The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Analysis of bispecific monoclonal antibody binding to immobilized antigens using an optical biosensor

The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K _ass) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K _ass of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k _diss) for anti-HRP shoulder of Babs was 21 times higher than k _diss for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody–enzyme conjugates in the case of binding of bivalent Mabs.     

Magnetic nanocomposite of anti-human IgG/COOH-multiwalled carbon nanotubes/Fe₃O₄ as a platform for electrochemical immunoassay

An electrochemical immunosensing method was developed based on a magnetic nanocomposite. The multiwalled carbon nanotubes (MWCNTs) were treated with nitric acid to produce carboxyl groups at the open ends. Then, Fe₃O₄ nanoparticles were deposited on COOH–MWCNTs by chemical coprecipitation of Fe²⁺ and Fe³⁺ salts in an alkaline solution. Goat anti-human IgG (anti-hIgG) was covalently attached to magnetic nanocomposite through amide bond formation between the carboxylic groups of MWCNTs and the amine groups of anti-hIgG. The prepared bio-nanocomposite was used for electrochemical sensing of human tetanus IgG (hIgG) as a model antigen. The anti-hIgG magnetic nanocomposite was fixed on the surface of a gold plate electrode using a permanent magnet. The hIgG was detected using horseradish peroxidase (HRP)-conjugated anti-hIgG in a sandwich model. Electrochemical detection of hIgG was carried out in the presence of H₂O₂ and KI as substrates of HRP. Using this method, hIgG was detected in a concentration range from 30 to 1000 ng ml^?1 with a correlation coefficient of 0.998 and a detection limit of 25 ng ml^?1 (signal/noise = 3). The designed immunosensor was stable for 1 month.     

Detection of polyclonal antibody against any area of the protein-antigen using immobilized protein-antigens: the critical role of the immobilization protocol

Antigens immobilized on solid supports may be used to detect or purify their corresponding antibodies (Ab) from serum. Direct immobilization of antigens on support surfaces (through short spacer arms) may promote interesting stabilizing effects on the immobilized antigen. However, the proximity of the support may prevent the interaction of some fractions of polyclonal Ab with some regions of the antigen (those placed in close contact with the support surface). Horseradish peroxidase (HRP) was immobilized on agarose by different protocols of multipoint covalent immobilization involving different regions of the antigen surface. Glyoxyl-agarose, BrCN-agarose, and glutaraldehyde-agarose were used as activated supports. Each HRP-immobilized preparation was much more stable than the soluble enzyme, but it was only able to adsorb up to 60-70% of a mixture of polyclonal anti-HRP antibodies. On the other hand, HRP was also immobilized on agarose through a very long, flexible, and hydrophilic spacer arm (dextran). This immobilized HRP was hardly stabilized, but it was able to adsorb 100% of the polyclonal anti-HRP. The absence of steric hindrances seems to play a critical role favoring the complete recognition of all classes of polyclonal Ab. Another solution to achieve a complete adsorption of polyclonal Ab on immobilized-stabilized antigens has been also reached by using a mixture of the differently immobilized and stabilized HRP-agarose preparations. In this case, an improved storage and operational stabilities of the immobilized antigens can be combined with the complete adsorption of any class of antibody.     

A new approach to the design of hybrid hybridomas based on the use of an actinomycin D-resistant line of murine myeloma]

The hybrid hybridomas (tetradomas) were produced from the fusion of the double mutant actinomycin Dr (ADr)/HATs hybridoma to horseradish peroxidase (HRP) and wild type hybridoma to alpha-endorphin (EP). The double mutant phenotype was constructed using the new strategy, based on the fusion of immune mouse splenocytes with mouse myeloma (X63.Ag8, 653) cell variants, made resistant to 30 ng/ml of AD by stepwise selection. This allowed the direct introduction of the dominant selective marker (ADr) into the hybrid cells. Tetradomas secreted the bispecific monoclonal antibodies (bi Mabs), simultaneously binding to EP and HRP in double antigen ELISA, the ELISA plates covered with EP-bovine serum albumin conjugate. Using rat pituitary the bi Mabs were shown to be effective for immunostaining of EP-producing cells. EP-producing cells.     

Polymyxin B-horseradish peroxidase conjugates as tools in endotoxin research.

The peptide antibiotic Polymyxin B (PMB) binds to bacterial endotoxin (lipopolysaccharide, LPS). We prepared covalent conjugates of PMB and horseradish peroxidase (HRP) by periodation of HRP-linked oligosaccharides followed by direct condensation with PMB. In addition we prepared monoclonal antibodies (Mabs) to PMB. The PMB-HRP conjugates and anti-PMB Mabs were used to study in ELISA the binding of PMB to LPS from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, PMB-HRP was used to quantify lipid A in ELISA, and to stain gram-negative bacteria histochemically. For the study of PMB-LPS interaction, PMB-HRP proved to be superior to the anti-PMB Mabs. PMB-HRP conjugates are useful general probes to detect or measure lipid A and LPS of various species using very simple methods and to stain bacteria, and they may obviate the need for many specific antisera. Thus, PMB-HRP conjugates are useful probes for endotoxin research.     

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.     

Monoclonal antibodies targeted to alpha-oligonucleotides. Characterisation and application in nucleic acid detection.

The aim of the present study was to test the antigenicity of alpha-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize alpha-deoxyribonucleotides. Two were raised against a poly(alpha-dT) sequence and specifically recognized the alpha-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as alpha-nucleotides and, surprisingly, only recognized the alpha-dG nucleotide. For all four Mabs, no cross reactivity was observed with beta-oligonucleotides. These Mabs were reactive with alpha-oligonucleotide sequences whether these sequences were single-stranded or hybridized to DNA or RNA. The four Mabs were tested in a sandwich hybridization assay that consisted of an alpha-oligonucleotide (for target sequence recognition), one of the four Mabs (for recognition of the hybridized alpha-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxidase (HRP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to detect PCR fragments of HPV 18 DNA. The sensitivity of this reaction was 1 pg of plasmid DNA containing the HPV 18 fragment. The specificity of the detection was demonstrated using HPV 6/11 and 16 DNA sequences.     

Immobilization of Horseradish Peroxidase on Multi-Wall Carbon Nanotubes and its Electrochemical Properties

Horseradish peroxidase (HRP) was immobilized on carboxylated multi-wall carbon nanotubes in the presence of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The immobilized HRP maintained its oxidative activity for guaiacol over a broad range of pH values (4–9). An electrode of graphite rod, 6 mm diam. was fabricated using the immobilized HRP. Cyclic voltammetry of the enzyme electrode confirmed electron transfer between the immobilized HRP and the electrode in the presence of H₂O₂ but without an added mediator or a reducing substrate.     

Silver ion microplates for immunoassays.

Microplate wells can be coated with silver ions using glutaraldehyde as a spacer molecule and thiourea as a complexing ligand. Microwells containing surface silver ions are shown to immobilize biotin-labeled horseradish peroxidase (HRP) in active form, while showing very little affinity for the unlabeled enzyme. These plates can also immobilize biotin-labeled antibodies that exhibit bioactivity after immobilization. Silver ions are needed for the complexation of the biotinylated enzyme or antibody because microwells modified to contain surface amine or thiourea molecules do not immobilize appreciable amounts of the labeled proteins. A maximum surface coverage for biotin-labeled HRP of 40 ng/cm2 and an immobilization binding constant of Km = 8 x 10(9)/M are determined from serial dilutions in a microplate. Detection of as little as 6.7 fmol HRP is achieved using antibodies immobilized on the silver ion-modified microplates. Active antibody surface densities were estimated to be between 130 and 260 nm2/antibody molecule. Background binding of HRP to the modified silver ion microplates was very low, allowing for reasonably accurate detection between 10(-14) and 10(-11) mol HRP.     

Removal of pentachlorophenol by immobilized horseradish peroxidase

Researches on the polymerization of aqueous pentachlorophenol (PCP) by the catalysis of horseradish peroxidase (HRP) with the existence of hydrogen peroxide (H₂O₂) were conducted. Factors, such as acidity, temperature, enzyme activity, and initial concentration of PCP and H₂O₂ that could influence the degradation were studied. Results showed that the optimum pH value for free enzyme was 5–6; relative higher temperature could accelerate the reaction greatly; PCP removal increased with an increase of enzyme concentration, and PCP (initial concentration 12.6 mg/L) removal percentage could reach nearly 70% under the highest enzyme concentration (about 0.05 u/ml) adopted in the experiment; removal percentage increased slightly with an increase of initial concentration of PCP, and when initial PCP concentrations were 13.0 and 0.7 mg/L, the removal percentages were about 73.7% and 35.7%, respectively; the molar ratio of the reaction between PCP and H₂O₂ was about 1:2.Based on the above results, researches on the removal of PCP by the immobilized HRP were conducted. The free HRP was immobilized on the polyacrylamide gel prepared by gamma-ray radiation method; then the immobilized HRP was filled into a column, and PCP was successfully removed by the immobilized HRP column. The results were compared with results using free HRP enzyme, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP, and when pH=5.15, the immobilized HRP could reduce PCP with initial concentration 13.4 mg/L to the concentration of 4.9 mg/L within 1 h, and the immobilized HRP column could be used to repeatedly.     

Suppression of growth defects of α-amylase secreting Escherichia coli by signal sequence fusion

Abstract In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae . In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.     

Unusual cross-reactions among monoclonal antibodies to bacterial antigens: idiotypic and competitive binding analysis

In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae. In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.     

A carbon nanotube/silica sol–gel architecture for immobilization of horseradish peroxidase for electrochemical biosensor

A novel third-generation biosensor for hydrogen peroxide (H₂O₂) has been constructed based on horseradish peroxidase (HRP) immobilized by the sol–gel (SG) technology on carbon nanotube (CNT)-modified electrode. CNT has good promotion effects on the direct electron transfer between HRP and the electrode surface and the SG network provides a biocompatible microenvironment for enzyme. The immobilized HRP retained its bioelectrocatalytic activity for the reduction of hydrogen peroxide and can respond to the change of concentration of H₂O₂ rapidly. The heterogeneous electron transfer rate constant was evaluated to be 2.8 ± 0.4 s⁻¹. The amperometric response to H₂O₂ shows a linear relation in the range from 0.5 to 300 μmol l⁻¹ and a detection limit of 0.1 μmol l⁻¹ (S/N = 3). The K _M^app value of HRP immobilized on the electrode surface was found to be 1.35 mmol l⁻¹. The biosensor exhibited high sensitivity, rapid response and excellent long-term stability.     

Generic Method for Attaching Biomolecules via Avidin-Biotin Complexes Immobilized on Films of Regenerated and Nanofibrillar Cellulose

We investigated the adsorption and chemical conjugation of avidin and its deglycosylated form, neutravidin, on films of regenerated and nanofibrillar cellulose. The dynamics and extent of biomolecular attachment were monitored in situ by quartz crystal microbalance microgravimetry and ex situ via surface analyses with atomic force microscopy and X-ray photoelectron spectroscopy. The installation of carboxyl groups on cellulose after modification with carboxymethylated cellulose (CMC) or TEMPO-oxidation significantly increases physisorption of avidins, which can be then covalently conjugated by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDS/NHS) coupling chemistries. The developed cellulose-avidin biointerfaces are able to scavenge biotinylated molecules from solution as demonstrated by successful surface complexation of biotinylated bovine serum albumin (Biotin-BSA) and antihuman immunoglobulin G (Biotin-anti-hIgG). Finally, we show that cellulose substrates carrying immobilized anti-hIgG are effective in detecting human immunoglobulin G (hIgG) from fluid matrices.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

Immunocytochemical evidence for the presence of enzymes synthesizing GABA and GHB in the same neuron

A specific and sensitive immunocytochemical double staining for visualization of glutamate decarboxylase (GAD) and semialdehyde succinate reductase (SSR₂) in the same brain section has been developed. SSR₂ is the enzyme responsible for the transformation of succinic semialdehyde into γ-hydroxybutyrate (GHB). GAD was detected using specific rabbit GAD-antibodies and unlabeled antibody enzyme peroxidase antiperoxidase, and SSR₂ using specific guinea-pig SSR₂ antibodies conjugate to a fluorescein-labeled second antibody. The coexistence of GAD and SSR₂ in the same neuron was demonstrated by a peroxidase reaction superimposed on fluorescent compounds. Cell bodies containing both antigens were observed in the cerebellum, dorso-median hypothalamus and raphe nuclei. GHB is present in most GABA containing neurons. Some neurons contain only SSR₂; these neurons may synthesize GHB by an active uptake of GABA.     

Electron microscopic investigation of cell surface antigens using a multivalent hybrid antibody with double specificity

Multivalent hybrid antibody (MHA) complexes with double specificity were prepared by combining two different antibodies, one specific against ferritin (Fer), the other against horseradish peroxidase using protein A from Staphylococcus aureus (SpA). Electron microscopy of mouse spleen lymphocytes and thymocytes (the latter coated with mouse anti-Thy-1 antibody) reacted with anti-HRP/SpA/anti-mIg complex and HRP showed the pattern of surface Ig receptors visualized by the specific peroxidase labelling. When IgG anti-Fer/SpA/IgG-anti-mIg complex was applied to the same cellular stuff, better resolution and higher accuracy were obtained although the distribution was similar. Surface Thy-1 alloantigen and Fe receptors (charged with hIgG) were simultaneously detected on the thymocyte surface by using in the same way a mixture of anti-Fer/SpA/anti-Thy-1 and anti-HRP/SpA/anti-Fab. The concomitant ultrastructural visualization of Thy-1 (with Fer) and Fc (with HRP) on mouse thymocytes clearly showed that their distribution was mainly independent and that the amount of Thy-1 antigen prevailed. These data show that electron microscopy with a mixture of MHA may be a useful technique for the concomitant location of two antigenic determinants on the cell surface.     

Evidence for cellular but not serologic cross-reactivity between keyhole limpet hemocyanin and sperm-whale myoglobin.

The addition of keyhole limpet hemocyanin (KLH) to cultures of rabbit lymph node cells (LNC) primed with KLH and sperm-whale myoglobin (Mb) induced the synthesis of antibody to Mb as well as to KLH. Several mechanisms for this heterologous induction were considered. It was established that KLH does not nonspecifically activate rabbit T or B lymphocytes. It was also shown that KLH and Mb do not cross-react serologically by several sensitive and specific criteria. Therefore, it was surmised that heterologous induction of Mb antibody synthesis by KLH was due to cellular cross-reactivity between these proteins. Rabbits were primed by the injection of Mb-complete Freund's adjuvant (CFA), alum-Mb, or alum-KLH, and their LNC challenged with KLH, Mb, and synthetic antigenic sites of Mb. These experiments yielded much and diverse evidence for cellular cross-reactivity between KLH and Mb, and especially between KLH and the Mb peptides: KLH plus Mb-primed LNC evoked enhanced anti-KLH and anti-Mb syntheses. KLH plus KLH-sensitized LNC resulted in a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either enhanced or inhibited the anti-KLH antibody response, depending on whether the priming adjuvant was CFA or alum. The addition of Mb to KLH-primed cells enhanced or inhibited the ensuing anti-Mb antibody synthesis; KLH did not affect or inhibit anti-KLH antibody synthesis. Addition of synthetic Mb antigenic sites to Mb-sensitized LNC elevated or suppressed anti-KLH antibody production, depending on the length of time between priming and in vitro challenge. A mixture of KLH and Mb peptide lowered the anti-Mb antibody response of Mb-educated LNC compared to KLH alone. A combination of KLH and Mb peptide also reduced the anti-KLH antibody synthesis of KLH-primed cells compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced their uptake of tritiated thymidine, and their transport of tritiated cyclic AMP and protein synthesis. Added Mb induced the synthesis of protein and nonspecific IgG by KLH-primed LNC; Mb peptides evoked protein synthesis by these cells. It is postulated that cross-reactivity at the T-cell level is responsible for the induction of Mb antibody synthesis by adding KLH to either Mb-primed or KLH/Mb-primed LNC. The implications of these findings with respect to cellular and humoral immunity are discussed.     

Preparation of high-density Concanavalin A adsorbent and its use for rapid, high-yield purification of peroxidase from horseradish roots

Preparation of Concanavalin A-adsorbents by immobilization on Sepharose activated with 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP-reagent) is reported. High immobilization yields of lectin (above 90%) were attained using an optimized CDAP-activating protocol. The effect of ligand density on the performance of the adsorbent for specific binding of glycoproteins was studied using horseradish peroxidase (HRP) as a model. Adsorption yields of pure HRP exceeding 90% were obtained with Con A-derivatives containing not < 20 mg of immobilized Con A/ml of packed gel. With lectin content of 2 mg/(ml of packed gel), only 20% of HRP was adsorbed. Purification of peroxidase from horseradish roots extract was successfully accomplished on Con A-Sepharose with high Con A content.