against candidiasis. Nevertheless recent evidence demonstrates that antibodies with defined specificities could act with different degrees showing protection against systemic and mucosal candidiasis. Mycograb is a human recombinant monoclonal antibody against heat shock protein 90 (Hsp90) that was revealed to have synergy when combined with fluconazole, caspofungin, and amphotericin B against a broad spectrum of Candida species. Furthermore, recent studies have established an important role for Hsp90 in mediating Candida resistance to echinocandins, giving to this antibody molecule even more attractive biological properties. In response to the failure of marketing authorization by the CHMP (Committee for Medicinal Products for Human Use) a new formulation of Mycograb, named Mycograb C28Y variant, with an amino acid substitution was developed in recent years. First data on Mycograb C28Y variant indicate that this monoclonal antibody lacked efficacy in a murine candidiasis model. PMID:23878583

Combination of treatment with death receptor 5-specific antibody with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects

PubMed Central

Tseng, Chih-Wen; Trimble, Cornelia; Monie, Archana; Alvarez, Ronald D.; Huh, Warner K.; Buchsbaum, Donald J.; Straughn, J. Michael; Wang, Mei-Cheng; Yagita, Hideo; Hung, Chien-Fu; Wu, T.-C.

2008-01-01

There is currently a vital need for the development of novel therapeutic strategies for the control of advanced stage cancers. Antigen-specific immunotherapy and the employment of antibodies against the death receptor 5 (DR5) have emerged as two potentially promising strategies for cancer treatment. In the current study, we hypothesize that the combination of treatment with the anti-DR5 monoclonal antibody, MD5-1 with a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7(detox)) administered via gene gun would lead to further enhancement of E7-specific immune responses as well as antitumor effects. Our results indicated that mice bearing the E7-expressing tumor, TC-1 treated with MD5-1 monoclonal antibody followed by CRT/E7(detox) DNA vaccination generated the most potent therapeutic anti-tumor effects as well as highest levels of E7-specific CD8+ T cells among all the groups tested. In addition, treatment with MD5-1 monoclonal antibody was capable of rendering the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic T lymphocytes. Our findings serve as an important foundation for future clinical translation. PMID:18598733

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part II. Comparison of immunoreactivity and biodistribution of monoclonal antibodies labeled with the 67Ga-chelate or with 131I.

PubMed

Matzku, S; Schuhmacher, J; Kirchgessner, H; Brüggen, J

1986-01-01

Coupling of the 67Ga-P-EDDHA chelate via carbodiimide to the anti-melanoma monoclonal antibody (Mab) M.2.9.4 resulted in a low degree of oligomerization, but a considerable degree of intra-molecular (inter-chain) cross-linking. However, this did not impair immunoreactivity, nor did the half-life in vivo differ substantially from that of 131I-M.2.9.4. Biodistribution analysis in normal mice showed Ga:I ratios near 1 in the blood and other tissues not involved in degradation and label excretion. In tissues of the reticulo-endothelial system (RES) and the kidneys, Ga:I ratios up to 2.51 were reached within 4 days of administration. In antigen-positive MeWo tumor tissue, retention of 67Ga also excreted that of 131I, so that tumor; organ ratios (except tumor:liver) were superior for the 67Ga-labeled MAb. It is concluded that the method of coupling pre-established 67Ga-P-EDDHA chelate to antibody results in a functionally intact tracer molecule, whose persistence in vivo is not significantly impaired. The major difference to I-labeled MAbs may be a prolonged retention of Ga in tissues (cells) physiologically involved in antibody catabolism.

Characterization of Sicilian strains of spotted fever group rickettsiae by using monoclonal antibodies.

PubMed Central

Vitale, G; Di Stefano, R; Damiani, G; Mansueto, S

1989-01-01

Twenty-two hybridomas producing anti-Rickettsia conorii monoclonal antibodies were obtained by nine fusion experiments. The strain chosen for immunization of mice was MAVI, an R. conorii strain isolated from a Sicilian patient with Boutonneuse fever. When tested for immunoglobulin isotype by an indirect immunofluorescence (IIF) assay, 46.6% of supernatants from the 22 hybridomas were immunoglobulin M. The supernatants were tested in the IIF assay for binding to the MAVI strain and four spotted fever group rickettsia strains isolated from Sicilian ticks (two virulent and two nonpathogenic when inoculated intraperitoneally in male guinea pigs). Only five of the supernatants showed a positive IIF result on all tested strains, although they produced different titers to the various strains, possibly an indication that they recognized an antigen common to spotted fever group rickettsiae. Immunodominant epitopes for humans were determined by using patient sera to analyze inhibition of binding to the MAVI strain. Although a limited number of serum samples were screened, a high percentage of Boutonneuse fever patients produced antibodies recognizing the same epitopes as were recognized by the mouse monoclonal antibodies. A striking heterogeneity was found both in the expression of mouse-recognized epitopes on the five rickettsial strains and in the serum antibody responses of Boutonneuse fever patients to these epitopes. PMID:2473092

Antibody-mediated reduction of {alpha}-ketoamides

DOEpatents

Schultz, P.G.; Gallop, M.A.

1998-06-09

Monoclonal antibodies raised against a 4-nitrophenyl phosphonate hapten catalyze the stereospecific reduction of an {alpha}-ketoamide to the corresponding {alpha}-hydroxyamide in the presence of an appropriate reducing agent.

Antibody-mediated reduction of .alpha.-ketoamides

DOEpatents

Schultz, Peter G.; Gallop, Mark A.

1998-01-01

Monoclonal antibodies raised against a 4-nitrophenyl phosphonate hapten catalyze the stereospecific reduction of an .alpha.-ketoamide to the corresponding .alpha.-hydroxyamide in the presence of an appropriate reducing agent.

[Progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases].

PubMed

Yao, Yuan; Yu, Chuan-xin

2013-08-01

Antibody has extensive application prospects in the biomedical field. The inherent disadvantages of traditional polyclonal antibody and monoclonal antibody limit their application values. The humanized and fragmented antibody remodeling has given a rise to a series of genetic engineered antibody variant. This paper reviews the progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases.

Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

1986-01-01

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin.

PubMed Central

Little, S F; Leppla, S H; Cora, E

1988-01-01

Thirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin have been characterized for affinity, antibody subtype, competitive binding to antigenic regions, and ability to neutralize lethal and edema toxin activities. At least 23 antigenic regions were detected on protective antigen by a blocking, enzyme-linked immunosorbent assay. Two clones, 3B6 and 14B7, competed for a single antigenic region and neutralized the activity of both the lethal toxin in vivo (Fisher 344 rat) and the edema toxin in vitro (CHO cells). These two antibodies blocked the binding of 125I-labeled protective antigen to FRL-103 cells. Our results support the proposal that binding of protective antigen to cell receptors is required for expression of toxicity. Images PMID:3384478

Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings

NASA Technical Reports Server (NTRS)

Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.

1988-01-01

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

A monoclonal antibody inhibits calcium accumulation and skeleton formation in cultured embryonic cells of the sea urchin.

PubMed

Carson, D D; Farach, M C; Earles, D S; Decker, G L; Lennarz, W J

1985-06-01

The assembly of the spicules (primitive skeleton) of the sea urchin embryo is being studied in primary mesenchyme cells cultured in vitro. A monoclonal antibody (1223) has been prepared that inhibits the deposition of CaCO3 into the spicules. This antibody reacts with a 130,000 Mr cell-surface protein that is concentrated on the surface of approximately 5% of the cells of dissociated gastrula stage embryos. When primary mesenchyme cells in the embryo or cells cultured in vitro are examined, the 1223 antigen is detected on the surface of the cells and on the extracellular material associated with the spicule. We conclude that the 1223 antibody recognizes a cell-surface protein that plays an essential role in spicule formation.

Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

PubMed

Ranade, V V

1989-10-01

Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera for studies of chromosome structure and function, gene mapping, embryogenesis, characterization and biosynthesis of developmental and differentiation antigens. These antigens are those that are specific for various cell types and tissues, species specific antigen, antigens involved in chemotaxis, immunogenetics and clinical genetics including genetically inherited disorders, chromosome aberrations and transplantation antigens. Besides these monoclonal antibodies, their complexes have recently been investigated as exquisitely sensitive probes to be guided to target cells or organs. They have been used to deliver cytotoxic drugs to malignant cells or enzymes to specific cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

[Establishment of A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and its preliminary application].

PubMed

Cai, Yu-Chun; Chen, Shao-Hong; Tian, Li-Guang; Chu, Yan-Hong; Lu, Yan; Chen, Mu-Xin; Ai, Lin; Zhou, Yang; Chen, Jia-Xu

2014-02-01

To establish A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. The characteristics of A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentration of A1E3-HRP. Under the optimal conditions, the serum samples of 20 acute schistosomiasis cases, 46 chronic schistosomiasis cases, and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy-two antibody positive serum samples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit, to evaluate the detection effects of this method. The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 contained a clear heavy chain with molecular weight of 88,000 and 52,000 respectively and had the same light chain with molecular weight of 20,000; while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schistosomiasis cases. The SEA-based ELISA demonstrated the titers of B1C4 and A1E3 were 1:10(5) and 1:30,000, respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA for detecting the circulating antigen were 45.8% and 43.1% respectively, and there was no significant difference between the results of the two methods. A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.

Seeing is believing: anti-PD-1/PD-L1 monoclonal antibodies in action for checkpoint blockade tumor immunotherapy

PubMed Central

Tan, Shuguang; Zhang, Catherine W-H; Gao, George F

2016-01-01

Structural immunology, focusing on structures of host immune related molecules, enables the immunologists to see what the molecules look like, and more importantly, how they work together. Antibody-based PD-1/PD-L1 blockade therapy has achieved brilliant successes in clinical applications. The recent breakthrough of the complex structures of checkpoint blockade antibodies with their counterparts, pembrolizumab with PD-1 and avelumab with PD-L1, have made it clear how these monoclonal antibodies compete the binding of PD-1/PD-L1 and function to blockade the receptor-ligand interaction. Herein, we summarize the structural findings of these two reports and look into the future for how this information would facilitate the development of more efficient PD-1/PD-L1 targeting antibodies, small molecule drugs, and other protein or non-protein inhibitors. PMID:29263905

Serodiagnosis of parasitic diseases.

PubMed Central

Maddison, S E

1991-01-01

In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens. PMID:1747862

Generation and Characterization of a New Monoclonal Antibody Against CXCL4.

PubMed

Gao, Jing; Wu, Mingyuan; Gao, Jin; Wang, Xia; Zhang, Yang; Zhu, Shunying; Yu, Yan; Han, Wei

2015-04-01

CXCL4 plays important roles in numerous disease processes, which makes the CXCL4 signaling pathway a potential therapeutic target. In this study, we aimed to develop a neutralizing antibody against both human and mouse CXCL4. Rats were immunized with recombinant human CXCL4 (rhCXCL4). Hybridoma clones were created by fusion of the immunized rat spleen cells with mouse myeloma SP2/0 cells and screened using recombinant mouse CXCL4 (rmCXCL4) and rhCXCL4. The CXCL4 monoclonal antibody (CXCL4 MAb) produced by the 16D6-3 hybridoma clone was sequenced and characterized by Western blot and Biacore assays. It recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in vitro. Thus, the antibody may be used in the studies of CXCL4 in murine disease models and as a template in the antibody humanization for clinical developments.

Production of monoclonal antibody for the detection of meat and bone meal in animal feed.

PubMed

Kim, Shin-Hee; Huang, Tung-Shi; Seymour, Thomas A; Wei, Cheng-i; Kempf, Stephen C; Bridgman, C Roger; Clemens, Roger A; An, Haejung

2004-12-15

For the detection of prohibited meat and bone meal (MBM) in animal feed, monoclonal antibodies (MAbs) were raised against heat-stable h-caldesmon purified from bovine intestinal smooth muscle. The obtained hybridoma cells were screened against extracts of the bovine MBM and heat-treated smooth muscle, and MAb 5E12 was identified as having the best performance. Antibody 5E12 did not react with animal feed, milk product, plant proteins, and other ingredients used for commercial animal feed except for the gelatin. This antibody diluted to 100-fold was able to detect MBM mixed in animal feed at 0.05% in an ELISA, and it showed strong affinity toward bovine smooth muscle autoclaved at 130 degrees C. Therefore, this antibody can be used in the ELISA system for field testing of the presence of MBM in animal feed.

Demonstration of the salmonid humoral response to Renibacterium salmoninarum using a monoclonal antibody against salmonid immunoglobulin

USGS Publications Warehouse

Bartholomew, J.L.; Arkoosh , M.R.; Rohovec, J.S.

1991-01-01

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytschawas compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarumpreparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarumantibody.

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

PubMed

Saito, Maiko; Kurosawa, Yae; Okuyama, Tsuneo

2012-02-01

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.

Insulin Action is Blocked by a Monoclonal Antibody That Inhibits the Insulin Receptor Kinase

NASA Astrophysics Data System (ADS)

Morgan, David O.; Ho, Lisa; Korn, Laurence J.; Roth, Richard A.

1986-01-01

Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay.

PubMed

Shankaran, Dhesingh Ravi; Kawaguchi, Toshikazu; Kim, Sook Jin; Matsumoto, Kiyoshi; Toko, Kiyoshi; Miura, Norio

2006-11-01

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNPh-KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh-KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.

Generation and Partial Characterization of Rabbit Monoclonal Antibody to Amyloid-β Peptide 1-37 (Aβ37).

PubMed

Mehta, Pankaj D; Blain, Jean-Francois; Freeman, Emily A; Patrick, Bruce A; Barshatzky, Marc; Hrdlicka, Lori A; Mehta, Sangita P; Frackowiak, Janusz; Mazur-Kolecka, Bozena; Wegiel, Jerzy; Patzke, Holger; Miller, David L

2017-01-01

Secreted soluble amyloid-β 1-37 (Aβ37) peptide is one of the prominent Aβ forms next to Aβ40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aβ37 levels in combination with Aβ38, Aβ40, and Aβ42 to support the diagnosis of patients with probable Alzheimer's disease (AD), and the value of antibody to Aβ37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aβ37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aβ37, and 2) to determine whether the antibody detects changes in Aβ37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aβ37 was found to be specific to Aβ37, since it did not react with Aβ36, Aβ38, Aβ39, Aβ40, and Aβ42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aβ37. The antibody was sensitive enough to measure CSF and plasma Aβ37 levels in ELISA. Immunohistological studies showed the presence of Aβ37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aβ37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.

[Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice].

PubMed

Wang, Xiaolin; Chi, Xiangyang; Liu, Ju; Liu, Weicen; Liu, Shuling; Qiu, Shunfang; Wen, Zhonghua; Fan, Pengfei; Liu, Kun; Song, Xiaohong; Fu, Ling; Zhang, Jun; Yu, Changming

2016-11-25

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

The Clinical Proteomic Technologies for Cancer | Antibody Portal

Cancer.gov

An objective of the Reagents and Resources component of NCI's Clinical Proteomic Technologies for Cancer Initiative is to generate highly characterized monoclonal antibodies to human proteins associated with cancer.