A reliable,sensitive, and convenient radioactive assay for benzpyrene monooxygenase

A new radioactive assay for benzpyrene monooxygenase has been developed, characterized, and compared to the fluorescent assay generally employed. The radioactive assay is based on a single extraction which effectively separates metabolites from remaining substrate. This assay is linear within reasonable ranges of time and protein concentration and responds as expected to inhibitors and an inducer of benzpyrene monooxygenase activity. The activity measured with the present assay is about twice that measured with the fluorescent assay. Furthermore, the radioactive assay can be scaled down so that it is at least as sensitive as the fluorescent assay.     

Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.     

Modeling longitudinal biomarker data from multiple assays that have different known detection limits

Summary .   Assays to measure biomarkers are commonly subject to large amounts of measurement error and known detection limits. Studies with longitudinal biomarker measurements may use multiple assays in assessing outcome. I propose an approach for jointly modeling repeated measures of multiple assays when these assays are subject to measurement error and known lower detection limits. A commonly used approach is to perform an initial assay with a larger lower detection limit on all repeated samples, followed by only performing a second more expensive assay with a lower minimum level of detection when the initial assay value is below its lower limit of detection. I show how simply replacing the initial assay measurement with the second assay measurement may be a biased approach and investigate the performance of the proposed joint model in this situation. Additionally, I compare the performance of the joint model with an approach that only uses the initial assay measurements in analysis. Further, I consider alternative designs to only performing the second assay when the initial assay measurement is below its lower detection limit. Specifically, I show that one only needs to perform the second assay on a fraction of assays that are above the lower detection limit on the first assay to substantially increase the efficiency. Further, I show the efficiency advantages of performing the second assay at random without regard to the initial assay measurement over a design in which the second assay is only performed when the initial assay is below its lower limit of detection. The methodology is illustrated with a recent study examining the use of a vaccine in treating macaques with simian immunodeficiency virus.     

Advances in assays of matrix metalloproteinases (MMPs) and their inhibitors

Matrix metalloproteinases (MMPs) play an important role in many physiological and pathological processes. To assay the activities of MMPs is important in diagnosis and therapy of the MMPs associated diseases, such as neoplastic, rheumatic and cardiovascular diseases. Several assay systems have been developed, which include bioassay, zymography assay, immunoassay, fluorimetric assay, radio isotopic assay, phage-displayed assay, multiple-enzyme/multiple-reagent assay and activity-based profiling assay. The principle, application, advantage and disadvantage of these assays have been reviewed in this article.     

Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance During the Purification Of Mammalian Cell Substrate Derived Biotherapeutics

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.     

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.     

Advances in assays of matrix metalloproteinases (MMPs) and their inhibitors

Matrix metalloproteinases (MMPs) play an important role in many physiological and pathological processes. To assay the activities of MMPs is important in diagnosis and therapy of the MMPs associated diseases, such as neoplastic, rheumatic and cardiovascular diseases. Several assay systems have been developed, which include bioassay, zymography assay, immunoassay, fluorimetric assay, radio isotopic assay, phage-displayed assay, multiple-enzyme/multiple-reagent assay and activity-based profiling assay. The principle, application, advantage and disadvantage of these assays have been reviewed in this article.     

Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay.

The quantitative PCR infectivity assay is a combination of virus propagation and quantitative PCR. Previously [Schalk JAC, van den Elzen C, Ovelgonne H, Baas C, Jongen PMJM. Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR. J Virol Methods 2004;117:179-87.], we used this assay to estimate the titer of infectious measles virus in trivalent, live, measles, mumps, rubella vaccines (MMR). Here we describe the further improvement and development of the assay for simultaneous potency estimation of measles, mumps and rubella viruses. The potency of measles and mumps virus is estimated within one assay after 1 day of cell culture. The potency of rubella virus is estimated in a separate assay after 2 days of cell culture. Compared to conventional CCID50 and plaque assays, the quantitative PCR infectivity assay has the advantage in being fast because the assay is not dependent on the formation of cytopathic effect. Furthermore assay design is simplified: serological neutralization can be omitted because PCR is virus-specific and, under the conditions used, the individual components of trivalent measles, mumps, rubella vaccines do not interfere with each other. The assay was validated and compared to the performance of a plaque assay.     

In vivo mutagenicity studies with iodinated glycerol azeo

Previous studies have shown that iodinated glycerol azeo is positive in a number of in vitro mutagenicity assays including the Ames assay (TA100; TA1535), mouse lymphoma assay, Chinese hamster ovary (cytogenetic) assay and in one in vivo study, the sex-linked-recessive-lethal assay in Drosophila. Prior studies have also shown that the drug is negative in the mouse micronucleus assay. We now report that the drug is also negative for mutagenic activity in a number of other in vivo tests. Single intraperitoneal doses of 25, 125 and 250 mg/kg were without effect in the rat bone marrow chromosomal aberration assay. Single oral doses of 30, 75, 150 and 300 were negative in the rat hepatocyte DNA-repair assay. Single intraperitoneal doses of 30 and 100 mg/kg were without effect in the sister chromatid exchange (SCE) assay in the mouse. Statistically significant effects were seen at 200 and 300 mg/kg in the initial SCE assay and at 300 and 350 mg/kg in the confirmatory SCE assay. The rationale for considering the SCE results to be anomalous and thus not relative to the overall safety evaluation of the drug is presented.     

A rapid and simple chemiluminescent assay for Escherichia coli beta-galactosidase.

We describe a chemiluminescent assay for E. coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate. Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer. Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay. Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay. As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay.     

Dominant lethal cell mutants detected by the autoradiographic assay for exotoxin A resistance

The autoradiographic assay (AR assay) for P. aeruginosa exotoxin A (PE) resistance in cultured mouse fibroblasts detects mutants able to synthesize proteins in the presence of the toxin, presumably due to mutations in the structural gene for elongation factor 2 (EF-2). Detection by the AR assay of PER cells is independent of their ability to divide. The frequencies of both spontaneous and mutagen-induced PER cells are higher than those detected by the conventional colony assay. Examination of phenotypic expression times in the PER cells, and of their in situ proliferation, reveals that this higher sensitivity of the AR assay is due to its ability to detect cells in which the PER mutation prevents proliferation, thus escaping detection by the colony assay. Expression of the mutant phenotype in the PER cells detected in the AR assay after mutagenesis with ethyl methanesulfonate (EMS) follows a pattern similar to that observed in the colony assay, reaching a maximum in 3 days, and then remaining constant for at least 4 more. After treatment with X rays (which fail to induce PER mutants in the colony assay), the frequency of PER cells detected in the AR assay also reaches a maximum on day 3, but then declines sharply, returning to the spontaneous level on day 7. In the absence of PE, the majority of the spontaneous or mutagen-induced PER cells detected in the AR assay are either incapable of dividing at all, or capable of undergoing a limited number of cell divisions to produce micro-colonies. Only few of them may continue to grow into 'full-size' colonies comparable to those detected in the colony assay. In the presence of the toxin, the proportion of PER cells which are able to divide is even smaller, and that of cells able to form full-sized PER colonies detectable in the AR assay is comparable to the results obtained in the conventional colony assay. We presume that the lethality of the PER mutations in the cells detected by the AR assay is due to abnormal protein synthesis resulting from the same mutational change that made these cells resistant to PE. While incapable of supporting colony formation, and hence detection by the colony assay, such abnormal protein synthesis still allows the detection of the mutant cells by the AR assay.     

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.     

Development of a peroxidase-coupled fluorometric assay for lysyl oxidase

Lysyl oxidase catalyzes the oxidation of peptidyl lysine in elastin and collagen and also acts upon nonpeptidyl amines, although the enzyme becomes slowly inactivated while processing nonpeptidyl substrates. In spite of this complexity, it has been possible to devise a continuously monitored peroxidase-coupled fluorometric assay for the oxidation of simple amines by lysyl oxidase. In the present study, optimal assay conditions have been explored and found to include assay temperatures of 50 to 60°C, the presence of urea in the assay, and the use of diaminopentane as substrate. Although the assay is subject to interference by contaminating macromolecules in enzyme fractions, a linear assay response to enzyme concentration is obtained with highly purified lysyl oxidase with a limiting sensitivity of 0.3 μg of enzyme per assay.     

Yeast DEL assay detects clastogens

Chromosomal rearrangements, including DNA deletions are involved in carcinogenesis. The deletion (DEL) assay scoring for DNA deletions in the yeast Saccharomyces cerevisiae is able to detect a wide range of carcinogens. Among approximately 60 compounds of known carcinogenic activity, the DEL assay detected 86% correctly whereas the Ames Salmonella assay detected only 30% correctly [R.J. Brennan, R.H. Schiestl, Detecting carcinogens with the yeast DEL assay, Methods Mol. Biol. 262 (2004) 111-124]. Since the DEL assay is highly inducible by DNA double strand breaks, this study examined the utility of the DEL assay for detecting clastogens. Ten model compounds, with varied mechanisms of genotoxicity, were examined for their effect on the frequency of DNA deletions with the DEL assay. The compounds tested were: actinomycin D, camptothecin, methotrexate and 5-fluorodeoxyuridine, which are anticancer agents, noscapine and furosemide are therapeutics, acridine, methyl acrylate and resorcinol are industrial chemicals and diazinon is an insecticide. The in vitro micronucleus assay (IVMN) in CHO cells, a commonly used tool for detection of clastogens, was performed on the same compounds and the results of the two assays were compared. The results of our study show that there is 70% concordance in the presence of metabolic activation (rat liver S9) and 80% concordance in the absence of metabolic activation between the DEL assay and the standard in vitro micronucleus assay. The lack of cytotoxicity observed for four of the ten compounds examined indicates limited diffusion of lipophilic compounds across the yeast cell wall. Thus, the development of a more permeable yeast tester strain is expected to greatly improve concordance of the DEL assay with the IVMN assay. The yeast DEL assay is inexpensive, amenable to automation and requires less expertise to perform than the IVMN. Thus, it has a strong potential as a robust, fast and economical screen for detecting clastogens in vitro.     

Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.     

Modification of the quantitative DNA assay using adriamycin.

A modification of the adriamycin quantitative assay for DNA is presented which is sensitive to 1–20 μg DNA. The assay is simple, rapid, and highly reproducible but, contrary to previous claims, not specific for DNA, RNA interferes with the assay while protein does not. The mechanism and sensitivity of the assay is interpreted with reference to published data on adriamycin DNA binding.     

Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model

The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.     

New separation-free assay technique for SNPs using two-photon excitation fluorometry

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia™ TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot™ assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.     

A sensitive fluorimetric assay for pyruvate

A sensitive and specific fluorimetric assay for the determination of pyruvate is reported here. This assay is based on the oxidation of pyruvate in the presence of pyruvate oxidase. Hydrogen peroxide generated by pyruvate oxidase reacts with nonfluorescent Amplex Red at a 1:1 stoichiometry to form the fluorescent product, resorufin. The assay is optimized with respect to pH of reaction buffer, enzyme concentration, dye concentration, and the time course. The usefulness of the assay is demonstrated by the accurate measurement of intracellular and extracellular pyruvate concentrations. The limit of detection of the assay is 5 nM.     

A simple method for hyaluronic acid quantification in culture broth

The carbazole assay has been used for determination of the percentage of hyaluronic acid in biological fluids. However, it is difficult to measure the concentration of hyaluronic acid in culture broth because glucose and polysaccharides remaining after cultures can react with sulfuric acid and carbazole. The glucose and polysaccharide remnants must be completely removed in order to get the correct value for hyaluronic acid. The turbidity assay, another method for estimating the concentration of hyaluronic acid, is based on the formation of insoluble complexes between hyaluronic acid and cetyltrimethylammonium bromide. This method is very easy and fast compared with the carbazole assay. Because concentrations of hyaluronic acid measured by the turbidity assay were ranged around 100% of those measured by the carbazole assay, the content of hyaluronic acid in culture broth can be determined by the turbidity assay. The turbidity method also has the advantage of being safer than the carbazole assay.