Quantitation of tigecycline, a novel glycylcycline [corrected] by liquid chromatography

An ion-paired HPLC assay was developed to determine tigecycline (GAR-936) concentrations in Hank's balanced salts solution, tigecycline intra-cellular concentrations in human polymorphonuclear neutrophils (PMNs) and tigecycline concentrations in human serum. Minocycline was used as internal standard, 5% trichloroacetic acid was added to lyse PMNs and also precipitate proteins in PMNs and serum. The top aqueous layer was aspirated for HPLC assay. The chromatograms were performed with a reversed-phase C18 column with UV detector. The mobile phase consisted of acetonitrile, phosphate buffer (pH 3) and 1-octanesulfonic acid at a flow rate of 1 ml/min. Good linearity and recovery were achieved over the range of standard curves. The relative standard deviations of three quality controls for intra- and inter-day precision were less than 6.4%, and the relative errors of the intra- and inter-day accuracy were less than 7.0%. Tigecycline in Hank's buffer, PMNs and serum was stable under different test conditions. This new liquid chromatography assay is a simple, accurate and reproducible method for determining tigecycline in different matrix.     

Adsorbed soluble [3H]elastin as substrate for proteinase activity. A new microassay technique.

A microassay for elastase activity has been designed. Microtubes are coated with soluble elastin substrate by passive adsorption to the tube walls. On contact with enzymes in test samples, radioactivity is released into the sample buffer, which is then transferred to vials containing scintillation fluid without further processing. Porcine pancreatic elastase (EC 3.4.21.11) is easily detected in concentrations down to 8 micrograms/litre. The interassay coefficient of variation (CV) was below 10% for values above 50 micrograms/litre. The assay is rapid, precise, economical and sensitive.     

Simple Method for Rapid Determination of Antibiotic Blood Levels

A simple method for the rapid determination of antibiotic blood levels is described. Results are readily obtainable within 4 to 6 hr. The reported procedure is based upon determination of the highest dilution of a patient's serum capable of inhibiting dextrose fermentation by a standardized inoculum of a staphylococcus strain or of an Escherichia coli strain, when colymycin or polymyxin are involved, as compared to previously determined endpoints in this regard of defined concentrations of the different antibiotics dissolved in normal human serum. Excellent correlation was observed with 36 serum specimens that contained different antimicrobial agents in varied concentrations when simultaneously assayed by the standard method and the subject procedure of this report.     

Development of an avidin-biotin competitive inhibition assay and validation of its use for the quantitation of human intervertebral disc serine proteinase inhibitory proteins.

A simple convenient method has been developed for the quantitation of serine proteinase inhibitors (SPIs) in tissue extracts. The method is based on the competitive binding to trypsin and chymotrypsin immobilized using glutaraldehyde on 96-well microtiter plate wells of native SPIs and a biotinylated secretory proteinase inhibitor (SLPI) standard. The bound SLPI standard was visualized using an avidin-alkaline phosphatase conjugate and inhibition curves were determined using absorbancy measurements at 405 nm. The standard assay had a range between 0.02 and 1 microgram SLPI/well and a lower detection limit of 20 ng SLPI/well; an improved microassay had a detection limit of 2 ng SLPI/well. Only active free inhibitor was detected in the assay since denatured and/or enzyme-inhibitor complexes did not bind to the plates. A range of SPI species was demonstrable in human bronchial mucus and intervertebral disc SPI samples using this technique. Quantitation of SPI levels in a number of intervertebral disc samples indicated that the SPIs were depleted in degenerate discs compared to nondegenerate discs (P less than 0.05, n = 12). Since the immobilized trypsin and chymotrypsin microplates used in this assay may be prepared in advance (and are stable at 4 degrees C for at least 1 month) the remaining two steps of the assay (the inhibition step and visualization) may be completed in 2-3 h; thus the assay is simple, convenient, and fast. All reagents (other than the biotinylated SLPI standard) are readily available commercially, and in principle the assay could be adapted to other systems provided defined biotinylated standards were available.     

A quantitative electrochemiluminescence assay for Clostridium perfringens alpha toxin

Described is a rapid direct sandwich format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru)-labeled detection antibodies. The ruthenium chelate of detection antibodies chemically reacted in the presence of tripropylamine and upon electronic stimulation emitted photons (electrochemiluminescence) that were detected by the photodiode of the detector. Elevated toxin concentrations increased toxin immunoadsorption and the specific immunoadsorption of Ru-labeled antibodies to alpha toxin, which resulted in increased dose-dependent electrochemiluminescent signals. The standardized assay was rapid (single 2.5-h coincubation of all reagents), required no wash steps, and had a sensitivity of about 1 ng/ml of toxin. The assay had excellent accuracy and precision and was validated in buffer, serum, and urine with no apparent matrix effects.     

A microwell assay for anchorage independent cell growth

We describe modifications of the conventional assay for anchorage independent growth of fibroblasts that enable the assay to be carried out in microwell plates, as opposed to the conventional Petri dishes. The microwell assay is a good discriminator of final EGF concentrations in the range 10-100 picograms/ml, and can be used to detect absolute amounts of EGF below 2 pg. Addition of TGF beta to EGF enhances colony formation in the microassay in the usual manner. We describe the use of this microassay to identify and map local production of transforming growth factor activity by the component pieces of individual chick embryos. Transforming activity was identified in all the stages tested (Hamburger and Hamilton stages 13-23). Highest levels were found near the mid-line of the embryo. No clear differences in the cranio-caudal axis have so far been identified. This technique will enable the spatial and temporal distribution of transforming activity throughout vertebrate embryos to be completely mapped. It seems likely that this mapping process will help elucidate the normal role of transforming growth factors in embryos.     

Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP

Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (-)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2M), and stored at -80 degrees C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1-20 microg/ml, assay precision was 1-5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 microg/ml with 0.2-0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.     

Quantitation of electrophoretic eluted proteins

Quantitation of stained, electroeluted proteins by the classical Lowry and Bradford protein assay is not possible because of some different interferences. In particular we have found that the substance interfering in the Lowry method cannot be removed by trichloroacetic acid precipitation nor can be compensated for by the appropriate blank. Interferences in the Bradford protein assay are due to detergents and pH of the protein buffer as well as to Coomassie brilliant blue R250 electroeluted with the protein sample. However, while these interferences can be compensated for by appropriate blank and standard curves, others (probably due to acrylamide fines) cannot be corrected. All these problems can be overcome by concentration and dialysis of electroeluted samples which permit the removal of interfering substances and the use of Bradford and Lowry protein assay in the 1-20 micrograms range, respectively. Successful applications are described for electroeluted bovine serum albumin, human hexokinase and phosphoglucomutase.     

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.     

Use of a Tabletop Computer for Antibiotic Assays

The increasing need for rapid and accurate assay of antimicrobial agents in body fluids requires technical improvement of skills in these areas. A method for using a tabletop computer to simplify and shorten the statistical analysis of the laboratory data obtained by bioassay with the Olivetti Underwood Programma 101 has been developed so that a secretary or laboratory helper can rapidly develop the standard curves for each day's assays.     

Optical chip immunoassay for hCG in human whole blood

We report on the development of an integrated optic chip sensor for performing rapid and sensitive immunoassays with human whole blood using human chorionic gonadotropin (hCG) as the model system. The optical chip is based on the Hartman interferometer, which uses a single planar lightbeam to address multiple interferometers, each comprising a signal/reference pair of sensing regions. The binding of antigen to specific capture antibodies on the signal sensing region causes a change in the refractive index of the surface layer, which is detectable by its effect on the evanescent field of the guided lightbeam. The reference-sensing region is coated with an irrelevant antibody, which optically cancels a large fraction of the non-specific adsorption that occurs on the specific-sensing region when the sensor is tested with clinical specimens. This work extends previous experiments with buffer and human serum to measurements in undiluted whole human blood. Optical chips were stored dry after surface functionalization, and rehydrated with blood. Colloidal gold nanoparticles conjugated to a second anti-hCG monoclonal antibody were used to provide signal amplification, thereby enhancing assay sensitivity, in a one-step procedure with the gold conjugate added to the test sample immediately prior to measurement. Background signals due to non-specific binding (NSB) in blood were found to be higher than those previously reported with human serum. In addition, a high level of background signal was found with the gold conjugate, which had not been observed in experiments with either buffer or serum. Nevertheless, hCG could be detected at 0.5 ng/ml within 10 min of sample application. The sensor response was linear over the concentration range 0.5-5 ng/ml hCG, as compared with the clinically-relevant range 0.3-1.5 ng/ml. Detection at higher concentrations was affected by scattering from large amounts of bound gold nanoparticles. However, initial binding rate measurements could be used to maintain assay quantitation.     

Studies on staphylococcal enterotoxin B. 1. Quantitative assay by capillary tube single diffusion test (author's transl)]

A method for rapidly identifying and quantitating small amount of staphylococcal enterotoxin B was developed on capillary tube single diffusion test. The high sensitivity of method has the advantages to save antiserum and incubation period. Some factors such as concentration of antigen and antiserum, incubation temperature, buffer systems, and position of capillary tube all might affect the test accuracy. If all those affecting factors were concerned, the toxin would probably be standardized. The most accessible quantitative estimation was accomplished by means of (1) plotting a precipitation curve from the different concentrations of standardized staphylococcal enterotoxin B; (2) the sample to be test was diluted by serial two-fold method with the same buffer system; (3) select suitable 3--5 dilutions to perform capillary tube diffusion test, taking the values of precipitation bands as abcissa and sample dilutions as longitudinal axis, rendered the precipitation curve of sample exquisitely paralled to that of the standard; (4) calibrate the figure values of sample exactly on the precipitation curve, and reading the toxin content of specimen to be tested from standard curve.     

One-step immunoassay for measuring protein concentrations in plasma, based on precipitate-enhanced ellipsometry

Standard enzyme immunoassays (EIAs) require washing steps to remove excess enzyme-antibody complexes. Such washing is laborious, lengthens assay time, and increases assay scatter. Recently, so-called precipitate-enhanced immunoassays (PEIAs) were introduced. Instead of color formation due to enzymatic conversion of a chromogenic substrate, this technique measures the rate of precipitate formation due to conversion of a substrate with a precipitating product. Such precipitation can be measured in the presence of active enzyme-antibody complexes in the buffer and no washing is required. In the present study this technique was used in a one-step PEIA, without washing steps, for the measurement of plasma concentrations of fatty-acid-binding protein. Horseradish peroxidase was used as tagging enzyme and diaminobenzidine as precipitating substrate. Precipitate formation was measured by ellipsometry. Assay time of the one-step PEIA was much shorter than that for an existing standard EIA. Test results can be obtained within minutes, depending on the sensitivity required. Assay precision of the one-step PEIA was better than that of the standard EIA. In the one-step assay, loss of surface-bound conjugate due to washing is prevented, which could explain part of the improved sensitivity compared to that of the two-step PEIA. More importantly, the presence of substrate-converting enzyme-antibody complexes in the buffer caused a large enhancement of precipitation.     

NASBA荧光分子信标技术定量检测丙型肝炎病毒

建立NASBA荧光分子信标探针检测技术,并对国家HCV标准品、人工构建HCVRNA野生株及HCV抗体阳性不同人群进行检测。实验结果:该方法检测HCV的灵敏度为103拷贝ml血清,阴性参比品的符合率为100%;检测的线性范围为103拷贝～109拷贝ml血清;精密性(CV值)小于6%,在HCV抗体阳性人群中HCVRNA的检出率在45%～65%之间。结论:该方法在HCVRNA临床定量检测中具有良好的灵敏度、特异性、重复性与实用性。     

The in vitro effects of EDTA-tris, EDTA-tris-lysozyme, and antimicrobial agents on equine genital isolants of Pseudomonas aeruginosa

Five isolants of Pseudomonas aeruginosa collected from clinical cases of equine genital infection and one standard strain of P. aeruginosa were exposed to various concentrations of ethylene-diaminetetraacetic acid (EDTA) and tris (hydroxymethyl) aminomethane (tris buffer pH 8) and EDTA-tris lysozyme. Colony forming units of the isolants and minimal inhibitory concentrations for 11 antimicrobial agents were determined with each isolant before and after exposure to the EDTA solutions. Decreased cellular viability was found with all six isolants after exposure to the EDTA-tris solutions. Reversal of antimicrobial resistance was variable and unpredictable. These effects were not enhanced by the addition of lysozyme. The results suggest that EDTA-tris could be a useful adjunct in treating equine genital infections caused by Pseudomonas aeruginosa .     

Determination of 5-methyltetrahydrofolic acid in human serum by stable-isotope dilution high-performance liquid chromatography-mass spectrometry.

The need for specific and sensitive methods for the determination of distinct serum folates is of high priority in clinical research settings. A stable-isotope liquid chromatography-mass spectrometry (LC/ESI-MS) assay was developed for the quantitative determination of the monoglutamyl form of 5-methyltetrahydrofolic acid (5-MTHFA) in human serum. Serum samples (0.5 ml) were amended with the internal standard, [5-13C5]MTHFA that had been labeled on the glutamic acid portion of the molecule and allowed to equilibrate. The analyte was trapped onto a solid-phase cartridge and then eluted with the HPLC mobile phase. Forty microliters was taken for LC/ESI-MS analysis using electrospray ionization operated in the positive ion mode. Using the standard method of addition of 5-MTHFA to serum, a linear dilution curve (y = 12.777x - 1.404; range 0.94-97 ng x ml(-1)) was constructed. The precision of the method was 5.3% (CV) based on the analysis of four sample replicates. The mass spectrum produced upon collision induced dissociation of the analyte in serum was used to confirm the identity of the 5-MTHFA. The method was applied to the analysis of a set of serum samples that contained standardized concentrations of 5-MTHFA. The determinations of 5-MTHFA in these samples using the LC/ESI-MS procedure were found to be in good agreement with other folate methods. A highly accurate and specific method for the analysis of 5-MTHFA in serum has been developed utilizing stable isotope dilution mass spectrometry.     

Validated capillary gas chromatographic-mass spectrometric assay to determine 2-methylcitric acid I and II levels in human serum by using a pulsed splitless injection procedure

BACKGROUND: Despite some clinical applications of 2-methylcitric acid (2-MCA) determination in urine and amniotic fluid, a diagnostic use of 2-MCA levels in serum is not common practice. This could be related to the complexity of the assay, or possibly to unawareness of other feasible clinical applications. METHODS: The levels of the diastereomers 2-MCA I and II in human serum were determined by GC-MS based on a method using a pulsed splitless injection technique. A stable isotope dilution principle was modified considering the diastereomer ratio and impurities of the internal standard. Precision parameters as well as recovery rates of the assay were determined. Reference intervals for 2-MCA(total), 2-MCA I and II levels were obtained in 52 healthy volunteers (31 female, 21 male, mean age 41.7+/-14.4 years). RESULTS: 2-MCA was readily detected in each sample of serum, as well as in urine, cerebrospinal fluid and amniotic fluid. The limit of detection was 10 nmol/l for 2-MCA(total). The internal standard showed a diastereomer ratio of 2-MCA II-d3 to 2-MCA I-d3 of 0.83+/-0.05, its chemical purity had to be corrected to 90.5+/-0.5%. In concentrations of 446, 750 and 1256 nmol/l 2-MCA(total), recovery rates of 98.5, 93.7 and 88% with a mean intra-assay RSD of 1.5% were determined. The day-to-day precision was 10% RSD (SD 40 nmol/l) for 2-MCA(total) obtained with a pooled serum sample at a concentration of 401 nmol/l 2-MCA(total) over a period of 5 months (n=17). The normal range for 2-MCA(total) in human serum was calculated as 81-266 nmol/l confirming previous findings. CONCLUSIONS: The GC-MS assay using a pulsed splitless injection procedure ensures a good response to differing concentrations of 2-MCA in various specimens. Considering exact determination of the diastereomer ratio as well as the purity of the internal standard, the assay offers good precision and recovery for 2-MCA I and II levels in serum.     

A high sensitivity assay for the inflammatory marker C-Reactive protein employing acoustic biosensing

C-Reactive Protein (CRP) is an acute phase reactant routinely used as a biomarker to assess either infection or inflammatory processes such as autoimmune diseases. CRP also has demonstrated utility as a predictive marker of future risk of cardiovascular disease. A new method of immunoassay for the detection of C-Reactive Protein has been developed using Resonant Acoustic Profiling™ (RAP™) with comparable sensitivity to a high sensitivity CRP ELISA (hsCRP) but with considerable time efficiency (12 minutes turnaround time to result). In one method, standard solutions of CRP (0 to 231 ng/mL) or diluted spiked horse serum sample are injected through two sensor channels of a RAP™ biosensor. One contains a surface with sheep antibody to CRP, the other a control surface containing purified Sheep IgG. At the end of a 5-minute injection the initial rate of change in resonant frequency was proportional to CRP concentration. The initial rates of a second sandwich step of anti-CRP binding were also proportional to the sample CRP concentration and provided a more sensitive method for quantification of CRP. The lower limit of detection for the direct assay and the homogenous sandwich assay were both 20 ng/mL whereas for the direct sandwich assay the lower limit was 3 ng/mL. In a step towards a rapid clinical assay, diluted horse blood spiked with human CRP was passed over one sensor channel whilst a reference standard solution at the borderline cardiovascular risk level was passed over the other. A semi-quantities ratio was thus obtained indicative of sample CRP status. Overall, the present study revealed that CRP concentrations in serum that might be expected in both normal and pathological conditions can be detected in a time-efficient, label-free immunoassay with RAP™ detection technology with determined CRP concentrations in close agreement with those determined using a commercially available high sensitivity ELISA.     

An enzymatic microassay for lactose

An enzymatic microassay for lactose using a lactase enzyme derived from Saccharomyces fragilis is described. The assay uses 50-μl samples, provides 100% hydrolysis of lactose, and is sensitive within the range of 12.5–500 nmol per sample. The assay has been validated against an assay for ¹⁴C lactose which involves thin-layer chromatographic isolation of lactose. The assay is sufficiently sensitive for use in physiologic studies.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.