Clonogenic assay of cells in vitro

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo "unlimited" division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1-3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose-survival curves is included.     

A sensitive immunochromatographic assay using gold nanoparticles for the semiquantitative detection of prostate-specific antigen in serum

In prostate cancer screening, prostate-specific antigen (PSA) has been utilized as a valuable biomarker. There are routinely used procedures based on enzyme-linked immunosorbent assay (ELISA) for PSA detection. The procedures based on ELISA, however, are time consuming, complicated, and costly. We have developed a rapid, very simple, cost effective and sensitive immunochromatographic assay using gold nanoparticles and evaluated its applications for first screening of prostate cancer in serum samples. The sensitive immunochromatographic assay requires only 40 μL of the serum sample. The assay used is rapid and simple, that it totally takes approx 15 min to complete. The method for sensitive immunochromatographic assay has the other advantage of decreasing the antibody concentration that is used for the test line. In this study, we show the advantage to decrease the antibody concentration and the evaluation of our sensitive immunochromatographic assay for the semiquantitative detection of PSA in serum. The results obtained from 163 serum samples using sensitive immunochromatographic assay are compared with the results obtained using the chemiluminescent enzyme immunoassay (CLEIA) and normal immunochromatographic assay. The results obtained in the sensitive immunochromatographic assay correlated well with the values obtained in CLEIA. We concluded that our sensitive immunochromatographic assay is applicable to the first screening test for the diagnosis of prostate cancer. Our developed sensitive immunochromatographic assay is a promising candidate for diagnosis or research use, which may become commercially available in the near future.     

微量法与试管比色法检测ALT的比较

经实验建立了一种用于检测血浆中丙氨酸氨基转移酶(ALT)含量的微量测定法,并与比色法（传统赖氏法)进行了比较。用两种方法检测定值血清、室内质控及样品并比较标准曲线后,结果无显著性差异,同时微量法重复性较好,结果表明微量法测定ALT酶活力可以替代比色法测定血浆中ALT含量,适合大批量血浆ALT含量的快速检测。     

Assay for cellobiose dehydrogenase in the presence of laccase

The currently used assay for cellobiose dehydrogenase (CDH), an enzyme produced by many wood degrading fungi, lacks specificity and can give false results. The presence of laccase interferes with the standard assay. We have developed an assay for CDH that is insensitive to the presence of both laccase and other phenoloxidases. The method is based on the decrease of reducing end groups in lactose determined by the DNS method. Ferricyanide is present as electron acceptor. Advantages and drawbacks of CDH assay methods are discussed     

Bioluminescent methods of analysis in microbiology

The prospects for application of bioluminescent ATP-metry in microbiology are considered. A bioluminescent assay is proposed to analyse biomass by measuring the content of intracellular ATP by means of immobilized firefly luciferase after ATP extraction with dimethylsulfoxide. The assay can be used for plotting the growth curves of microorganisms and for determining the sensitivity of microorganisms to antibiotics. The detection limit of the assay is 700 cells per ml of the measured solution.     

A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues

The conversion of xanthine dehydrogenase to a free radical producing oxidase is an important component of oxygen-mediated tissue injury. Current assays for these enzymes are of limited sensitivity, making it difficult to analyze activities in organ biopsies or cultured cells. The xanthine oxidase-catalyzed conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a fluorometric assay which is 100-500 times more sensitive than the traditional spectrophotometric assay of urate formation from xanthine. Enzyme activity as low as 0.1 pmol min-1 ml-1 can be measured with the fluorometric pterin assay. Xanthine oxidase is assayed in the presence of pterin only, while combined xanthine dehydrogenase plus oxidase activity is determined with methylene blue which replaces NAD+ as an electron acceptor. The relative proportions and specific activities of xanthine oxidase and dehydrogenase determined by the fluorometric pterin assay are comparable with the spectrophotometric measurement of activities present in rat liver, intestine, kidney, and plasma. The assay has been successfully applied to brain, human kidney, and cultured mammalian cells, where xanthine dehydrogenase and oxidase activities are too low to detect spectrophotometrically.     

果胶酶分离纯化及分析方法的研究进展

果胶酶能降解果胶质,在果汁制造、果酒酿造等方面有着广泛应用。果胶酶分子生物学的迅速发展极大地促进了分离纯化与分析方法的研究。由于不同菌种产生的果胶酶成分复杂程度不同,分离纯化手段和分析方法也不相同。本文对果胶酶分离纯化手段及其分析方法进行了综述。     

A non-invasive micronucleus assay in the rat liver

The potent rat-liver mitogen 4-acetylaminofluorene (4AAF) is shown here to provide an effective replacement for the surgical procedure of 2/3 partial hepatectomy (2/3PH) in the in vivo rat-liver micronucleus assay described by Tates and his colleagues. This protocol modification enables the assay to be conducted on a routine basis. Control observations for both 2/3PH and 4AAF-treated rats are presented, together with evidence indicating 4AAF itself to be without activity in the assay, irrespective of the mitogenic stimulus. The activities of the rat carcinogens DMN, 2AAF, DMH and 6BT, and of the non-carcinogens 4AAF and 4N are demonstrated. Recommendations for the conduct of the modified assay are made.     

Artifacts in the assay of maize leaf phosphoenolpyruvate carboxylase activity due to its instability

When the assay of maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity is started with phosphoenolpyruvate, much lower reaction rates are obtained as compared to the enzyme-initiated reaction. The difference is due to the lability of the dilute enzyme in the absence of its substrate and is increased with incubation time in the absence of substrate or stabilizers. The activation of the enzyme by glucose-6-phosphate is overestimated with the substrate-initiated assay since a part of the apparent activation is due to stabilization of the enzymic activity by this effector during the minus-substrate preincubation. In contrast, the inhibitory effect of malate is underestimated when the reaction is started with the substrate. The enzyme-initiated assay is recommended provided that the necessary corrections for apparent activity in the absence of substrate and for inactivation during the assay at low substrate levels are made.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone     

Potencies of naturally-occurring AKH/RPCH peptides in Locusta migratoria in the acetate uptake assay in vitro and comparison with their potencies in the lipid mobilisation assay in vivo

The biological potencies of a number of naturally-occurring octa- and decapeptides of the large AKH/RPCH family of peptides were determined in Locusta migratoria using the lipid-mobilising assay in vivo and the acetate uptake assay in vitro. The most potent of the newly-tested peptides in the in vitro assay, Phl-CC, differs from the endogenous major locust peptide, Lom-AKH-I, only by an exchange of serine versus threonine at position 10. However, the most active peptide in the in vitro assay remains Lom-AKH-III. At the other extreme is the peptide Mem-CC which contains a tyrosine residue at position 4 rather than the more typical phenylalanine. This peptide is over 20,000 times less potent than Lom-AKH-III in the in vitro assay, and also results in an unusual dose-response curve in the in vivo assay. Only a few peptides are approximately equipotent in both assays, but mostly the bioanalogues have a higher potency in vitro. The majority of them are 2- to 10-fold more potent in vitro, but Ani-AKH and Lom-AKH-III are 19- and 48-fold more potent. The results are discussed in relation to either the actions of proteases or of possible preferential binding of different receptors involved in the different assays.     

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays

The ability to identify active compounds (3hits2) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the 3suitability2 of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called 3Z-factor,2 is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.     

Fluorescence-microscopy-based image analysis for analyte-dependent particle doublet detection in a single-step immunoagglutination assay

A novel fluorescence-microscopy-based image analysis method for classification of singlet and doublet latex particles is demonstrated and applied to a particle-based immunoagglutination assay for quantification of biomolecules in microliter-volume bulk samples. The image analysis method, verified by flow cytometric agglutination analysis, is based on a pattern recognition algorithm employing Gaussian-base-function fitting which allows robust identification and counting of singlets, doublets, and higher agglomerates of fluorescent microparticles. The immunoagglutination assay is experimentally modeled by a biotin-streptavidin interaction, with the goal of both theoretically and experimentally investigating the performance of a general immunoagglutination-based assay. For this purpose a theoretical model of the initial agglutination kinetics, based on particle diffusion combined with a steric factor determined by the level of specific and nonspecific agglutination, was developed. The theoretical model combined with the experimental data can be used to optimize an agglutination-based assay with regard to sensitivity and dynamic range and to estimate the affinity, receptor surface density, molecular and binding site sizes, and level of nonspecific binding that is present in the assay. The experimental results are in good agreement with the theoretical model, indicating the usefulness of the model for immunoagglutination assay optimization.     

Monitoring modulators of platelet aggregation in a microtiter plate assay

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.     

An assessment of the in vivo rat hepatocyte DNA-repair assay

The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested.     

Acetylene reduction in the rhizosphere of rice: Methods of assay

Summary A method is described for thein situ assay of acetylene reduction activity in the rhizosphere of rice involving the use of inexpensive, lightweight equipment. The assay is quick to set up and thus many replicates can be run simultaneously. Control of any acetylene-reducing blue-green algae in the assay chamber is achieved with a photosynthetically active herbicide which has no measurable effect on rhizosphere activity. Results using this method are compared with those from an excised root assay of the same plants. Only a very weak correlation between the two methods was found.     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

Fluorometric assays for succinyl-CoA and propionyl-CoA in mitochondrial extracts

Fluorometric assay procedures are described for the quantitative measurements of succinyl-CoA and propionyl-CoA down to concentrations of 0.1 μm in the reaction mixture. The enzymatic assay for succinyl-CoA couples the reaction of 3-ketoacid CoA transferase (succinyl-CoA transferase) to β-OH butyryl-CoA dehydrogenase. A simple purification procedure is described for the isolation of succinyl-CoA transferase from beef heart. Two enzyme assays for propionyl-CoA are described. In the first, CoA, acetyl-CoA and propionyl-CoA are assayed by sequential addition of α-ketoglutarate dehydrogenase, citrate synthase and phosphotransacetylase. The second assay for propionyl-CoA utilized propionyl-CoA carboxylase to convert propionyl-CoA to methylmalonyl-CoA in the presence of ATP and bicarbonate, and the ADP formed was assayed by coupling pyruvate kinase with lactate dehydrogenase. Illustrations are given for the application of these assay procedures to measurements of succinyl-CoA and propionyl-CoA in neutralized perchloric acid extracts prepared from rat heart and liver mitochondria incubated under a variety of conditions.     

Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic samples

Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are "canonical triplexes". Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.     

The comet assay in male reproductive toxicology

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.     

A novel semiautomated method for the estimation of free fatty acid in serum or plasma

A modification of the semiautomated assay method of Antonis (1965. J. Lipid Res. 6:307-312) for free fatty acid is presented. Free fatty acids are extracted from serum or plasma into di-n-butyl ether-2-methoxyethanol; the extract is almost free from phospholipids. The acids are analyzed in a portion of extract by a copper soap method using diphenylcarbazide. The extractant, being less dense than water, is easily separated from an aqueous phase both in the extraction of samples and in the assay of copper soaps. The assay is comparable in accuracy with well-tried titrimetric methods and is quicker and easier to operate.