Potentiation of the in vitro cytotoxicity of chlorambucil by monoclonal antibodies

It had previously been shown by using polyclonal antibodies that chlorambucil (CBL) and antibody did not have to be covalently bound to selectively inhibit tumor cell growth. This paper has reexamined this phenomena by using monoclonal antibodies (MoAb) and has sought to determine how antibody may serve to focus and to increase the cytotoxic effect of non-covalently bound CBL. In vitro, it was shown that MoAb per se had no effect, the MoAb had to be reactive with and to bind to tumor target cells for CBL to have an enhanced cytotoxicity. Furthermore, not all MoAb were effective; IgM and IgG2a subclasses enhanced CBL cytotoxicity, but IgG3 did not. At the cell surface, studies clearly showed that endocytosis of CBL and MoAb did not occur, as metabolic inhibitors, and lowered temperatures, which both inhibit endocytosis, had no effect on their cytotoxicity. In addition, NH4Cl an inhibitor of lysosomal enzymes did not reduce the cytotoxicity of CBL and MoAb. Thus CBL that is non-covalently bound to MoAb enters the cell independently of the MoAb. We conclude therefore that cell-bound MoAb is able to concentrate CBL on the surface of tumor cells by an unknown mechanism thereby enabling CBL to selectively alkylate their cell membranes and subsequently inhibit additional tumor cell growth.     

Photochemical attachment of biomolecules onto fibre-optics for construction of a chemiluminescent immunosensor.

We report herein a simple and effective way to photochemically immobilize biomolecules onto a fibre-optic silica surface. The system is based on a photoreactive benzophenone derivative that is bound to SiO2 surfaces of the optical fibre via a silane anchor. The benzophenone derivative was 4-allyloxybenzophenone, synthesized by standard procedures that were later used to synthesize the 4-(3'-chlorodimethylsilyl) propyloxybenzophenone and 4-(3'-dichloromethylsilyl) propyloxybenzophenone by regular hydrosilation procedures. After silanization with the benzophenone derivatives, the fibres were immersed in a cholera toxin B subunit solution and illuminated with UV light (wavelength > 345 nm). As a result of the photochemical reaction, a thin layer of the antigen was covalently bound to the benzophenone-modified surface. The photochemically modified fibre-optics were then tested as immunosensors in the detection of cholera anti-toxin antibody and revealed through chemiluminescence measurements. A secondary antibody labelled with horseradish peroxidase acted as the marker for the cholera toxin antibody. A photo-electronic set-up was designed specifically to monitor the signal. The immunosensor system was shown to be both specific and sensitive. The lowest rabbit serum titre detected was 1:1 700,000.     

Electron transfer between soluble and immobilized mammalian cytochrome c. Equilibrium and kinetic studies on immobilized cytochrome c.

Horse heart cytochrome c was covalently bound to Sepharose 4B and its redox properties were measured under various experimental conditions. The equilibrium constant for the electron exchange between the oxidized and the reduced form of cytochrome c when one of the two forms was in the semi-solid state and the other one in solution was close to 1. Matrix-bound ferrocytochrome c is very stable to autoxidation and is not oxidized by O2 even in the presence of mammalian cytochrome oxidase. Oxidation occurs if catalytic amounts of soluble cytochrome c are added to the reaction mixture. The rate of oxidation of matrix-bound ferrocytochrome c in the presence of cytochrome oxidase and catalytic amounts of soluble cytochrome c may be correlated with the rate of electron transfer between soluble and matrix-bound cytochrome c. This rate is more than two orders of magnitude lower than that reported for the homonuclear (between identical species) electron transfer in solution.     

Kinetics of binding reactions of an antibody molecule with haptens on a membrane surface

Direct measurement has been made of the reaction rate of binding of a bivalent antibody and fluorescent haptens, which were covalently bound on a model membrane surface, by a method of stopped-flow fluorometry. The result was interpreted as indicating that the reaction takes place in two steps: (i) binding of a hapten with one of the two antigen-combining sites of an antibody molecule, and (ii) binding of another hapten with the other site of the antibody molecule in question. The rate of the second step was found to depend on the fluidity of the membrane.     

Anti-rabbit immunoglobulin G detection in complex medium by PM-RAIRS and QCM Influence of the antibody immobilisation method

Two antibody immobilisation procedures were compared to set up an immunosensor for goat anti-rabbit immunoglobulin (anti-rIgG), i.e. rIgG covalently bound or immobilised via affinity to protein A (PrA). In both cases, the first layer of protein was covalently bound to a mixed self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) and mercaptohexanol (C6OH) on a gold surface. The elaboration of the sensitive surfaces, as well as their selectivity and sensitivity were studied step by step by polarization modulation-reflection absorption infra-red spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM) with impedance measurement. QCM measurements showed that the viscoelastic properties of the antibody layer were markedly modified during the antigen recognition when the antibody was bound by affinity to PrA. The specific detection of antigen within a complex medium was assessed by PM-RAIRS thanks to the grafting of cobalt-carbonyl probes. Affinity constants between the immobilised rIgG and the anti-rIgG were determined from PM-RAIRS analysis.     

Sarcosine oxidase: structure,function, and the application to creatinine determination

Summary Determination of creatinine is important in the clinical laboratory. Jaffé reaction has long been used to determine creatinine, but the method suffers from various interferences. To overcome this problem, the enzymatic methods were invented and have been used widely. Sarcosine oxidase has a critical role in the enzymatic method. Of sarcosine oxidases,Corynebacterium enzyme has been studied extensively in kinetic and structural aspects. The enzyme contains noncovalently bound and covalently bound FADs, and consists of 4 non-identical subunits (A, B, C, D). The covalently bound FAD is bound to the subunit B. The rate of oxidation of sarcosine was explained by the rates of the oxidation and reduction of the bound FADs. From the chemical modification of the enzyme with iodoacetamide, the amino acid sequence around the non-covalently bound FAD is suggested and the modification changed the enzyme so that the only noncovalently bound FAD functions in the oxidation of sarcosine.     

Surface engineering: optimization of antigen presentation in self-assembled monolayers.

The formation of self-assembled monolayers (SAMs) on gold surfaces containing an antigenic peptide (NANP)6 and HS(CH2)11OH, and the specific binding of a monoclonal antibody to these layers were investigated by surface plasmon resonance (SPR). Peptides were synthesized by solid-state phase synthesis and were linked either to cysteine or to an alkyl-thiol to allow covalent attachment to gold. The content of the peptide in the SAMs was systematically varied, and the binding properties of the monoclonal antibody were compared with those measured by microcalorimetry in solution. At a critical peptide concentration in the SAM an optimal antibody binding and complete surface coverage was attained. At lower peptide concentrations, the amount of adsorbed antibody decreased; at higher peptide concentrations, the binding constant decreased. These effects can be explained if the accessibility of the antigenic epitopes depends on the peptide density. Addition of free antigen induced the desorption of bound antibodies and allowed accurate measurements of the dissociation rate constant. Binding constants obtained from steady-state measurements and from measurements of the kinetic rate constants were compared.     

Hydrolysis of human high-molecular-mass kininogen by human plasma kallikrein. Proposal of a new model concept for the course of reaction in presence and absence of C1(-)-inhibitor

Hydrolysis of high-molecular-mass kininogen was studied by following the changes in the amounts of substrate, intermediates and products as a function of time using quantitative polyacrylamide-gel electrophoresis (silver staining). The experimental data was analysed on the basis of the concept that the overall reaction is composed of three hydrolysis reactions, two positional-change processes of intermediates at the active site, and two product-substrate exchange processes. It is proposed C1(-)-inhibitor to form two types of complexes with kallikrein, one with non-covalent and one with covalent bonds. With an adequately chosen set of reaction-partner concentrations and four different kinds of experimental conditions with respect to kininogen and inhibitor addition to kallikrein, the following results were obtained: 1) Non-covalently bound inhibitor has no effect on the first and the second hydrolysis reaction, but efficiently interferes with the third hydrolysis reaction; 2) Nicked kininogen (first intermediate; one of the two bradykinin bonds split) for the second bond to be hydrolysed undergoes a positional change during which it remains strongly bound to the enzyme, never exchanges with kininogen, and is not displaced by non-covalently bound inhibitor; 3) Intermediate kinin-free kininogen (second intermediate; both bradykinin bonds split and bradykinin released) prior to turning over into stable kinin-free kininogen (final product; histidine-rich fragment split off and released) undergoes a positional change involving dissociation and reassociation so that non-covalently bound inhibitor finds access to the active site; 4) Intermediate kinin-free kininogen to sustain multiple turnovers exchanges with kininogen via a stable complex of such structure that during this process non-covalently bound inhibitor cannot or can only slightly interfere; 5) Stable kinin-free kininogen to sustain multiple turnovers exchanges with intermediate kinin-free kininogen via free enzyme with the effect that non-covalently bound inhibitor efficiently interferes; 6) As hydrolysis proceeds more and more inhibitor becomes covalently bound, gradually leading to complete inactivation of the enzyme.     

Force-based analysis of multidimensional energy landscapes: application of dynamic force spectroscopy and steered molecular dynamics simulations to an antibody fragment-peptide complex

Multidimensional energy landscapes are an intrinsic property of proteins and define their dynamic behavior as well as their response to external stimuli. In order to explore the energy landscape and its implications on the dynamic function of proteins dynamic force spectroscopy and steered molecular dynamics (SMD) simulations have proved to be important tools. In this study, these techniques have been employed to analyze the influence of the direction of the probing forces on the complex of an antibody fragment with its peptide antigen. Using an atomic force microscope, experiments were performed where the attachment points of the 12 amino acid long peptide antigen were varied. These measurements yielded clearly distinguishable basal dissociation rates and potential widths, proving that the direction of the applied force determines the unbinding pathway. Complementary atomistic SMD simulations were performed, which also show that the unbinding pathways of the system are dependent on the pulling direction. However, the main barrier to be crossed was independent of the pulling direction and is represented by a backbone hydrogen bond between Gly^H-H40 of the antibody fragment and Glu^Oε-6_peptide of the peptide. For each pulling direction, the observed barriers can be correlated with the rupture of specific interactions, which stabilize the bound complex. Furthermore, although the SMD simulations were performed at loading rates exceeding the experimental rates by orders of magnitude due to computational limitations, a detailed comparison of the barriers that were overcome in the SMD simulations with the data obtained from the atomic force microscope unbinding experiments show excellent agreement.     

Acetylcholinesterase-based biosensor electrodes for organophosphate pesticide detection. I. Modification of carbon surface for immobilization of acetylcholinesterase

Screen-printed carbon electrodes modified with the dialdehydes, glutaraldehyde and terephthaldicarboxaldehyde, and then polyethyleneimine have been utilized for production of pesticide biosensors based on acetylcholinesterase. To improve the extent of dialdehyde modification, the electrodes were NH2-derivatized, initially by electrochemical reduction of 4-nitrobenzenediazonium to a nitroaryl radical permitting attachment to the carbon surface. Subsequent reduction of the 4-nitrobenzene yields a 4-aminobenzene modified carbon surface. Drosophila melanogaster acetylcholinesterase was immobilized either covalently onto dialdehyde modified electrodes or non-covalently onto polyethyleneimine modified electrodes. Internal diffusion limitations due to the dialdehyde and polyethyleneimine modifications increased the apparent Km of the immobilized enzyme. The thiocholine sensitivity was about 90% for dialdehyde modified electrodes and about 10% for polyethyleneimine modified electrodes as compared with non-modified carbon electrodes. The detection limit of the biosensors produced by non-covalent immobilization of acetylcholinesterase onto polyethyleneimine modified carbon electrodes was found to be about 10(-10) M for the organophosphate pesticide dichlorvos.     

A novel glycosyl-amino acid linkage: rabbit-muscle glycogen is covalently linked to a protein via tyrosine

A recent review summarizes our identification in rabbit-muscle glycogen of a protein that resists all attempts at removal by means that should displace non-covalently bound protein [Kennedy et al. (1985) In Membranes and Muscle (Berman, M.C., Gevers, W. and Opie, L.H. eds.) pp. 65-84, ICSU Press/IRL Press, Oxford]. Here we confirm that the glycogen is covalently bonded to the protein and report that the attachment is via a novel glycosidic linkage involving the hydroxyl group of tyrosine.     

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: kinetic studies

The photoaffinity analogue ethidium monoazide was used to prepare samples of poly(dGdC).poly(dGdC) containing covalently attached ethidium. The effects of both noncovalently and covalently bound ethidium on the kinetics of the NaCl-induced B to Z transition in poly(dGdC).poly(dGdC) was examined using absorbance and fluorescence spectroscopy to monitor the reaction. Covalently and noncovalently attached ethidium were equal in the extent to which they reduce the rate of the B to Z transition. By using fluorescence to selectively monitor the fate of noncovalently bound ethidium over the course of the transition, we found that ethidium completely dissociates as the reaction proceeds, but at a rate that lags behind the conversion of the polymer to the Z form. These experiments provide evidence for the redistribution of noncovalently bound ethidium over the course of the B to Z transition, leading to the development of biphasic reaction kinetics. The observed kinetics suggest that the primary effect of both covalently and noncovalently bound ethidium is on the nucleation step of the B to Z transition. The reduction in the rate of the B to Z transition by noncovalently or covalently bound ethidium may be quantitatively explained as resulting from the reduced probability of finding a drug-free length of helix long enough for nucleation to occur. As necessary ancillary experiments, the defined length deoxyoligonucleotides (dGdC)4, (dGdC)5, and (dGdC)6 were synthesized and used in kinetic experiments designed to determine the nucleation length of the B to Z transition, which was found to be 6 bp. The activation energy of the B to Z transition was demonstrated to be independent of the amount of covalently bound ethidium and was found to be 21.2 +/- 1.1 kcal mol-1. Covalent attachment of ethidium was observed to increase the rate of the reverse Z to B transition, presumably by locking regions of the polymer into a right-handed conformation and thereby providing nucleation sites from which the Z to B conversion may propagate.     

Infrared optical immunosensor: application to the measurement of the herbicide atrazine

A new approach to optically transduce antigen-antibody association, needing no label, is described herein, taking advantage of the ability of reflection-absorption infrared (IR) spectroscopy to analyze organic thin films at the surface of reflective materials with high sensitivity. As a proof-of-principle, this new technique was applied to the immunodetection of the herbicide atrazine. Gold-coated chips were covered with a capture layer consisting of a protein derivative of the herbicide atrazine covalently bound to a self-assembled monolayer containing a carboxy-terminated thiolate. Successive binding of anti-atrazine antibody and secondary anti-rabbit immunoglobulin G antibody resulted in a change of the IR absorption properties of the organic film at the sensor surface. The two prominent amide I and II bands observed on the surface IR spectra were taken for semiquantitative analysis of the adsorbed protein amount. The presence of increasing amounts of atrazine resulted in the progressive inhibition of antibodies binding to the sensors, yielding a relative lower increase of the IR signals. The deduced standard curves displayed a sigmoidal shape typical of competitive inhibition assays. The test midpoint (IC(50)) and the limit of detection (IC(80)) were found to be in the nanomolar range and very close to those measured by an in-house enzyme-linked immunosorbent assay using the same antibody and the same antigen competitor.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein

The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.     

Electrogenerated indium tin oxide-coated glass surface with photosensitive interfaces: surface analysis

We present herein a photo-immobilization technique for the localized and specific conjugation of biochip platforms with different proteinaceous bioreceptors, such as antigen or antibodies. This methodology based on a photoactivable electrogenerated polymer film, pyrrole-benzophenone, allows the covalent immobilization of biomolecules through light mediation. The surface-conductive glass platform electropolymerized with poly(pyrrole-benzophenone) thin film may then be used to affinity-coat the chip with molecular recognition probes. This glass chip electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide (ITO). Thereafter, pyrrole-benzophenone monomers are electropolymerized onto the conductive metal oxide surface and then exposed to an antigen Staphylococcal Enterotoxin B (SEB)) solution and illuminated with UV light (wavelength approximately 345 nm) through a mask. As a result of the photochemical reaction, a pattern thin layer of the antigen was covalently bound to the benzophenone-modified surface. Then the sample to be analyzed, along with its specific target antibody (anti-SEB antibodies), is introduced onto the glass surface and left to react with the previously photo-immobilized antigen. When the immuno-reaction is completed, the specifically attached immunoglobulin analytes are detected by using secondary antibodies conjugated with Fluorescein isothiocyanate (FITC). The fluorescence signal emanating from the biochip surface is then quantified by two methods, using a filtered intensified charge-coupled device (CCD) camera and a grating spectrometer.     

Functional cholesteryl binding agents: evaluation of viability and specificity of antibodies bound to modified phospholipid vesicles

A cholesterol derivative was incorporated into small unilamellar phospholipid vesicles, and antibodies were bound covalently to the vesicles. More than one antibody was bound to each vesicle. The antigen binding viability and specificity were determined using a modified radioimmunoassay and an in vitro cell assay. Both of these tests showed good antibody activity and specificity. The antigen affinity of the bound antibodies was higher than for the unbound antibody due to more than one viable antibody being bound to each vesicle. The modified vesicles can be used as immunodirected drug delivery systems for both diagnosis and therapy.     

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.     

Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

Surface membrane biotinylation efficiently mediates the endocytosis of avidin bioconjugates into nucleated cells

Here we demonstrate that biotin covalently attached to cell surface obligates existing receptors to endocytose avidin bioconjugates into nucleated cells. Incubation of fluorescein-labeled avidin with biotinylated cell lines resulted in uniform and rapid surface attachment and endocytosis compared with no detectable association of the avidin-conjugated dye with unbiotinylated cells. Uptake was detected within minutes with efficiencies approaching 100% in cell lines and freshly obtained peripheral blood mononuclear cells. After 24 h, avidin was barely detectable on the surface of the nucleated cells. In marked contrast, fluorescent avidin remained exclusively on the external membrane of erythrocytes after 24 h. To investigate biotin-mediated endocytosis for the delivery of DNA, we prepared polyethylenimine-avidin (PEI-avidin) conjugates. Surface biotinylation significantly increased the transfection efficiencies of PEI-avidin condensed plasmid DNA coding green fluorescent protein (GFP) to the level of transferrin-receptor targeted gene delivery (15-20% GFP positive cells in culture after 48 h). The increase in transfection efficiency was blocked by the addition of free avidin or biotin to the culture medium. Biotin covalently bound to cell surface membrane proteins efficiently mediates the entry of avidin bioconjugates into nucleated cells.