Reduction of albumin adsorption onto silicon surfaces by Tween 20

The ability of Tween 20 to reduce the adsorption of albumin on silicon surfaces of different hydrophobicity was investigated by ellipsometry. As expected, protein adsorption was found to depend on the degree of hydrophobicity of the surfaces and on the concentration of the surfactant. A reduction of 90% in albumin adsorption on hydrophobic methylated surfaces by 0.05% Tween 20 was achieved, whereas a reduction of only 15% on hydrophilic surfaces was observed. Experiments of time-dependent protein adsorption in both pure protein and protein-surfactant mixtures were conducted to ascertain the stability of physically adsorbed Tween 20 films on intermediate silicon surfaces. It was found that the adsorbed Tween 20 film was robust and there was no evidence of exchange of the Tween molecules with albumin for up to 240 min exposure. Adsorption minima were confirmed to correlate with minima in contact angle and critical micelle concentration (CMC). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 618-625, 1997.     

Development of immunosensors for the analysis of 1-naphthol in organic media

Immunosensor systems have been developed for the rapid determination of 1-naphthol. In this work, the comparison of performance of immunosensors working in aqueous and organic media was done. Direct, indirect and capture formats were studied. Immunoreagents were immobilized on controlled pore glass (CPG), hidroxysuccinimide agarose gel or on azlactone Protein A/G supports. The Protein A/G-based sensor showed the best performance. In aqueous media, a LOD of 16.2 microg l(-1) and a DR of 33.7-586.6 microg l(-1) were achieved employing Tween 20 at a concentration ranging from 0.01 to 0.05% v/v. Maximum sensitivity was reached with 0.025% of surfactant. Binary mixtures of methanol or acetonitrile with aqueous buffer and ternary mixtures of methanol/isopropanol or ethyl acetate/methanol with the same buffer were studied as organic media. The mixture 50% MeOH-50% 20 mM sodium phosphate, pH 8, with 0.05% (v/v) Tween 20 resulted to be the best. A detection limit of 12.0 microg l(-1) and a dynamic range of 53.6-17,756.0 microg l(-1) were reached. The recycling of Protein A/G-based sensor working in this media was about 300 assays. Preconcentration factors around 250 were achieved using methanol as extracting solvent. It has been demonstrated that the technique can be successful in carrying out the analysis of low solubility in water analytes, such as 1-naphthol. The sensors developed can use higher concentrations of organic solvent (up to 50% methanol) compared to ELISA. On the other hand, the advantage of preconcentration can also be taken for the use of the same procedure as recommended for standard sample treatments.     

Use of Horseradish-Peroxidase Labelled Antibodies in ELISA for Plant Virus Detection

Detection of plant viruses by ELISA using horseradish peroxidase for antibody labelling (ELISA-peroxidase) has been standardized by evaluating variants of the procedure, regarding composition and concentration of buffers and additives. Immunoglobulins (IgG) are isolated from antisera by precipitation with ammonium-sulphate and by purification with DEAE-52 (Whatman) cellulose. IgG are conjugated with horseradish peroxidase by a modified oxidation-periodate method. In ELISA-peroxidase 0.05 M carbonate-bicarbonate coating buffer pH 9.6 has been substituted by 0.01 M carbonate buffer pH 9.2. Extraction buffer is used with 0.5% bovine serum albumin (BSA), without polyvinylpyrrolidone (PVP). Samples are diluted in, phosphate buffered saline (PBS) pH 7.2 with 0.05% Tween 20 and 0.5% BSA. IgG are conjugated with horseradish peroxidase, diluted in 0.1 M Tris-HCl, pH 7.4 with 0.05% Tween 20 and 1% BSA. The substrate is incubated in the darkness for 20 min at room temperature. ELISA-peroxidase proved to be equivalent in sensitivity and specificity with ELISA using alkaline phosphatase for antibody labelling. Its advantage is a lower cost of chemicals used in the test.     

Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells

ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 microg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 microg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.     

An investigation of the antibacterial effect of carrot on Listeria monocytogenes.

Carrot slices immersed in a potassium phosphate buffer (0.1 mol/l, pH 7.0) or carrot tissue macerated in the buffer had a lethal effect on Listeria monocytogenes. This antilisterial activity was suppressed by anaerobiosis, thiol compounds (1 mmol/l) and bovine serum albumin (0.05%) but was not affected by sodium ascorbate (200 mmol/l), propyl gallate (25 mmol/l), catalase (1100 U/ml), superoxide dismutase (357 U/ml), or chelating agents (10 mmol/l). Free-radical scavengers had no effect at 10 mmol or 50 mmol/l but histidine and diazabenzocyclooctane at 100 mmol/l reduced the antilisterial activity. The addition of Tween 20, 0.05% (v/v), to carrot macerates improved the recovery of the activity in the supernatant liquid after centrifugation at 10,000 g for 2 min. The addition of higher concentrations of the detergent to the macerate reduced the antilisterial activity.     

The prevention of adsorption of interferents to radiolabelled protein by Tween 20

Radiolabels are often used to quantitatively determine the amount of protein immobilized on chromatographic supports, immunochemical plates and biosensor surfaces. Bovine serum albumin (BSA) was chosen as a model protein for quantitative deposition studies. BSA was radioiodinated (125I-) or fluorescently labelled (fluorescein), then incubated with the following surfaces: quartz, quartz derivatized by 3-aminopropyltriethoxysilane (Qz-APTES), and Qz-APTES reacted with glutaraldehyde or tresyl chloride. The amounts of BSA immobilized to the different surfaces were compared using data from radioactivity and fluorescence assays. Irreproducible results were obtained with radioiodinated BSA due to adsorption/desorption behaviour of an unidentified radioactive species. When the non-ionic detergent Tween 20 was added to the protein/surface incubation mixture, radiolabelled BSA gave reproducible protein binding results which agreed with fluorescent protein binding patterns. The effect of Tween 20 was due to either the binding to BSA displacing the interferent and/or the solubilization of the interferent.     

An investigation of the antibacterial effect of carrot on Listeria monocytogenes

Carrot slices immersed in a potassium phosphate buffer (0.1 mol/l, pH 7.0) or carrot tissue macerated in the buffer had a lethal effect on Listeria monocytogenes. This antilisterial activity was suppressed by anaerobiosis, thiol compounds (1 mmol/l) and bovine serum albumin (0.05%) but was not affected by sodium ascorbate (200 mmol/l), propyl gallate (25 mmol/l), catalase (1100 U/ml), superoxide dismutase (357 U/ml), or chelating agents (10 mmol/l). Free-radical scavengers had no effect at 10 mmol or 50 mmol/l but histidine and diazabenzocyclooctane at 100 mmol/l reduced the antilisterial activity. The addition of Tween 20, 0.05% (v/v), to carrot macerates improved the recovery of the activity in the supernatant liquid after centrifugation at 10 000 g for 2 min. The addition of higher concentrations of the detergent to the macerate reduced the antilisterial activity.     

Enzyme immunoassay for the determination of hexestrol in meat

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an anabolic hormone forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC₁₀) equaled 0.01 ng/ml, IC₅₀ equaled 0.17 ng/ml, and the working range (IC₂₀–IC₈₀) equaled 0.03–0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05, 2.9, and 0.26–32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.     

R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens.

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.     

Use of an immunoaffinity column for tetrachlorodibenzo-p-dioxin serum sample cleanup

Covalently linking 1-amino-3,7,8-trichlorodibenzo-p-dioxin with either keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) provided antigens that generated antibodies in chickens. Competitive ELISA analysis demonstrated that the antibodies isolated from egg yolk (IgY) bound with 1,3,7,8-tetrachlorodibenzo-p-dioxin (1,3,7,8-TCDD). The antibodies were linked to CNBr-Sepharose to generate an immunoaffinity column. Radiolabeled 1,3,7,8-TCDD in a 0.05% Tween 20 solution was retained by the column and could be eluted by increasing the Tween 20 concentration. The binding efficiency for 10.7 ng per ml gel matrix ranged from 85 to 97%. Immunoaffinity columns generated by this method did not effectively bind ¹⁴C-1,3,7,8-TCDD from serum samples. Diluting the serum 1:20 with 0.05% Tween 20 increased the binding efficiency. Alternately, ethanol–hexane extraction followed by solid phase extraction on a carbon column using a fat removal protocol also provided an appropriate preaffinity column cleanup for serum samples. After this preaffinity column cleanup, spiked serum samples applied to the immunoaffinity column showed binding efficiencies of over 90%.     

Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.     

表面活性剂对出芽短梗霉多糖生产影响的研究

研究了表面活性剂对出芽短梗霉细胞培养过程中多糖释放的影响。在摇瓶中,比较添加0.05%(w/v)的Tween 80、Tween 60、Tween 40,结果显示几种表面活性剂均能促进细胞释放多糖,其中以Tween 80的效果最佳。在5L发酵罐中,以100g/L玉米粉水解液做碳源的出芽短梗霉细胞培养液中分别添加了表面活性剂Tween 80 0.01%、0.05%、0.1%,其中以添加Tween 800.05%时的效果最好,与不添加表面活性剂相比多糖产量提高25%左右,发酵周期缩短了将近2d。     

The effect of cultural conditions on the production of lipase by Acremonium strictum

Summary The optimum cultural conditions for the production of lipase byA. strictum under stationary condition are: period of incubation, 7 days; temperature, 30°C; xylose at a concentration of 2% (w/v) and 3.5% (w/v) soyabean meal as carbon and nitrogen sources respectively. Incorporation of 1% (v/v) of Tween 80 in culture medium enhanced enzyme production while the presence of fatty acids reduced both fungal growth and lipase production. The enzyme showed broad substrate specificity.     

A simple procedure for identification ofClostridium botulinum colonies

For direct identification of toxigenic colonies ofClostridium botulinum type E, suspected colonies are uniformly suspended in a phosphate buffer containing 0.5% (w/v) gelatin and 0.05% (w/v) Tween 20. After centrifuging, the supernatant is tested for botulinal toxin by an enzyme-linked immunosorbent assay (ELISA). The assay is specific for this type as it did not react with culture filtrates of otherClostridium species, including non-toxigenic E-like organisms.     

Development of an IMS-PCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water

AIMS: To develop a sensitive detection method for Mycobacterium avium ssp. paratuberculosis (Map) in water by modifying and optimizing an existing immunomagnetic separation polymerase chain reaction (IMS-PCR) technique. METHODS AND RESULTS: Sterile distilled water (50 ml) spiked with 10(6) Map ml(-1) was subjected to either filtration (0.45 microm pore size) followed directly by IS900 PCR (method 1) or centrifugation (2500 g for 20 min) followed by IMS and IS900 PCR (method 2). Method 2 permitted the detection of Map, whereas method 1 did not. Method 2 was then optimized by adding different concentrations of Tween 80 (0.05, 0.1, 0.2, 0.4 and 0.6% v/v) to water samples spiked with Map (10(6)-1 CFU ml(-1)) prior to centrifugation, and assessing the impact of this action on the detection sensitivity of subsequent IMS-PCR. The optimum Tween 80 concentration was found to be 0.4%, which permitted the detection of 10 Map CFU ml(-1) in spiked water samples by IMS-PCR. CONCLUSIONS: This method will be used to determine the incidence of Map in water destined for domestic use in future studies. SIGNIFICANCE AND IMPACT OF THE STUDY: A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.     

Enantioselective resolution of racemic styrene oxide at high concentration using recombinant Pichia pastoris expressing epoxide hydrolase of Rhodotorula glutinis in the presence of surfactant and glycerol

The reaction medium was optimized to accomplish epoxide hydrolase-catalyzed, batch enantioselective hydrolysis of racemic styrene oxide at high initial substrate concentrations. The recombinant Pichia pastoris containing the epoxide hydrolase gene of Rhodotorula glutinis was used as the biocatalyst. Enantiopure (S)-styrene oxide with 98% ee was obtained with 41% yield (maximum yield = 50%) from 1.8 M racemic styrene oxide at pH 8.0, 4 degrees C in the presence of 40% (v/v) Tween 20 and 5% (v/v) glycerol.     

Anti-Ulcerogenic Effect of Methanolic Extracts from Enicosanthellum pulchrum (King) Heusden against Ethanol-Induced Acute Gastric Lesion in Animal Models

A natural source of medicine, Enicosanthellum pulchrum is a tropical plant which belongs to the family Annonaceae. In this study, methanol extract from the leaves and stems of this species was evaluated for its gastroprotective potential against mucosal lesions induced by ethanol in rats. Seven groups of rats were assigned, groups 1 and 2 were given Tween 20 (10% v/v) orally. Group 3 was administered omeprazole 20 mg/kg (10% Tween 20) whilst the remaining groups received the leaf and stem extracts at doses of 150 and 300 mg/kg, respectively. After an additional hour, the rats in groups 2–7 received ethanol (95% v/v; 8 mL/kg) orally while group 1 received Tween 20 (10% v/v) instead. Rats were sacrificed after 1 h and their stomachs subjected to further studies. Macroscopically and histologically, group 2 rats showed extremely severe disruption of the gastric mucosa compared to rats pre-treated with the E. pulchrum extracts based on the ulcer index, where remarkable protection was noticed. Meanwhile, a significant percentage of inhibition was shown with the stem extract at 62% (150 mg/kg) and 65% (300 mg/kg), whilst the percentage with the leaf extract at doses of 150 and 300 mg/kg was 63% and 75%, respectively. An increase in mucus content, nitric oxide, glutathione, prostaglandin E2, superoxide dismutase, protein and catalase, and a decrease in malondialdehyde level compared to group 2 were also obtained. Furthermore, immunohistochemical staining of groups 4–7 exhibited down-regulation of Bax and up-regulation of Hsp70 proteins. The methanol extract from the leaves and the stems showed notable gastroprotective potential against ethanol.     

The effect of Tween 20 on indirect immunoperoxidase staining of blood group antigen A in human urothelium

The effect of the nonionic detergent Tween 20 on background staining, sensitivity, and specificity in the indirect immunoperoxidase staining for blood group antigen A was investigated histologically and spectrophotometrically. Pretreatment of dewaxed formalin-fixed Paraplast-embedded tissue sections from human ureters with 2% Tween 20 and dilution of the first and second layer antisera with 0.05 or 2% Tween 20 significantly reduced background staining of the urothelial cell cytoplasm, ureteral stroma, and musculature. Spectrophotometrical analysis of tissue sections from hypernephroma (rich in cytoplasm), cervix (fibrous stroma), and myometrium (musculature) underlined the histological results with a significant reduction of the maximum absorbance of Tween 20-modified indirect immunoperoxidase-stained tissue sections. Sensitivity, evaluated histologically by the endpoint titers of urothelial cell membrane staining, endothelial cell staining, and focal cytoplasmic staining of urothelial cells, was not influenced by the Tween 20 treatment. The specificity was improved as the staining was highly reduced or absent in control sections subjected to Tween 20.     

A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies

An extracellular, thermostable, alkaline lipase was partially purified from a thermophilic Bacillus strain J 33. It was optimally active at pH 8.0 at 60°C, retaining 50% activity at 70°C for 30 min. It had native molecular mass of 45 kDa. The lipase was stable in 90% (v/v) hexane or benzene mixtures in water. It converted 66% oleic acid at 0.25 M with 0.4 M methanol in hexane to methyl oleate at 60°C in 16 h. Activity was stimulated by Mg2 (10 mM) but inhibited by EDTA (10 mM) and PMSF (10 mM). It was stable in Triton X-100, Tween 20 and Tween 80 (0.1% v/v). © Rapid Science Ltd. 1998     

Loss of plasma membrane proteins of bull spermatozoa through the freezing-thawing process

The widespread application of A. I. and realization of its full potential depends largely on the use of frozen semen. However, fertility resulting from A. I. is poorer than that from fresh semen in most species. The objective of this study was to compare the protein composition of fresh and frozen-thawed bull sperm plasma membrane surface. The effect of Tween 20 on protein removal from fresh and frozen sperm plasma membrane surface was studied and compared. The effect of incubation with different detergent concentrations on sperm motility and viability was examined. Approximately 2 x 10(8) frozen-thawed bull spermatozoa washed through a discontinuous Percoll gradient were incubated for 15 min at 20 degrees C with 0.01, 0.03 and 0.05% Tween 20. Sperm motility was completely eliminated at all 3 assayed detergent concentrations, while the initial sperm viability of 52% was decreased to 26, 10 and 5%, respectively, at the 3 concentrations. The removal of sperm plasma membrane proteins also increased from 0.72 mg to 2 mg with 0.05% Tween 20. Similar results were found with fresh semen samples. Although the amount of extracted proteins was significantly lower than that obtained with frozen spermatozoa, fresh sperm motility was likewise eliminated by the detergent treatment, and sperm viability was decreased. A semen sample with an initial sperm viability of 59% had a value of only 8% after treatment with 0.05% Tween 20. Comparative SDS-PAGE analysis of the extracted fractions from fresh and frozen-thawed semen treated with Tween 20 showed that the higher amount of extracted proteins in the frozen semen samples corresponded to the egg yolk lipoproteins in the cryoprotectant medium. However, it is worth noting that 4 more bands were found in the sample obtained from fresh semen than from frozen semen. These results indicate that some cell membrane proteins are lost through the freezing-thawing process.