An aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection

An aptamer-based chromatographic strip assay method for rapid toxin detection was developed. The aptamer-based strip assay was based on the competition for the aptamer between ochratoxin A and DNA probes. The sensing results indicated that the sensitivity of the aptamer-based strip was better than that of conventional antibody-based strips. The visual limit of detection of the strip for qualitative detection was 1 ng/mL while the LOD for semi-quantitative detection could down to 0.18 ng/mL by using scanning reader. The recoveries of test samples were from 96% to 110%. All detections could be achieved in less than 10 min, indicating that the aptamer-based strip could be a potential useful tool for rapid on-site detections.     

An enzyme-immobilized microplate for determination of linamarin for large number of samples

Summary An enzyme-immobilized microplate for determination of linamarin was prepared by covalently linking cassava leaf linamarase to the microplate. For linamarin determination, cassava roots were homogenised in 0.1 Mo-phosphoric acid and the filtrate adjusted to pH 6 with NaOH prior to adding into the wells. The cyanide released was then determined spectrophotometrically. One nmol linamarin can be detected. The microplate method is suitable for analysis of large number of samples and is useful for screening purposes.     

Nylon-linamarase electrode for rapid determination of linamarin

Summary An enzyme electrode was constructed using cassava leaf linamarase covalently linked via polyethyleneimine to Hybond-N nylon. The nylon-enzyme electrode response was Nerstian for linamarin range of 0.1 to 20 mM. A steady state reading could be obtained within 4 to 6 mins. The nylon-enzyme discs could be reused. Compared to the previously reported enzyme electrode prepared by entrappment of linamarase in ENT-4000 prepolymer resins, the nylon-enzyme electrode gave faster response and could save analysis time by 60%.     

Stable paper indicator systems for rapid identification of microorganisms]

The authors prepared under experimental-industrial conditions paper indicator systems for the express identification of microbes, including carbohydrate discs (by the method of Nikitin et al.), and newly worked out types for the determination of the activity of cytochromoxidase, urease, indol formation, and indicator amino acid decarboxylases (lysin, ornithin, arginine). The use of paper indicator discs proved to be expedient for rationalization of laboratory diagnosis of bacterial intestinal infections.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

An adaptation of the filter paper disk technique for the differential estimation of DNA and RNA of rat liver

A method is described for the differential estimation of DNA and RNA from rat liver on filter paper disks. To differentiate between DNA and RNA, samples are subjected to alkaline hydrolysis in microtitration plates prior to absorption on the disks. This procedure eliminates the loss of DNA which is observed if the disks are subjected to RNA hydrolysis after the sample has been absorbed on disks. The procedure is directly comparable to standard methods for the measurement of nucleic acids.     

Use of paper indicator discs for determining the minimal inhibitory concentration of antibiotics]

Sensitivity of clinical strains of Staphylococcus and some Enterobacteriaceae to a number of widely used antibiotics was compared simultaneously with the use of two methods, i. e. the agar diffusion method and the method of serial dilutions. Regularities in distribution of the staphylococcal strains according to their sensitivity to antibiotics, such as erythromycin, benzylpenicillin, levomycetin and others were also studied with respect to every year using indicator paper discs. Interrelation observed during the comparison of the microbial sensitivity with the use of the two assay methods provided elaboration of the criteria for classification of the strains as "resistant" or "sensitive". The differentiation boarder for these two groups was determined according to the principle of the assay error minimization. A necessity of using standard dry media for specification of individual characteristics of various drugs in estimation of the microbial sensitivity to them by the agar diffusion method is emphasized.     

A modified ferrozine method for the measurement of enzyme-bound iron

A general procedure for the determination of the iron content of enzymes by digestion with methanesulfonic acid to release protein-bound iron has been developed. This procedure replaces the tedious and potentially hazardous method of wet ashing with concentrated nitric-sulfuric-perchloric acids. The method has been used to determine the stoichiometry of iron for nanomole quantities of heme-iron proteins, iron-sulfur proteins, complex iron-sulfur proteins, as well as in phenylalanine hydroxylase, an enzyme with iron in an undetermined coordination.     

Implications of paper vs stainless steel biological indicator substrates for formaldehyde gas decontamination

Aims: This study was undertaken to determine the effectiveness of biological indicators currently being employed during formaldehyde decontamination. Data suggest that detectable amounts of formaldehyde are absorbed into the paper strips contained in currently used biological indicators. Absorbed formaldehyde has the potential to inhibit the growth of indicator spores, thus leading to false negative results. Indicators composed of either stainless steel carriers or paper strips were investigated to determine whether stainless steel carriers can be used as an alternative to paper strip indicators. Methods and Results: Biological indicators were exposed to formaldehyde gas and were tested for the presence of formaldehyde and any possible inhibition of spore growth. Absorbed formaldehyde was detected in the paper strip carriers while no formaldehyde was detected from any of the stainless steel carriers. Exposed paper strips were found to inhibit growth of up to 1 × 10⁶ spores while the stainless steel carriers did not inhibit the growth of spores. Conclusions: During decontamination, biological indicators composed of paper spore strips absorb formaldehyde and inhibit growth of any surviving spores. Stainless steel carriers do not absorb formaldehyde and are an ideal alternative substrate for biological indicators. Significance and Impact of the Study: The popular paper strip biological indicator can lead to false negative results during decontamination and is unsuitable for validating formaldehyde decontamination.     

Determination of the toxigenicity of Corynebacterium diphtheriae by using paper indicator disks

The method for the determination of the toxigenicity of C. diphtheriae by means of discs impregnated with diphtheria antitoxin is proposed. The discs can be prepared long before use and stored for a year. The determination of toxigenicity with these discs is not inferior in its sensitivity to the routine procedure of the immunoprecipitation test. The disc method decreases the consumption of unavailable diphtheria antitoxin by 10 times, that of culture media by 2-2.5 times: besides, it is less labor consuming.     

Quantitative estimation of gibberellic acid by paper chromatography

Метод для количественного определения от gibberellic кислоты в процессе брожения средства массовой информации, с использованием бумаги по убыванию хроматографии в butylacetate воды описана. Образца корректируется, чтобы рН 2.5-3.0, добыто с н-бутанола, и 0,05 мл. органического слоя пятнами на Хроматографический бумагу. После equilibration от Атмосфера в банке, chromatogram Разработана в butylacetate насыщенных с водой, за 7 часов, и растворитель разрешено покинуть капельного нижней части листа. Обнаружение осуществляется путем опрыскивания с 3% раствор серной кислоты в метаноле и после сушки бумаги, пятна с синий u.v. флуоресценции наблюдается. Определенный артикль площадь пятна оценивается с помощью калибровочной кривой, заговор с ценностей, стандартов, соответствующих 20, 60 и 120 μ g. gibberellic кислоты. Погрешность оценки составляет ± 10-15% когда оценки выполняются тщательно. Низкий предел чувствительности 5 μg.