A sensitive radioimmunoassay for adenosine in biological samples

A simple, sensitive, specific, and reproducible radioimmunoassay for the measurement of adenosine in biological materials has been developed. Adenosine antibody was obtained by immunizing rabbits with an immunogen prepared by conjugating 2′,3′-disuccinyladenosine to human serum albumin. By succinylating adenosine in samples at the 2′- and 3′-O positions with a premixed reagent consisting of succinic anhydride, triethylamine, and dioxane, the assay became sensitive enough to detect less than picomole amounts of adenosine in minute quantities of tissues. The corss-reactivity of structurally related compounds with the antibody was mostly negligible except for 2′-deoxyadenosine, whose usual concentration was very low. The use of this method made it possible to measure adenosine without any prior purification procedure. The immunoreactive materials in various biological samples disappeared during incubation of the samples with adenosine deaminase.     

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-l-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.     

A sensitive method for the quantitation of lysophosphatidylcholine in canine heart

We have developed a procedure for the determination of small amounts of lysophosphatidylcholine in cardiac tissue. Lysophosphatidylcholine from canine heart was separated from the major phospholipids by column chromatography, and then acetylated with labeled acetic anhydride. The acetylated lysophosphatidylcholine was isolated by thin-layer chromatography and the lysophosphatidylcholine content was calculated from the radioactivity associated with the acetylated product. Although the sensitivity of the assay depends on the specific radioactivity of the acetic anhydride used, as low as 0.5 nmol of lysophospholipid in tissue samples can be readily quantitated. The results obtained from the control and ischemic canine cardiac tissues by this assay compares favorably with those obtained by lipid-phosphorus assay. The sensitivity and specificity of the present procedure allows us and other investigators to assay for lysophosphatidylcholine content in very small (10 mg wet weight) tissue samples.     

A novel sensitive method for the detection of user-defined compositional bias in biological sequences

MOTIVATION: Most biological sequences contain compositionally biased segments in which one or more residue types are significantly overrepresented. The function and evolution of these segments are poorly understood. Usually, all types of compositionally biased segments are masked and ignored during sequence analysis. However, it has been shown for a number of proteins that biased segments that contain amino acids with similar chemical properties are involved in a variety of molecular functions and human diseases. A detailed large-scale analysis of the functional implications and evolutionary conservation of different compositionally biased segments requires a sensitive method capable of detecting user-specified types of compositional bias. RESULTS: We present BIAS, a novel sensitive method for the detection of compositionally biased segments composed of a user-specified set of residue types. BIAS uses the discrete scan statistics that provides a highly accurate correction for multiple tests to compute analytical estimates of the significance of each compositionally biased segment. The method can take into account global compositional bias when computing analytical estimates of the significance of local clusters. BIAS is benchmarked against SEG, SAPS and CAST programs. We also use BIAS to show that groups of proteins with the same biological function are significantly associated with particular types of compositionally biased segments.     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

A rapid and sensitive method for the detection of Yersinia enterocolitica strains from clinical samples

AIMS: To compare three culture methods to detect Yersinia enterocolitica from oral or rectal swabs from experimentally infected pigs. METHODS AND RESULTS: The three methods used were: direct plating on Cefsulodin-Irgasan-Novobiocin (CIN) agar, cold enrichment in phosphate buffered saline (PBS) followed by plating on CIN agar and selective enrichment with Luria-Bertani-Bile Salts Irgasan (LB-BSI) followed by plating on CIN agar. Selective enrichment with LB-BSI produced the highest recovery rate (63%), when compared with cold enrichment (52%) and plating on CIN agar alone (43%). Selective enrichment with LB-BSI was significantly (P < 0.02) more sensitive than direct plating on CIN agar and more sensitive than cold enrichment (P < 0.1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Selective enrichment with LB-BSI was more sensitive than the widely accepted method of cold enrichment and it reduced the time required for detection of Y. enterocolitica by three weeks. Selective enrichment with LB-BSI was also compatible with a multiplex PCR technique.     

A sensitive method for quantitative analysis of hydrogen peroxide and oxidase substrates in biological samples]

The sensitive method for peroxidative assays of hydrogen peroxide (up to 5 nmole in 1 ml) and oxidases' substrates (glucose, ethanol) has been developed by use of a non-cancerogenic chromogen--3,3', 5,5'-tetramethylbenzidine (TMB), horseradish peroxidase and corresponding oxidases. The proposed modification of the known method consists in using decreased concentration of TMB (0.05-0.1 mM) and stopping the reaction by adding of acid to pH 1.4-2.1. This approach allowed to enhance the sensitivity of assays of corresponding analytes (7-10 times) and decrease the costs of analysis.     

A multi-well filtration assay for quantitation of inositol phosphates in biological samples

The quantitation of inositol phosphates (IPs), mediators of certain signal transduction processes, typically involves laborious and time consuming conventional ion-exchange chromatography procedures. We have developed a high throughput microtiter plate-based IP assay that utilizes vacuum rather than gravitational flow and has significant advantages over existing methods. The response of recombinant HEK-293 cells expressing human LHRH receptor cDNA to LHRH agonists was used as a model system to develop the assay conditions. Cell lysates containing labeled IPs were applied in 96-well plates fitted with filtration discs containing regenerated Dowex AGI-X8 resin. Specifically bound inositol phosphates were eluted with 1 M ammonium formate in 0.1 M formic acid directly into a fresh 96-well plate and an aliquot of the eluate from each well is transferred into a 96-well plate and counted. The results were comparable to those obtained with the conventional column method and the variation among replicates was significantly improved. This assay facilitates rapid quantitation of inositol phosphates from a large number of samples with relative ease and reduced generation of radioactive waste.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

High-performance cation-exchange chromatography and pulsed amperometric detection for the separation, detection, and quantitation of N-alkylated imino sugars in biological samples

The use of imino sugars for the potential treatment of lysosomal glycolipid storage diseases and hepatitis virus infections requires accurate, quantitative measurement of these compounds in biological samples. We demonstrate here the versatility of cation-exchange chromatography and pulsed amperometric detection of a range of compounds that differ in both isometric structure and N-alkyl chain length. Although column retention appears dependent upon residual charge on the imine function, successful isocratic separation can be achieved by secondary hydrophobic interactions. A series of N-alkylated deoxynojirimycin compounds containing C(1-10) alkyl chains are readily separated and detected by pulsed amperometry after cation suppression. Using experimentally derived response factors for imino sugars and measurement of peak areas we have developed a reliable method for quantitatively determining concentrations in solution. A rapid protocol for the removal of protein and contaminants in biological samples is described. This has allowed the successful measurement of imino sugars in animal tissues and will be useful for understanding the factors involved in compound bioavailability and in the design of novel therapeutics.     

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids.     

Extractionless and sensitive method for high-throughput quantitation of cetirizine in human plasma samples by liquid chromatography-tandem mass spectrometry

Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.     

Radioimmunoassay on polycarbonate membranes: a sensitive and simplified method for the detection and quantitation of antibody

[This corrects the article on p. 475 in vol. 27.].     

A sensitive automated method for adenosine triphosphatase kinetics.

An automated method for measuring adenosine triphosphatase (ATPase) activity is described. A modified version of a Technicon Autoanalyzer utilizing a sensitive colourimetric technique for determining inorganic phosphate concentrations (1 nmol/ml) allows investigations on enzymes of low specific activities. Dialysis may be used for measuring tissue homogenate activities or bypassed by examining purified enzyme preparations. When combined with a gradient apparatus, the proposed technique is particularly well suited for the study of enzyme kinetics in relation to cation or anion concentrations.     

A sensitive method for detection and estimation of juvenile hormones from biological samples by glass capillary combined gas chromatography-selected ion monitoring mass spectrometry

Addition of n-1H,1H,2H,2H-perfluorohexanol (NFH-OH) to the epoxide ring of juvenile hormone (JH)-I, -II, -III, and ethyl-10,11-epoxyfarnesoate (JH-III-Et) yields a stable 10-hydroxy-11-NFH derivative, the mass spectrum of which contains an NFH fragment as base peak. Combination of capillary column gas chromatography-selected ion monitoring mass spectrometry makes it possible to detect and to quantify all the naturally occurring juvenile hormones from biological probes in the femtomole range. Besides high selectivity and sensitivity the method as described works faster than other procedures as extensive purification of the samples is unnecessary. By use of JH-III-Et as internal standard, interference with hormone signals in the selected ion current profile can be avoided and all the homologous juvenile hormones measured simultaneously.     

A rapid and sensitive method for the detection of histone peptides

Fluorescamine has been used to obtain a peptide map of a mixture of histones (H₃, H₂A, H₂B, and H₄) prepared from oocytes of Xenopus laevis. Fluorescamine was found to be more sensitive than o-phthalaldehyde or ninhydrin-Cd for the detection of peptide fragments obtained from tryptic digestion of oocyte histones of X. laevis and the peptic digestion of the β chain of insulin. Using the β chain of insulin for a comparison, the 8 major peptide fragments could be separated by electrophoresis within 30 min and were detectable at the picomols level. Some 70 peptide spots of X. laevis oocyte histones were resolved, thus permitting the analysis of this complex mixture of polypeptides without the need for prior separation.     

A rapid and sensitive method for the identification and quantitation of sub-picomolar amounts of neurohypophysial peptides

A simple, rapid and sensitive method, using isocratic reversed-phase HPLC, is described for the concomitant identification and quantitation of low levels of the various neurohypophysial peptides in biological tissues and fluids. The method requires little or no sample preparation and utilises UV peak detection at 215 nm with a serial signal amplification system to achieve a usable maximum sensitivity of less than 200 fmol of peptide.