Altered activity in cultured cells caused by contaminants in tubes widely used for blood collection and serum preparation

Summary Cell culture has been recognized as an extremely sensitive system for measuring the toxicity of various materials. A study was done to determine whether the type of tube used to collect blood or store human serum might affect results in experiments requiring blood drawn into such tubes. In order to test tubes for contaminants that might alter cellular activity, a variety of commercially available tubes used for collection of blood and storage of serum were shaken while containing culture medium with fetal bovine serum. The medium was then applied to 3T3 fibroblasts in culture. Measuring incorporation of tritiated thymidine into DNA in log phase cells as an index of cellular proliferation, it was found that medium containing serum preincubated in tubes routinely used for blood collection could be extremely toxic. The same types of tube were also used to prepare human serum. When serum from some of the tubes was applied to 3T3 fibroblasts, a stimulatory effect was observed, perhaps caused by selective adsorption of inhibitory components of the blood or serum by various tubes. It is, therefore, crucial in a properly controlled experiment using serum in vitro to collect blood in tubes that exert no toxic or stimulatory effects in the assay or, at least, to be consistent in one’s choice of tube. None of the tubes used for storage of serum showed significant effects in our assay.     

Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device

Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format.     

A sensitive, robust high-throughput electrochemiluminescence assay for rat insulin

In diabetes research, mouse and rat models are used for in vivo experiments, and quantification of insulin in serum samples under different pathophysiological conditions and after treatment with compounds is essential. There are few commercial radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) assay kits to determine the rat/mouse plasma levels of insulin. However, reliability in insulin measurements using the available biological assays is a great concern. The authors report a robust, extremely sensitive electrochemiluminescence (ECL) insulin assay using the Origen technology platform. The assay performance, as judged by the Z' value of 0.82+/-0.03 and the signal-to-background (S/B) ratio of 133, suggests that this is a robust and reliable assay. The intra-assay and interassay variation is less than 5%. The dynamic range of detection for insulin is 5 pg to 5 ng in the ECL assays. Recovery of insulin was about 100% when different volumes of serum were spiked with exogenous insulin. These results suggest that the ECL insulin assay is an extremely sensitive, robust, nonradioactive homogeneous assay and can be used successfully to determine the insulin levels in rodent serum samples.     

Antibody quantitation in seconds using affinity Perfusion Chromatography

An extremely rapid assay technique for antibodies has been developed utilizing protein A or protein G bound to Perfusion Chromatography support matrices. Either dilute or concentrated samples are directly injected on a column that selectively binds antibody, which is quantitated directly by elution and UV absorbance. Due to the unique mass transport characteristics of the supports, total assay cycle times are typically 1 minute or less, with assays as short as 15 seconds possible. The assay system can accurately quantitate a 100,000:1 or greater dynamic range in sample concentration without sample dilution, is extremely repeatable and is easy to automate with conventional HPLC systems. Assay of antibodies in a wide range of sample types has been demonstrated.     

A simple lipase assay using trichloroacetic acid

An extremely rapid and sensitive assay for lipoprotein lipase activity, suitable for routine determinations, is described. The substrate for the assay is emulsified [2-(3)H]glyceryl trioleate, activated by serum. The method is based on trichloroacetic acid precipitation of unreacted substrate and measurement of (3)H-labeled glycerol.     

Multiplex genotyping of PCR products with MassTag-labeled primers.

A simple mass spectrometric based assay, the PinPoint assay, has previously been described for typing single nucleotide polymorphisms. The identity of a polymorphism is determined by mass differences of single base extended genotyping primers as determined by MALDI-TOF mass spectrometry. A simple method for multiplexing the assay is described, employing multiple primers with 5'oligo(dT) sequences (MassTags) which serve to mass discriminate the peaks of multiple extended and non-extended primers. The assay is extremely rapid and requires no labeling reagents.     

Application of region-specific immunoassay for human chromogranin A: substantial clue for detection and measurement of chromogranin A in human plasma

Chromogranin A (CgA), a secretory protein, is co-released with catecholamines from storage vesicles. It is known to be elevated in the circulation of patients with neuroendocrine and endocrine tumors. For further investigation of the protein, especially in humans, it is essential to facilitate quantitative analysis of the protein in human biological materials. In order to introduce novel immunological methodology for this purpose, we purposely selected human CgA(344-374) for the synthetic immunogen to produce region-specific CgA antibodies. The anti-synthetic peptide antibody thus obtained made it possible to develop an immunological method for measurement and characterization of CgA in human plasma. The plasma CgA-immunoreactivity (LI) level measured by the method was 0.31+/-0.01 pmol/ml (mean+/-SEM) in normal subjects and 1.55+/-0.29 pmol/ml in pheochromocytoma. On gel chromatography and HPLC analysis of the plasma of patients with pheochromocytoma, the region-specific assay system enabled us to show the presence of N-terminal truncated CgA, besides CgA itself. By following up changes of plasma CgA-LI in a pheochromocytoma patient using samples that were collected consecutively over a two-year period, the present assay system using the region-specific antibody, anti-human CgA (344-374) serum, was confirmed to be extremely valuable for the measurement of CgA-LI in human plasma. The characteristic features and high sensitivity of the present assay system will give us a substantial clue to the detection and measurement of CgA to develop further investigation of the protein in humans.     

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m²/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.     

Radioactive method for the assay of glycogen phosphorylases

A new assay for glycogen phosphorylase (EC 2.4.1.1) is presented. This assay employs a filter paper technique in which newly formed glycogen-¹⁴C is precipitated on paper squares using 66% ethanol while other radioactivity is removed by washing. It is rapid and reproducible and may be employed with crude as well as purified enzyme preparations. An additional advantage of the method is the potential for increased sensitivity. It should find utility for assay of extremely small tissue samples and should be useful in kinetic studies.     

Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis

A sensitive method for the analysis of inorganic pyrophosphate (PPi) which utilizes the enzymes ATP sulfurylase and firefly luciferase is described. The assay is based on continuous monitoring of the ATP formed in the ATP sulfurylase reaction using purified firefly luciferase. The assay can be completed in less than 2 s and is not affected by inorganic phosphate. The method has been used for continuous monitoring of formation of PPi in Rhodospirillum rubrum chromatophores. The assay is extremely sensitive, the linear range of the assay being 1 X 10(-9) - 5 X 10(-7) M PPi. It is suitable for routine applications. It is also possible to use the method for determination of low amounts of adenosine 5'-phosphosulfate.     

Zebrafish models of the immune response: taking it on the ChIn

The zebrafish is proving to be an extremely versatile new experimental model for unraveling the mysteries of innate immunity and has considerable promise as a system for the identification of novel modulators of this crucial biological process. A rate-limiting factor, however, is the mechanical stimulus required to induce the inflammatory response. A new chemically induced inflammation assay ('ChIn' assay) published in BMC Biology obviates this requirement and seems set to accelerate progress in the field.     

Electrophoretically mediated microscale reaction of glycosidases: kinetic analysis of some glycosidases at the nanoliter scale

Capillary electrophoresis (CE) is one of the extremely important analytical techniques known for its high sensitivity and resolution. We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of some native glycosidases. Under optimized conditions, the enzymatic reactions of alpha-glucosidase, beta-galactosidase and beta-N-acetylglucosaminidase were carried out, and the Michaelis constants were obtained. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme and substrate required for a reaction.     

A qauntitative in vitro assay for the evaluation of phototoxic potential of topically applied materials

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2–500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.Abbreviations C centigrade - cm centimeter - cm² square centimeter - ml milliliters - mm millimeters - 8-MOP 8-methoxy psoralen - mW milliwatt - nm nanometer - UVA ultraviolet radiation, 320–400 nm The first in a series of research papers on alternatives to animal toxicity studies.     

Quantitative determination of phenolsulfotransferase using 4-methylumbelliferone

The method presented here offers a rapid and reproducible technique for assay of the enzymic formation of MUS from MU, as use is made of the extremely intensive fluorescence of MU and of the possibility of quantitative separation of the product formed from the substrate by use of ion-exchange chromatography.     

Ocular safety: a silent (in vitro) success story

Ocular irritation testing has been one of the animal test methods most criticised by animal welfare advocates. Additional criticism has arisen from within the scientific community, based on the variability of the animal test results and the questionable relevance of the extremely high dose levels employed. As a result, the Draize eye irritation test has been one of the main targets for in vitro replacement. Despite extensive efforts, however, there is still no in vitro method that is fully validated as a regulatory replacement. In spite of this, many individual companies are using diverse in vitro ocular irritation tests to gain important safety and efficacy information about their products and raw materials, eliminating the need for animal testing in the process. This is done in a safe fashion by applying intelligent testing paradigms. ECVAM has played a major role in this success, through its many programmes that have emphasised the importance of understanding the true toxicological need, and then using in vitro tests to provide that information. Thus, even in the absence of a successfully validated regulatory assay, the desired result of reducing animal testing is being met.     

A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes

A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.     

New tester strains of Salmonella typhimurium highly sensitive to mutagenic nitroarenes

The nitroreductase and acetyltransferase genes of Salmonella typhimurium TA1538 have been cloned into pBR322. When transformed into TA1538 derivatives, these plasmids provided the basis for an extremely sensitive assay for the detection of mutagenic nitroarenes.