A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent

A method is described for measuring lipid peroxides by means of the color reagent of a commercially available test kit for cholesterol estimation. In principle, this assay makes use of the oxidative capacity of lipid peroxides to convert iodide to iodine, which can be measured photometrically at 365 nm. Calibration curves were obtained using peroxides such as H2O2, t-butyl hydroperoxide, and cumene hydroperoxide. A stoichiometric relationship was observed between the amount of organic peroxides assayed and the concentration of iodine produced. Concentrations of lipid peroxides as small as 1 nmol/ml could be measured. The ability to estimate lipid peroxides of isolated low density lipoprotein was demonstrated.     

Use of a commercially available ELISA kit for detection of salmonellas in water

The accuracy and specificity of a commercially available ELISA kit (Locate, Rhone-Poulenc Diagnostics) were assessed when applied to enrichment cultures of naturally contaminated water and sewage. The kit showed 66/180 samples positive by both culture and ELISA. The ELISA showed six positives which could not be confirmed by culture.     

Affinity differences between some commercially available immobilized Con A with rat alpha-fetoprotein

Rat alpha-fetoprotein contains three Con A-affinity molecular variants evidenced by affino-immuno-electrophoresis, (a) Con A-non reactive (52 %), (b) Con A-weakly reactive (31 %), (c) Con A-reactive (17 %). Affinity chromatography on several commercially available Con A-linked agarose displays different alpha-fetoprotein affinity patterns. The Con A-weakly reactive variant can be either bound and eluted with the Con A-reactive fraction or unbound and eluted with the Con A-non reactive one. These differences of chromatographic behaviour should be taken into account in structural, biochemical and biological studies dealing with the Con A-affinity molecular variants of alpha-fetoprotein or of other glycoproteins.     

Efficacy of a commercially available post-thaw bovine semen sexing kit in both single-ovulating and hyperstimulated cows

Currently, there are few inexpensive, reliable, effective methods for commercially separating X- and Y-chromosome bearing fresh and frozen bovine sperm. The objective of these experiments was to determine the efficacy of a commercially available post-thaw bovine semen sexing kit, HeiferPlus™ (HP) which claims to alter the sex ratio in favor of female calves following artificial insemination. Three trials included the insemination of hyperstimulated cows with Control or HP-treated semen, non-surgical embryo collection on Day 7, and a combined PCR/dot blot assay to determine embryo sex. Chi-square analysis showed that the Control group produced a greater proportion (p < 0.0005) of female embryos than the HP group. There were no differences in the proportions of transferable compared with degenerate embryos or in number of ovulations, embryos, and unfertilized ova collected from Control compared with HP groups. When treatments were combined, one of the two bulls used in the hyperstimulation studies produced an overall greater proportion of females (p < 0.05), suggesting a bull effect.Another trial involved the insemination of cows synchronized via OvSynch^® with fetal sexing via ultrasonography. Results of these studies indicated that HP semen sexing kit did not alter the sex ratio in favor of females in either hyperstimulated or single-ovulating cows; however, potential bull effects may be further evaluated to understand the capacity of HP with semen from specific bulls. Additionally, perhaps the sex of the surviving embryo can be manipulated by the maternal side, through ovarian, hormonal, oviductal, or uterine influences.     

A sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration

Serum paraoxonase (PON) is associated with plasma high density lipoproteins, and prevents the oxidative modification of low density lipoproteins. We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA), using two monoclonal antibodies against PON, to measure serum PON concentration. The concentration of PON in healthy Japanese subjects was 59.3 +/- 1.3 microgram/mL (mean +/- SEM; n = 87). Serum PON concentrations in relation to the PON 192 genetic polymorphism were: 69.5 +/- 2.9 microgram/mL in the QQ genotype; 63.0 +/- 1.9 microgram/mL in the QR genotype; and 52.8 +/- 1.7 microgram/mL in the RR genotype. Concentrations were significantly lower in the RR than in the QQ genotype (P < 0.01). Serum paraoxonase specific activity was higher in RR than in QQ subjects (18.6 +/- 0.40 vs. 2. 56 +/- 0.05 nmol/min/microgram, P < 0.01), but arylesterase specific activity was unrelated to genotype. PON concentration was positively associated (P < 0.001) with both serum arylesterase activity and, after adjusting for the effect of the position 192 polymorphism, with serum paraoxonase activity. Subjects with angiographically verified coronary heart disease had significantly lower PON concentrations than the healthy controls (52.0 +/- 2.3 microgram/mL; n = 35, P < 0.01). This association was independent of the position 192 genotype. Our new ELISA should be of value for epidemiologic and clinical studies of serum PON concentration. immunosorbent assay for human serum paraoxonase concentration.     

A fluorimetric assay for available lysine in proteins

A two-dimensional fingerprinting gel system that provides sensitive analyses with high resolution of T1-resistant oligonucleotides of large RNA molecules is described. Unique oligonucleotides less than 30 bases in length are recovered quantitatively while longer oligonucleotides are recovered in very large (～90%) yields by active transfer of the fingerprint to DEAE paper. After elution of the oligonucleotides from DEAE paper, secondary analysis is performed by digestion of oligonucleotides with pancreatic RNase and separation of the products by high-voltage electrophoresis on polyethyleneimine cellulose. The complete analysis of up to 40 oligonucleotides can be accomplished within 4 days.     

A rapid microtiter plate serum bactericidal assay method for determining serum complement-mediated killing of Mannheimia haemolytica

In this study, we describe a rapid microtiter serum bactericidal assay (RMSBA) that can be used to measure the functionality of immune sera. It quantifies bactericidal activity of immune sera in the presence of complement against a homologous bacterium, M. haemolytica in this case. There is high correlation between data from RMSBA and standard complement-mediated bacterial killing assay (r=0.756; p<0.0001). The RMSBA activity of sera can be generated in less than 5 h instead of overnight incubation. RMSBA costs substantially less in terms of time, labor, and resources and is highly reproducible.     

Characteristics of aldosterone binding in rat and human serum

Binding of cortisol and corticosterone by serum proteins is well established, but discrepancies exist regarding aldosterone. We have observed that approximately 1% of 3H-aldosterone incubated with rat serum was bound in a time-dependent process, although it was not competed by a large excess of non-radioactive aldosterone, assessed by Florisil separation or gel filtration on Sephadex G-50 columns. After electrophoresis on cellulose acetate of rat serum incubated with 3H-aldosterone, specific or non-specific binding to protein fractions was not obtained. Further, a 10 000-fold molar excess of aldosterone (10 microM) displaced only 34% of the bound 3H-aldosterone to rat serum, preventing the calculation of the IC50 value. Increasing concentrations of aldosterone (3-83 nM) did not displace 3H-corticosterone bound in rat serum to presumably corticosterone binding globulin (CBG). In contrast, inhibition of this binding by 3-83 nM corticosterone was concentration dependent, showing an IC50 value of 10(-8) M. In normal human serum, binding of 3H-aldosterone demonstrated competition by a 100 and 1 000-fold excess of aldosterone. Displacement curves of 3H corticosterone bound to human serum by 1.7-75 nM corticosterone or 0.05-8.8 microM aldosterone yielded IC50 values in the range of 10(-8) M for corticosterone and 10(-6) M for aldosterone. With horse serum, aldosterone's binding affinity was three orders of magnitude lower than that of corticosterone. These studies suggest that in the rat aldosterone was loosely and weakly bound to a high capacity binder, possibly albumin. In agreement with the work of others, in humans aldosterone may be bound to both CBG and albumin. The current data do not substantiate for the presence of specific aldosterone binding proteins in serum.     

High-performance liquid chromatographic analysis of cyclosporin A in rat blood and liver using a commercially available internal standard

An automated and rapid method for quantifying malondialdehyde (MDA) in breath condensate was developed and validated. The method is based on derivatisation with thiobarbituric acid, HPLC separation and fluorescence detection and is optimised for determination of MDA in breath condensate. Sample collection is non-invasive and simple. The detection limit (4.1 nM) is low, precision is good and the analysis time is short. The response is linear in the concentration range of 0.020 to 1.0 microM. Samples could be stored for 1 month at -20 degrees C and for 3 months at -80 degrees C without losses. Using this method, there was no statistically significant difference between patients with asthma and patients without asthma. However, among females, subjects with asthma had higher MDA levels as compared to females without asthma (0.17 vs. 0.12 pmol/s, p=0.04). The use of the method when studying airway inflammation has to be further evaluated.     

A simple and sensitive assay for determining plasma tipranavir concentration in the clinical setting by new HPLC method

A simple method for the quantification of tipranavir, the first non-peptidic HIV protease inhibitor, was developed and validated. Quinoxaline, as internal standard, was added to 50 microl of plasma before a liquid-liquid extraction by 600 microl of protein precipitation solution. The extracts were diluted before being injected in the chromatographic system. Chromatographic separation was made on a C18 column using potassium phosphate buffer (pH 3.2) and acetonitrile with gradient. Detection was performed by an UV detector at 260 nm. Relative error at three control quality concentrations ranged from -1.81 to 1.72%. Intra-day (CV%) and inter-day (CV%) precision ranged from 0.94 to 2.55% and from 3.07 to 4.24%, respectively. LOQ and LOD were 0.090 microg/ml and 0.035 microg/ml, respectively. Mean recovery was 87.1%+/-2.4%. Calibration curve was linear up to 180 microg/ml. Concentration range when optimized (0.703-180 microg/ml) proved to be adequate to measure tipranavir concentration in HIV-1-positive patients, therefore this method could be suitable for therapeutic drug monitoring of this drug.     

Use of commercially available monoclonal antibodies for immunoenzyme double staining

Summary An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green-and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.     

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of praziquantel concentration in serum

A simple and reproducible enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of praziquantel in the serum was developed. Since praziquantel has no functional group to conjugate with carrier protein, praziquantel was first converted to a compound with an amino group similar to praziquantel. This compound was then conjugated to bovine serum albumin for use as an immunogen, and to horseradish peroxidase, as enzyme-labeled praziquantel, respectively. The conjugate of praziquantel-bovine serum albumin conjugate was used to raise anti-praziquantel antiserum in mice. The direct competitive ELISA was conducted by simultaneously incubating praziquantel and horseradish peroxidase-labeled praziquantel conjugate with anti-praziquantel antiserum over a second antibody and the enzyme activity of the remaining horseradish peroxidase-labeled praziquantel conjugate was measured. The intra- and inter-assay coefficient of variation was < 10% in the range of 1.0 to 30 ng ml(-1), and the limit of the detection was 0.3 ng ml(-1). The cross reactivities of anti-praziquantel antibody with compounds related to praziquantel were negligible. Using this ELISA, serum levels of praziquantel were easily determined in male Wistar rats up to 8 h following a single intraperitoneal injection at 2 mg kg(-1) of body weight.     

A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit

The seamless ligation cloning extract (SLiCE) method is a novel seamless DNA cloning tool that utilizes homologous recombination activities in Escherichia coli cell lysates to assemble DNA fragments into a vector. Several laboratory E. coli strains can be used as a source for the SLiCE extract; therefore, the SLiCE-method is highly cost-effective.The SLiCE has sufficient cloning ability to support conventional DNA cloning, and can simultaneously incorporate two unpurified DNA fragments into vector. Recently, many seamless DNA cloning kits have become commercially available; these are generally very convenient, but expensive. In this study, we evaluated the cloning efficiencies between a simple and highly cost-effective SLiCE-method and a commercial kit under various molar ratios of insert DNA fragments to vector DNA. This assessment identified that the SLiCE from a laboratory E. coli strain yielded 30?85% of the colony formation rate of a commercially available seamless DNA cloning kit. The cloning efficiencies of both methods were highly effective, exhibiting over 80% success rate under all conditions examined. These results suggest that SLiCE from a laboratory E. coli strain can efficiently function as an effective alternative to commercially available seamless DNA cloning kits.     

Functional polymorphisms in inbred rat strains and their allele frequencies in commercially available outbred stocks.

Polymorphisms that have been proven to influence gene functions are called functional polymorphisms. It is significant to know the distribution of functional polymorphisms in the rat, widely used in animal models for human diseases. In this study, we assessed 16 functional polymorphisms consisting of 3 coat color and 13 disease-associated genes in 136 rat strains, as a part of the genetic profiling program of the National Bio Resource Project for the Rat (NBRP-Rat). Polymorphisms of Cdkn1a, Fcgr3, Grp10, Lss, and Fdft1, which were proven to function in prostate tumorigenesis, glomerulonephritis, hyperphagia, and cholesterol biosynthesis, were shared among various inbred strains. These findings indicated that most rat strains harbored the disease-associated alleles and suggested that many unidentified functional polymorphisms might exist in inbred rat strains. The functional polymorphisms shared in inbred strains were also observed within outbred stocks available commercially. Therefore, this implies that experimental plans based on either rat inbred strains or outbred stocks need to be carefully designed with a full understanding of the genetic characteristics of the animals. To select the most suitable strains for experiments, the NBRP-Rat will periodically improve and update the genetic profiles of rat strains.