Preparation and characterization of active site protected poly(ethylene glycol)–avidin bioconjugates

Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin–PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin–PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin–PEG 10 and 20 kDa prepared in the presence of imminobiotin–PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79–85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin–PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.     

A solid-phase radioimmunoassay of serum dehydroepiandrosterone sulphate using a monoclonal antibody

A monoclonal antibody directed against dehydroepiandrosterone, but with high affinity for dehydroepiandrosterone sulphate (DHA-S), has been used to develop a solid phase radioimmunoassay for measuring serum DHA-S. The antibody was covalently linked to polyacrylamide microbeads with no change in binding characteristics. The procedure requires only the chromatography of serum on anion-exchange cellulose before assaying the equivalent of 0.25 microliter serum. The method is precise, accurate and specific and can detect 19.5 pg of DHA-S. Serum DHA-S levels measured by this method were in good agreement with those found in a validated radioimmunoassay method involving hydrolysis. The method is quick and one operator could assay 50 blood specimens per day. DHA-S levels in serum from 50 men and 86 women were in agreement with those in the literature. With the availability of theoretically limitless quantities of consistently high quality monoclonal antibodies the advantages of developing solid phase radioimmunoassays for steroids is discussed.     

Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.     

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.     

Optimisation of aqueous two-phase extraction of human antibodies

The purification of human antibodies in an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 6000 and phosphate was optimised by surface response methodology. A central composite design was used to evaluate the influence of phosphate, PEG and NaCl concentration and of the pH on the purity and extraction yield of IgG from a simulated serum medium. The conditions that maximise the partition of IgG into the upper phase were determined to be high concentrations of NaCl and PEG, low concentrations of phosphate and low pH values. An ATPS composed of 12% PEG, 10% phosphate, 15% NaCl at pH 6 was further used to purify human monoclonal antibodies from a Chinese Hamster Ovary (CHO) concentrated cell culture supernatant with a recovery yield of 88% in the upper PEG-rich phase and a purification factor of 4.3. This ATPS was also successfully used to purify antibodies from a hybridoma cell culture supernatant with a recovery yield of 90% and a purification factor of 4.1.     

Inhibition of 17 beta-hydroxysteroid dehydrogenase activity in human endometrium by adrenal androgens

The effect of dehydroepiandrosterone sulphate (DHA-S) and its metabolites dehydroepiandrosterone (DHA) and 5-androstene-3 beta, 17 beta-diol (ADIOL) on the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrial tissue was investigated by an isotope ratio technique. The apparent KM for oestradiol was 1.59 X 10(-6) M. All three androgens inhibited the metabolism of oestradiol and the apparent Ki values were: ADIOL, 2.05 X 10(-6) M; DHA-S and DHA, 1.59 X 10(-6) M. However, ADIOL acted by direct competition with oestradiol for the active enzyme site whereas inhibition by DHA and its sulphate was non-competitive. DHA-S and DHA were more potent inhibitors of oestradiol metabolism than was ADIOL. These results support the hypothesis that adrenal androgens could be involved in the development of endometrial hyperplasia and adenocarcinoma. Inhibition of oestradiol metabolism could increase the concentration of oestradiol in endometrial tissue and if unopposed by progesterone, e.g. after the menopause or in subjects with ovulatory defects, could stimulate abnormal endometrial growth.     

Monoclonal antibody-based quantitation of poly(ethylene glycol)-derivatized proteins, liposomes, and nanoparticles

Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.     

Effects of DHA-S on placental 3 beta-hydroxysteroid dehydrogenase activity, progesterone and 20 alpha-dihydroprogesterone concentrations in placenta and serum

To study the effects of dehydroepiandrosterone sulfate (DHA-S) on placental steroid metabolism and maternal steroidal profiles at term, the following in vivo and in vitro experiments were performed. Two hundred mg of DHA-S was given to five pregnant women 30 minutes prior to delivery. After delivery, the placenta was collected and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and sulfatase activity was determined by measuring the rate of conversion of pregnenolone to progesterone and DHA-S to DHA. The amount of C21-delta 4-steroid in the placental tissue was measured by gas chromatography mass spectrometry (GC-MS) and compared with the control groups. The maternal serum concentration of several steroids was also measured by GC-MS before and after the administration of DHA-S. 3 beta-HSD activity in the placentae from the mothers who received DHA-S before delivery was significantly lower than in the controls. On the other hand, no significant change was observed in the activity of sulfatase. The serum concentration of progesterone (P) and 20 alpha-dihydro-P (20-P) before DHA-S loading decreased following the administration whereas estradiol (E), DHA, and androstenedione (A) levels increased. To study the direct effect of DHA-S and its related steroids on placental 3 beta-HSD activity, placental tissue samples were incubated with pregnenolone in vitro. Several other steroids were added simultaneously into the medium. It was observed that placental 3 beta-HSD activity was directly inhibited by DHA-S. These results indicate that DHA-S inhibits 3 beta-HSD activity in the placenta and subsequently causes a reduction in P and 20-P.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family.

Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies. Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones. The sequences of the four classes of cDNA were determined to be 85-95% homologous. Restriction fragments were isolated from the cDNA in each class and used as class specific probes. Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT. Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT.     

Self-binding antibodies (autobodies) form specific complexes in solution

In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.     

Fluorescence microscopic study of cells and polykaryons in the early periods following polyethylene glycol treatment

A fluorescence-microscopical study is made of cultured murine fibroblasts (L-cells) in early periods after the treatment with polyethylene glycol (PEG). Optimal conditions of fusion procedure were found under which the effectiveness of fusion was the highest and the toxical effect of PEG the lowest. The number of dead cells after the treatment with PEG did not exceed 10%. No significant changes in chromatin cytochemical properties (Acridine Orange and Olivomycin binding) were observed in the early periods of PEG treatment, that allows to use PEG for studying chromatin properties in hybrid cells obtained by PEG fusion. By means of PEG fusion, the hybrid cells with prematurely condensed chromosomes and also hybrids between animal and yeast cells have been obtained.     

A production of monoclonal antibodies by a simple electrofusion technique induced by ac pulses

A simple and effective technique of electrofusion of the mouse myeloma cells and lymphocytes induced by AC pulses was investigated. Instead of pearl chain formation, we used a mild centrifugation of high-density cell suspensions to enhance the cell-cell contacts. The optimal frequency of the AC field used for cell fusion was 10 kHz. We confirmed the production of monoclonal antibodies against human serum albumin (HSA) by the hybridoma obtained by this method. Under the conditions used in the present work, the efficiency of hybridoma formation is several times higher than those obtained by the chemical method using polyethylene glycol (PEG). Hybridoma colonies were detected in as high as 95% of the wells and 33% of them proved positive against HSA.     

Radioimmunoassay of plasma androsterone

A method is described and evaluated for the determination of androsterone in peripheral venous plasma (1.0 ml) from men and women. The procedure involves addition of labelled internal standard and extraction with diethyl ether. Aliquots (10 %) are removed for radioimmunoassay. An antiserum to androsterone-17-carboxymethyl oxime-bovine serum albumin and tritiated androsterone complete the system. The practical systematic errors have been determined by replicate analyses. The range of values (mean ± S.D.) in plasma from 40 healthy men are 54 ± 32 ng/100 ml, and the corresponding values for women 46 ± 28.     

Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts

Large numbers of viable protoplasts of pea (Pisum sativum) and grass pea (Lathyrus sativus) were efficiently and reproducibly obtained and, for the first time, fused. Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm(-1)), or on the use of macro or micromethods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution. Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons). Both the division of heterokaryons and the formation of small calluses were observed.     

The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women

Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.     

Investigative strategy of hyperandrogenism in women

Investigative procedures in the assessment of female hyperandrogenism are reviewed. Based on their experience, the authors suggest an inexpensive investigative strategy in hyperandrogenic females consisting of the following: the first step depends upon the clinical symptoms--in cases of hirsutism with regular menstrual cycles, plasma testosterone (T) and plasma dehydroepiandrosterone sulfate (DHA-S) are assayed, and the basal body temperature chart is recorded. In cases of hirsutism with irregular or anovulatory menstrual cycles, in addition to T and DHA-S, plasma 17-hydroxyprogesterone and urinary-free cortisol are assayed. In case of anovulation without hirsutism, T and DHA-S are assayed, and the LHRH test is performed. The results of this first investigation allow to attribute to the woman one of the six following hormone profiles: (1) metabolic hyperadrenalism; (2) tumoral hyperandrogenism; (3) 21-hydroxylase defect; (4) nontumoral DHA-S increase; (5) nontumoral ovarian hyperandrogenism; (6) idiopathic hirsutism. The additional investigative procedures required in each of these groups are detailed.     

PEGylation of cholecystokinin prolongs its anorectic effect in rats

The anorectic compound CCK-9 was coupled to polyethylene glycol 5 kDa, 10 kDa, 20 kDa and 30 kDa, under different reaction conditions. Conjugates were purified by HPLC and characterized by MALDI-TOF MS. A 96% PEGylation yield was obtained in buffer pH 7.5 after 6h reaction at 20 degrees C. The anorectic activity was tested in vivo in rats. A single bolus intra-peritoneal injection of non-modified CCK-9 resulted in a significant initial food intake reduction 30 min after food presentation (87% compared to paired control group). When PEG-CCK-9 conjugates modified with polymers of molecular weight up to 20 kDa were injected, lower but statistically significant initial food intake reductions were obtained (76% for PEG 10 kDa-CCK-9 conjugate compared to control group). The cumulative food intake reduction of non-modified CCK-9 is normalized within 1-2h, whereas the PEG-CCK-9 molecules showed a prolonged anorectic activity lasting for 6h for PEG 5 kDa-CCK-9; 23 h for PEG 10 kDa-CCK-9 and between 8h and 23 h for PEG 20 kDa-CCK-9. For PEG 30 kDa-CCK-9 conjugate, neither an initial nor a cumulative FI reduction was observed. PEG-CCK-9 conjugates show a significantly prolonged anorectic activity in comparison to the non-modified peptide. This effect is most evident for the PEG 10 kDa-CCK-9 conjugate.     

Enhanced activity of 3alpha-hydroxysteroid dehydrogenase by addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate in aqueous phase of biphasic systems for reductive production of steroids

The enzyme activity of 3alpha-hydrosteroid dehydrogenase (HSDH) was enhanced by the addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate ([Bmim][lactate]) to 50 mM Tris-HCl buffer. When utilizing [Bmim][lactate], the reaction velocity of HSDH increased. Also, reductive production of androsterone was investigated in an aqueous-organic solvent biphasic system containing 5% [Bmim][lactate] as the co-solvent of aqueous phase. In a coupled-enzyme system comprising HSDH and formate dehydrogenase (FDH), a two-fold increase in production rate of androsterone was obtained when utilizing [Bmim][lactate] with NADH regeneration.     

Evidence for protein kinase C regulation of ovarian theca-interstitial cell androgen biosynthesis

The hypothesis that protein kinase C may be an important regulator of ovarian theca-interstitial cell steroidogenesis was tested by using phorbol-12-myristate-13-acetate (PMA) and phorbol-12, 13-dibutyrate (PDB) to directly stimulate protein kinase C activity. Collagenase-dispersed cells (4 x 10(5) viable cells/dish) form ovaries of hypophysectomized immature rats were cultured in serum-free medium in the presence and absence of 0-100 ng/ml of luteinizing hormone (LH), PMA (0-100 nM), and/or PDB (0-100 nM). Treatment with 100 ng/ml LH stimulated androsterone production 100-fold at Day 4 of culture. The presence of 100 nM PMA or PDB had no effect on basal androsterone production; however, treatment with increasing concentrations of PMA or PDB (0-100 nM) caused a dose-related inhibition (maximum 70%) of LH-stimulated androsterone synthesis (ID50 = 1.8 nM and 2.4 nM, respectively). PMA and PDB did not significantly alter DNA, protein, or cell viability, indicating that their inhibitory effects were not due to changes in cell number or viability. Cells treated with LH and 100 nM 4 alpha-phorbol didecanoate (4 alpha-PDD; a phorbol ester that does not activate protein kinase C) failed to show significant decreases in androsterone production. Time-course studies revealed that when PMA treatment was delayed until Day 2 or 4 of culture, dramatic inhibitory effects on LH-stimulated androsterone production were still observed. These results suggest that the biological activity of protein kinase C is retained after the cells have expressed their differentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)