Automated comet assay analysis.

BACKGROUND: Recently the "comet assay" or "single-cell gel electrophoresis assay" has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction. METHODS: The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (B?cker et al.: Phys Med Biol 1997;42:1981-1992) allows for faster analysis. RESULTS: Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage by low-dose radiation (0-2 Gy). The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. CONCLUSIONS: A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible.     

Spin-label assay for lysozyme.

A spin-label assay for lysozyme, which is based on the enzymatic hydrolysis of spin-labeled peptidoglycan, is described. Hydrolysis of this polymer by lysozyme results in sharpening of the esr spectrum. The rate of spectral sharpening is a function of enzyme concentration. When the activities of hen egg-white and human lysozymes are compared by this method, human lysozyme is 3.5 times as active as the hen enzyme. The pH optima for both enzymes are pH 5.0. At this pH, the maximal activity for the hen egg-white lysozyme is observed at an ionic strength of 0.09. This assay is suitable for measuring lysozyme levels in biological fluids. It is a sensitive, continuous assay that measures muramidase activity on a defined substrate.     

Fluorimetric assay of D-lactate.

A fluorimetric assay for D-lactate in human blood samples was developed using an endpoint enzymatic assay with D-lactate dehydrogenase from Staphylococcus epidermidis. The intrabatch and interbatch coefficients of variance were 8.7% (n = 4) and 16.6% (n = 4), respectively. The limit of detection in blood was 3.73 nmol/ml. The assay suffers minor interference from S-D-lactoylglutathione, which was also present in the blood samples. The concentration of D-lactate in blood was (mean +/- SE, nmol/ml) normal healthy individuals, 11.0 +/- 1.2 (n = 7); and diabetic patients, 20.0 +/- 1.3 (n = 55) (a significant increase in diabetes mellitus; P < 0.01, Mann-Whitney U test).     

Convenient assay for interferons.

A convenient assay for interferons based on reduction of cytopathic effect was developed. The number of manipulations and the lengths of the various incubation steps were reduced to a minimum. The assay is simple to perform and can be completed within 16 h. Moreover, it can be used with various types of cells and a variety of viruses.     

Chromogenic redox assay for beta-lactamases yielding water-insoluble products. II. Heterogeneous sandwich assay for hCG.

A very sensitive and rapid heterogeneous sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) is described. The assay is based on the application of the novel chromogenic redox substrate system for beta-lactamase which is used as label. The chromogen system consists of a thioacetylcephalosporin beta-lactamase substrate, which upon turnover by the enzyme label releases the thiolate with the concomitant reduction of the tetrazolium salt to a colored formazan. The concentration of the formazan is directly related to the amount of the hormone in the sample and is read spectrophotometrically. The enzyme-antibody conjugates, produced through use of heterobifunctional maleimide crosslinker, maintain 90% of the enzyme activity after 30 days at 25 degrees C. Concentrations of the hormone as low as 5 mIU/ml, equivalent to 25 fmol/ml, are detectable in 3 h.     

A rapid assay for ribokinase.

A rapid method capable of detecting low levels of ribokinase is given. [γ-³²P]ATP is converted to ribose 5-[³²P]phosphate which is not absorbable onto charcoal. The assay is linear in enzyme concentration to a lower limit of at least 4 × 10^?2 mg of enzyme/ml.     

Quantitative assay for algal chemotaxis.

A quantitative capillary assay is described for measuring chemoreception in the neritic and littoral unicellular alga Dunaliella tertiolecta. Lucite chemotaxis plates were used in the assay with 3-microliter capillaries. A Coulter Counter was employed to determine algal cell numbers. D. tertiolecta is attracted to ammonium ion with a maximum positive response at 10(-3) M. Inclusion of calcium and L-methionine in the chemotaxis medium stimulates algal chemoreception, although neither chemical is essential for motility. Attraction of the chlorophyte to ammonium is dependent on time of incubation, cell density, and pH. The optimum pH for attraction was found to be 6.25.     

Fluorimetric assay of sialic acids.

A fluorimetric assay has been developed for sialic acids in which sialic acids react with pyridoxamine to give fluorescent compounds in the presence of zinc ion and pyridine. This assay method is specific for unbound sialic acids and is a simple and sensitive procedure compared with the thiobarbituric acid assay of sialic acids.     

Cholinesterase assay by gas--solid chromatography.

The determination of serum cholinesterase activity was based on the hydrolysis of butyrylcholine chloride, solvent extraction, and the quantitative estimation of the hydrolytic product, butyric acid, by gas-solid chromatography. Using this procedure, eptimum conditions of pH and buffer strength for the cholinesterase activity were pH 8.0 and 0.05 m Tris-HCl, respectively. The cholinesterase activities in blood samples from healthy subjects, as determined by this method, were in the range of normal values reported in the literature. The effects of eserine and other drugs on the cholinesterase activity were also reported. The high accuracy (1.1%) and high sensitivity which permit the determination of butyric acid at levels down to 1 μg/ml make this procedure attractive as a serum cholinesterase assay.     

Fluorometric assay of tubulin-colchicine complex.

A fluorometric assay method for the tubulin-colchicine complex has been developed. This assay method is based on the fact that the binding of colchicine to tubulin leads to a 300-fold enhancement of fluorescence intensity of colchicine at 430 nm. Since the excitation wavelength can be set at 350 nm, far from an absorption band of tubulin, the assay does not necessitate the separation of the tubulin-colchicine complex from free colchicine. Fluorescence intensity is linear up to 2 mg/ml of the tubulin-colchicine complex.