Dissociation of monoclonal antibody-antigen complexes: Implications for ELISA procedures

A single step solid phase radioimmunoassay (SS-SPRIA) has been developed for human chorionic,gonadotropin (hCG) using monoclonal antibodies (MAb) from culture media adsorbed immunochemically on plastic tubes. The assays have been found to be very simple in terms of operation and do not demand purification of MAbs. Several MAbs which do not show any displacement in liquid phase RIA and ELISA provide a satisfactory SS-SPRIA. Our investigations revealed that the assumption regarding the stability of the primary Mab-Ag complex during incubation and washing steps in ELISAs is not strictly valid for dissociable MAbs. A comparison of different assay systems suggests that the single step SPRIA offers additional advantages over conventionally used multistep ELISA procedures and provides a quantitative probe for the analysis of epitope-paratope interactions.

Realtime kinetic analysis of antigen-antibody interaction using solid phase binding: Transformation of hCG-monoclonal antibody complex

Kinetic constants of MAb-hCG interactions have been determined using solid phase binding of I-125[hCG] to immobilized MAb. While association has been shown to follow the expected pattern, dissociation consists of at least two reversible steps, one with a rate constant of 0.0025 min(-1), and a second with a rate constant of 0.00023 min(-1). Validity of affinity constant measurements in the light of the complex reaction kinetics is discussed, A comparison between the method of surface plasmon resonance technology (BIAcore) and solid phase binding (SPB) for determination of kinetic parameters shows that SPB provides not only a cost-effective approach for determination of realtime kinetic parameters of macromolecular ligand-ligate interaction but also a method with several advantages over the BIAcore system in investigating the mechanism of antigen-antibody interaction.

Production of biosurfactant “Biosur-Pm” by Pseudomonas maltophila CSV89: characterization and role in hydrocarbon uptake

Pseudomonas maltophilia CSV89, a soil bacterium, produces an extracellular biosurfactant, ''Biosur-Pm''. The partially purified product is nondialyzable and chemically composed of 50% protein and 12-15% sugar, which indicates the complex nature of Biosur-Pm. It reduces the surface tension of water from 73 to 53 x 10(-3) N m(-1) and has a critical micellar concentration of 80 mg/l. Compared to aliphatic hydrocarbons, Biosur-Pm shows good activity against aromatic hydrocarbons. The emulsion formed is stable and does not require any metal ions for emulsification. The kinetics of Biosur-Pm production suggest that its synthesis isa growth-associated and pH-dependent process. At pH 7.0, cells produced more Biosur-Pm with less cell surface hydrophobicity. At pH 8.0, however, the cells produced less Biosur-Pm with more cell surface hydrophobicity and showed a twofold higher affinity for aromatic hydrocarbons compared to the cells grown at pH 7.0. The Biosur-Pm showed a pH-dependent release, stimulated growth of the producer strain on mineral salts medium with 1-naphthoic acid when added externally, and facilitated the conversion of salicylate to catechol. All these results suggest that Biosur-Pm is probably a cell-wall component and helps in hydrocarbon assimilation/uptake.

Molecular mimicry by antiidiotypic monoclonal antibody to gonadotropin releasing hormone

Antipeptide and antiidiotypic antibodies to several receptors are known to mimic their respective ligands in transducing signals on binding their receptors. In our attempts to study gonadotropin releasing hormone receptor, antipeptide and antiidiotypic monoclonal antibodies specific to the receptor were established earlier. The antipeptide mAb F1G4 was to a synthetic peptide corresponding to the extracellular domain of human GnRH receptor and the antiidiotypic mAb 4D10C1 was to the idiotype of a GnRH specific mAb. Here we report the physiological effects of the two mAbs on binding the receptor, as investigated using in vitro cultures of(a) human term placental villi and (b) rat pituitaries. The mAb 4D10C1 exerted a dose-dependent release of human chorionic gonadonopin in cultures of human term placental villi as well as luteinising and follicle stimulating hormones in cultures of rat pituitaries.

Real-time kinetic analysis of hCG-monoclonal antibody interaction using radiolabeled hCG probe: presence of two forms of Ag-mAb complex as revealed by solid phase dissociation studies

Real-time kinetics of ligand-ligate interaction has predominantly been studied by either fluorescence or surface plasmon resonance based methods. Almost all such studies are based on association between the ligand and the ligate. This paper reports our analysis of dissociation data of monoclonal antibody-antigen (hCG) system using radio-iodinated hCG as a probe and nitrocellulose as a solid support to immobilize mAb. The data was analyzed quantitatively for a one-step and a two-step model. The data fits well into the two-step model. We also found that a fraction of what is bound is non-dissociable (tight-binding portion (TBP)). The TBP was neither an artifact of immobilization nor does it interfere with analysis. It was present when the reaction was carried out in homogeneous solution in liquid phase. The rate constants obtained from the two methods were comparable. The work reported here shows that real-time kinetics of other ligand-ligate interaction can be studied using nitrocellulose as a solid support. (C) 2002 Elsevier Science B.V. All rights reserved.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV AP The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavagereligation equilibrium, increasing single-strand nicks and proteinDNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.

A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

Uptake of permanganate from aqueous environment by surfactant modified montmorillonite batch and fixed bed studies

Organo-clay was prepared by incorporating different amounts (in terms of CEC, ranging from 134-840 mg of quaternary ammonium cation (QACs) such as hexadecytrimethylammonium bromide (C19H42N]Br) into the montmorillonite clay. Prepared organo-clays are characterized by CHN analyser and XRD to measure the amount of elemental content and interlayer spacing of surfactant modified clay. The batch experiments of sorption of permanganate from aqueous media by organo-clays was studied at different acidic strengths (pH 1-7). The experimental results show that the rate and amount of adsorption of permanganate was higher at lower pH compared to raw montmorillonite. Laboratory fixed bed experiments were conducted to evaluate the breakthrough time and nature of breakthrough curves. The shape of the breakthrough curves shows that the initial cationic surfactant loadings at 1.0 CEC of the clay is enough to enter the permanganate ions in to the interlamellar region of the surfactant modified smectile clays. These fixed bed studies were also applied to quantify the effect of bed-depth and breakthrough time during the uptake of permanganate. Calculation of thermodynamic parameters shows that the sorption of permanganate is spontaneous and follows the first order kinetics.

IgG2 subclass isotype antibody and intrauterine infections

The foetus is dependent on its mother for passive immunity involving receptor-mediated specific transport of antibodies. IgG antibody is present in highest concentration in serum and is the only antibody type that can cross the placenta efficiently, except for its IgG2 subclass. Most of the pathogenic manifestations affecting the foetus involve capsular antigens and polysaccharides of pathogens and it is known that immune response to these antigens is primed to the predominant production of IgG2 type of antibody. Paradoxically, the IgG2 subclass cannot cross the placenta and neutralize such antigens; therefore, infections related to these antigens may persist and can lead to serious conditions like miscarriage and stillbirth. This article describes in brief the properties of IgG subclasses, intrauterine infections seen during pregnancy and discusses possible IgG-based strategies to manage infections to afford protection to the foetus.

Adsorption on Edge-Functionalized Bilayer Graphene Nanoribbons: Assessing the Role of Functional Groups in Methane Uptake

A combination of ab initio and classical Monte Carlo simulations is used to investigate the effects of functional groups on methane binding. Using Moller-Plesset (MP2) calculations, we obtain the binding energies for benzene functionalized with NH2, OH, CH3, COOH, and H2PO3 and identify the methane binding sites. In all cases, the preferred binding sites are located above the benzene plane in the vicinity of the benzene carbon atom attached to the functional group. Functional groups enhance methane binding relative to benzene (-6.39 kJ/mol), with the largest enhancement observed for H2PO3 (-8.37 kJ/mol) followed by COOH and CH3 (-7.77 kJ/mol). Adsorption isotherms are obtained for edge-functionalized bilayer graphene nanoribbons using grand canonical Monte Carlo simulations with a five-site methane model. Adsorbed excess and heats of adsorption for pressures up to 40 bar and 298 K are obtained with functional group concentrations ranging from 3.125 to 6.25 mol 96 for graphene edges functionalized with OH, NH2, and COOH. The functional groups are found to act as preferred adsorption sites, and in the case of COOH the local methane density in the vicinity of the functional group is found to exceed that of bare graphene. The largest enhancement of 44.5% in the methane excess adsorbed is observed for COOH-functionalized nanoribbons when compared to H terminated ribbons. The corresponding enhancements for OH- and NH2-functionalized ribbons are 10.5% and 3.7%, respectively. The excess adsorption across functional groups reflects the trends observed in the binding energies from MP2 calculations. Our study reveals that specific site functionalization can have a significant effect on the local adsorption characteristics and can be used as a design strategy to tailor materials with enhanced methane storage capacity.

Cellular uptake and remarkable photocytotoxicity of pyrenylter pyridine oxovanadium(IV) complexes of dipyridophenazine bases

Pyrenylterpyridine (pytpy) oxovanadium(IV) complexes VO(pytpy)(L)]Cl-2 (1-6) of the dipyridophenazine bases (L), viz., dipyrido-6,7,8,9-tetrahydrophenazine (dpqC in 1), dipyrido3,2-a:2',3'-c]phenazine-2-carboxylic acid (dppzc in 2), dipyrido3,2-a:2',3'-c]phenazine-11-sulfonic acid (dppzs in 3), 7-aminodipyrido3,2-a:2',3'-c]phenazine (dppza in 4), benzo-i]dipyrido3,2-a:2',3'-c]phenazine (dppn in 5) and dipyrido3,2-a:2',3'-c]phenazine (dppz in 6) were prepared, characterized and their DNA binding, photocleavage activity and photocytotoxicity studied. The complexes which showed a d-d band near 750 nm in DMF are efficient binders to calf thymus DNA (K-b: 3.2 x 10(5)-2.9 x 10(6) M-1). The complexes showed significant pUC19 DNA cleavage in near-IR light of 785 nm forming center dot OH radicals and photocytotoxicity in HeLa cells in visible light with the benzo-i] dipyrido3,2-a:2',3'-c]phenazine complex 5 showing a remarkably low IC50 value of 0.036 mu M. Flow-cytometric analysis shows a high sub-G1 phase cell cycle arrest in HeLa cells by the complexes on photo-irradiation. The photocytotoxicity correlates well with the hydrophobicity, photosensitizing ability and DNA binding propensity of the complexes. (C) 2012 Elsevier B.V. All rights reserved.

Isolation of a High Affinity Neutralizing Monoclonal Antibody against 2009 Pandemic H1N1 Virus That Binds at the `Sa' Antigenic Site

Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs) have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K-D = 2.1 +/- 0.4 pM) murine MAb, MA2077 that binds specifically to the hemagglutinin (HA) surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC50 = 0.08 mu g/ml). MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 mu g/ml) against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic) on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein `Sa' and `Sb' sites were independently mutated, localized the binding site of MA2077 within the `Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

Antibody-mediated catalysis: Induction and therapeutic relevance

Abzymes are immunoglobulins endowed with enzymatic activities. The catalytic activity of an abzyme resides in the variable domain of the antibody, which is constituted by the close spatial arrangement of amino acid residues involved in catalysis. The origin of abzymes is conferred by the innate diversity of the immunoglobulin gene repertoire. Under deregulated immune conditions, as in autoimmune diseases, the generation of abzymes to self-antigens could be deleterious. Technical advancement in the ability to generate monoclonal antibodies has been exploited in the generation of abzymes with defined specificities and activities. Therapeutic applications of abzymes are being investigated with the generation of monoclonal abzymes against several pathogenesis-associated antigens. Here, we review the different contexts in which abzymes are generated, and we discuss the relevance of monoclonal abzymes for the treatment of human diseases.

Mechanistic Insights into the Neutralization of Cytotoxic Abrin by the Monoclonal Antibody D6F10

Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1: 10 molar concentration of abrin: antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.