Acid pH promotes bispecific antibody formation by the redox procedure.

Bispecific antibodies (BsAbs), are potential theranostics. Chemical procedures of preparation of BsAbs, in which two monospecific antibodies are split into half molecules and heterodimerized, continue to attract attention in view of their simplicity. Poor dissociation of antibodies with reduced inter-heavy chain disulfides into half molecules under neutral conditions however restricts the BsAbs formation. In this study, we report that the heterodimerization of antibodies can be improved leading to over 6-fold increase in the yield of BsAbs, by carrying out the redox procedure at pH 4.0. In view of improvement in heterodimerization, BsAbs could be conveniently prepared starting from partially purified ion-exchange fraction of the antiserum and purified by twin affinity chromatography on antigen supports. The UV, CD, intrinsic and extrinsic fluorescence spectral analysis of BsAbs prepared by the modified redox procedure were comparable with the native IgG, which suggest the absence of significant acid-pH-induced damage. ThT binding studies and native size exclusion chromatography ruled out amyloid fibril formation.

Augmenting the cytotoxicity of oleic acid-protein complexes: Potential of target-specific antibodies.

HAMLET (Human Alpha-Lactalbumin Made LEthal to Tumor cells), a complex of oleic acid (OA) with partially unfolded human α-lactalbumin, shows remarkable toxicity towards a spectrum of tumor cells as well as few differentiated cells including mammalian erythrocytes. Human erythrocytes, for this reason, have been used as convenient model cells to study toxic properties of the OA complexes. The toxicity of HAMLET-like complexes, prepared using immunoglobulin gamma (IgG) isolated from the sera of rabbits immunized with human erythrocytes as well as those unimmunized, towards the red cells was investigated. The OA complex of the IgG prepared by the heat-treatment procedure comprised of protein monomers and oligomers with bound OA. The IgG in the complexes retained most secondary but only partial tertiary structure and complex formation with OA did not abolish the ability of anti-erythrocyte IgG to bind to the erythrocytes. Anti-erythrocyte IgG-OA complexes were remarkably more hemolytic than those prepared using non-specific IgG, while complexes prepared using affinity purified anti-erythrocyte IgG were most effective in hemolyzing the cells. The work suggests that antibodies that exhibit affinity towards target cells may be useful in the preparation of selective and highly toxic OA complexes for the cells.

A detergent-based procedure for the preparation of IgG-like bispecific antibodies in high yield.

Bispecific antibodies (BsAbs), with the ability to recognize two different epitopes simultaneously, offer remarkable advantages in bioassays, cancer therapy, biosensors, and enzyme electrodes. Preparation and purification of BsAbs in adequate quantities remains a major hurdle in their use in various applications. Poor yield is also the principal limitation in the preparation of BsAbs by the redox procedure. IgG with reduced inter-heavy chain disulfides do not dissociate into half molecules at neutral pH. In this study, we report that the dissociation occurs in presence of sodium dodecyl sulphate (SDS) and inclusion of the detergent during the redox procedure results in remarkable increase in the formation of the BsAbs. Exposure of antibodies to 0.1% (w/v) SDS causes only minor loss in secondary/tertiary structure and the ability to bind the antigen. The BsAbs prepared using the modified redox procedure that recognize the antigens HRP and α-LA were prepared and successfully employed for detecting α-LA in milk/dairy products by ELISA and dot blot techniques. BsAbs were also prepared from partially purified immunoglobulin gamma (IgG). This work shows for the first time that SDS, by dissociating IgG with reduced inter-heavy chain disulfides into half molecules, markedly enhances the formation of BsAbs by the redox procedure.

Behaviour of oleic acid-depleted bovine alpha-lactalbumin made LEthal to tumor cells (BAMLET).

Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effective in the killing of a variety of tumor cells. While debate on whether the key component of the complexes is the OA or protein continues, studies probing the mechanism of action of the complexes at the tumor cell surface or in the cell interior assume the action of a molecule in the form of an undissociated complex. Recent evidence however suggests that OA complexes of protein are stripped of bound OA on interaction with artificial or natural membranes before entering the cell. Properties of BAMLET stripped of its OA by exposure to erythrocytes (ET-BAMLET) were investigated in the study. ET-BAMLET resembled α-LA in its inability to induce hemolysis of erythrocytes and behaviour in a gel filtration column. Spectroscopy techniques-fluorescence, far- and near UV CD as well as calorimetry and proteolysis however suggest the molecule to be different both from native α-LA and the apo form. Remarkably, unlike native α-LA and apo-α-LA, ET-BAMLET binds OA and turns hemolytic by simple mixing with the fatty acid around neutral pH. Since BAMLET/HAMLET incubated cells take up large amounts of OA, the study suggests the possibility of ET-BAMLET combining with OA and reforming the complex inside the cells.

Oleic acid complex of bovine α-lactalbumin induces eryptosis in human and other erythrocytes by a Ca(2+)-independent mechanism.

BACKGROUND: Complexes of oleic acid (OA) with milk α-lactalbumin, received remarkable attention in view of their selective toxicity towards a spectrum of tumors during the last two decades. OA complexes of some structurally related/unrelated proteins are also tumoricidal. Erythrocytes are among the few differentiated cells that are sensitive and undergo hemolysis when exposed to the complexes. METHODS: The effects of OA complex of bovine α-lactalbumin (Bovine Alpha-lactalbumin Made LEthal to Tumor cells, BAMLET) on human, goat and chicken erythrocytes on calcein leakage, phosphatidylserine exposure, morphological changes and hemolysis were studied by confocal microscopy, FACS analysis, scanning electron microscopy and measuring hemoglobin release. RESULTS: Erythrocytes exposed to BAMLET undergo eryptosis-like alterations as revealed by calcein leakage, surface phosphatidylserine exposure and transformation to echinocytes at low concentrations and hemolysis when the concentration of the complex was raised. Ca(2+) was not essential and restricted the alterations when included in the medium. The BAMLET-induced alterations in human erythrocytes were prevented by the cation channel inhibitors, amiloride and BaCl2 but not by inhibitors of thiol proteases, sphingomyelinase and by the antioxidant N-acetyl cysteine. CONCLUSIONS: The work shows for the first time that low concentrations of BAMLET induces eryptosis in erythrocytes by a novel mechanism not requiring Ca(2+) and hemolysis by detergent-like action by the released OA at higher concentrations. GENERAL SIGNIFICANCE: The study points out to the need for a comprehensive evaluation of the toxicity of OA complexes of α-lactalbumin and other proteins towards erythrocytes and other differentiated cells before being considered for therapy.

A rabbit eye model for in vivo transformation of progenetic metacercariae of Clinostomum complanatum into ovigerous adult worms.

Clinostomum complanatum is a digenetic trematode that causes yellow grub disease in some fish species and also shows zoonotic potential by sporadically infecting humans. In this study, progenetic metacercariae of C. complanatum were obtained from the fish Trichogaster fasciatus, and were aseptically placed in conjunctival incisions made in the superior and inferior fornices of the eye of rabbits, which served as the experimental hosts. Worms were harvested without necropsy of the host on days 4 and 8 post infection, to observe in vivo transformation of the progenetic metacercariae into ovigerous adult worms. The worms appeared to cause minimal damage to the host although they were tenaciously attached. In vivo maturation was evident by the development of the vitellaria, enlargement of gonads, the presence of a large number of shelled eggs in a distended uterus and ramifications of the intestinal caeca. Obtaining mature ovigerous worms without sacrificing the host clearly gives the rabbit eye model an advantage over those described previously. Due to the relative advantage of the short time required for maturation and the prolific egg production by C. complanatum, it is suggested that this host-parasite system could be used as an excellent model for classroom teaching of trematode biology and to investigate the cues involved in in vivo transformation and host-parasite interactions.

Plasma beads loaded with Candida albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice.

The development of a prophylactic vaccine against systemic candidiasis, employing Candida albicans cytosolic proteins (Cp) as antigen and fibrin cross-linked plasma beads as an antigen bearing dual delivery system is described. Groups of mice were administered either with free Cp, or Cp entrapped in plasma beads, Cp entrapped in liposomes or liposome encapsulated Cp further entrapped in plasma beads. Humoral immunity was studied by measuring the anti-Cp antibody titers in the sera of the immunized animals. Induction of cell-mediated immunity was assessed by delayed type hypersensitivity (DTH), NO production, up-regulation of co-stimulatory molecules viz. CD80, CD86 on APCs on one hand and T-cells proliferation as well as induction of IFN-γ and IL-4 on the other. The efficacy of various vaccine formulations in protecting mice against a lethal challenge with C. albicans, was assessed by determining animal survival rate and fungal burden in the systemic circulation and vital organs. Among various Cp-based vaccines investigated, the preparation containing liposomized Cp entrapped in plasma beads imparted superior protection in the immunized mice as compared to other antigens delivery systems.

Abandoning the ship: spontaneous mass exodus of Clinostomum complanatum (Rudolphi, 1814) progenetic metecercariae from the dying intermediate host Trichogaster fasciatus (Bloch & Schneider, 1801).

The dramatic and spontaneous exodus of live Clinostomum complanatum progenetic metacercaria from the gill slits of the dying intermediate host, Trichogaster fasciatus is reported. Basic water parameter tests for dissolved oxygen, pH and temperature revealed slightly lower level of dissolved oxygen in tank water used for water change. To the best of our knowledge, it is the first report of a digenean metacercariae, en mass leaving their intermediate host, upon its death in search of an alternative host to support their survival and help in continuing their life cycle.

Observations on in vitro and in vivo antimicrofilarial effects of Bishop's weed (Trachispermum ammi).

The antimicrofilarial efficacy of Trachispermum ammi extacts in vitro and in vivo using Setaria cervi as a model, was investigated. T. ammi seed extracts were prepared using different solvents (with increasing order of polarity of the solvent) including petroleum ether, diethyl ether, chloroform, ethyl acetate, acetone, ethanol, and methanol. The extracts were tested for in vitro antimicrofilarial activity. The ethanolic and the methanolic extracts showed maximum activity in causing flaccidity in the microfilariae. The extracts were potent even at concentrations as low as 5 µl/ml. When orally administered to experimentally infected rats, the extracts eliminated circulating microfilariae within 2 weeks. It is inferred that the antimicrofilarial molecule(s), are polar in nature. They induce flaccidity in the microfilariae, by possibly inhibiting monoamine oxidase. This communication supplements the ethnopharmacological information for the use of T. ammi as an antihelminthic, and indicates that T. ammi could be used as a potential source of antimicrofilarial drugs.

Levels of some antioxidant molecules and lipid peroxidation during in vivo transformation of the progenetic metacercaria of Clinostomum complanatum to ovigerous adult worms.

The levels of oxidative stress markers are an important indicator of the physiological state of the parasite and its host. In the present study levels of lipid peroxidation, glutathione S transferase, glutathione, superoxide dismutase and catalase were determined in the Clinostomum complanatum progenetic metacercaria, obtained from the fish peritoneum (a hypoxic habitat). The in vivo transformed ovigerous adult worms were obtained from the aerobic environment of the buccopharyngeal region of experimentally infected chickens. Levels of antioxidant molecules were also determined in the blood of experimentally infected chickens. An increase in the levels of lipid peroxidation, and a significant decrease in the levels of glutathione S transferase, glutathione, superoxide dismutase and catalase was observed in the infected host as compared to the controls. In the ovigerous worms, the levels of lipid peroxidation, glutathione S transferase, glutathione, superoxide dismutase were found to be significantly less than the levels observed in the progenetic metacercaria. Since the establishment of worm in the buccal cavity of the avian host would lead to its exposure to oxygen and the haematophagous nature of the parasite also exposes it to the free radicals in the host blood, the progenetic metacercaria has evolved to produce excess free radical scavenging molecules reserved to combat the oxidative stress encountered within the microhabitat of the definitive host.

A partially folded state of ovalbumin at low pH tends to aggregate.

At pH 2, ovalbumin retains native-like secondary structure as seen by far-UV CD and FTIR, but lacks well-defined tertiary structure as seen by the fluorescence and near-UV CD spectra. Addition of 20 mM Trifluoroacetic acid (TFA) or 30 mM Trichloroacetic acid (TCA) on acid-induced state results in protein aggregation. This aggregated state possesses extensive ß-sheet structure as revealed by far-UV CD and FTIR spectroscopy. Furthermore, the aggregates exhibit decreased ANS fluorescence and increased thioflavin T fluorescence. The presence of aggregates was confirmed by size exclusion chromatography. Such a formation of ß-sheet structure is found in the amyloid of a number of neurological diseases such as Alzheimer's and scrapie. Ovalbumin at low pH, in the presence of K(2)SO(4), exists in partially folded state characterized by native-like secondary structure and tertiary folds.

Compact acid-induced state of Clitoria ternatea agglutinin retains its biological activity.

The effects of pH on Clitoria ternatea agglutinin (CTA) were studied by spectroscopy, size-exclusion chromatography, and by measuring carbohydrate specificity. At pH 2.6, CTA lacks well-defined tertiary structure, as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of 50% native-like secondary structure. The mean residue ellipticity at 217 nm plotted against pH showed a transition around pH 4.0 with loss of secondary structure leading to the formation of an acid-unfolded state. This state is relatively less denatured than the state induced by 6 M guanidine hydrochloride. With a further decrease in pH, this unfolded state regains ~75% secondary structure at pH 1.2, leading to the formation of the A-state with native-like near-UV CD spectral features. Enhanced 8-anilino-1-naphthalene-sulfonate binding was observed in A-state, indicating a "molten-globule" like conformation with exposed hydrophobic residues. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a compact conformation at low pH. Size-exclusion chromatography shows the presence of a compact intermediate with hydrodynamic size corresponding to a monomer. Thermal denaturation of the native state was cooperative single-step transition and of the A-state was non-cooperative two-step transition. A-State regains 72% of the carbohydrate-binding activity.

Complexing of glucose oxidase with anti-glucose oxidase antibodies or the F(ab)'(2)/F(ab)' fragments derived therefrom protects both the enzyme and antibody/antibody fragments against glycation.

Incubation of Aspergillus niger glucose oxidase with glucose, fructose, or ribose results in remarkable inactivation of the enzyme. Glucose oxidase incubated with the sugars migrated as a diffuse band of low intensity and silver stained poorly after SDS-PAGE. Purified anti-glucose oxidase antibodies and F(ab)'(2) or F(ab)' derived therefrom were effective in restricting the inactivation of the enzyme induced by the sugars, providing up to 90% protection. The sugars also caused remarkable changes in the electrophoretic behavior of anti-glucose oxidase antibodies and the fragments, but complexing with glucose oxidase restricted the changes both in the enzyme and the antibody/antibody fragments.

A procedure for the rapid screening of Maillard reaction inhibitors.

A procedure for the rapid screening of inhibitors of glycation reaction, based on their ability to protect RNase against sugar induced inactivation of the enzyme is described. Glycation is implicated in variety of disorders including diabetes, atherosclerosis various micropathies yet is a slow process both in vivo and in vitro. In order to speed up glycation, the reaction was carried out at 60 degrees C using a thermostable protein RNase and ribose, a sugar that is known to react rapidly than glucose in the glycation reaction. It was observed that incubation of RNase with ribose at 60 degrees C in rapid inactivation of the enzyme with a parallel decrease in tyrosine fluorescence, enhancement in new fluorescence and hyperchromicity in the UV-region. No such alterations in the enzyme activity were observed when the incubation was carried out in absence of the sugar. Compounds and drugs that are known to act as inhibitors of glycation reaction restricted the ribose-induced inactivation of RNase. RNase immobilized on CNBr-activated Sepharose was also sensitive to exposure to ribose and appeared a better system to screen inhibitors of glycation from natural sources that contain substances that interfere with the assay of enzyme as well as in the study of post Amadori inhibitors of glycation.

Glycoprotein targeting and other applications of lectins in biotechnology.

Glycoconjugates comprise a variety of structures, include glycoproteins and glycolipids and are found on the surfaces of animal and plant cells, as well as on the surface of microorganisms. Determination of the structure and the distribution of glycoconjugates on cell surfaces are important for the understanding their biological function. Lectins are useful to investigate protein-carbohydrate interactions, because they have specificity for defined carbohydrate structure. They have been implicated in cell-to-cell recognition and signaling, blood group typing, in immune recognition process, and various other biological processes, such as viral, bacterial, mycoplasmal and parasitic infections, fertilization, cancer metastasis, growth and differentiation. Once thought to be confined to plant seeds, lectins are now recognized as ubiquitous in virtually all living systems, ranging from viruses and bacteria to animals. Plant lectins provide a rich source of carbohydrate-recognizing protein reagents for glycobiologists and biotechnologists. Biotechnology offers the therapeutic use of lectin against certain life threatening diseases such as human immunodeficiency virus and cancer. This review presents a comprehensive summary of research efforts that focus on the actual and potential uses and advantages of using lectins to target glycoproteins and also glycoproteins to target lectins.

Additional stabilization of stem bromelain coupled to a thermosensitive polymer by uniform orientation and using polyclonal antibodies.

Stem bromelain was covalently coupled to a thermosensitive polymer of N-isopropylacrylamide (p(NIPAm)) either through the amino groups of the enzyme (randomly coupled) or via the lone oligosaccharide chain (uniformly coupled). The enzyme coupled via the oligosaccharide chain exhibited better access to the substrate casein as compared to the preparation in which the amino groups formed the point of contact between the enzyme and the polymer. Native bromelain exhibited a pH optimum of 8.0 and a broad pH-activity profile. The polymer-coupled preparations exhibited broader pH-activity profiles and shifting of pH optimum to 10.0 at 35 degrees C. At 25 degrees C, the shifting of pH optimum was observed for the randomly coupled enzyme only. The temperature-activity profiles of bromelain coupled to p(NIPAm) also showed appreciable broadening and the preparations retained greater fraction of maximum activity above the temperature optimum. The optimum temperature of the uniformly oriented preparation also rose to 70 degrees C. Inactivation rates of the polymer-coupled bromelain were remarkably low at 60 degrees C as compared to the native protease, and binding of antibromelain antibodies improved the resistance to inactivation of the polymer-coupled preparations. The cleavage patterns of hemoglobin and IgG by the native bromelain and the polymer-coupled preparations were comparable.

Inactivation and modification of superoxide dismutase by glyoxal: prevention by antibodies.

Glyoxal is an endogenous compound, the levels of which are increased in various pathologies associated with hyperglycaemia and other related disorders. It has been reported to inactivate critical cellular enzymes by promoting their cross-linking and perpetuates advanced glycation end-product (AGE) formation. In this study, we used superoxide dismutase (SOD) as a model to investigate the ability of specific anti-enzyme antibodies and monomer Fab fragments to protect against glyoxal-induced deactivation and aggregate formation. We found that glyoxal deactivated SOD, in a concentration and time-dependent fashion. The enzymatic activity was monitored spectrophotometrically and it was found that enzyme lost approximately 95% of its original activity, when exposed to 10 mM glyoxal for 120 h. SDS-polyacrylamide gel electrophoresis demonstrated the formation of high molecular weight aggregates in SOD samples exposed to glyoxal. Surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) showed increase in relative molecular mass (M(r)), upon exposure to glyoxal. Specific anti-enzyme antibodies and monomer Fab fragments markedly inhibited SOD deactivation caused by glyoxal and decreased the extent of cross-linking or formation of aggregates. This protection by the antibodies or Fab fragments was specific since, other non-specific antibodies were not able to protect SOD. Previously, antibodies have been used to prevent aggregation of beta-amyloid peptides in Alzheimer and prion-protein disease. Our findings provide a new perspective, for use of antibodies to prevent the biomolecules against glycation-induced deactivation and alteration.

Immunoglobulin glycation with fructose: a comparative study.

BACKGROUND: Immunoglobulins undergo non-enzymatic glycation reaction with sugars both in vivo and in vitro. Effects of glycation on the ability of the antibodies to bind antigens are contradictory. Antibodies raised in various animals may also be exposed to high concentration of sugars that are added during freeze-drying/pasteurization for preservation. METHODS: IgG isolated from the sera of goat, human, rabbit, mouse, buffalo as well as IgY from hen egg yolk was subjected to in vitro glycation with fructose. The behavior of glycated IgG was investigated by SDS-PAGE, hyperchromicity at 280 nm, tryptophan fluorescence and new fluorescence. RESULTS: Marked variations were observed in the response of the immunoglobulins derived from various animals to incubation with fructose. Also, incubation of anti-glucoseoxidase (GOD) antibodies with fructose resulted in a rapid loss of their ability to bind the enzyme antigen as revealed by immunodiffusion and ELISA. DETAPAC and EDTA were quite protective but were unable to completely prevent the fructose-induced alterations. CONCLUSIONS: Immunoglobulins derived from goat, human, rabbit, mouse, buffalo and hen egg yolk undergo remarkable structural alterations on incubation with fructose. The susceptibility of the immunoglobulins to the modification however differed remarkably. The goat IgG was most recalcitrant while hen egg yolk IgY was most susceptible to the alterations. DETAPAC or EDTA restricted the fructose-induced alterations remarkably.

Bioaffinity based oriented immobilization of stem bromelain.

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 degrees C for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K (m) values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V (max) values remained unaffected on immobilization.

Investigation of conformational changes induced by binding of pancreatic RNase to anti-RNase IgG derived Fab monomer using optical procedures.

The conformational changes induced in Fab fragments of polyclonal anti-RNase antibody molecules obtained by digestion with papain as a result of binding of pancreatic RNase have been studied. The RNase-Fab complex (RN-Fab), being soluble, could be subjected to thermodynamic investigations using optical strategies, also because of the absence of tryptophan in RNase. Internalization of the chromophores (tryptophans and tyrosines) of Fab occurs when it binds to RNase, suggesting an increase in the compactness of Fab due to the binding of RNase.