Development of a robust microtiter plate-based assay method for assessment of bioactivity

A microtiter plate-based assay was developed for the quantitative monitoring of bioactive compound production in Streptomyces hygroscopicus fermentation samples. The method reported demonstrates the successful application of the theories of disk diffusion based methods of bioactivity assessment, to a microtiter assay for high throughput analysis. The assay method facilitates the generation of the dose-response curve of test organisms (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) to a bioactive compound. Using this dose-response curve, the method facilitates definition of three distinct Minimum Inhibitory Concentration (MIC) values for use in the characterisation of the bioactive attributes of a sample. The assay uses established standard procedures to facilitate adaptation of the assay for use with a wider range of test microorganisms. Errors due to the assumption of a linear relationship between turbidity and biomass concentration are also reduced, due to incorporation of a step to convert turbidity to biomass concentration, for use in the calculation of bioactivity.     

A new, sensitive method for enzyme kinetic studies of scarce glucosides

The maize β-glucosidase Zm-p60.1 is important for the regulation of plant development through its role in the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. Enzyme kinetics studies using these scarce substrates close to physiological concentrations are difficult due to two reasons: (a) Available methods are mainly suited for end-point kinetics. (b) These methods are not sufficiently sensitive when using scarce glucoside substrates.We developed a glucose assay using a system comprising three enzymes β-glucosidase, glucose oxidase and horseradish peroxidase, with the new substrate N-acetyl-3,7-dihydroxyphenoxazine-Amplex Ultra Red reagent (Molecular Probes). A calibration curve was constructed for resorufin and validation was carried out by comparing our method with the standard spectrophotometric method using p-nitrophenyl-β-d-glucopyranoside. In comparison with the other methods, this method is more sensitive, precise and accurate. The assay is rapid and hence suited for continuous kinetics, it is readily adapted to suit automated procedures, and potential applications include its use in studying the physiological role(s) of enzymes that cleave scarce glucoside substrates.     

Validated methods for the quantification of biologically active constituents of poplar-type propolis

The validation of rapid, low-cost spectrophotometric procedures for the quantification of the three main groups of bioactive substances (flavones and flavonols, flavanones and dihydroflavonols, and total phenolics) in poplar-type propolis has been performed. A spectrophotometric assay based on the formation of an aluminium chloride complex was applied for the quantification of total flavones and flavonols using galangin as standard. Because of the high amount of flavanones and dihydroflavonols in "poplar type" propolis, the introduction of a distinct procedure for their quantification was considered of special significance and the DAB9 colorimetric method was applied for the purpose. Total phenolic content was measured by the Folin-Ciocalteu procedure using a mixture of pinocembrin and galangin as a reference. The procedures were validated using a model mixture of compounds representing the poplar-type propolis composition as found in previous studies. The accuracy (recovery) varied in the range 84-109%, and the relative standard deviation was 0.5-6.2%. The developed spectrophotometric procedures were applied to six poplar type propolis samples. The results were verified independently by a HPLC procedure. The two sets of results agreed satisfactory, as proven by Student's t-test.     

Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system.

The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.     

Improved procedures for the determination of lipid phosphorus by malachite green.

We have developed two procedures for the measurement of lipid phosphorus based on interaction between phosphomolybdenum and malachite green. One method, the "micro" assay uses 50-200 microliters of HClO4 and has a sensitivity range of 0.01-1.5 micrograms phosphorus. The second method, the "macro" assay, has a sensitivity range of 0.03-5.0 micrograms phosphorus with 100-500 microliters HClO4. Both assays are very reproducible with day to day standard deviations of less than 6% between triplicates irrespective of the HClO4 content used. At different concentrations of HClO4, each method was successfully used to determine the phosphorus content in phosphatidylethanolamine and sphingomyelin standards that covered the proposed sensitivity ranges. The increased range, sensitivity and greater volumes of HClO4 permitted in the procedures represent significant improvements over existing methods.     

Assays for amino acid decarboxylase enzymes using ion-exchange cartridges

A general radiochemical method for estimating the activity of amino acid decarboxylases is reported. This method utilizes ion-exchange cartridges to separate unreacted radiolabeled amino acid substrates from product amines, which can then readily be quantitated by liquid scintillation counting. The assay is simple, rapid, and more sensitive than standard 14CO2 trapping procedures if uniformly labeled amino acid substrates are utilized. Acidic, basic, and aromatic amino acid decarboxylases can be assayed with the appropriate choice of cation or anion exchangers. The utility of the method is demonstrated for aspartate-alpha-decarboxylase, tyrosine decarboxylase, and lysine decarboxylase where kinetic parameters are comparable to values obtained by standard radiochemical 14CO2 trapping assays.     

Assay for the enzymic hydrolysis of penicillin and acylhydroxamates

A rapid and easily reproducible assay for penicillin acylase and aliphatic amidase activity is described. The method, which is based on the colorimetric estimation of proton release, is very sensitive and rates as low as 0.5 nmoles/minute can be measured readily. Determination of the kinetic constants of the enzymes gave results as good as or better than standard procedures.     

Statistical analysis of in vivo rodent micronucleus assay

The in vivo rodent micronucleus assay (MNC) is widely used as a cytogenetic assay to detect the clastogenic activity of a chemical in vivo. MNC is one of three tests in a battery recommended by the fourth International Conference on Harmonization (ICH4) of Genotoxicity Guidelines. As such it has been accepted by many regulatory authorities. However, the determination of a positive result in a genotoxicity test, including MNC, has been an issue of debate among toxicologists and biometricians. In this presentation we compare several statistical procedures that have been suggested for the analysis of MNC data and indicate which one is the most powerful. The standard protocol of MNC has at least three dose levels plus the control dose and uses at least four animals per group. For each animal, 2000 polychromatic erythrocytes (PCE) are counted. Two statistical procedures can be employed, either alone or jointly, for the analysis of the MNC dose-response curve. These are the Cochran-Armitage (C-A) trend test and the Dunnett type test. For performing Dunnett type tests, toxicologists often use negative historical control rate for the estimate of the concurrent negative control rate. Some toxicologists emphasize the reproducibility of assay results instead of the dose-response relationship for the important criterion [J. Ashby, H. Tinwell, Mutat. Res. 327 (1995) 49-55; for the rebuttal see M. Hayashi, T. Sofuni, Mutat. Res. 331 (1995) 173-174]. The following three procedures are currently employed in toxicology labs for the evaluation of MNC result. The assay response is deemed positive if it is detected by (i) the C-A trend test alone, (ii) both the C-A trend test and the Dunnett type test and (iii) either the C-A trend test or the Dunnett type test. Using Monte Carlo simulation, we first find for each procedure, sizes of tests which yield the experiment-wise type I error rate of 0.05 and show that the procedure (ii) is the most powerful against the alternatives of monotone increase. The procedure (ii) which originated from Hayashi's three-step procedure was coded in C and termed 'MNC'. The MNC software program is available in the public domain through the ftp.     

The detection of antibodies to recombinant interferon alfa-2a in human serum

Three different procedures have been used for detecting antibodies to Roferon-A (recombinant human interferon alfa-2a, rHuIFN alpha-2a) in the serum of patients who received this interferon as part of ongoing clinical trials: an antiviral neutralization bioassay (ANB), the standard method recommended by the World Health Organization (WHO), and the more recently developed radioimmunoassay (RIA) and enzymeimmunoassay (EIA). Although the three tests are based on different principles, the correlation among them was excellent. The assays show differences in sensitivities with the ANB being the least sensitive of the three. The EIA equals the RIA in sensitivity, reproducibility, accuracy and labor and provides the advantage of safety and convenience in the use of non-radioactive materials. Therefore, the EIA has been selected as the most suitable assay for initial screening of the sera of patients receiving Roferon-A for the presence of antibodies to this interferon. EIA positive sera are then tested in the ANB to determine whether or not neutralizing activities are present.     

Improved efficiency in determining the titer of the Autographa californica baculovirus nonoccluded virus.

An economical and time-efficient method for titering the Autographa californica multiple-embedded nuclear polyhedrosis virus (AcMNPV) by the endpoint method is described. The method uses an electronic pipetting device to perform dilutions in the same 60-well microplate as is used for the assay, thus eliminating the need for test tubes or vials for making dilutions. Additionally, since small volumes are used for the dilutions, a substantial savings in medium is achieved. The effect of using three different lepidopteran cell lines in this assay for AcMNPV is also described. This test revealed that one line (the Trichoplusia ni IPLB-TN-R2 line) is at least 1.5 logs more sensitive to AcMNPV when using occlusion body formation as the measure of infection. The titer was about 6- to 12-fold higher in the IPLB-TN-R2 cell line than the other two lines when plaque assay procedures were used. The titer of a recombinant baculovirus with a bacterial beta-galactosidase gene was also measured in the three cell lines using X-gal as an indicator and showed the IPLB-TN-R2 line to be fourfold more sensitive to this virus.     

Determining the sensitivity of Pseudomonas to chemotherapeutic preparations by the micromethod in liquid synthetic medium

Ever increasing interest is being displayed lately to simple, economic and standard systems for assay of antibiotic sensitivity of microbes with microtechniques in nutrient media requiring no raw materials in short supply. For determining sensitivity of Pseudomonas spp. to chemotherapeutics a liquid synthetic medium balanced by its cationic composition and containing no competing agents of sulfanylamides was used. Three procedures were comparatively estimated: the method of serial dilutions in the liquid medium with using immunological trays, the method of serial dilutions in agar and the diffusion test. In the estimation 185 strains of various Pseudomonas species were used: P. aeruginosa, P. cepacia, P. fluorescens, P. stutzeri, P. putida and P. pseudomallei. The method using the liquid synthetic medium and trays provided more precise interpretation of the results of the assay of the Pseudomonas spp. sensitivity to aminoglycosides, tetracycline, polymyxin and sulfamonomethoxine that the routine procedures. It showed some other advantages such as simplicity, low cost, low medium requirement and glassware economy. The application of the method allowed to exclude the use of expensive imported nutrient media in assay of sulfanylamide sensitivity.     

Synthesis of 4-nitrophenyl caffeate and its use in assays of caffeoyl esterases

We have prepared 4-nitrophenyl caffeate by a combination of standard procedures of organic synthesis and enzymatic deacetylation. Based on hydrolysis of 4-nitrophenyl caffeate, a convenient spectrophotometric assay was developed for specific monitoring of caffeoyl esterase. The method is fast and easy to perform, and it requires no expensive equipment. Its reliability was tested on eight enzyme preparations comprising various combinations of caffeoyl, feruloyl, and acetyl esterase as well as protease activities.     

Rapid and Sensitive Single-Step Radiochemical Assay for Catechol-O-Methyltransferase

Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol- O -methyltransferase activity is described. The method is based on the conversion of catechol to [³H]guaiacol by catechol- O -methyltransferase in the presence of Mg²⁺, adenosine deaminase and S -adenosyl l -[methyl-³H]methionine. Incubation and direct extraction of [³H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol- O -methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.     

Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution

We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

A selective ion monitoring assay for probenecid

A selective ion monitoring assay for probenecid (p-(dipropylsulfamyl)benzoic acid) using m-(di-isobutylsulfamyl)benzoic acid as an internal standard is described. The method has been applied to the determination of probenecid concentrations in human cerebrospinal fluid and lends itself to incorporation into the selective ion monitoring procedures used to quantitate other acidic and neutral compounds in such samples.     

Real time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification

Chinese hamster ovary cells used for pharmaceutical protein production express noninfectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing processes to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell-based infectivity assay has been the standard virus quantification method. In this article, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantification. This method provides accurate and reproducible quantification of X-MuLV particle RNA (pRNA) over a linear dynamic range of at least 100,000-fold with a quantification limit of approximately 1.5 pRNA copies microL(-1). It is about 100-fold more sensitive than the cell-based infectivity assay. High concentrations of protein and cellular DNA present in test samples have been demonstrated to have no impact on X-MuLV quantification. The X-MuLV clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. X-MuLV clearance measured by both methods showed that anion exchange chromatography (QSFF) and DV50 viral filtration are robust retroviral removal steps. In addition, combination of the two methods was able to distinguish the viral removal from inactivation by the Protein A chromatography, and fully recognize the viral clearance capacity of this step. This new method offers significant advantages over cell-based infectivity assays. It could be used to substitute cell-based infectivity assays for process validation of viral removal procedures, but not inactivation steps. Its availability should greatly facilitate and reduce the cost of viral clearance evaluations for new biologic product development.     

Insulin degradation. XV. Use of different assay methods for the study of mechanism of action of glutathione-insulin transhydrogenase.

Insulin degradation by glutathione-insulin transhydrogenase has been studied using three different assay procedures: the measurement of the change in insulin immunoreactivity; the formation of 5% trichloroacetic acid-soluble radioactivity from 125 I-labeled insulin and the formation of GSSG via coupling to the oxidation of NADPH with the use of glutathione reductase. The extent of reaction as measured by each assay was different, and the ratios between the assays were not constant with time. Kinetic experiments with the NADPH-coupled assay and the trichloroacetic acid assay yielded similar results: Line-weaver-Burke plots with insulin as variable and GSH as fixed substrate gave a set of straight, intersecting lines, and such plots with GSH as variable and insulin as fixed substrate were parabolic. Apparent Km values for insulin at 1 mM GSH were found to be quite similar by three assay techniques; however, the V values per unit of enzyme protein varied considerably with different procedures. The results are interpreted as indicating that immunoreactivity is lost after reduction of only one of the disulfide bonds of insulin whereas the two interchain disulfide linkages must be broken to produce the trichloroacetic acid-soluble A chain. The results of the NADPH-coupled assay suggest that all three disulfide bonds of insulin are possible substrates for the enzyme. The trichloroacetic acid precipitation assay seems to be the most practicable technique for general use because of the greater ease in performing large number of samples, precision and sensitivity.     

A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp™Legionella system and seroagglutination

Modifications to the EnviroAmp^™ Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila , this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila , permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 °C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.     

Assays for allantoinase

Allantoinase hydrolyzes allantoin, a purine metabolite and a nitrogen transport molecule in plants, to form allantoic acid. The standard enzyme assay involves acid-catalyzed product decomposition to form urea and glyoxylate, reaction of glyoxylate with phenylhydrazine, and oxidative conversion of phenylhydrazone to 1, 5-diphenylformazan that is measured colorimetrically. When used with crude cell extracts this assay is problematic and its complexity is a hindrance to detailed enzyme characterization; thus, three alternative assays were developed. In the first assay, 2, 4-dinitrophenylhydrazine was reacted with allantoate-derived glyoxylate and the concentration of hydrazone was measured directly by its absorbance at 450 nm. This assay exhibited enhanced reproducibility compared to the standard method and entailed fewer steps, but was 3-fold less sensitive. The second assay combined allantoate decomposition and glyoxylate reaction with o-phenylenediamine to yield a quinoxalone that was detected by its absorbance at 340 nm. This one-step method was the least error prone of those examined, but was more than 10-fold less sensitive than the standard assay. The third assay involved urease-catalyzed hydrolysis of allantoate-derived urea, followed by reaction of the released ammonia to form indophenol. This was the most laborious of the assays, but was more sensitive than the standard method.