A monoclonal antibody recognizing the C-terminal region of chicken egg white riboflavin carrier protein terminates early pregnancy in mice

In order to identify the functionally relevant epitopes on chicken riboflavin carrier protein, we have raised monoclonal antibodies to the vitamin carrier. One of these, 6B2C12, was found to interact specifically with a synthetic oligopeptide corresponding to the C-terminal 17 amino acid residues of the chicken egg white riboflavin carrier protein, which is missing in part in the egg yolk riboflavin carrier protein. This epitope is conserved through evolution in mammals including humans. Administration of the ascites fluid of 6B2C12 to pregnant mice intraperitoneally, resulted in the termination of pregnancy indicating that this epitope is involved in or closely associated with the transplacental transport of the vitamin from the maternal circulation to the growing fetus.

Functional Mimics of Glutathione Peroxidase: Bioinspired Synthetic Antioxidants

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the biological system's ability to detoxify these reactive intermediates. Mammalian cells have elaborate antioxidant defense mechanisms to control the damaging effects of ROS. Glutathione peroxidase (GPx), a selenoenzyme, plays a key role in protecting the organism from oxidative damage by catalyzing the reduction of harmful hydroperoxides with thiol a ``catalytic triad'' with tryptophan and glutamine, which cofactors. The selenocysteine residue at the active site forms activates the selenium moiety for an efficient reduction of peroxides. After the discovery that ebselen, a synthetic organoselenium compound, mimics the catalytic activity of GPx both in vitro and in vivo, several research groups developed a number of small-molecule selenium compounds as functional mimics of GPx, either by modifying the basic structure of ebselen or by incorporating some structural features of the native enzyme. The synthetic mimics reported in the literature can be classified in three major categories: (i) cyclic selenenyl amides having a Se-N bond, (ii) diaryl diselenides, and (iii) aromatic or aliphatic monoselenides. Recent studies show that ebselen exhibits very poor GPx activity when aryl or benzylic thiols such as PhSH or BnSH are used as cosubstrates. Because the catalytic activity of each GPx mimic largely depends on the thiol cosubstrates used, the difference in the thiols causes the discrepancies observed in different studies. In this Account, we demonstrate the effect of amide and amine substituents on the GPx activity of various organoselenium compounds. The existence of strong Se ... O/N interactions in the selenenyl sulfide intermediates significantly reduces the GPx activity. These interactions facilitate an attack of thiol at selenium rather than at sulfur, leading to thiol exchange reactions that hamper the formation of catalytically active selenol. Therefore, any substituent capable of enhancing the nucleophilic attack of thiol at sulfur in the selenenyl sulfide state would enhance the antioxidant potency of organoselenium compounds. Interestingly, replacement of the sec-amide substituent by a tert-amide group leads to a weakening of Se ... 0 interactions in the selenenyl sulfide intermediates. This modification results in 10- to 20-fold enhancements in the catalytic activities. Another strategy involving the replacement of tert-amide moieties by tert-amino substituents further increases the activity by 3- to 4-fold. The most effective modification so far in benzylamine-based GPx mimics appears to be either the replacement of a tert-amino substituent by a sec-amino group or the introduction of an additional 6-methoxy group in the phenyl ring. These strategies can contribute to a remarkable enhancement in the GPx activity. In addition to enhancing catalytic activity, a change in the substituents near the selenium moiety alters the catalytic mechanisms. The mechanistic investigations of functional mimics are useful not only for understanding the complex chemistry at the active site of GPx but also for designing and synthesizing novel antioxidants and anti-inflammatory agents.

Effect of a specific estrogen antibody on pregnancy establishment in the bonnet monkey (Macaca radiata)

The requirement for estrogen for pregnancy establishment has not been conclusively demonstrated in primates. Selective neutralization of estrogens was achieved in mated female monkeys during preimplantation and postimplantation periods by injecting characterized estrogen antiserum from either day 14 to 18 or day 28 to 32 of cycle. While estrogen deprivation during preimplantation period in 5 animals exposed to 14 ovulatory cycles resulted in only one pregnancy, only 3 of 13 monkeys treated during postimplantation period continued pregnancy to term. In comparison with controls (4 of 5 monkeys becoming pregnant), the percent protection against pregnancy in animals treated during preimplantation period was 93. The pregnancy termination in 10 of 13 monkeys treated during postimplantation period when compared with normal postimplantation pregnancy wastage in our colony (2%) is also highly significant (P less than 0.01). The present study demonstrates a critical need for estrogen during the peri-implantation period for a successful pregnancy establishment in primates.

Detection of IgG, IgA, IgM and IgE Antibodies in Invasive Amoebiasis in Endemic Areas

Entamoeba histolytica-specific serum IgG, IgA, IgM and IgE antibodies were assayed in cases of amoebiasis in an endemic area. Patient groups consisted of amoebic liver abscess (n=18), pre-abscess hepatic amoebiasis (n=22) and amoebic colitis (n=30). Control subjects comprised 26 asymptomatic cyst passers, 13 giardiasis cases, 20 typhoid patients and 24 non-amoebic individuals. Serum IgG was assayed by ELISA, using a monoclonal anti IgG β- galactosidase (IgG β-gal) conjugate, a polyclonal avidin biotin horse radish peroxidase (AB-HRP), and a polyclonal anti IgG horse radish peroxidase (IgG HRP) conjugate. IgA and IgM were assayed by the β-gal ELISA and IgE by AB-HRP. Diagnostically significant IgG and IgA while lower IgM and IgE antibody levels were seen in extraintestinal cases. About 40% of suspected pre-abscess hepatic amoebiasis cases were confirmed by antibody estimation. All isotype levels in most dysentery cases were in the range of the controls.

Supermacroporous Polymer-Based Cryogel Bioreactor for Monoclonal Antibody Production in Continuous Culture Using Hybridoma Cells

Cryogel matrices composed of different polymeric blends were synthesized, yielding a unique combination of hydrophilicity and hydrophobicity with the presence or absence of charged surface. Four such cryogel matrices composed of polyacrylamide-chitosan (PAAC), poly(N-isopropylacrylamide)-chitosan, polyacrylonitrile (PAN), and poly(N-isopropylacrylamide) were tested for growth of different hybridoma cell lines and production of antibody in static culture. All the matrices were capable for the adherence of hybridoma cell lines 6A4D7, B7B10, and H9E10 to the polymeric surfaces as well as for the efficient monoclonal antibody (mAb) production. PAAC proved to be relatively better in terms of both mAb production and cell growth. Further, PAAC cryogel was designed into three different formats, monolith, disks, and beads, and used as packing material for packed-bed bioreactor. Longterm cultivation of 6A4D7 cell line on PAAC cryogel scaffold in all the three formats could be successfully done for a period of 6 weeks under static conditions. Continuous packed-bed bioreactor was setup using 6A4D7 hybridoma cell line in the three reactor formats. The reactors ran continuously for a period of 60 days during which mAb production and metabolism of cells in the bioreactors were monitored periodically. The monolith bioreactor performed most efficiently over a period of 60 days and produced a total of 57.5 mg of antibody in the first 30 days (in 500 mL) with a highest concentration of 115 mu g mL(-1), which is fourfold higher than t-flask culture. The results demonstrate that appropriate chemistry and geometry of the bioreactor matrix for cell growth and immobilization can enhance the reactor productivity. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 170-180, 2011

Augmentation of murine natural killer cell and antibody dependent cellular cytotoxicity activities by phyllanthus emblica, a new immunomodulator

When administered orally, Phyllanthus emblica, an excellent source of vitamin C (ascorbate), has been found to enhance natural killer (NK) cell activity and antibody dependent cellular cytotoxicity (ADCC) in syngeneic BALB/c mice, bearing Dalton's lymphoma ascites (DLA) tumor. P. emblica elicited a 2-fold increase in splenic NK cell activity on day 3 post tumor inoculation. Enhanced activity was highly significant on days 3, 5, 7 and 9 after tumor inoculation with respect to the untreated tumor bearing control. A significant enhancement in ADCC was documented on days 3, 7, 9, 11 and 13 in drug treated mice as compared to the control. An increase in life span (ILS) of 35% was recorded in tumor bearing mice treated with P. emblica. This increased survival was completely abrogated when NK cell and killer (K) cell activities were depleted either by cyclophosphamide or anti-asialo-GM, antibody treatment. These results indicate: (a) an absolute requirement for a functional NK cell or K cell population in order that P. emblica can exert its effect on tumor bearing animals, and (b) the antitumor activity of P. emblica is mediated primarily through the ability of the drug to augment natural cell mediated cytotoxicity.

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish peroxidase and H2O2 and decreased the ratio of scopoletin (consumed):H2O2 (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH: O-2 of 2:1. On step-wise addition of a small concentration of H2O2 a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H2O2 ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound;d accumulated during oxidation of NADH. The effectiveness of a small concentration of H2O2 in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H2O2 during scopolerin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab Ea-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small nonnuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex. (C) 1994 Academic Press, Inc.

Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

Antigenic determinants at the carboxy terminus of chicken egg white riboflavin carrier protein (RCP): Epitope mapping and antibody-mediated pregnancy curtailment in rodents

The epitopic core sequences recognized by three monoclonal antibodies raised to chicken riboflavin carrier protein (RCP) were mapped to the C-terminal tail-end of the protein using the pepscan method A 21-residue synthetic peptide corresponding to residues 200-219 of the protein and comprising the regions corresponding to the antibodies was synthesized. Administration of polyclonal antibodies specific to this peptide led to termination of early pregnancy in mice. Also, active immunization of rats with the peptide-purified protein derivative conjugate inhibited establishment of pregnancy. These results demonstrate the functional importance of the C-terminal 200-219 region of chicken RCP. Copyright (C) 1996 Elsevier Science Ltd.

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.

Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: Results of a pilot study

A study of 140 days duration was performed to examine if human male volunteers (n = 5) respond to ovine follicle stimulating hormone (oFSH) immunization (administered adsorbed on Alugel on days 1, 20, 40 and 70) by producing antibodies capable of both binding and neutralizing bioactivity of human FSH. The kinetics of antibody production for both the immunogen (oFSH) and the cross-reactive antigen (hFSH) were essentially similar, The volunteers responded only to the first two immunizations, The boosters given on days 40 and 70 were ineffective, probably because of the presence of substantial amounts of circulating antibody to oFSH. Of the antibodies generated to oFSH, 25-45% bound hFSH with a mean binding affinity of 0.65 x 10(9) +/- 0.53 M(-1). The binding capacities at the time of high (30-80 days of immunization) and low (>110 days) titres were 346 +/- 185 and 10.5 +/- 5.8 ng hFSH/ml respectively, During the period of high titre, free serum FSH (value in normal males 1-5 ng/ml) was not monitorable, A 50 mu l aliquot of the antiserum obtained from different volunteers between days 30 and 80 and on day 140 blocked binding of I-125-labelled hFSH to its receptor by 82 +/- 9.7 and 53 +/- 12.2% respectively, The antibody produced was specific for FSH, and no significant change in the values of related glycoprotein hormones (luteinizing hormone/testosterone and thyroid stimulating hormone/thyroxine) were recorded, Seminal plasma transferrin, a marker of Sertoli cell as well as of seminiferous tubular function, showed marked reduction (30-90%) following immunization with oFSH. Considering that endogenous FSH remained neutralized for approximately one sperm cycle only (65 days), the reduction in sperm counts (30-74%) exhibited by some volunteers is encouraging, Immunization with oFSH did not result in any significant changes in haematology, serum biochemistry or hormonal profiles, There was no production of antibodies capable of interacting with non-specific tissues, It is concluded that it should be possible to obtain a sustained long-term blockade of endogenous FSH action in men by using oFSH as an immunogen, This is a prerequisite for obtaining significant reduction in the quality and quantity of spermatozoa produced, thus leading to infertility.

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.