High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction.

A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and beta-Gal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specific monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate co-amplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays by approximately three orders of magnitude. Detection limits for hTSH, beta-Gal and hCG were respectively 1 x 10(-19), 1 x 10(-17) and 1 x 10(-17) mol. Given the enormous amplification afforded by PCR and the existing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays.     

Characterization of a novel beta-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel beta-galactosidase (beta-Gal II). In cells grown on TOS, in addition to the lactose-degrading beta-Gal (beta-Gal I), another beta-Gal (beta-Gal II) was detected and it showed activity towards TOS but not towards lactose. beta-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. Beta-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. Beta-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35 degrees C. The enzyme showed highest V(max) values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. Beta-Gal II was active towards beta-galactosyl residues that were 1-->4, 1-->6, 1-->3, and 1 <--> 1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.     

Transgalactosylation in a water-solvent biphasic reaction system with beta-galactosidase displayed on the surfaces of Bacillus subtilis spores

The ever-increasing industrial demand for biocatalysis necessitates innovations in the preparation and stabilization of biocatalysts. In this study, we demonstrated that beta-galactosidase (beta-Gal) displayed on Bacillus spores by fusion to the spore coat proteins (CotG) may be used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. The resulting spores had a specific hydrolytic activity of 5 x 10(3) U/g (dry weight) of spores. The beta-Gal was tightly attached to the spore surface and was more stable in the presence of various organic solvents than its native form was. The thermostability of the spore-displayed enzyme was also increased, and the enzyme was further stabilized by chemically cross-linking it with glutaraldehyde. With spore-displayed beta-Gal, octyl-beta-D-galactopyranoside was synthesized at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively. Interestingly, the spores were found to partition mainly at the interface between the water and solvent phases, and they were more available to catalysis between the two phases, as determined by light microscopy and confocal fluorescence microscopy. We propose that spore display not only offers a new and facile way to construct robust biocatalysts but also provides a novel basis for phase transfer biocatalytic processes.     

Syntheses of model compounds related to an antigenic epitope in pectic polysaccharides from Bupleurum falcatum L

The beta-galactosidases from Xanthomonas manihotis (beta-Gal Xmn) and Bacillus circulans (beta-Gal-3 Bcir) are retaining glycosidases that hydrolyze glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The mechanism-based inactivator 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside was shown to inactivate beta-Gal Xmn and beta-Gal-3 Bcir through the accumulation of 2-deoxy-2-fluorogalactosyl enzyme intermediates with half lives of 40 and 625 h, respectively. Peptic digestion of these labeled enzymes and analysis by LC-MS identified Glu(260) and Glu(233) as the catalytic nucleophiles involved in the formation of the glycosyl-enzyme intermediate during catalysis by beta-Gal Xmn and beta-Gal-3 Bcir, respectively. These findings confirm the previous prediction of the position of these residues based on primary sequence similarities to other members of the glycoside hydrolase family 35.     

A new radioimmunoassay for human chorionic gonadotropin using monoclonal antibody

A new radioimmunoassay (RIA) for human Chorionic Gonadotropin (hCG) was developed using murine monoclonal antibody to the beta-subunit of hCG (beta-hCG). The IgG fraction of the monoclonal antibody which did not react with 125I-beta-hCG was purified from hybridoma ascites, and covalently coupled to Sepharose 4B. This solid-phase antibody was incubated with standard hCG or serum sampled for 48 hours. The reaction medium was then removed by centrifugation and 125I-beta-hCG and anti-beta-hCG rabbit polyclonal antibody were added to the precipitate. The alcohol precipitation method was used for separating "bound" and "free" forms in the second reaction. The sensitivity for hCG in this assay system was 0.5 mIU/ml serum and the cross-reactivity with human Luteinizing Hormone (hLH) was 0.4%. This assay system was shown to be clinically applicable. Serial serum samples from two patients with trophoblastic disease were assayed and minute amounts of hCG, which could not be determined by conventional assay methods, could be assayed by this new RIA.     

Effect of partitioning equilibria on the activity of beta-galactosidase in heterogeneous media

We had demonstrated that membrane adsorption or penetration differentially modulated beta-Galactosidase (beta-Gal) activity against soluble substrates (Coll. and Surf., 24, 21, 2002). In a heterogeneous media, not only the enzyme but also the rest of the chemical species taking part in a chemical reaction would eventually interact with the available surfaces. The aim of the present work was to investigate if, in addition to changes in the intrinsic mechanism of the reaction at the lipid-water interface, the kinetics of enzyme-catalyzed reactions could be significantly affected by the partitioning of the substrate (ortho-nitro-phenyl galactopyranoside (ONPG)), the product (ortho-nitro-phenol (ONP)) and the enzyme (E. coli beta-Gal) towards the membrane. Multilamellar vesicles of sPC were used as model membranes. Membrane-water partition coefficients (Pm/w) were determined according to the theory and methodology developed previously (J. Neurosci. Meth. 36, 203, 1991). The values of Pm/w obtained (PONPG =0, PONP =50 and P beta-Gal = 118) were applied to a two-compartment model, which assumed a free access of the substrate to the enzyme and a nucleophile-like activatory effect exerted, within the membrane compartment, by the lipid-water interface. This model: (i) reproduced the lipid concentration-dependence we had observed previously in Vmax, (ii) predicted the values of k4 = 3.54 x 10(7) s-1 and the extinction coefficient of the aglycone in the membrane phase, 4012 M(-1) cm-1, with p < 0.0001 and p < 0.02, respectively, as well as for P beta-Gal =117 (which was poor (p=0.6716) but gave a numerical value within the same order of magnitude that the experimental value) and (iii) emphasized the importance of the more efficient reaction mechanism in the membrane phase compared with that in the aqueous phase (k4>k3).     

Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique

A simple direct enzymeimmunoassay (EIA) on microtiter plates for plasma progesterone using the second antibody coating technique and horseradish peroxidase (HRP) as the enzyme label (EIA-HRP) is described and compared with an identical EIA procedure which employed alkaline phosphatase (AP) as the enzyme label (EIA-AP). The assays used antiserum raised against progesterone-7-carboxyethlthioether-BSA in rabbits. Both systems were further compared with the conventional direct progesterone radioimmunoassay (RIA) in regular use. The enzymes HRP and AP were coupled to progesterone-6 beta-hydroxy-hemisuccinate by a mixed anhydride method. While the precision of EIA-HRP was comparable to RIA, the sensitivity in terms of the lowest detection limit obtained in EIA-HRP was about 10 times better than that seen in RIA. Progesterone estimates from plasma samples in EIA-HRP showed good correlation (r = 0.94) with the RIA values and the levels measured in the two systems were identical. Progesterone estimates from plasma samples in EIA-AP were at least three times higher than those obtained by either EIA-HRP or RIA. Thus, only the EIA-HRP but not the EIA-AP was suitable for the reliable direct measurement of progesterone in plasma.     

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.     

The clinical evaluation of the simultaneous measurements of human chorionic gonadotropin (hCG) and its alpha-subunit in sera of patients with trophoblastic diseases

The concentrations of human chorionic gonadotropin (hCG) and its free immunoreactive alpha-subunit (hCG-alpha) in the sera of patients with trophoblastic diseases were measured by hCG and hCG-alpha radioimmunoassay (RIA), respectively. In the sera of 12 women with hydatidiform mole large amounts of hCG and considerably high level of hCG-alpha were detected in all cases. After the evacuation of mole the serum level of these glycoproteins decreased, the leve of hCG-alpha declined more rapidly than hcg. in the sera of patients with destructive mole the concentration of hCG-alpha was usually lower than that of hCG. After hysterectomy and chemotherapy the levels of hCG-alpha declined practically paralleling that of hCG. However, when hCG had decreased to undetectable level, hCG-alpha could no longer be detected in all cases. Although in the serum of patient with choriocarcinoma involving the uterus and lungs the concentration of hCG-alpha was almost as high as that of hCG, the secretory pattern of hCG and hCG-alpha might not be closely related. The changes in the serum level of free hCG-alpha as well as that of hCG parelled the clinical course of the patients examined in this study. The present results suggest that measurements of the serum free hCG-alpha may be a useful parameter to follow the clinical course and to evaluate the efficacy of treatments of trophoblastic diseases.     

Photolithographic fabrication of poly(ethylene glycol) microstructures for hydrogel-based microreactors and spatially addressed microarrays

We describe the fabrication of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures with a high aspect ratio and the use of hydrogel microstructures containing the enzyme beta-galactosidase (beta-Gal) or glucose oxidase (GOx)/horseradish peroxidase (HRP) as biosensing components for the simultaneous detection of multiple analytes. The diameters of the hydrogel microstructures were almost the same at the top and at the bottom, indicating that no differential curing occurred through the thickness of the hydrogel microstructure. Using the hydrogel microstructures as microreactors, beta-Gal or GOx/HRP was trapped in the hydrogel array, and the time-dependent fluorescence intensities of the hydrogel array were investigated to determine the dynamic uptake of substrates into the PEG-DA hydrogel. The time required to reach steady-state fluorescence by glucose diffusing into the hydrogel and its enzymatic reactions with GOx and HRP was half the time required for resorufin beta-D-galactopyranoside (RGB) when used as the substrate for beta-Gal. Spatially addressed hydrogel microarrays containing different enzymes were micropatterned for the simultaneous detection of multiple analytes, and glucose and RGB solutions were incubated as substrates. These results indicate that there was no cross-talk between the beta-Gal-immobilizing hydrogel micropatches and the GOx/HRP-immobilizing micropatches.     

Identification of epitopes associated with hCG and the beta hCG carboxyl terminus by monoclonal antibodies produced against a synthetic peptide

We have produced a library of monoclonal antibodies directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl terminus of hCG. Five antibodies, designated FB01, FB02, FB03, FB04, and FB00, were developed and analyzed with respect to affinity and specificity for epitopes on human chorionic gonadotropin (hCG) and beta hCG by enzyme-linked immunoabsorbent and radioimmunoassays (RIA). All monoclonal antibodies demonstrated low affinity constants (approximately 10(-7) liters/mol) compared with those obtained by immunization with native beta hCG. One antibody, namely FB00, bound only to the synthetic peptide, whereas all other monoclonal antibodies recognized either free native beta hCG or both beta hCG and HCG. Antibodies produced against the synthetic peptide did not cross-react with other glycoprotein hormones such as LH, TSH, and FSH. Characterization of the monoclonal antibody-binding sites revealed the presence of at least four separate and distinct epitopes on the last 35 amino acids of beta hCG. Indeed, one epitope recognized by FB01 is located between residues 109 and 118, whereas another antigenic region recognized by FB04 appears to be present on the 109-121 portion of the molecule near or at position 118. One additional antigenic site was localized between residues 118 and 136. Finally, FB00 recognized an epitope located on the last 10 amino acids (136-145) of beta hCG. With the use of such antibodies, two- and three-site monoclonal RIA were developed and employed to detect free beta hCG and hCG in sera of patients with choriocarcinoma. These assays may be useful in the detection of beta hCG- and hCG-producing tumors and subsequent monitoring of patients in response to surgery and/or chemotherapy.     

Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting.

Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM-1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross-linking protein that facilitates anti-PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM-directed vascular immunotargeting of a reporter enzyme (beta-galactosidase, beta-Gal) in intact animals. Intravenous injection of [125I]SA-beta-Gal conjugated with either anti-PECAM or IgG led to a high 125I uptake in liver and spleen, yet beta-Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti-PECAM/[125I]SA-beta-Gal, but not IgG/[125I]SA-beta-Gal, accumulated in the lungs (36.0+/-1.3 vs. 3.9+/-0.6% injected dose/g) and induced a marked elevation of beta-Gal activity in the lung tissue persisting for up to 8 h after injection (10-fold elevation 4 h postinjection). Using histochemical detection, the beta-Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti-PECAM carrier also provided 125I uptake and beta-Gal activity in the renal glomeruli. Predominant intracellular localization of anti-PECAM/SA-beta-Gal was documented in the PECAM-expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell-selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.     

Heterologous radioimmunoassay of monkey luteinizing hormone: a critical assessment

Available reagents for the immunoassay (RIA) of luteinizing hormone (LH) in monkeys, including a cynomolgus (cynLH) tracer, an antiserum against human chorionic gonadotropin (hCG), and a rhesus standard (rhLH), were assessed using an in vitro bioassay and the RIA in connection with fractionation by high-resolution isoelectrofocusing. The data presented indicate that the RIA system represents a significant improvement over the ovine-antiovine system. Since, however, the sensitivity of the RIA is some 50 times less than that of the in vitro bioassay, and since the rhLH standard is heavily contaminated with FSH, it is felt that until the advent of a homologous RIA, the in vitro bioassay is the method of choice.     

Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary

These studies were done to examine the effect of gonadotropin on rat luteal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (the rate-limiting step in cholesterol biosynthesis) in ovaries of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) primed rats. Administration of hCG stimulated HMG CoA reductase activity in a time- and dose-dependent manner: significant increases were noted within 4 h, with maximum effects (30-40-fold increases) seen 24 h after hCG (25 IU) administration. This effect was specific in that only LH, of several hormones tested, was as effective as hCG in stimulating HMG CoA reductase activity, and no change in the activity of either liver microsomal HMG CoA reductase or luteal microsomal NADPH-cytochrome c reductase was seen after hCG. The gonadotropin-induced increase in HMG CoA reductase activity seemed to be due to a net increase in enzyme activity, not to a change in the phosphorylated/dephosphorylated state of the enzyme. Pretreatment of animals with aminoglutethimide, an inhibitor of the conversion of cholesterol to steroid (pregnenolone), prevented the hCG-induced rise in HMG CoA reductase activity, whereas treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), which depletes cellular cholesterol content, led to striking increases in enzyme activity. However, the combined effects of 4-APP and hCG were additive, suggesting that the stimulating effect of hCG on HMG CoA reductase activity is not entirely due to a depletion of cellular sterol content of luteinized ovaries. Similarly, cholesteryl ester and cholesterol syntheses as measured by [14C]acetate conversion were also increased by hCG and 4-APP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human chorionic gonadotropin causes an estrogen-mediated induction of rat ovarian carbonyl reductase.

We earlier reported that human chorionic gonadotropin (hCG) stimulates rat ovarian carbonyl reductase (CR) activity and content, and that estrogen enhances the stimulatory effect. The present study was performed to determine the mode of action of the gonadotropin. Cycloheximide (CHX) and actinomycin D (AD) were given to estradiol-pretreated immature rats 6 h before hCG treatment. The enzyme activity was measured with three substrates, and the enzyme content was determined by the method of Western-blot analysis using anti-rat ovarian CR anti-serum as the first antibody. Both protein inhibitors significantly prevented hCG from increasing the enzyme activity and content in estradiol-pretreated ovary. These results indicate that rat ovarian CR is induced by LH via the action of estrogen.     

人胎盘滋养层细胞培养与体外hCG释放的研究

本研究的目的是了解细胞滋养层细胞和合胞体滋养层细胞体外分化和生物学特性。方法:采用酶消化和Percoll密度梯度离心法,对人足月胎盘细胞滋养层细胞进行分离、纯化和体外培养。采用放射免疫法(RIA)检测细胞培养上清液hCG含量的变化。结果:经分离和纯化的细胞滋养层细胞在体外培养中生长良好,通过细胞分裂和融合形成合胞体滋养层细胞,随着合胞体滋养层细胞的生长,细胞培养上清液中hCG含量显著升高。我们认为从胎盘中分离和纯化的细胞滋养层细胞在体外培养中可分化和融合形成合胞体滋养层细胞,体外hCG含量的增加与合胞体滋养层细胞生长有关。     

Testosterone and dihydrotestosterone levels in epididymis, vas deferens, seminal vesicle and preputial gland of mice after hCG injection

Temporal changes of testosterone (T) and dihydrotestosterone (DHT) levels were measured by RIA in epididymis, vas deferens, seminal vesicle and preputial gland of adult male mice after a single injection of hCG. The response of circulating T to hCG stimulation was rapid and persisted over a period of 48 h. The temporal changes of androgen content of target organs paralleled the modifications of circulating T. In all organs the high androgen levels attained at 1 or 4 h plateaued until 24 h, decreased thereafter and returned to basal values at 72 h. The concentration of T by sex accessory organs was more accelerated by hCG injection than its conversion into DHT.     

The relationship between ovarian progesterone and proteolytic enzyme activity during ovulation in the gonadotropin-treated immature rat.

To clarify the mechanisms by which progesterone acts as a mediator in the ovulatory process, ovulation rate and proteolytic enzyme activities were investigated in immature 22-day-old rats treated with PMSG/hCG, RU486 (10 mg/kg), synthetic antiprogesterone, and RU486 (10 mg/kg) + progesterone (10 mg/kg). The number of ova was significantly decreased when RU486 (10 mg/kg) was given from 2 h before to 4 h after the hCG injection. In addition, its inhibitory action on ovulation was reversed by exogenous progesterone (10 mg/kg) at 2 or 4 h after the hCG injection. Serum progesterone and estradiol concentrations in the rats treated with RU486 did not show any significant differences compared to controls. The proteolytic enzyme activities were measured by using the synthetic substrates alpha-N-benzoyl-DL-Arg-beta naphthylamide (BANA) and dinitrophenyl peptide (DNP). Activities were significantly increased after hCG injection in the control group during 8-9 h for BANA hydrolase and 7-10 h for DNP peptidase. The preovulatory increase of these activities was totally suppressed by RU486 with hCG. After administration of progesterone (10 mg/kg) following hCG and RU486 injection, the elevation of proteolytic, enzyme activities in the preovulatory phase was effectively reversed, and levels became similar to those in the control group. These results suggest that progesterone plays an indispensable role in the first 4 h of the ovulatory process by regulating proteolytic enzyme activities.     

Identification of insulin in the human pancreatic juice

In this study, we purified insulin-like substance (ILS) in the human pancreatic juice by the combined use of affinity chromatography and radioimmunoassay (RIA). The amino acid sequence of ILS in the N-terminal region is the same as that of human insulin. The influence of the enzymes present in the pancreatic juice on the RIA procedure, was examined. Trypsin, chymotrypsin and amylase showed steep influences on radioactivity. The addition of enzyme inhibitors could not reduce pseudo-activity, but the elimination of enzymes in the pancreatic juice by ultrafiltration with the Mole-Cut (Millipore, Japan) resulted in a lowering of the pseudo-insulin activity. Affinity chromatography on Sepharose 4B coupled with anti-porcine insulin was used to capture ILS. ILS was eluted by 1 M acetic acid from the affinity column monitoring pH and the insulin activity by RIA. The amino acid sequences of two components of ILS in amino terminal region were Phe-Val and Gly-Ile-Val. This indicates that ILS obtained from human pancreatic juice was the insulin derived from endocrine secretion of pancreas.