Chemiluminescence Detection Method Research Articles

A sensitive chemiluminescent enzyme immunoassay for the bioanalysis of carboxyl-terminal B-chain analogues of human insulin.

Quantification of analogues of human insulin in biological matrices is complicated by differences in their immunoreactivity and the presence of both the analogue and endogenous concentrations of insulin in test samples. To facilitate pharmacokinetic comparisons of carboxyl-terminal B-chain analogues of human insulin, we undertook development of a sensitive ELISA. The ELISA detection method was optimized systematically to permit routine analysis of 10-microl serum samples. Accordingly, a noncompetitive 'sandwich' chemiluminescent ELISA was validated for the quantification of carboxyl-terminal B-chain insulin analogues in human serum over a concentration range from 5 to 3125 pM. The mean bias (RE%) within the validated range varied from -10.3 to 4.3%, with an intermediate precision (inter-assay CV%) from 4.2 to 11.5%. The two-sided 90% expectation tolerance interval for total measurement error was within +/-25% of the nominal concentration for all levels of validation samples. Insulin lispro, human insulin, proinsulin, despentapeptide insulin (DPI) and porcine insulin displayed comparable crossreactivity in the ELISA. Potential utility of the new assay for insulin bioanalysis in nonhuman species was investigated by assessing the pharmacokinetic profile of DPI in rats following administration of a single subcutaneous dose. The sensitive chemiluminescent detection method is simple to perform and should be readily adaptable for ELISAs of other therapeutic proteins.

Detection of trace amounts of hidden allergens: hazelnut and almond proteins in chocolate

Many patients with immediate type allergy to tree pollen also suffer from intolerance to hazelnuts and almonds. Since rather low levels of hazelnut and almond proteins can provoke an allergic reaction in sensitized individuals, an immunoblot technique has been developed for the detection of potentially allergenic hazelnut and almond proteins in chocolate. Initially, IgE binding hazelnut and almond proteins were detected by immunoprobing with allergic patients’ sera. For routine analysis, patients’ sera were substituted with polyclonal rabbit antisera, and sensitivity was enhanced by the use of a chemiluminescent detection method. This technique allowed the detection of less than 0.5 mg of hazelnut or almond proteins per 100 g of chocolate (=5 ppm). It was applied for routine screening purposes in product quality control as well as for optimization of cleaning steps of filling facilities to minimize cross contamination during production.

Yeast V-ATPase Complexes Containing Different Isoforms of the 100-kDa a-subunit Differ in Coupling Efficiency and in VivoDissociation

The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.

Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production and Clinical Application: Improved Safety and High Titer

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

Effect of arthrocentesis and sodium hyaluronate injection on nitrite, nitrate, and thiobarbituric acid-reactive substance levels in the synovial fluid

To evaluate the effect of arthrocentesis and sodium hyaluronate (SH) injections on nitrite, nitrate, and thiobarbituric acid-reactive substance (TBA-RS) levels in temporomandibular joint internal derangements. Arthrocentesis was performed on 10 patients, and 15 patients received a supplemental injection of SH after arthrocentesis. All these patients received an SH injection 15 days after the first intervention. The synovial fluid samples were obtained before arthrocentesis on the first appointment and before the SH injection 15 days later. Nitrite and nitrate levels were measured with a highly sensitive and specific chemiluminescence detection method, and the concentration of lipid peroxidation products was assessed by means of the thiobarbituric acid reaction. Symptomatic improvement was seen in both groups. Nitrite, nitrate, and TBA-RS levels only decreased significantly (P <.05) with a supplemental SH injection after arthrocentesis. Intra-articular injections of SH may reduce nitrite, nitrate, and TBA-RS levels that play a role in the pathogenesis of various temporomandibular disorders.

Capillary electrophoresis of monoamines and catechol with indirect chemiluminescence detection.

A capillary electrophoresis (CE)/indirect chemiluminescence (CL) detection method is described for monoamines, viz., serotonin (5-HT), dopamine (DA), epinephrine (EP), and norepinephrine (NE) and for catechol (CA). Optimal separation and detection were obtained with an electrophoretic buffer of 10 mM sodium borate (pH 9.5) containing 5 mM luminol and 25 mM H2O2, and a catalyst solution of 30 microM CuSO4 in 30 mM borate buffer (pH 10.0). Complete separation of 5-HT, DA, EP, NE and CA was achieved in less than 5 min. The Cu(II)-catalyzed luminol CL reaction was employed to provide the high and constant background. Since monoamines and catechol can form stable complexes with Cu(II), inverted analyte peaks due to decreased catalytic activity of Cu(II) can be detected. The degree of CL suppression is proportional to the analyte concentrations. Linearity (r> or =20.99) over two orders of magnitude was generally obtained. The concentration limits of detection (CLODs) for the monoamines and catechol studied were between 0.5 and 3.1 uM. The relative standard deviation (RSD) values on peak size and migration time were in the ranges 3.2-4.4% and 0.4-0.5%, respectively. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.

Capillary electrophoresis of monoamines and catechol with indirect chemiluminescence detection

A capillary electrophoresis (CE)/indirect chemiluminescence (CL) detection method is described for monoamines, viz., serotonin (5-HT), dopamine (DA), epinephrine (EP), and norepinephrine (NE) and for catechol (CA). Optimal separation and detection were obtained with an electrophoretic buffer of 10 mM sodium borate (pH 9.5) containing 5 mM luminol and 25 mM H2O2, and a catalyst solution of 30 microM CuSO4 in 30 mM borate buffer (pH 10.0). Complete separation of 5-HT, DA, EP, NE and CA was achieved in less than 5 min. The Cu(II)-catalyzed luminol CL reaction was employed to provide the high and constant background. Since monoamines and catechol can form stable complexes with Cu(II), inverted analyte peaks due to decreased catalytic activity of Cu(II) can be detected. The degree of CL suppression is proportional to the analyte concentrations. Linearity (r> or =20.99) over two orders of magnitude was generally obtained. The concentration limits of detection (CLODs) for the monoamines and catechol studied were between 0.5 and 3.1 uM. The relative standard deviation (RSD) values on peak size and migration time were in the ranges 3.2-4.4% and 0.4-0.5%, respectively. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.

Association between arthroscopic diagnosis of temporomandibular joint osteoarthritis and synovial fluid nitric oxide levels

Objective. The purpose of this study was to determine whether there is a relationship between synovial fluid levels of nitric oxide and clinical and arthroscopic findings of synovitis or cartilaginous degeneration. Study design. Arthroscopic surgery was performed on 20 joints in 15 female patients with internal derangement and osteoarthritis of the temporomandibular joint. Synovial fluid aspirates were obtained immediately before arthroscopy. Synovial fluid was also obtained from 14 joints of 11 female asymptomatic volunteers. The concentration of nitrite in the fluid recovered from each temporomandibular joint was measured through use of a highly sensitive and specific chemiluminescence detection method, calibrated per 1 mg of synovial fluid protein and expressed as nitric oxide; the result was then compared with clinical and arthroscopic findings of synovitis and cartilaginous degeneration. Results. Significantly higher levels of nitric oxide (median, 0.331 μmol/mg) were seen in the patients with internal derangement and osteoarthritis than in the control group (median, 0.001 μmol/mg; P < .0001). Synovial fluid from joints with pain in the joint area had significantly higher levels of nitric oxide than did fluid from joints without such pain. Synovial fluid from joints with degenerative changes (median, 0.467 μmol/mg) had significantly higher levels of nitric oxide than did fluid from joints without osteoarthritis (median, 0.057 μmol/mg; P < .05). Although the levels of nitric oxide in synovial fluid aspirates were markedly elevated in some joints with synovitis, there was no correlation between the levels of nitric oxide and the presence of synovitis. Conclusions. The findings indicate that increased levels of nitric oxide are involved in the pathogenesis of cartilaginous degeneration of the temporomandibular joint. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;88:129-36)

Chemiluminescence Determination of Sulfite in Sugar and Sulfur Dioxide in Air Using Ru(bipy)32+-K2S2O8 System

A chemiluminescence (CL) detection method for the determination of sulfite is described. The light produced in the reaction of Ru(bipy)32+(bipy=2,2′-bipyridyl)-SO32--K2S2O8 is detected. The concentration of sulfite is proportional to the CL intensity in the range 1.5×10-7 - 1.O×10-4 mol/l. The limit of detection is 4.1×10-8 mol/l and the relative standard deviation is 4.3% for 2×10-5 mol/l sulfite solution in 9 repeated measurements. This method has been successfully applied to the determination of sulfite in sugar and sulfur dioxide in air by using triethanolamine (TEA) as the absorbent material.

Chemiluminescent detection of amines and amino acids using in situ generated Ru(bpy) 33+ following separation by capillary electrophoresis

A new sensitive chemiluminescent detection method for capillary electrophoresis is described. Underivatized amines and amino acids were detected following capillary electrophoresis separation by their chemiluminescent reaction with Ru(bpy) 3 3+ generated in situ at 35 μm carbon fibers. Detection limits for triethylamine and proline were 5 and 3 fmol, respectively at a SNR of three. The noise limiting the detectability of separated analytes was determined to exist at the level of the dark noise limit of the PMT used for these studies and additional noise reduction strategies are expected to improve the quantitative aspects of the method. Theoretical plate numbers for proline were approximately 20 000. End column addition of Ru(bpy) 3 2+ coupled with in situ generation of Ru(bpy) 3 3+, has been shown to be compatible with the nanoliter elution volumes characteristic of capillary electrophoresis.

Western immunoblot analysis.

AbstractSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1) combined with Western immunoblot analysis (2,3) is a widely used technique for quantitatively and qualitatively evaluating specific protein expression. This chapter describes a Western immunoblot technique routinely used in our laboratories for protein analysis with representative data shown in Fig. 1. The protocols for SDS-PAGE and electrophoretic transfer are based on using the Mini-PROTEAN II system (Bio-Rad, Hercules, CA). Protein detection is based on using the enhanced chemiluminescence (ECL) detection method (Amersham, Arlington Heights, IL). In this technique, mixtures of solubilized proteins under denaturing conditions (in the presence of detergent [SDS], and reducing agent [mercaptoenthanol]) are separated by electrophoresis based on their molecular weights, followed by transferring proteins to PVDF membrane and identifying a protein of interest using a specific antibody. It is highly recommended that, before beginning the protocol, the operator read Subheading 4. Western immunoblot analysis of protein expression of eNOS in ovine fetoplacental artery endothelial cells in culture: Proteins (2 μg protein/lane) were separated by electrophoresis (7.5% SDS-PAGE gels). After transfer to the membrane, immunoblotting was performed using a mouse monoclonal ecNOS antibody (1∶750 [0.33 μg/mL]; Transduction Laboratories, Lexington, KY) with ECL dectection at 5-min exposure, as described. Lanes 1–2, controls; lanes 3–8, cells treated with basic fibroblast growth factor (1 or 10 ng/mL) for 24 h. KeywordsGlass PlateTris BaseAmmonium PersulfateTransfer BufferElectrophoresis BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Determination of sulfite in sugar and sulfur dioxide in air by chemiluminescence using the Ru(bipy)32+–KBrO3 system

A chemiluminescence (CL) detection method for the determination of sulfite using the reaction of Ru(bipy)32+ (bipy = 2,2′-bipyridyl)–SO32––KBrO3 is described. The concentration of sulfite is proportional to the CL intensity in the range 2.5 × 10–8–9.5 × 10–5 mol l–1. The limit of detection is 3.8 × 10–9 mol l–1 and the relative standard deviation is 4.6% for 5 × 10–5 mol l–1 sulfite solution with nine repeated measurements. This method was successfully applied to the determination of sulfite in sugar and sulfur dioxide in air by using triethanolamine as the absorbent material.

Chemiluminescent biosensors based on porous supports with immobilized peroxidase

Combining a sensitive and fast chemiluminescence detection method with novel immobilization methods for horseradish peroxidase (HRP) has enabled us to develop sensitive sensors for hydrogen peroxide and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). HRP biospecific immobilization was shown to have well-defined advantages in terms of a higher specific activity of the enzyme on the surface and a greater sensitivity in the enhanced chemiluminescence (ECL) reaction. The lower detection limit for hydrogen peroxide was 0·025 nmol with HRP immobilized on nitrocellulose membrane through avidin-biotin linkage. An immunosensor for the detection of 2,4-D provides a lower detection limit of 0·2 μg/l with specific antibodies covalently immobilized on photoactivated nylon. The assay with chemiluminescent detection was also characterized with a wider concentration range when compared with the colorimetric assay.

Methods and reagents: Chemiluminescent detection methods

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the internet. This month's column discusses some tips for using chemiluminescent detection methods. For details on how to partake in the newsgroup, see the accompanying box.

Off-column chemiluminescence detection in capillary electrophoresis

An off-column chemiluminescence (CL) detection method for capillary electrophoresis (CE) is described. A cellulose acetate-coated porous polymer joint was created on-column near the end of the separation capillary to electrically isolate the CL detector from the CE system. This off-column detection scheme was found necessary in cases where the CL reagent or catalyst added at the column end will electrophoretically migrate into the separation capillary and decompose the analytes prior to their detection. Loss of efficiency due to the presence of a porous polymer joint was minimal. Application of off-column CL detection to CE of serotonin, catecholamines and catechol was demonstrated.

Ca 2+-Dependent changes of acetylcholine release and IP 3 mass in Torpedo cholinergic synaptosomes

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP 3) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP 3 mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP 3 mass levels under resting conditions. The IP 3 mass was neither modified by changes in external Ca 2+ nor by a Ca 2+-free medium containing EGTA. IP 3 mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K + and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca 2+ emphasizing that Ca 2+ entry, independently of the influx mechanism involved, leads to an IP 3 increase. The phospholipase C β inhibitors U-73122 and U-73343 reduced K +-stimulated IP 3 levels while K +-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization–neurotransmitter secretion coupling. The effect reported showing an increase of IP 3 by agents that stimulate ACh release may suggest a possible link between IP 3 metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C.

Ca2+-DEPENDENT CHANGES OF ACETYLCHOLINE RELEASE AND IP3 MASS IN TORPEDO CHOLINERGIC SYNAPTOSOMES

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP3) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP3 mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP3 mass levels under resting conditions. The IP3 mass was neither modified by changes in external Ca2+ nor by a Ca2+-free medium containing EGTA. IP3 mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K+ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca2+ emphasizing that Ca2+ entry, independently of the influx mechanism involved, leads to an IP3 increase. The phospholipase Cβ inhibitors U-73122 and U-73343 reduced K+-stimulated IP3 levels while K+-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP3 by agents that stimulate ACh release may suggest a possible link between IP3 metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C. Copyright © 1996 Elsevier Science Ltd

Elevated levels of nitric oxide in synovial fluid from patients with temporomandibular disorders

The objective of this study was to measure nitric oxide activity in synovial fluid samples taken from patients with temporomandibular disorders. We investigated six volunteers without symptoms and 56 patients with temporomandibular disorders. Nitric oxide activity was measured in temporomandibular joint synovial fluid with a highly sensitive and specific chemiluminescence detection method. A detectable nitrite concentration was found in only 1 of 10 joints in the control group. Measurable levels (> 0.001 mol/l) of nitrites in the synovial fluid were found in 8 (72.7%) of 11 joints with disk derangement with clicking, 24 (85.7%) of 28 joints with disk derangement with locking, and all 26 (100%) joints with osteoarthritis. The mean nitrite concentrations in the temporomandibular joint synovial fluid in the disk derangement with clicking, disk derangement with locking, and osteoarthritis groups were significantly higher than that in the control group (p < 0.05, p < 0.001, p < 0.001, respectively). These data clearly show increased nitric oxide production in certain temporomandibular disorders and suggest that nitric oxide may play a role in the pathogenesis of synovitis and degenerative changes of cartilaginous tissue and bone of the temporomandibular joint.

Determination of inorganic anions in water samples by ion-exchange chromatography with chemiluminescence detection based on the neutralization reaction of nitric acid and potassium hydroxide

A faint chemiluminescence (CL) from the neutralization reaction of nitric acid and potassium hydroxide was enhanced by addition of iron(III) to the acid. The enhanced CL emission was suppressed by adding inorganic anions such as chloride, bromide, nitrite, nitrate and sulfate to the base. Based on the findings, a new post-column CL detection method was developed for the determination of the anions. Several kinds of anions were separated by anion-exchange chromatography using a potassium hydroxide solution as eluent. The effluent from the anion-exchange column was mixed with nitric acid containing iron(III) ions, and the CL was suppressed by the eluting anions. For each anion, linear calibration ranges extending from 100 ng ml −1 to 100 μg ml −1 were obtained. The relative standard deviations for five replicate measurements of 10 μg ml −1 were 1.3–4.3%. The present method was successfully applied to determine inorganic anions in water samples.

Anti-melanoma monoclonal antibody HMB-45 on enhanced chemiluminescence-western blotting recognizes a 30-35 kDa melanosome-associated sialated glycoprotein.

HMB-45 is an anti-melanoma monoclonal antibody widely used in diagnostic pathology owing to its great specificity in identifying poorly differentiated melanomas. In this study, by a series of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots with the enhanced chemiluminescent (ECL) detection method on the HU-214 melanoma cell line, we identified the antigen of HMB-45 in a protein or proteins of 30-35 kDa. Although this result is in discrepancy with the previous literature which identified the antigen as a protein of 7 or 10 kDa, a family of proteins of 25-70 kDa of as a protein of 100 kDa (gp100), the present data indicate that the antigen signal we found might be specific. Furthermore, immunoblots on neuraminidase-treated cell lysates show, in agreement with already published data, that the antigen might be a sialated glycoprotein with the sialic acid involved in the epitope. Immunoblots on partially purified melanosomes confirmed the presence of the antigen in these organelles.