Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.     

The role of pH and its control on effective conjugation of bovine hemoglobin and human serum albumin

Human serum albumin (HSA) and bovine hemoglobin (Hb) conjugate is a promising candidate as a blood substitute. However, preparation of the conjugate is problematic because both proteins tend to conjugate between themselves rather than crosslink each other. In this work, a facile process for conjugation of Hb and HSA was developed through control strategy of the reaction. The reaction was carried out in a buffer containing borax-borate and mannite. The borax-borate was used for pH buffering while mannite was used as a pH switch and a reaction promoter. As a result, self-conjugation of Hb and self-conjugation of HSA were minimized. After the one-step conjugation reaction in aqueous solution, followed by the one-step purification by ion-exchange chromatography, the conjugate of HSA and Hb was obtained with the total yield about 50%. The P₅₀ and the Hill coefficient for the product were 16.1 mmHg and 1.82, respectively.     

Characterization of a yeast D-mannan with an alpha-D-glucosyl phosphate residue as an important immunochemical determinant

Conditions for the reaction of concanavalin A and dextranase with glutaraldehyde have been established to give a soluble, intermolecularly cross-linked conjugate possessing both dextranase and concanavalin activities. Evidence is presented that the dextranase and concanavalin molecules are linked to each other in the conjugate. The conjugate gives a different pattern of hydrolysis products on incubation with dextran than does dextranase.     

On the conjugate hand reaction: Communication IV. Changing rates of conjugate hand reaction to sound and light modality signals upon surgical interventions in the subcortical brain structures in humans

The results of the study on conjugate reaction time (RT) of hands (the time of simple mental reaction) of 16 patients with Parkinsonism, cerebral palsy, and spastic torticollis before and after surgery are presented. Conjugation of the left hand and right hand RTs to sound and light modality signals with a warning signal has been analyzed to detect the morphological structures that influence the conjugate reaction. In some of the patients, no disturbance of RT conjugation was shown; in other patients, the coefficient of correlation used for assessment of the left and right hand RT conjugation significantly changed. The coefficient of correlation between the left hand and right hand RTs decreased in response either to the sound signal or simultaneously to the sound and light signals. Disturbances of the conjugate hand reaction were observed in the case of ventral-lateral thalamotomy, subthalamotomy, and pallidotomy.     

A simple and sensitive enzyme-mediated assay of biotin.

Free biotin was quantitated by a competition by coating biotin-bovine serum albumin conjugate on a polystyrene microplate for binding to avidin-beta-galactosidase conjugate. The enzyme conjugate remaining on the plate surface as a result of the competition was detected by reaction with one of the following fluorogenic substrates, resorufin beta-D-galactoside and fluorescein di-beta-D-galactoside, in a fluorescence plate reader. Free biotin as little as 0.1 nmol can be routinely detected.     

Immunoprotective potential of polysaccharide-tetanus toxoid conjugate in Klebsiella pneumoniae induced lobar pneumonia in rats

The polysaccharide (PS) derived from K. pneumoniae NCTC 5055 lipopolysaccharide (LPS) was covalently linked to tetanus toxoid by using carbodimide with adipic acid dihydrazide as a spacer molecule. The conjugate was found to be non-toxic and non-pyrogenic at 100 microg dose level. At a similar dose, the conjugate did not elicit any local skin reaction on intradermal preparatory injection in rabbits. The conjugate was immunoprotective as was evident from the decrease in relative colonization of bacteria in lungs of immunized rats as compared to the control animals. Immunization with the conjugate resulted in alveolar macrophage activation in terms of their ability to phagocytose bacteria in vitro.     

Synthesis, pulse radiolysis, and in vitro radioprotection studies of melatoninolipoamide, a novel conjugate of melatonin and alpha-lipoic acid

A novel conjugate of melatonin 2 and alpha-lipoic acid 4 has been prepared using DCC mediated coupling. The conjugate named melatoninolipoamide has been assigned its structure 1 on the basis of spectral analysis (UV, IR, NMR, and EI-MS). Pulse radiolysis studies of the conjugate were carried out in aqueous solutions with both oxidizing and reducing radicals. The results indicate that the melatonin moiety of the conjugate reacts preferably with oxidizing radicals and the lipoic acid moiety exhibits preferential reaction with reducing radicals. The in vitro radioprotection ability of 1 was examined by gamma-radiation induced lipid peroxidation in liposomes and hemolysis of erythrocytes, and compared the results with those of melatonin and alpha-lipoic acid. The studies suggest that the conjugate can be explored as a probable radioprotector.     

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.     

Improved functional properties of the ovoinhibitor by conjugating with galactomannan

The chicken egg white ovoinhibitor, a multi-type proteinase inhibitor, was conjugated with galactomannan through the Maillard reaction in a controlled dry heating state at 60 degrees C and 65% relative humidity. The formation of an ovoinhibitor-galactomannan conjugate during dry heating was confirmed by SDS-PAGE. The resulting ovoinhibitor-galactomannan conjugate showed almost the same inhibitory activity toward trypsin, chymotrypsin and elastase as that of the untreated ovoinhibitor, while the conjugate showed stronger heat stability and better emulsifying properties than the untreated ovoinhibitor. These results suggest that the ovoinhibitor-galactomannan conjugate can be used as a protease inhibitor having heat stability and outstanding emulsifying properties for industrial application.     

Inducible alkylation of DNA by a quinone methide-peptide nucleic acid conjugate

The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate, but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alternatively, alkylation of noncomplementary sequences is only possible when a template strand is present to colocalize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self-adducts formed between the PNA and its attached QM remained active and reversible over more than 8 days in aqueous solution prior to reaction with a chosen target added subsequently.     

A new chitosan-thymine conjugate: synthesis, characterization and biological activity

Conjugation of chitosan with nucleobases is expected to expand its not only antimicrobial activity but also anti-cancer activity. Here, we report the synthesis of a novel chitosan-thymine conjugate by the reaction between chitosan and thymine-1-yl-acetic acid followed by acylation. The synthesized conjugate was characterized by FTIR, XRD, (1)H NMR, TGA and SEM. The microbiological screening results demonstrated the antimicrobial activity of the conjugate against bacteria viz., Escherichia coli, Staphylococcus aureus, and fungi viz., Aspergillus niger. The chitosan-thymine conjugate also inhibited (p<0.05) the proliferation of human liver cancer cells (HepG2) in a dose-dependent manner but had no cellular toxicity in non-cancerous mouse embryonal fibroblast cells (NIH 3T3). Thus, the chitosan-nucleobase conjugate may open a new perspective in biomedical applications.     

Solid phase enzyme-linked competitive binding assay for riboflavin

A new solid-phase enzyme-linked assay for riboflavin (vitamin B2) is described. The assay is based on the competition between analyte vitamin molecules and a glucose-6-phosphate dehydrogenase-3-carboxymethylriboflavin conjugate for a limited number of riboflavin-binding protein sites immobilized on Sepharose particles. Significant improvements in conjugate catalytic activity and thus detectability are achieved by optimizing the reaction conditions used to covalently link 3-carboxymethylriboflavin to the enzyme. Optimization experiments include studying the effects of reaction pH and organic solvent composition. Final assay detection limits and the sensitivity of the dose-response curves are dependent on the ratio of conjugate to binding protein sites utilized in an equilibrium assay protocol. Selectivity of the method correlates well with that predicted based on the known association constants of riboflavin-binding protein with flavin analogs. The assay is shown to offer adequate detection limits and selectivity for direct measurement of riboflavin in urine, infant formula, and vitamin capsules.     

One pot conjugation of the polypeptides directed by phosphorus oxychloride.

A series of homopeptides and their conjugates were synthesized in one pot reaction in the presence of phosphorus oxychloride and the conjugate yield was structurally dependent. Menthol and benzylamine conjugated to the homopeptides quantitatively. Homopeptides when treated with diisopropyloxyphosphite (DIPPH) and NaClO yield the corresponding N-phosphoryl peptides. Electrospray ionization-mass spectrometry (ESI-MS)/MS was used to study the structure of peptide conjugates. This paper reports a simple method to synthesize the homopeptides and their conjugate derivatives and the fact that phosphoryl peptides could also be obtained by one pot reaction.     

Formation of a bioconjugate composed of hemin, smectite, and quaternary ammonium chloride that is soluble and active in hydrophobic media

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) intercalated with a quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA), to form a hemin-smectite-DOA conjugate. The hemin-smectite-DOA conjugate was soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with a light yellow color. Its absorption spectrum in benzene showed two bands, 600 and 568 nm, in the visible region and a sharp Soret band at 400 nm with the molar extinction coefficient of 7.5 x 10(4) M(-1) cm(-1). The formation of the conjugate of smectite and DOA was confirmed by X-ray diffraction analysis: the basal spacing, d(001), of hemin-smectite-DOA conjugate was 19 A which is an expansion of the interlayer space by 5 A based upon the basal spacing of smectite of 14 A. Hemin-smectite-DOA conjugate catalyzed the peroxidase-like reaction in organic solvents using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The temperature-dependent peroxidase-like activity of the conjugate was compared with peroxidase activity of horseradish peroxidase. The hemin-smectite-DOA conjugate exhibited higher activity as the temperature was increased from 30 to 70 degrees C, while horseradish peroxidase activity was reduced as the temperature was increased.     

Invertase immobilization via its carbohydrate moiety

After periodate oxidation of its glycosidic component, invertase was covalently bound onto three types of modified solid supports: glycidyl methacrylate, styrene-divinylbenzene copolymers, and bead cellulose. Direct reaction of the invertase aldehyde groups that were formed with amino groups of the support and use of the modified Ugi reaction have been employed as immobilization procedures. Apart from binding methods, the important effects of the buffer, support, conditions of periodate oxidation, and the length of the spacer on the activity of the enzyme conjugate have been investigated. Superior conjugate activity was obtained, via modified Ugi reaction, by the immobilization of a suitably oxidized invertase to a styrene-divinylbenzene copolymer having free amino groups.     

Dextran–estrone conjugate: synthesis and in vitro release study

A novel hormone conjugate has been prepared by a coupling reaction between modified estrone and dextran. In order to provide a suitable reactive estrone derivative for coupling with dextran and a spacer between the drug carrier and the hormone, the steroidal sex hormone was succinylated by reaction with succinic anhydride. Subsequently, the carboxylic acid terminal of succinylated estrone was further reacted with thionyl chloride to replace the hydroxy group with chlorine to make a better leaving group. The ester bond was employed as labile linkage between the hormone and the biopolymer carrier backbone so that the coupled estrogen could be released from the conjugate via ester hydrolysis. Structures of the modified estrone and dextran–estrone conjugate were determined by elemental analysis and by FTIR, ¹H and ¹³C NMR spectroscopies. The degree of substitution (D.S.) per anhydroglucose (AHG) unit was 0.33 (11.0 mol-% of estrone moieties), as calculated from the ¹H NMR spectrum. In vitro hydrolysis of the conjugate in aqueous phosphate buffer/ethanol solutions at pH 8.0 and 7.4 and 37°C released estrone was completed within a few days and followed zero-order kinetics.     

Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.     

Single-stranded DNA modification by an oligonucleotide-phthalocyanine Fe(II) conjugate: the kinetic features and the process mechanism

Catalytic oxidative modification of a single-stranded DNA with hydrogen peroxide and molecular oxygen in the presence of a conjugate containing an oligonucleotide complementary to the DNA fragment and tetra-4-carboxyphthalocyanine Fe(II) was studied. The conjugate examined was found to be active in the reaction of oxidative DNA cleavage in the presence of hydrogen peroxide, like the earlier studied oligonucleotide conjugates containing metallocomplexes tetra-4-carboxyphthalocyanine Co(II) and 2,4-di-[2-(2-hydroxyethyl)]deuteroporphyrin IX Fe(III) generating active oxygen forms. The new conjugate was more active in the case of oxidation with molecular oxygen. Kinetic features and optimal regimes of DNA oxidation with hydrogen peroxide were found.     

Direct immunocytochemistry with a horseradish peroxidase-conjugated monoclonal antibody against substance P

The procedure for the isolation and conjugation of the anti-substance P monoclonal antibody NC1/34 with the enzyme horseradish peroxidase (HRP) is described. This resulted in a molecular complex of monoclonal antibody/HRP of 1:1. This conjugate was of approximately 400,000 daltons, as estimated by gel chromatography. Practically all the isolated antibody was coupled to HRP. The conjugate was tested both in a model system where CNBr-activated Sepharose beads were coupled to substance P and on fixed tissue preparations from the rat spinal cord and medulla oblongata. The conjugate revealed staining in nerve fibers in areas known to contain substance P. The best immunohistochemical results were obtained by prolonged incubations at 12 degrees C in the presence of 0.1% Triton X-100. The preabsorption of the conjugate with substance P obliterated the reaction.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.