ELISA assay optimization using hyperbolic regression

The enzyme-linked immunosorbent assay (ELISA) is routinely usedfor estimation of specific protein concentration in researchand in manufacturing. To optimize both the sensitivity of theassay and its usable dynamic range, it is necessary to adjustthe concentrations of reagents, as well as other experimentalconditions. Hyperbolic regression is a novel mathematical techniquewhich gives the investigator a quantitative analysis of theassay's kinetic response to variations in reagents and conditions.The equations for hyperbolic regression are easily programmedon a computer spreadsheet or even on a scientific pocket calculator.Hyperbolic regression can be used to facilitate the initialoptimization of the assay conditions, or to estimate the concentrationsof unknowns on a routine basis directly from the standard curve,or to monitor the long-term degradation in activity of antibodyreagents and antigen standards. Received on August 15, 1990; accepted on October 10, 1990     

A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in TR activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes; therefore, quantification of thioredoxin activity can be a valuable indicator of clinical conditions. Here we describe a new direct assay, termed the SC–TR assay, to determine the activity of TR based on the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as in older methods, the SC–TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC–TR assay can be used in a cuvette using traditional spectrophotometry or as a 96-well plate-based format using a plate reader. In addition, the SC–TR assay is compatible with the use of nonionic detergents, making it more versatile than other methods using cell lysates.     

Protease and protease inhibitor assays using biotinylated casein coated on a solid phase

A new type of solid-phase assay for proteases and protease inhibitors has been developed using biotinylated casein. The assay involves coating of titer plate wells with biotinylated casein, hydrolysis of this substrate with a protease such as trypsin, reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase-streptavidin complex, and finally quantification of the amount of casein remaining on the plate using alkaline phosphatase activity as the indicator. The activity of the bound indicator enzyme is oppositely related to the protease activity of the sample. In addition, the assay can be modified for quantitating the corresponding amount of protease inhibitor in the sample. The assay is simple, sensitive, accurate, inexpensive, and amenable to automation.     

Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide

In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as     

Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid

Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.     

A sensitive assay for proteolytic enzymes using bacterial luciferase as a substrate

A general assay for proteolytic enzymes using bacterial luciferase as a substrate is described. This luciferase is rapidly inactivated by unusually small amounts of different proteases having a wide spectrum of specificities. The activity is lost exponentially; the pseudo-first-order rate constant for inactivation is proportional to the amount of protease. Since luciferase activity can be measured by a very simple and rapid assay, it affords an accurate, sensitive, and convenient assay for proteolytic activity. This technique is capable of detecting as little as 20 ng of trypsin, requires no centrifugation, and is not hampered by the presence of contaminating pigments in the protease preparation. It is compared at pH 6, 7, and 8 to the phenol color assay and the azocoll assay using seven different proteases.     

Selection of the Best Blood Compartment to Measure Cytidine Deaminase Activity to Stratify for Optimal Gemcitabine or Cytarabine Treatment

Cytidine deaminase (CDA) plays a crucial role in the degradation of cytidine analogs, such as gemcitabine and cytarabine. Several studies showed that a low CDA activity is associated with more toxicity but a higher efficacy, while a high activity will lead to a lower efficacy but less toxicity. A stratified dosing strategy based on the relative CDA activity would increase efficiency. In order to predict these events, a reliable measurement of CDA with a validated method is crucial. We aimed to determine which phenotype assay would be most suitable; a spectrophotometric assay using cytidine as a substrate, or an HPLC assay using gemcitabine as a substrate. In serum and whole blood of 26 volunteers, both assays showed an excellent correlation (R > 0.999), but not in plasma nor in red blood cells. Moreover, there was no difference between males and females.

In conclusion, the spectrophotometric assay seems the most simple and cost-effective test. It should be performed in serum, while it should be normalized on protein content as measured by the Bicinchoninic Acid.     

A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5' end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.     

Determination of peptidylglycine alpha-amidating monooxygenase activity in human serum by thin-layer chromatography

We developed a simple assay system for the quantitative evaluation of peptidylglycine alpha-amidating monooxygenase activity using as substrate a 125I-labeled synthetic tripeptide, 125I-D-Tyr-Val-Gly, thin-layer chromatography, and a radiochromatoscanner. The basic principle of this method is that thin-layer chromatography separates the reaction product, 125I-D-Tyr-Val-NH2, from the substrate in an assay mixture. The 125I activities of both substrate and product separated from each other on a thin-layer chromatography plate were quantified with a radiochromatoscanner and the rate of conversion of the substrate to the product was calculated from their counts. Human serum was used as an enzyme source and the values of alpha-amidation activity obtained by our method under optimal conditions were almost identical to those of the published method using ion-exchange chromatography (sulphopropyl-Sephadex C-50 column) and a gamma-counter. Our method makes it possible to estimate the 10-pmol level of the product using 10 microliters of human serum and to assay a large number of samples rapidly and easily. It is therefore thought to be very useful for screening various tissues for alpha-amidation activity.     

Functional characterization of 102-amino acid-deleted form of human aromatase (delta102-aromatase).

A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325-329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an 'In-cell' assay. A strong aromatase activity, however, was observed for the delta102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted delta102-aromatase was 6.8 min(-1). The results of the immunosuppression assay suggested that delta102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC50 values of MAb3-2C2 were determined to be 24 and 23 microg/ml for the whole homogenate and the 105,000 x g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC50 of 1.3 microg/ml was shown for the full-length human aromatase. These results indicate that the delta102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of delta102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an 'In-cell' assay.     

Assessing the binding and endocytosis activity of cellular receptors using GFP-ligand fusions

We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells. Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands. We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins. A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity. Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors. To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells. Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.     

Assaying Arabidopsis lipase activity

A low lipase activity from a crude extract of Arabidopsis seedlings was assayed using three sensitive methods (radiolabelled triacylglycerols, commercial resorufin ester and triacylglycerols containing the naturally fluorescent parinaric acid as substrates). The specific activity of the extract was found to be similar using the three methods. However, the plant lipase activity measured using the radioactivity and the fluorescence assays could be abolished by heating the extract, contrary to the apparent activity measured using the commercial colorimetric assay. Unlike the radioactivity assay, the fluorescence assay can be monitored continuously. The parinaric acid-based method is therefore the only one to provide a sensitive, specific and continuous assay.     

Measurement of ferredoxin-dependent sulfite reductase activity in crude extracts from leaves using O-acetyl-L-serine sulfhydrylase in a coupled assay system to measure the sulfide formed

Ferredoxin-dependent sulfite reductase (EC 1.8.7.1) catalyses the reduction of sulfite to sulfide, using reduced ferredoxin as an electron donor. An assay system was developed for measuring this enzyme activity in crude extracts and broken chloroplast preparations from leaves. The assay consists of a coupled system in which the sulfide formed is used for cysteine synthesis by added O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8). Cysteine thus formed is determined with ninhydrin under conditions where O-acetylserine does not react and serves as a measure for ferredoxin-dependent sulfite reductase activity. Cysteine synthesized in the assay can be determined from 10 to 200 nmol. One assay per minute can be performed.     

Activity of urethane and N,N-dimethylurethane in the mouse bone-marrow micronucleus assay: equivalence of oral and intraperitoneal routes of exposure

Urethane is shown to be active in the mouse bone-marrow micronucleus assay when administered as a single dose by either gavage or intraperitoneal injection. The magnitude of the response using the two routes was not statistically significantly different. N,N-Dimethylurethane (DMU) is shown to be mutagenic to Salmonella and active in the bone-marrow micronucleus assay by both routes of administration. The activity of DMU in the bone marrow precludes elimination of ethanol, yielding cyanate ion, as an explanation for the micronucleus-inducing activity of urethane.     

Evaluating the utility of the HTRF Transcreener ADP assay technology: A comparison with the standard HTRF assay technology

The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay technology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detecting the formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we compare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as a case study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constants and inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic window and lower Z′ factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect both kinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenues for assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this can speed up the drug discovery process. However, caution must be exercised because any contaminating ATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP in the assay will also limit the application of this technology, especially in compound mechanistic studies such as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool to complement existing assay technologies for drug discovery.     

Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection

An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC(50) value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples.     

Assay of disulfide oxidase and isomerase based on the model of hirudin folding

Oxidative folding of fully reduced hirudin (R-Hir, six cysteines) undergoes two distinct stages. A first stage of nonspecific disulfide formation promoted by oxidase converts R-Hir to form 3-disulfide scrambled hirudins (X-Hir) as obligatory intermediates. A second stage of disulfide shuffling catalyzed by isomerase converts X-Hir to the native hirudin (N-Hir). The model of hirudin folding is utilized here to develop an assay system for measuring the activity of disulfide oxidase and isomerase, using high-performance liquid chromatography (HPLC) quantification of R-Hir, X-Hir, and N-Hir. The oxidase assay measures the ability of an oxidase to promote R-HirX-Hir conversion. The molar specific activity is expressed as mol ofR-Hir decrease per mol of oxidase per min. The isomerase assay measures the ability of an isomerase to catalyze X-HirN-Hir transformation. The molar specific activity is expressed as mol ofN-Hir increase per mol of isomerase per min. Alternatively, the recovery of N-Hir in the isomerase assay can be determined by its alpha-thrombin inhibitory activity. Using both HPLC and activity-based assay, we have measured the relative oxidase and isomerase activity of reduced and oxidized glutathione, Cys, Cys-Cys, and reduced and oxidized protein disulfide isomerase (PDI). The molar specific activity of reduced PDI was shown to be 0.1+/-0.01 U, which is consistent with documented data obtained by the scrambled RNase-A-based assay. These proposed assay methods provide alternatives to the limited option of methodologies currently available for measuring oxidase and isomerase activities. A major merit of the proposed assay system is the potential to accommodate the analysis of biological samples.     

A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc

Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC(50) values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC(50) values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity.     

Measurement of the phospholipase activity of endothelial lipase in mouse plasma

Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL''s hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface.