Synthesis of tocopheryl succinate phospholipid conjugates and monitoring of phospholipase A₂ activity

Tocopheryl succinates (TOSs) are, in contrast to tocopherols, highly cytotoxic against many cancer cells. In this study the enzyme activity of secretory phospholipase A(2) towards various succinate-phospholipid conjugates has been investigated. The synthesis of six novel phospholipids is described, including two TOS phospholipids conjugates. The studies revealed that the TOS conjugates are poor substrates for the enzyme whereas the phospholipids with alkyl and phenyl succinate moieties were hydrolyzed by the enzyme to a high extent.     

Conjugation of proteins and enzymes with hydrophilic polymers and their applications.

The efficacy of a number of therapeutically active proteins and peptides is severely limited due to their instability in circulation. Of the various approaches used to stabilise these proteins, the one more successful is covalent modification of the protein or enzyme with some hydrophilic polymers such as dextran or PEG. These conjugates are more stable than the native protein both in vitro as well as in vivo. They exhibit enhanced resistant to proteolytic degradation, have a long-life in circulation and exhibit reduced immunogenicity. The therapeutic efficacy of these conjugates is also greatly enhanced compared to the native protein or enzyme.     

Immunoenzyme demonstration of viruses and virus-specific antibodies by using conjugates based on gene engineering-produced beta-lactamase

Enzyme immunoassays for the detection of viral antigens and virus-specific antibodies in biological samples have been described. Molecular complexes of antibodies and beta-lactamase (penicillinase) have been used as anti-specific conjugates. To synthesize the conjugate, the enzyme obtained with the aid of genetic engineering has been used. Enzyme immunoassays have been tested for the indication of the influenza virus and virus-induced specific antibodies. Enzyme immunoassays were shown to possess certain advantages (e.g., the use of simple and nontoxic substrate) along with the sensitivity identical to that of other methods, employing peroxidase-based conjugates.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

Development of homogeneous enzyme immunoassay for the organophosphorus insecticide fenthion

A rapid, convenient homogeneous competitive enzyme immunoassay for estimating the amount of fenthion is described. The assay utilizes glucose-6-phosphate dehydrogenase-hapten conjugates that are inhibited in solution by antibodies obtained from bovine serum albumin-hapten conjugates. In order to investigate the effects of bridging group recognition on the sensitivity of dose response characteristics, the bridging groups of varying alkyl chain length were attached at the phosphate position of fenthion. Among the antibodies used, the one obtained from the use of hapten (fenthion analog) with the same bridging group structure that was used in preparing the enzyme-fenthion conjugates showed maximum inhibition (up to 51.8%) in the absence of fenthion. In the presence of fenthion, the activity of the enzyme-hapten conjugate is regained in an amount proportional to the fenthion concentration. Under the optimized condition, the ED50 value for fenthion was 0.809 microg/ml. The assay developed in this study is a rapid effective screening method for fenthion prior to precise analysis.     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

Electron immunohistochemical analysis of localization of neutral Mn2+-dependent DNAase. I. Synthesis of ferritin and colloidal gold conjugates with monospecific antibodies against neutral Mn2+-dependent DNAase

Rabbit antibodies against a neutral Mn(2+)-dependent rat liver DNAse were obtained, whose specificity towards DNAse was ascertained by suppression of the enzyme activity both in vitro system and immunoblotting assays. Procedures of synthesis of ferritin and colloidal gold conjugates with antibodies are described. The biological activity of the conjugates proved to be similar to that of the original antibodies.     

Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.     

Biological activities of melanotropic peptide fatty acid conjugates.

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the alpha-MSH fragment analog, H-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a "creeping" potency in the lizard skin bioassay-that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10-100 times more active than alpha-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Asp5, D-Phe7, Lys10]alpha-MSH5-10-NH2, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.     

Enzymatic synthesis of disaccharide-serine and peptide conjugates

We describe enzymatic transglycosylations between an appropriate glycosyl donor and galactosyl (or glucosyl)-serine and -peptide conjugates to obtain diglycosyl-serine or -peptide derivatives. The reactions are catalyzed by β-galactosidase (from E. coli or from Aspergillus oryzae) and β-glucosidase (from Almonds). The enzymatic reactions give, preferentially, β(1 å6) linked diglycosyl-serine (or -peptide) conjugates. However, in the case of the digalactosyl derivatives, β(1 å3) linkages are mainly observed. By changing the source of the enzyme (E. coli or Aspergillus oryzae) the regioselectivity can be reversed for these digalactosyl derivatives. Deprotection of the aminoacid of the diglycosyl-peptides under mild conditions is also described.     

Charge-directed targeting of antimicrobial protein-nanoparticle conjugates

Use of antimicrobial enzymes covalently attached to nanoparticles is of great interest as an antibiotic-free approach to treat microbial infections. Intrinsic properties of nanoparticles can also be used to add functionality to their conjugates with biomolecules. Here, we show in a model system that nanoparticle charge can be used to enhance delivery and increase bactericidal activity of an antimicrobial enzyme, lysozyme. Hen egg lysozyme was covalently attached to two types of polystyrene latex nanoparticles: positively charged, containing aliphatic amine surface groups, and negatively charged, containing sulfate and chloromethyl surface groups. In the case of bacterial lysis assay with a Gram-positive bacteria Micrococcus lysodeikticus, activity of lysozyme conjugated to positively charged nanoparticles was approximately twice as large as that of free lysozyme, while lysozyme conjugated to negatively charged nanoparticles showed little detectable activity. At the same time, when assayed using a low-molecular weight oligosaccharide substrate, lysozyme attached to both positively and negatively charged nanoparticles showed slightly lower activity than free enzyme. A possible explanation of these results is that lysozyme attached to negatively charged nanoparticles cannot be effectively targeted to the bacteria because of the electrostatic Coulombic repulsion from the negatively charged bacterial cell walls, whereas lysozyme conjugated to positively charged nanoparticles was targeted better than free enzyme due to stronger electrostatic attraction to bacteria. Zeta potential measurements confirmed the validity of this hypothesis. Thus, nanoparticle charge is an important factor that can be used to control targeting and activity of protein-nanoparticle conjugates.     

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.     

Enzyme leakage and multipoint attachment of agarose-bound enzyme preparations

The solvolytic detachment of leucine aminopeptidase from Sepharose-enzyme conjugates with multiple and single anchoring bonds has been studied under a variety of conditions by radiochemical and enzymological methods. The release of the single-point-fixed conjugate could be described by a leakage function, derived previously, yielding the first-order rate constant of the cleavage of the enzyme-matrix bond. The nucleophile hydroxylamine increased the detachment rate considerably. The release of the immobilized enzyme was incomplete in all experiments even after prolonged times. The enzyme leakage from multipoint-attached conjugates was still high enough to prohibit a long-term use of such preparations in routine work at room temperature.     

Catalytic activity of steroid conjugates of various types of peroxidases in aqueous and micellar media

The horseradish peroxidase (HRP) conjugates with the sheep antirabbit antibodies and cortisol (COR) or progesterone (PROG) containing 9 to 40 steroid molecules per HRP molecule were synthesized. In aqueous media all the conjugates have lower catalytic activity in the o-phenylenediamine oxidation than the native enzyme. In reversed Aerosol OT micelles in heptane the HRP-COR and HRP-PROG conjugates containing 12 and 9 steroid molecules, respectively, have catalytic constants 2.6 and 2.7 times higher than the unmodified enzyme. The influence of the HRP hydrophobisation and its inactivation on the course of modification on catalytic properties of the enzyme is discussed.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

The development and application of an enzyme immunoassay for urinary estrone conjugates

An enzyme immunoassay (EIA) for estrone conjugates is described and applied to urine samples from a female Indian rhinoceros, a female gorilla, and a female lion-tailed macaque. Concomitant measures of estrone conjugates in the same sample are compared to the values obtained with radioimmunoassay. High correlation coefficients for values obtained from each assay indicate that EIA measurements provide information that is comparable to values obtained by radioimmunoassay. EIA methods for urinary steroid conjugates can provide a practical tool to evaluate female reproductive status of zoo species without the need for a traditional endocrine laboratory.     

Immunoenzyme method of sequential saturation for determining antigens using the model of insulin

The method for the determination of insulin by means of the enzyme immunoassay, based on the use of insulin-peroxidase conjugates, has been developed. In this assay the scheme of the successive saturation of the active sites of antibodies is used. The antigenic properties of two conjugates differing in the method of their preparation are compared. The conjugates were obtained by the covalent binding of peroxidase, oxidized in its carbohydrate component, with insulin (conjugate 1) or hexamethylene-diamine-modified insulin (conjugate 2). The conjugates represented a mixture of oligomers differing in their molecular weight. Conjugate 1 possessed higher affinity to antibodies and higher enzymatic activity than conjugate 2. The method for evaluating the quality of antisera to insulin used in the assay has been proposed. The time of the insulin assay is 5-16 hours, the limit of insulin detection is 5 microU/ml, the variation factor is 3-12%.     

Homogeneous enzyme immunoassay of estradiol using estradiol-3-O-carboxymethyl ether as hapten

A homogeneous enzyme immunoassay for estradiol estimation has been developed, which can be extended to other steroids. A new procedure for the preparation of estradiol -3-0- carboxymethyl ether by a simple one step reaction in high yield (90%) has been described. This hapten has been used for raising highly specific anti-estradiol antibody in rabbits and for preparation of enzyme conjugates. Two different enzymes, lysozyme and glucose -6- phosphate dehydrogenase have been studied for their suitability as enzyme labels. Our results indicate that lysozyme-conjugate meets the essential requirement for a practical enzyme immunoassay. The advantage of the present nonradioactive procedure is the overall simplicity, low cost and high stability of the reagents.     

Exploring the cellular activity of camptothecin-triple-helix-forming oligonucleotide conjugates

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.     

A direct antigen heterologous enzyme immunoassay for measuring progesterone in serum without using displacer

An antigen heterologous enzyme-linked immunosorbent assay (ELISA) for directly measuring progesterone in serum is described. Six combinations of antigens and enzyme conjugates were tested; the enzyme conjugate 17-alphaOH-progesterone-3-O-carboxymethyloxime-alkalinephosphatase (17-alphaOH-P-3-CMO-ALP) and the immunogen progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) were found to be best. Fifty microliters of standard or serum sample and 100 microL of the 17-alphaOH-P-3-CMO-ALP enzyme conjugate were added to the antibody coated wells, and incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using p-nitrophenyl phosphate as substrate. The sensitivity of the assay was 0.11 ng/mL, and intra- and inter-assay CVs ranged from 5.1% to 9.6%. The analytical recoveries were 97-105%. The serum progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r=0.97 (n=44). Moreover, in this ELISA no displacing agent was used or special means was required to displace progesterone from corticosteroid binding globulin (CBG). Serum progesterone concentrations of subjects, with histories of recurrent spontaneous abortions were also measured, and correlated well with clinical history.