Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.     

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 new rapid assay for measuring deoxycytidylate- and deoxythymidylate-kinase activities

A rapid assay for deoxycytidylate- and deoxythymidylate-kinase has been developed that is applicable also to the assay of other kinase enzymes with minor modifications. The method is based on the ability of a radioactive triphosphate nucleotide to adhere to a DEAE disc under conditions in which the corresponding radioactive monophosphate nucleotide is readily removed. This is accomplished by pretreatment of DE81 paper with dTMP for the assay of thymidylate kinase and subsequent washing with a solution composed of 4 formic acid and 1 m ammonium formate for assay of either enzyme. No pretreatment of DE81 paper is required for assay of dCMP-kinase.     

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.     

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.     

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 microM) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 microM and initial rates could be determined. The apparent Km was about 12 microM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.     

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.     

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.     

Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions

The bincinchoninic acid protein assay has been adapted for use with microtiter plates and a plate reader to reduce the time needed for analyses. The total volume of the assay was reduced to 0.21 ml with various ratios of sample to reagent volume being used. The assay is sensitive to a limit of less than 1 microgram of bovine serum albumin. The assay is insensitive to the presence of the detergents sodium dodecyl sulfate, Triton X-100, and deoxycholate. In addition, the method has been adapted for determining the protein concentrations of sucrose density gradient fractions, which eliminates the need for removing the sucrose prior to assay.     

A novel in-gel assay and an improved kinetic assay for determining in vitro sulfite reductase activity in plants

Sulfite reductase (SiR; EC 1.8.7.1), an essential enzyme in the sulfate reduction pathway, catalyzes the reduction of sulfite to sulfide, as an intermediate for cysteine biosynthesis. The commonly used kinetic assay for the detection of in vitro SiR activity in plants is based on a coupled reaction, in which the sulfide produced is converted to cysteine through the presence, in the assay medium, of O-acetylserine sulfhydralase (EC 2.5.1.47) and its substrate, O-acetylserine. An improved kinetic assay for SiR activity in crude desalted protein extracts was developed. The improvement was based on pre-treatment of the protein with tungstate, which improved SiR activity in Arabidopsis and tomato leaf by 29 and 12%, respectively, and the addition of NADPH to the reaction medium, which increased SiR activity by 1.6- and 2.8-fold in Arabidopsis and tomato, respectively, in comparison with the current protocols. Despite the availability and reliability of the kinetic assay, there is currently no assay that enables the direct detection of SiR in relatively large numbers of samples. To meet this need, we developed a novel in-gel assay to detect SiR activity in crude extracts. The method is based on the detection of a brownish-black precipitated band of lead sulfide, formed by the reaction of lead acetate with sulfide. The in-gel assay for SiR activity is reliable, sensitive and technically simpler than the kinetic assay, and opens up the possibility for detecting active SiR isoenzymes and splice variants.     

Radiometric assay for minute quantities of l-fucose

A radiometric assay has been developed for l-fucose which is capable of measuring quantities of this deoxysugar as low as 25 pmol. The assay couples l-fucose dehydrogenase to l-glutamate dehydrogenase and l-glutamate decarboxylase to yield radioactive CO₂ which is collected and counted as a measure of l-fucose content. The assay eliminates high backgrounds observed with fluorescence assays.     

Development of an in vitro activity assay as an alternative to the mouse bioassay for Clostridium botulinum neurotoxin type A

Currently, the only accepted assay with which to detect active Clostridium botulinum neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow and not practical in many settings, and it results in the death of animals. Here, we describe an in vitro cleavage assay for SNAP-25 (synaptosome-associated proteins of 25 kDa) for measuring the toxin activity with the same sensitivity as that of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many laboratory settings, and has the potential to be used for toxin typing. The assay has two main steps. The first step consists of immunoseparation and concentration of the toxin, using immunomagnetic beads with monoclonal antibodies directed against the 100-kDa heavy chain subunit, and the second step consists of a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a fluorescence resonance energy transfer assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and this test can detect the activity of the toxin in carrot juice and beef. These results suggest that the assay has a potential use as an alternative to the mouse bioassay for analysis of C. botulinum type A neurotoxin.     

A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy

A fluorescence microscopic assay for the activity of complement, perforin, and other cytolytic proteins which form transmembrane pores in cellular membranes is described. The assay was worked out and tested with red blood cell membranes (ghosts) and was then applied to intact hemoglobin-free cells. Resealed human erythrocyte ghosts were incubated with complement or perforin. A small polar fluorescent probe (fluorescein-labeled 1-kDa dextran, FD1) which permeates through complement and perforin pores but not through normal cell membranes was added to the samples. The capability of the confocal laser scanning microscope (CLSM) to generate thin optical sections was exploited to visualize and quantitate fluorescence inside single ghosts and thus determine the fraction of ghosts which had become permeable for FD1. The activity of complement or perforin was quantitated by plotting the fraction of permeable cells versus the concentration of the pore-forming protein. The results were in good agreement with those of a conventional hemolytic assay. The CLSM-based assay was then applied to intact hemoglobin-free cells for which only few alternative assays are available. Compared to conventional hemolytic assays for the activity of pore-forming proteins the assay described here can be applied to a large variety of natural and artificial membrane systems. The assay can be performed under nonlysing conditions. Furthermore, the assay is simple, relatively fast, and requires only extremely small amounts of cells and pore-forming proteins.     

A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes

We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.     

Protein assay sensitive at nanogram levels

A simple, fast protein assay which utilizes the affinity of colloidal gold for proteins is described. This assay is sensitive at the 20-ng level when a visible light spectrophotometer is used to measure absorbance. Few chemicals interfere with the assay. Interfering reagents include those that are strongly alkaline, contain high levels of salt, or contain sodium dodecyl sulfate. The problem of alkaline interference can be overcome by acidifying the protein solution before performing the assay. Purified proteins have different capacities to interact with the colloidal gold but this variability is not greater than that seen with the Bradford protein assay.     

Assay of sulfotransferases.

Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3′-phosphoadenosine–5′-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [³⁵S]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.     

Quantitative detection of residual E. coli host cell DNA by real-time PCR

E. coli has been widely used as a host system to manufacture recombinant proteins for human therapeutic use. Among impurities to be eliminated during the downstream process, residual host cell DNA is a major interest for safety. Residual E. coli host cell DNA in the final products are usually determined using conventional slot blot hybridization assay or total DNA Threshold assay, although these methods are time consuming, expensive, and relatively insensitive. Therefore a sensitive real-time PCR assay for specific detection of residual E. coli DNA was developed and compared with slot blot hybridization assay and Threshold assay to validate the overall capability of these methods. Specific primer pair for amplification of the E. coli 16S rRNA gene was selected to improve the sensitivity, and E. coli host cell DNA was quantified by use of SYBR Green 1. The detection limit of real-time PCR assay in the optimized condition was calculated to be 0.042 pg genomic DNA, which is much higher than those of slot blot hybridization assay and Threshold assay of which detection limit were 2.42 and 3.73 pg genomic DNA, respectively. The real-time PCR assay was validated to be more reproducible, accurate, and precise than slot blot hybridization assay and Threshold assay. The real-time PCR assay may be a useful tool for quantitative detection and clearance validation of residual E. coli DNA during the manufacturing process for recombinant therapeutics.     

Bioelectrochemical immunoassay for human chorionic gonadotrophin in serum using an electrode-immobilised capture antibody

An amperometric technique for the quantification of an enzyme immunoassay which utilises a capture antibody covalently attached to a carbon electrode is described. The electrode is used both to separate the assay and to monitor the activity of the bound enzyme label. A ‘two-site’ immunometric assay with monoclonal antibodies directed against human chorionic gonadotrophin (HCG) was used as the model system. The activity of the enzyme bound to the electrode is determined electrochemically by the use of an electron transfer mediator (dimethylaminomethyl ferrocene) permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the assay is 9mIU HCG ml⁻¹ in serum (1st International Reference Preparation). The correlation between the amperometric measurement of serum HCG and data for an immunoradiometric assay was r = 0·988. The assay is rapid requiring a total assay time of 20 min per sample, which includes 15 min for antibody—antigen binding.     

New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.