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.     

A high-throughput soft agar assay for identification of anticancer compound

A 384-well soft agar assay was developed to identify potential novel anticancer compounds. Normally used to detect cell transformation, the assay is used here to quantitate cell proliferation in a 3-dimensional (3-D) anchorage-independent format. HCC827 cells, which are highly sensitive to epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors, were used to develop the method and a set of 9600 compounds used to validate the assay. Results were compared to a monolayer assay using the same compound set. The assay provides a robust method to discover compounds that could be missed using traditional monolayer formats.     

Accuracy of the endpoint assay for virus titration

The statistics of estimators used with the endpoint assay for virus titration were investigated. For a standard assay with 10 wells/dilution, the graphical estimator traditionally used was found to produce estimates with significant positive bias and a relatively low accuracy. Furthermore, the graphical estimator was found to be inconsistent. A superior estimator based on the maximum likelihood principle was developed. The results are discussed in relation to the choice between the endpoint titration assay and the plaque assay, and an alternative two-stage assay is presented.     

Characterization of strain specific typing antisera for genetic monitoring of inbred strains of rats

Strain-specific typing antisera (SSTA) were prepared for six inbred strains of rats by using a pooled immunization protocol. The SSTA were used in both a haemagglutination assay and a complement dependent microcytotoxicity assay to compare the usefulness of the two test systems. Both assays were simple, reliable and repeatable, and each system had distinct advantages and disadvantages. The haemagglutination assay was faster and required less specialized equipment than the microcytotoxicity assay. On the other hand, interpretation of results in the microcytotoxicity assay was easier and more objective. It was concluded that SSTA could be used with the microcytotoxicity assay and/or the haemagglutination assay to provide a simple and effective genetic monitoring method for inbred strain of rats.     

Modified Microbiological Assay for Rapid Estimation of Antibiotic Concentrations in Human Sera

Antibiotic concentrations in human sera were estimated in 5 to 6 hr by a modified microbiological assay. By using Staphylococcus aureus and Streptococcus pyogenes as the assay organisms, the seeded assay plates were preincubated for 2 to 6 hr and then were stored at 4 C until used for assay. Paper discs saturated with the specimen were placed on the preincubated assay plates with reference discs saturated with known concentrations of antibiotic. After 5 to 6 hr of incubation, zones of antibacterial activity were measured and compared with a standard curve for estimation of antibiotic concentration. Results from this rapid assay method compared favorably with those from the commonly used 24-hr assay.     

A high-throughput hybridization method for titer determination of viruses and gene therapy vectors.

One of the challenges facing researchers working with viruses and gene therapy vectors is the need to rapidly assay for infectious virus. Current methods used to titer many viruses are cumbersome and are not amenable to handling large numbers of samples. Here we describe the development of an assay that can rapidly quantify infectious viruses and gene therapy vectors. The assay relies on biological amplification of viral sequences and hybridization of labeled probes to immobilized nucleic acid from infected cells. The amplification of the viral genome makes this a highly sensitive method. The assay is configured in a high-throughput format that has been used to detect recombinant adeno-associated virus (AAV), wild-type AAV and infectious adenovirus. The assay is quantitative, and can be used to titer virus preparations with or without a known standard.     

Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes

An on-line assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of I mg l⁻¹ together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.     

Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (microM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIAassays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of K(i) = 0.01 nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.     

An enzyme-linked immunosorbent assay for the detection of cytoplasmic polyhedrosis virus

An enzyme-linked immunosorbent assay (ELISA) for the detection of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV) is described. Antisera to EsCPV, produced in rabbits and guinea pigs, are specific to EsCPVs when used in an indirect assay. This indirect assay approach permits the detection of homologous antigens at a concentration of about 1 μg/ml; however, this procedure is not suitable to test large numbers of unpurified specimens. For this type of analysis we used a double antibody sandwich assay which can detect 10 ng/ml of homologous antigen in unpurified material without nonspecific reactions. This assay is used to diagnose EsCPV infections in field and laboratory studies.     

A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.     

The bacterial tryptophan reverse mutation assay with Escherichia coli WP2

The Escherichia coli WP2 tryptophan reverse mutation assay detects trp(-) to trp(+) reversion at a site blocking a step in the biosynthesis of tryptophan prior to the formation of anthranilic acid. The different WP2 strains all carry the same AT base pair at the critical mutation site within the trpE gene. The assay is currently used by many laboratories in conjunction with the Ames Salmonella assay for screening chemicals for mutagenic activity. In general the WP2 strains are used as a substitute for, or as an addition to Salmonella strain TA102 which also carries an AT base pair at the mutation site. The assay is also recommended together with the Ames assay for data submission to regulatory agencies. National and international guidelines have been established for performing these mutagenicity assays.The E. coli WP2 assay procedures are the same as those described elsewhere in this volume for the Ames Salmonella assay (Mortelmans and Zeiger, 2000) with the exception that limited tryptophan instead of limited histidine is used. This chapter is an addendum to the previous chapter and the reader should refer to the previous chapter for details regarding experimental procedures and assay design.     

Fluorometric assay for urea in urine, plasma, and tubular fluid

The variation in the sensitivity of proteins to some commonly used protein assay procedures was estimated by a calculation of the ideal titer per unit weight of protein for a sample of 350 proteins. On the basis of such calculations, the variation expected of nitrogen-based assay procedures is expected to be small, and such procedures are considered to give a more consistent quantification of a variety of proteins than other commonly used assay procedure.     

A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway.     

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals

The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.     

Quantitation of plasma apolipoprotein A-I using two monoclonal antibodies in an enzyme-linked immunosorbent assay

A rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitation of human apolipoprotein (apo) A-I was developed. The assay uses a pair of noncompeting purified monoclonal antibodies to detect apoA-I in plasma. The antibodies used in this assay were selected because they bind greater than 90% of radioiodinated high density lipoprotein (HDL), they identify "fresh" nondeamidated epitopes on apoA-I, and they have comparable binding affinities for isolated HDL and HDL in plasma. The assay was standardized with a plasma secondary standard composed of lyophilized human serum. The assay was used to measure the apoA-I levels in normal subjects, patients with coronary artery disease, and patients with familial hypercholesterolemia. The results indicate that certain monoclonal antibodies can be used to reliably measure plasma levels of apoA-I in diverse groups of subjects.     

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD⁺ as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application.     

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.     

Quantitative determination of myeloperoxidase using tetramethylbenzidine as substrate

Tetramethylbenzidine, a noncarcinogenic, nonmutagenic derivative of benzidine, has been used as a substrate to assay myeloperoxidase. The assay is sensitive to 0.1 μg of enzyme and can be used to quantitate myeloperoxidase over a pH range of 4.4 to 7.4.     

Development of a time-resolved fluorescent assay for measuring tyrosine-phosphorylated proteins in cells

We have developed a time-resolved fluorescent assay using Wallac's DELFIA system (DELFIA assay) to monitor changes in the phosphorylation level of insulin receptor from rat hepatoma (KRC-7) cells in response to ligand and the nonspecific, protein-tyrosine phosphatase inhibitor pervanadate. In this system, a biotinylated antiinsulin receptor antibody was used to capture the insulin receptor and an europium-labeled antiphosphotyrosine antibody was used to assess tyrosine phosphorylation. This assay provides a highly sensitive, nonradioactive readout of receptor phosphorylation. We have validated the DELFIA assay by directly comparing receptor phosphorylation using the well-established technique of immunoblotting. The utility of the DELFIA assay in measuring the phosphorylation status of other receptors has also been demonstrated using epidermal growth factor receptor from A431 cells.     

Analysis of glycoprotein hormone receptor extracellular domain interactions using a solid-phase capture assay

The receptors for the glycoprotein hormones are unique in having a large extracellular domain that is responsible for mediating ligand binding. We describe the characterization, validation, and application of a solid-phase radioligand binding assay that can be used to assess the interaction of peptides and small molecules at the extracellular domain (ECD) of the follicle-stimulating hormone receptor (FSHR). The assay utilizes a C-terminal tag on the FSHR-ECD, which is used to capture the ECD and position it in a sterically favorable orientation on a solid-phase platform. Competition experiments with the cognate ligand, FSH, indicated that the interaction at the FSHR-ECD using the solid-phase assay was comparable to the full-length receptor assayed using a standard filtration assay. The utility of the assay was evaluated by competing several peptides and a small molecule for both the full-length FSHR and the FSHR-ECD. The solid-phase capture format allowed for the establishment of an assay to specifically evaluate compounds that interact at the ECD or require the full-length receptor, thereby facilitating structure-activity studies. This assay format should be applicable to the other receptors of this family.