Enzyme-linked immunosorbent assay-based selection and optimization of elution buffer for TAG72-affinity chromatography

An enzyme-linked immunosorbent assay (ELISA)-elution assay was developed to screen a large variety of elution buffers for selection of a suitable one for purification of the fusion protein FV/TNF-α by affinity chromatography. Various commonly used buffer systems utilizing widely differing conditions such as extreme pH, denaturants, chaotropic ions and polarity reducing reagents were investigated. Ammonia solution (1 M, pH 11.5) proved to exert the most suitable influence on dissociation of the FV/TNF-α/TAG72 complex while having a minimal protein denaturing effect on FV/TNF-α. The total yield of purified FV/TNF-α using the TAG72-affinity column with this elution system was 300-fold higher than that using the common elution buffer, 0.1 M glycine, 0.5 M NaCl, pH 2.7. Our study indicates that the ELISA-elution assay will be most useful in the selection of suitable elution buffers for affinity chromatography.     

A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin

The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low-yield positive results. In this study, three different strategies including soluble antibody-capturing, pH-stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme-linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single-chain fragment variable (scFv) fragments were purified by nickel-nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody-capturing method yielded 15 positive phage-scFv clones against diphtheria toxoid. Conventional panning and pH-stepwise elution model resulted from nine and five positive phage-scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody-capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two-fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody-capturing method is an efficient method for isolation of specific scFv fragments.     

Sensitive high-performance liquid chromatographic determination with fluorescence detection of phenol and chlorophenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent

A sensitive HPLC method for the determination of phenol and chlorophenols was developed. The fluorescence labeling reaction of phenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was completed in 30 min at 60°C. The separation of DIB-derivatives of five representative phenols, i.e., phenol, o-, p-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, was achieved within 35 min with an ODS column using isocratic elution. The detection limits of these DIB derivatives at a signal-to-noise ratio (S/N) of 3 were in the range of 0.024 to 0.08 μM (0.12–0.45 pmol/20 μl injection). Twelve kinds of DIB derivatives with phenols containing mono-, di-, tri-, tetra- and penta-chlorophenol were also well separated within 208 min by changing the elution conditions. The derivatives were stable for at least for 24 h when they were placed at room temperature in the dark. The proposed method was applied to the assay of human urine samples and free and total phenol were determined. The relative standard deviations (RSDs) of the proposed method for within and between-day assay were <7.0% and <14.2%, respectively. The average concentrations of free and total phenol found in urine (n=6) were 4.3±2.5 and 29.5±14.0 μM, respectively.     

Improvement of production,assay and purification of streptavidin

Summary The production of streptavidin byStreptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay. This assay appears to give useful estimates of streptavidin production. Depending upon the medium employed, streptavidin yields ranged from 0.5 mg/l to 53 mg/l. Production was successfully scaled up to ten liter fermentors. Streptavidin was purified in a one step process from centrifuged, concentrated fermentation broths by binding the protein to an iminobiotin column at pH 11 followed by elution at pH 4.0. Recovery percentages varied depending upon the solubility of the fermentation media ingredients.     

Trehalose determination in linseed subjected to osmotic stress. HPAEC‐PAD analysis: an inappropriate method

Trehalose is a non‐reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co‐elution of trehalose in these two species by a trehalase assay and liquid chromatography‐high resolution mass spectrometry analysis, gas chromatography–mass spectrometry (GC‐MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC‐PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC‐MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (?0.30 MPa), the quantity (31.49 nmol g^–1 dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.     

Evaluation of cyst loss in standard procedural steps for detecting ofGiardia lamblia andCryptosporidium parvum in water

The standard procedure outlined by the United States Environmental Protection Agency (US EPA) in Method 1623 for analyzingGiardia lamblia cysts andCryptosporidium parvum oocysts in water samples consists of filtration, elution, centrifugal concentration, immunomagnetic separation (IMS), and immunofluorescence assay (IFA) followed by microscopic examination. In this study, the extent of (oo)cyst loss in each step of this procedure was evaluated by comparing recovery yields in segmented analyses: (i) IMS+IFA, (ii) concentration +IMS+IFA, and (iii) filtration/elution + concentration +IMS+IFA. The complete (oo)cyst recovery by the full procedure was 52–57%. The (oo)cyst loss in the IMS step was only 0–6%, implying that IMS is a fairly reliable method for (oo)cyst purification. Centrifugal concentration of the eluted sample and pellet collection before IMS resulted in a loss of 8–14% of the (oo)cysts. The largest (oo)cyst loss occurred in the elution step, with 68–71% of the total loss. The permeated loss of (oo)cysts was negligible during filtration of the water sample with a 1.0-μm pore polyethersulfone (PES) capsule. These results demonstrated that the largest fraction of (oo)cyst loss in this procedure occurred due to poor elution from the filter matrix. Improvements in the elution methodology are therefore required to enhance the overall recovery yield and the reliability of the detection of these parasitic protozoa.     

Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond

A dinoflagellate bloom was found associated with a fish kill event in a South Carolina brackish water retention pond. A multi-analytical approach was used to confirm the identity of the bloom dinoflagellate and evaluate its potential toxicity. Karlodinium micrum was confirmed through light microscopy, pigment profile comparisons, species-specific PCR, and gene sequence data. Necropsy findings on several fish were suggestive of an acute kill event. Toxicity of filtrate from bloom samples was tested by a hemolytic assay using rainbow trout (Oncorhynchus mykis) erythrocytes and an ichthyotoxicity assay using larval zebrafish (Danio rerio). Hemolytic activity was measurably high (>80% hemolysis) in both whole filtrate and fractionated filtrate (from the 80% MeOH C₁₈ column elution). This fraction also demonstrated high ichthyotoxic activity as exposed fish experienced rapid death. These results implicate toxic K. micrum as a causative factor in fish death in a non-aquaculture brackish pond associated with a housing development, and extend recent findings linking this species to fish kills in aquaculture ponds.     

Determination of flunarizine in rat brain by liquid chromatography–electrospray mass spectrometry

A rapid liquid chromatography–electrospray mass spectrometry (LC–ES-MS) assay for the determination of flunarizine (FZ) in rat brain has been developed. A C₁₈ column and an isocratic elution were employed for the separation. Using post-column split, 64% of the eluent was introduced into the ES-MS system for detection. The [M+H]⁺ (m/z 406) and a fragmented ion (m/z 203) were detected using selected ion monitoring. The linear range of this assay was good, ranging from 0.05 to 5 μM (r²=0.99). The intra- and inter-day precisions showed relative standard deviations ranging from 1.4% to 2.0% and 1.3% to 2.9%, respectively. The application of this newly developed method was demonstrated by examining the pharmacokinetics of FZ in rat brain.     

Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa

Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.     

Increased sensitivity and discrimination in screening through an immobilized-resin microbiological assay method

Summary Problems with present bioactive microbial product screening techniques include low sensitivity and insufficient discrimination capabilities. These problems are addressed by our new immobilized-resin microbiological assay. This technique concentrates bioactive samples on macroporous polymeric resins that are immobilized in hydrogel beads. These beads are then subjected to elution in the wells of an agar diffusion microbiological assay medium. With a strong base anion exchanger, the sensitivity to ampicillin of the -lactam-supersensitiveEscherichia coli mutant ESS-22-31 was increased 10-fold. Similar increases in sensitivity were obtained in the detection of streptomycin using a weak acid cation exchanger withBacillus subtilis and for cycloheximide by a neutral resin andSaccharomyces cerevisiae NRRL-Y-139. A judicious choice of resin type and eluent permitted a selective sensitivity increase based on the charge or hydrophobic nature of the desired product. This selectivity imparts a discrimination capability to the technique.     

Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non‐resistant plants

Phytochelatin (PC) synthesis is considered necessary for Cd tolerance in non‐resistant plants, but roles for PCs in hyper‐accumulating species are currently unknown. In the present study, the relationship between PC synthesis and Cd accumulation was investigated in the Cd hyperaccumulator Sedum alfredii Hance. PCs were most abundant in leaves followed by stems, but hardly detected by the reversed‐phase high‐performance liquid chromatography (HPLC) in roots. Both PC synthesis and Cd accumulation were time‐dependent and a linear correlation between the two was established with about 1:15 PCs : Cd stoichiometry in leaves. PCs were found in the elution fractions, which were responsible for Cd peaks in the anion exchange chromatograph assay. About 5% of the total Cd was detected in these elution fractions as PCs were found. Most Cd was observed in the cell wall and intercellular space of leaf vascular cells. These results suggest that PCs do not detoxify Cd in roots of S. alfredii. However, like in non‐resistant plants, PCs might act as the major intracellular Cd detoxification mechanism in shoots of S. alfredii.     

Biochemical Studies on “Bakanae” Fungus

The production of gibberellin A₇ by Gibberella fujikuroi was studied by using newly devised assay method. Gibberellin A₇ increased at preferable temperature range between 32°C and 34°C at the controlled pH(6.0~7.5). The improved isolation process by using column chromatography composed of granular charcoal was found to be extremely convenient, because of its quick elution with satisfactory separation from gibberellic acid which is always accompanied by gibberellin A₇ in culture medium.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor

To facilitate selection of monoclonal antibodies for immunoaffinity chromatography, an ELISA screening procedure was developed. The assay is based on the avidin-biotin system and provides a profile of the monoclonal antibody which is based on the binding characteristics of the antigen binding site when exposed to different elution reagents. The elution profiles of 5 monoclonal antibodies to soy bean trypsin inhibitor (SBTI) were determined and for 2 of the antibodies the results obtained in the ELISA were verified using column experiments. The affinity constants were determined for the same 5 monoclonal antibodies and no correlation was seen with the ease of elution. The elution profiles presented here are easily obtained and the results indicate that a general screening procedure for suitable combinations of antibodies and elution conditions can be carried out using an elution ELISA assay when modified as described herein.     

Isolation of two arginine vasopressin-like factors from ganglia of Locusta migratoria

Tissues of Locusta migratoria are known to contain a material which crossreacts with an antibody against arginine vasopressin (AVP), and this factor has been correlated with the diuretic hormone of this species. In this paper, we report the isolation of two AVP-like factors from suboesophageal ganglia and thoracic ganglia of Locusta migratoria. The less abundant, more hydrophobic of these AVP-like factors shows diuretic activity in an assay where excretion of amaranth dye from Locusta migratoria hemolymph is used as the scoring criterion. After extracting a total of ～ 51,000 ganglia with an acidic solvent, the crude extract was prepurified by batch adsorption/elution from disposable reversed-phase cartridges. The prepurified extract was then sequentially purified by reversed-phase liquid chromatography using solvent programs of substantially differing selectivity. The more abundant factor was isolated to apparent homogeneity in three steps, while the less abundant factor required four or five steps. Use of a C₄ reversed-phase column minimized losses of the minor, more hydrophobic factor.     

Use of short high-performance liquid chromatography columns and tandem-mass spectrometry for the rapid analysis of a prostaglandin analog, fluprostenol, in rat plasma

A short reversed-phase HPLC column and a tandem mass spectrometer were used to develop a stable-isotope-dilution assay for the rapid and sensitive analysis of fluprostenol, a prostaglandin analog, in rat plasma. A Waters Symmetry ODS column (2.1×10 mm) afforded rapid isocratic elution of fluprostenol (t_R=40 s) but still provided a relatively large k′ value of 4. The use of tandem mass spectrometry allowed the interference-free detection of fluprostenol under the rapid elution conditions, with a limit of quantitation of 25 pg ml⁻¹ fluprostenol, using 0.2 ml plasma sample volumes. The method was linear over three orders of magnitude, yielded accurate and precise results and allowed the pharmacokinetic profile of fluprostenol to be defined following intravenous administration in rats.     

Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage

In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.     

Induction of single-strand breaks and interstrand cross-links in liver DNA after the administration of 2-acetylaminofluorene and trans-4-acetylaminostilbene to rats

2-Acetylaminofluorene (AAF) or trans-4-acetylaminostilbene (AAS) was orally or intraperitoneally administered to female Wistar rats. DNA from liver cells was analyzed for single-strand breaks by the alkaline elution assay. Only borderline effects were observed with doses (100 μMol/kg) used in animal carcinogenesis experiments. Even high doses of AAF (1,000 μMol/kg) were not effective. Methyl methanesulfonate (MMS) in vivo and gamma irradiation in vitro were shown to produce dose-dependent DNA single-strand breaks (positive control). Only a marginal effect was obtained with 100 μMollkg MMS. The elution rate of DNA was increased by a factor of 34 in liver cells in vitro with 400 rad of gamma irradiation. Only a fraction of this rate could be demonstrated immediately after irradiation in vivo, and no lesions were found two hours later. This strongly indicates the rapid repair of single-strand breaks. Additional experiments showed that AAS, a nonhepatocarcinogen, produced more interstrand cross-links in the rat liver DNA than did AAF.     

The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays

A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of (1–4)galactosyltransferase, (1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated.     

Improved high-performance liquid chromatographic method for N-acetylgalactosamine-4-sulfate sulfatase (arylsulfatase B) activity determination using uridine diphosho-N-acetylgalactosamine-4-sulfate

UDP-N-acetylgalactosamine-4-sulfate (UDP-GalNAc-4-S) was isolated from hen oviduct (isthmus) with a yield of 31 μmol per 100 g of wet tissue and used for arylsulfatase B (ASB) activity determination. Two HPLC methods of separation and quantitation of the reaction product were described: (1) an original gradient elution method which makes it possible to determine the reaction product when only partially purified ASB was used and additional uridine derivatives were formed during incubation: (2) an improved, fast isocratic elution method which may be used in the case of purified ASB preparations, devoid of other nucleotide hydrolysing enzymes. For both methods the detection limit was 0.1 nmol of product with standard error of determination ?3%. Using the gradient elution method we have found that UDP-GalNAc-4-S was hydrolysed by bovine arylsulfatase B1 most efficiently at pH 5.0 and concentration 0.5 mM with K_m=85 μM.