A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

A quantitative method to evaluate neutralizer toxicity against Acanthamoeba castellanii.

A standard methodology for quantitatively evaluating neutralizer toxicity against Acanthamoeba castellanii does not exist. The objective of this study was to provide a quantitative method for evaluating neutralizer toxicity against A. castellanii. Two methods were evaluated. A quantitative microtiter method for enumerating A. castellanii was evaluated by a 50% lethal dose endpoint method. The microtiter method was compared with the hemacytometer count method. A method for determining the toxicity of neutralizers for antimicrobial agents to A. castellanii was also evaluated. The toxicity to A. castellanii of Dey-Engley neutralizing broth was compared with Page's saline. The microtiter viable cell counts were lower than predicted by the hemacytometer counts. However, the microtiter method gives more reliable counts of viable cells. Dey-Engley neutralizing medium was not toxic to A. castellanii. The method presented gives consistent, reliable results and is simple compared with previous methods.     

ADAPTATION OF A METHIONINE AUXOTROPH ESCHERICHIA COLI GROWTH ASSAY TO MICROTITER PLATES FOR QUANTITATING METHIONINE

A rapid microtiter methionine assay was developed using a methionine auxotroph E. coli strain. The bacterial strain was first grown on rich media to promote extensive bacterial growth and the cells were depleted to exhaust all endogenous methionine. After depletion, cells were transferred to minimal media with increasing concentrations of methionine and microtiter plates were incubated at 37C for 6 h. Methionine microtiter standard curves yielded linear growth responses to increasing concentrations of methionine in the range of 0 to 26.8 μM. Addition of different antibiotic and antifungal agents to the media did not significantly alter the linear growth response observed in the microtiter assay. This microtiter plate E. coli methionine assay has potential as a rapid in vitro assay method for quantifying methionine.     

Microtiter Method for the Evaluation of Viable Cells in Bacterial Cultures

A microtiter technique was investigated as a means of evaluating viable cells in bacterial cultures. Parallel experiments were performed employing the conventional agar plate method along with the microtiter procedure. Statistical analyses showed that the correlation between the two methods was highly significant. With this new method, many samples were analyzed simultaneously, and readable results were obtained in 12 to 15 hr. Other advantages of this method were substantial savings of time, space, and materials. Also, the applicability of this method to estimates of mixed bacterial populations was demonstrated by studying the associative growth of two bacterial cultures.     

In vitro antichlamydial activity of antimicrobials and quinolones using a rapid microtiter screening test system

The activities of twelve antimicrobials against Chlamydia trachomatis were compared when determined in a cell culture system by shell vial and by a rapid microtiter assay. The minimal inhibitory concentration of nine of the twelve were identical in both assays and ranged from 0.03 to > 512 μg/ml. The other three differed by one two-fold dilution from shell vial to microtiter assay and had MICs which ranged from 0.015 to 0.8 μg/ml. The activities of eleven quinolones against Chlamydia trachomatis were measured in vitro by microtiter assay. The minimum inhibitory concentrations of the eleven obtained by microtiter assay ranged from 0.5 to > 1024 μg/ml and were comparable to published shell vial values. The microtiter method, which is easier and less time consuming to carry out, gives comparable activities to the longer shell vial system.     

Statistical Evaluation of Diluents and Automatic Diluting and Pipetting Machines in Influenza Serology

The use of three diluents (i.e., 0.01 m phosphate-buffered saline, PBS; PBS with 0.2% gelatin, PBS/GEL; and PBS with 0.4% bovine plasma albumin) and three methods (i.e., the standard tube macro-procedure, TUBE; the manual microtechnique, MANUAL; and the semiautomatic microtechnique, AUTO) were statistically compared for their reproducibility and sensitivity in determining hemagglutinin (HA) and hemagglutination-inhibition (HI) antibody titers. In the HA test, analyses of between-cell variances of the different methods showed the AUTO microtiter procedure to be more reproducible than the standard TUBE method. The MANUAL microtiter procedure was the least reproducible. In the HI test, the TUBE method was the most reproducible. No significant difference in the reproducibility of the diluents was observed in either the HA or HI test. When a comparison of the sensitivity of test methods and diluents was made for determining HA titers, the AUTO microtiter procedure and PBS/GEL diluent appeared to be the method and diluent of choice. Evaluation of another instrument, the autopipetter, which standardizes the volume of diluent to be added in the microtechnique, suggests that the reproducibility of the AUTO microtiter procedure might be further increased.     

Development of a microtiter plate-based method for determination of degradation profile of nitrophenolic compounds

A microtiter plate-based assay was developed for the automatic monitoring of degradation profile of the yellow-coloured nitrophenolic compounds. The method enables to reduce the intervals between measurements of substrate concentration to minutes and to overcome the problem of discontinuity of sampling typical for conventional methods. The concentrations of nitrophenolic compounds were calculated from the absorbance values determined automatically by BIOSCREEN C. Verification of the method was based on the comparison of results with the conventional HPLC method results. The values of the rate and saturation constants were comparable for both the microtiter plate-based assay and the conventional HPLC method. The automatic method described here seems to be efficient for the screening degradation studies, which requires the treatment of quantity of samples.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase.

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Comparison of Microtiter Procedures with the Plaque Technique for Assay of Vesicular Stomatitis Virus

This paper describes a microprocedure for the tissue culture assay of vesicular stomatitis virus (VSV) and compares the sensitivity of the method with the conventional plaque assay of viral concentration. Microtiter and plaque assay methods were used in titrations, neutralization tests, and thermoinactivation studies with this virus in chick embryo fibroblasts (CEF) and L, HeLa, and PK cell lines. Titration experiments with VSV by the microtiter procedure on preformed monolayers were significantly more sensitive than the plaque assay (P = 0.05). The results of the neutralization tests and thermoinactivation studies also showed greater ability to detect residual virus by the microtiter procedure (P = 0.10). In addition, the microtiter procedure was simpler, less costly, and more rapid than the plaque assay.     

Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.     

Development of a Micromethod for Identification of Anaerobic Bacteria

A microprocedure was described for determining the carbohydrate fermentation patterns of 48 anaerobic bacteria at one time in microtiter plates. The cultures were transferred into agar-filled wells of microtiter plates with a replicator inside an anaerobic glove box. Fermentation was measured both with a colorimetric indicator and with a small pH electrode. The method was approximately 97% accurate. It would be most useful for laboratories that need to identify large numbers of anaerobes at one time.     

A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity

We have developed a highly sensitive micro-gel well diffusion assay for the determination of antimicrobial activity. In essence, the normal radial diffusion type assay was adapted to perform it in a microtiter plate. We compared our micro-gel well diffusion assay to a radial diffusion assay and a microtiter broth dilution method, using gramicidin S as model antibiotic, and Micrococcus luteus as the indicator organism. The micro-gel well diffusion assay was as sensitive as the microtiter broth dilution method, and approximately twice as sensitive as the radial diffusion method. Data analysis to calculate minimum inhibitory concentration, 50% microbial growth inhibition and maximum inhibitory concentration was refined by generating dose-response curves with the software package Prism 3.0 (Graphpad Software Inc.). The minimum inhibitory concentrations, determined by the three methods, were significantly different (P<0. 001), highlighting the limitations involved in comparing data obtained from different methods.     

A microtiter plate assay for protein kinase C

We report the development of a microtiter plate assay for protein kinase C. Reaction components and enzyme samples (protein kinase C purified by phosphatidylserine/cholesterol affinity or DEAE-Sephacel ion-exchange chromatography) were added to wells of a 96-well microtiter plate. The assay was started by the addition of [gamma-32P]ATP with a repeating pipet. After a 3-min incubation at 30 degrees C the wells were sampled six at a time with a 12-channel pipet and spotted onto phosphocellulose filter paper rectangles which were washed with tap water and acetone and counted for radioactivity. The microtiter plate method was more rapid than but gave results similar to those of a standard assay performed in plastic test tubes individually incubated in a 30 degrees C water bath. The microtiter plate procedure gave an intraassay (within one plate) variation of less than 9% and an interassay (between plates) variation of less than 5%. It was linear with time of incubation for 20 min and with amount of enzyme. This method can be used to expedite the assaying of column chromatography fractions for protein kinase C (and other kinase) activity.     

Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.     

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.     

Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate

Abstract A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella . One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of HeLa cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. The described combination of microplate sandwich hybridisation and PCR seems to be a suitable method for rapid detection of Salmonella subspecies I. It only requires a thermal cycler and a conventional microtiter reader, and can be readily done on a large scale.     

Sequencing reactions in microtiter plates

We describe a simple and inexpensive method of performing sequencing reactions for 24 single-strand M13 DNA clones in microtiter plates. To simplify elevated temperature incubations during sequencing reactions, two heating blocks were designed to accommodate microtiter plates and fit within common laboratory heating modules. With only slight modification of standard fluorescent and radioisotopic sequencing methods, the sequencing reactions for 24 clones can be done in as little as 40 minutes.     

Colorimetric microtiter plate assay of glucose and fructose by enzyme-linked formazan production: applicability to the measurement of fructosyl transferase activity in higher plants

A rapid, enzyme-linked colorimetric assay, for the sequential determination of nanomole quantities of glucose and fructose in the same sample, has been developed for the measurement of fructosyl transferase activity in plant extracts. The assay extends the conventional dehydrogenase-linked assay for these sugars by utilizing the intermediary electron carrier, phenazine methosulfate, to couple NADP reduction to the production of a formazan dye from the tetrazolium salt, thiazolyl blue, in a form suitable for measurement using a microtiter plate reader. When the microtiter plate assay was used to measure the activities of yeast invertase and sucrose:sucrose fructosyl transferase from Lolium temulentum, results obtained were very similar to results obtained using the conventional procedure. The rapidity, small scale, and ease of execution of the method offers considerable advantages over the conventional hexose assay and is particularly suitable for screening of large numbers of small samples, exploiting both the speed of the microtiter plate reader and the facility of for microcomputer processing of data. The potential of this method for use with other enzyme systems and other metabolites is discussed.     

Rapid and sensitive detection of b-galactosidase-producing yeasts by using microtiter plate assay

Yeast b-galactosidase activity was detected by a microtiter plate assay using pNPG or Xgal as substrate in 30 minutes. The detection gave a clear result which is well correlated with the specific b-galactosidase activity present in each strain studied. The microtiter plate assay is an effective method to improve the detection and quantify the b-galactosidase gene in recombinant strains of Saccharomyces cerevisiae.