Bioluminescent assay of D-3-hydroxybutyrate in serum

An enzymatic assay of D -3-hydroxybutyrate in which the hydroxybutyrate dehydrogenase reaction is coupled to the bacterial oxidoreductase—uciferase system is described. The bioluminescent assay is based on either, end-point, or on initial velocity measurements. This simple and rapid assay requires a single serum sample of 10 μl. Its linear range covers two orders of magnitude from 10^?6 mol/I upwards. This assay is suitable for the routine determination of D -3-hydroxybutyrate in human blood with good accuracy.     

Bioluminescent assay of total bacterial contamination of drinking water.

A bioluminescent assay of total bacterial contamination (TBC) of drinking water (DW) with a detection limit of approximately 1 CFU/mL and duration of less than 7 h has been developed. The protocol of the TBC assay comprises: incubation of water sample in nutrition broth supplemented with salts mixture, up to 6 h; filtration of bacterial suspension obtained through membrane filter (pore size 0.45 microm); release of bacterial ATP by dimethyl sulphoxide; determination of bacterial ATP concentration using highly sensitive ATP reagent based on recombinant Luciola mingrelica luciferase. To simplify the assay, special luminometer microcuvette Filtravette (New Horizons Diagnostics Corp., USA) are used. A good correlation (R=0.98) between ATP concentration measured after 6 h incubation and initial bacterial titre in DW was observed. Semi-quantitative TBC assay of DW is also available. The TBC value in DW is assessed by the fixation of incubation time required to detect a measurable bioluminescent signal: 3, 4 and 6 h corresponds to 100-1000, 10-100 and 1-10 CFU/mL, respectively.     

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor™ filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 × 10³ cfu/mL of E. coli in the sample within 2 h with high accuracy (CV = 1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.     

A semi-automated protein assay for cell cultures

A semi-automated modification of the protein determination procedure of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275) is described. The assay is well suited to the analysis of the protein of adherent cultured cells. The procedure is carried out in 96-well microtest plates on protein solutions of 50 microliter or less, and can detect less than 0.5 micrograms of protein (equivalent to about 10(3) cultured cells). Optical densities are read and printed by an automatic microplate reader capable of processing 96 samples in less than 2 min.     

Evaluation of rapid immunochromatographic assay kit for HBsAg-screening using whole blood

A rapid immunochromatographic assay kit using whole blood to screen hepatitis B surface antigen was developed and evaluated by using sera from 240 patients. The reference diagnosis was based on the results obtained with GENEDIA Anti-HBs Rapid kit which is very similar to the above kit except for the use of serum. The test demonstrated a good correlation with the reference immunochromatographic assay kit, that is, the sensitivity and the specificity of the kit was 100%, respectively. The rapid test kit using whole blood should be more convenient and useful for the diagnosis of hepatitis B virus because the kit does not need machines and time to prepare serum. In addition, this kit is safe from inadvertent infection during sample treatment because the blood is sterilized with hydrogen peroxide, eliminates the procedure required to prepare serum and reduces the possibility of exposure to infectious agents.     

Bioluminometric assay of ATP in mouse brain: Determinant factors for enhanced test sensitivity

Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based onin vitro cell systems. This study reports an optimized FLB procedure for the analysis of ATP in small tissue samples. The results showed that the sensitivity of the FLB test can be enhanced several fold by using ultraturax homogenizer, perchloric acid extraction, neutralization of acid extract and its optimal dilution, before performing the assay reaction.     

Fibroblast monolayer cultures in scintillation counting vials: Metabolic and growth experiments using radioisotopes and a microfluorometric DNA assay

Summary We have developed a simple technique for the investigation of cellular metabolism and growth in cultured human fibroblasts which facilitates experiments using up to 3×10⁵ cells in each of 100 or more culture vessels. The method has been used to study cell growth, glucose utilization and oxidation, and protein, RNA and DNA synthesis. The use of radiolabeled substrates in tracer experiments is simplified since transfer of cell material is not required. Methods for measuring both total cellular protein and DNA have been adapted to this culture system. Although we have used this technique for fibroblast cultures, it also can be easily applied to experiments on any other type of cell that can be grown in a monolayer. Supported by PHS grants AM-02456, AM-05020 and AM-15312, and by the Kroc Foundation. Recipient of Research Career Development Award AM-47142 from NIAMDD     

Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87^N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.     

An enzymatic assay for acetate in spent bacterial culture supernatants

A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supernatants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et al. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 microM. The assay is cheaper than commercially available assays and is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assay to the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs.     

基于ATP生物发光法的微生物数量快速检测技术的研究进展

微生物数量的快速检测一直是工业生产与食品行业需要解决的问题,腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)生物发光法具有操作简便、检测周期短等优点,可满足一般微生物检测的需求。然而,ATP生物发光法的准确性也受到不同因素的影响,如微生物的ATP检测限值较高、微生物自身及其他因素(如非微生物ATP、提取剂种类、荧光素酶活性等)均对微生物数量的检测产生影响。本文简述了不同微生物数量检测方法的优缺点,介绍了ATP生物发光法的发展历程及原理,综述了非微生物ATP与游离ATP、微生物量、ATP提取剂、荧光素酶等因素对ATP生物发光法灵敏度与稳定性的影响,归纳总结了ATP生物发光法及检测设备在食品、医疗、污水处理等领域的应用现状,并就ATP生物发光法体系的优化及ATP在线检测的应用等方面进行了展望,以期为ATP生物发光法的高效应用提供新的思路。     

细菌生理特性快速检测试剂盒研制

选取各代谢途径中利用某些特定碳源,如蔗糖、葡萄糖、甘露醇等95种碳源作为测试碳源,分别加入96孔酶标板中.选用指示剂噻唑兰(MTT)来鉴定细菌利用特征碳源情况.同时选取不同种类和不同浓度的抗生素,不同起始pH、不同NaCl浓度加入96孔酶标板中,测定细胞是否生长,可以快速地获得该菌的抗生素耐受性谱.试验还从指示剂浓度、培养基的固化、接菌浓度、观察时间等方面对试剂盒进行优化,得到一种最佳的试剂盒鉴定系统BiobiqA(碳源利用谱)和BiobiqB(生理抗性谱).用模式菌株对试剂盒进行测试和验证,结果表明,试剂盒具有操作简便、结果准确、节省成本、节约时间等优点,可以进行细菌生理特性的快速检测.     

Development of a new procedure based on the energy charge measurement using ATP bioluminescence assay for the detection of living mould from graphic documents

Fungal contamination is a major cause of deterioration in libraries and archives. Curators and conservators increasingly need rapid microbiological analyses. This paper presents a rapid detection method for the fungal contaminants on documents. A previous study showed that the calculation of energy charge, using bioluminescence ATP assays, provides a useful indicator to determinate the viability of fungal strains. We argue that this sensitive and time‐saving method is better than traditional culture techniques. However, the procedure needs to be modified to make it usable for lay persons. An improved and simplified protocol is proposed here for the extraction of adenylate nucleotides (AN) from fungal spores and for their measurements. Our new procedure can detect the existence of viable fungal strains on documents, presenting suspect spots within minutes. The extraction is performed by filtration with DMSO–TE solution as extractant. The different step of the measurement of AN content is carried out successively in a single test tube instead of the three tubes necessary in the initial method. The new procedure was tested on 12 strains among those most frequently found in archives and libraries and validated on swab samples from real documents. Copyright © 2008 John Wiley & Sons, Ltd.     

Detection of bacterial contamination in cultures of eucaryotic cells by gas chromatography-mass spectrometry

The use of gas chromatography-mass spectrometry for early detection of bacterial contaminations in cultures of baker's yeast, Penicillium chrysogenum, and an animal cell line was evaluated; muramic acid and characteristic cellular fatty acids were used as analytes. By analyzing branched-chain and cyclopropane-substituted fatty acids as methyl esters, Staphylococcus epidermidis, Bacillus subtilis, Lactobacillus reuteri, Enterobacter cloacae, and Pseudomonas fluorescens were detected in a 500-fold excess (w/w) of baker's yeast; the amounts injected corresponded to 300 ng (dry mass) of the bacteria. Contamination with Bacillus was detected in cultures of Penicillium chrysogenum and animal cells by analyzing muramic acid, both as its alditol acetate derivative, using electron impact ionization, and its trifluoroacetyl methyl glycoside derivative, using negative ion-chemical ionization. The trifluoroacetylated derivative was detected in injected amounts corresponding to 1 x 10(3) bacterial cells in the contaminated animal cell line, whereas amounts corresponding to 1 x 10(5) bacteria were required for detection of the alditol acetate derivative; the amounts in the original samples were 5 x 10(5) and 5 x 10(6), respectively. However, the alditol acetate method exhibited lower chemical interferences than the trifluoroacetyl methyl glycoside procedure. The results show the potential of using gas chromatographic-mass spectrometric analysis of cellular constituents for the detection of bacterial contaminations in eucaryotic cultures as an alternative to conventional microbiological methods. (c) 1993 John Wiley & Sons, Inc.     

ATP bioluminescence rapid detection of total viable count in soy sauce

The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10²–3 × 10⁴ CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB‐941) and 96‐well plates and could analyse 50–100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation of ATP luminometry for rapid and accurate titration of Mycoplasma hyopneumoniae in Friis medium and a comparison with the color changing units assay

Limited reports are available on the growth response of Mycoplasma hyopneumoniae in Friis medium and the routinely used color changing units (CCU) assay has not yet been profoundly compared with other titration methods. Firstly, growth kinetics of 7 diverse M. hyopneumoniae isolates were followed by ATP luminometry in five Friis medium batches. Secondly, results of the CCU and ATP assays were compared hereby evaluating the methods.Growth curves of all isolates had log, stationary and senescence phases, and reached similar maximal titres when cultured in the same batch of Friis medium. Doubling times (Tds) of the isolates grown in slowly shaken cultures varied between 4.8 and 7.8 h. Maximal titres, Tds, growth phase in which the phenol red indicator turned from red to yellow due to acidification by mycoplasmal metabolism, and the length of the stationary phase varied depending on the Friis medium batch. The effect of static vs. shaking culture conditions on the Td depended on the isolate. ATP and CCU assays obtained similar growth curves, but when maximal levels were reached the CCU titre dropped earlier than the ATP titre. During log phase, CCU and ATP titres were strongly linearly linked. We developed a model enabling transformation of ATP into CCU titres or vice versa. The calculated amount of ATP per CCU (1.77 amol ATP/ml) indicated that the CCU assay likely underestimates the actual cell concentration. When titres were determined as means of 3 measurements, the ATP assay was 7 times more accurate and had 11-fold lower outliers than the CCU assay. Unlike the CCU assay, luminometry only requires one measurement to obtain sufficient accuracy.It was concluded that the ATP assay constitutes a valuable robust alternative for reproducible real-time titre assessment of freshly grown M. hyopneumoniae cultures. It is faster, more accurate and time, work and cost efficient compared to the CCU assay. The assay is preferred to better standardise and describe M. hyopneumoniae cultures used in various experiments.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Influence of two cell harvesting methods on intracellular ATP and amino acid concentrations in human fibroblast cultures

The intracellular ATP and amino acid concentrations were determined in human fibroblast cultures reaching confluence. The values obtained were very different, depending on the cell harvesting method: trypsinization or scraping. Trypsinization appeared to be the better method for measuring the ATP concentrations (21.25 +/- 0.96 nmol per mg cell protein), this level being much lower with scraping. On the contrary, scraping was the most appropriate method for amino acid measurement. This work underlines the importance of harvesting methods for metabolic studies in human cell cultures.     

Bioluminescent high-throughput assay for the bacteria adherence to the tissue culture cells

The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.