Thermometric enzyme linked immunosorbent assay: TELISA.

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.     

Chemiluminescent assay for detection of viable microorganisms

The redox reaction between quinone and viable microorganisms produces active oxygen species. In this study, the production rates of active oxygen species were determined by a luminol chemiluminescent assay, and the luminescence intensity was found to be proportional to the viable cell number. The high sensitivity of the luminol chemiluminescent assay was achieved with Mo-ethylenediaminetetraacetate complex and menadione or coenzyme Q1. The detectable cell densities of bacteria and yeasts were found to be approximately several thousand colony-forming units (CFU/ml) when assays were performed with a 96-well microplate luminometer. The chemiluminescent assay requires 10 min for incubation of quinone and microorganisms and 2s for photon counting. Single Escherichia coli was detected after 4h of cultivation and centrifugation (5 min x 2). This simple chemiluminescent assay is expected to be useful for the rapid detection of viable bacteria and yeast.     

Rat cerebral-cortex adenosine deaminase activity and its subcellular distribution

A radioisotopic assay for adenosine deaminase (EC 3.5.4.4) is described together with its application in investigating the activity of the enzyme in rat cerebral cortex. Activity of the adenosine deaminase was determined to be 115nmol/min per g of tissue, measured in isoosmotic sucrose dispersions of the neocortex, and to be 170nmol/min per g of tissue after treatment with Triton X-100. The enzyme was concluded to be largely cytoplasmic, with a K(m) of 54-57mum for adenosine. Action of the deaminase, and other aspects of the metabolism of adenosine in intact neocortical tissue, were quantitatively appraised on the basis of the newly determined characteristics.     

Rainbow trout egg quality determination by the relative weight increase during hardening: a practical standardization

The relative weight of fish eggs increases during water hardening; this increase is significantly correlated with egg viability in rainbow trout when determined after 120 min, providing an assay test for egg quality control. In the present paper, additional investigations on egg water uptake at different time intervals during the hardening process were performed to correlate these with egg viability and to improve the sensitivity of the assay while also reducing the handling time. Eggs of high quality had a significantly higher rate of water uptake than low quality eggs, with the highest rate occurring during the first 30 min of hardening. Bivariate correlation coefficients and regression models revealed that in addition to egg weight increase after 120 min of water hardening, the increases in weight after 30 min and 60 min were also significantly related to egg viability (P < 0.001). In contrast, the initial weight increase after 15 min of hardening revealed a lower correlation (P < 0.006) with egg viability. These results indicate the initial velocity of water uptake is not a valid criterion, but that the total amount of water imbibed during hardening provides the most reliable information on egg quality. The present study confirms the previously reported sensitivity of the assay, and also shows that a shorter incubation time of 30 min is adequate for reliable results. The shorter handling time is an improvement for testing large numbers of egg batches.     

Measurements of tumor tissue oxygen tension using a time-resolved luminescence-based optical oxylite probe: comparison with a paired survival assay

Recently, a system that measures tissue oxygen tension using time-resolved luminescence-based optical sensors has become available commercially (Oxford Optronix, Oxford, England). Two experiments were conducted using this system. First, the oxygen tension distribution was measured in two tumor lines: a spontaneous mouse fibrosarcoma, FSa-II, and a human squamous cell carcinoma xenograft, FaDu. The area in which the pO(2) was equal to or lower than 2.5 mmHg was defined as the hypoxic lesion, and the hypoxic cell fraction was taken as the fraction of these measurements in a tumor. The measured hypoxic cell fractions were compared with those determined by the paired cell survival assay for tumors of various sizes. Second, the tumor tissue pO(2) was measured continuously after administration of two different anesthetics to evaluate the effect of these drugs on tissue pO(2). Results indicated a good agreement between the hypoxic cell fractions measured by this system and those determined by the paired cell survival curve assay for tumors smaller than approximately 500 mm(3). For tumors larger than approximately 500 mm(3), the hypoxic cell fractions measured by the oxygen probe system were higher than those measured by the paired cell survival assay. This may suggest that the hypoxic cell fraction measured by the oxygen probes included both hypoxic and necrotic areas in large tumors where necrotic lesions occupied a significant portion of the tumor. Continuous measurements of pO(2) after anesthesia (Nembutal, or ketamine plus xylazine) showed a consistent rise in the pO(2) during the first 20-30 min of measurement. Subsequently, the pO(2) values became constant or continued to rise slowly. For comparison, the tumor cell survivals were assayed after a dose of 20 Gy given in air at 5, 20 and 60 min after anesthesia. The result showed a decrease in cell survival only in tumors irradiated 20 min after an injection of Nembutal.     

Pharmacokinetics Study of Recombinant Hirudin in the Plasma of Rats Using Chromogenic Substrate,ELISA, and Radioisotope Assays

Aim

To compare the analytical methods used to study the pharmacokinetics of recombinant hirudin in the plasma of rats that had been injected with ¹²⁵I-recombinant hirudin.

Methods

2.0 mg/kg ¹²⁵I-recombinant hirudin were injected intravenously into rats. The recombinant hirudins in the plasma was analyzed by chromogenic substrate assay, enzyme-linked immunosorbent assay (ELISA), total radioisotope assay (RA) and trichloroacetic acid pre-treated total radioisotope assay (TCA-RA).

Results

The chromogenic substrate assay standard curve was linear over the concentration range from 3.12 to 40.00 ng/ml for the recombinant hirudin in plasma. The relative standard deviations (RSD) for the intra- and inter-day variation were 5.0 to 6.3% and 11.9 to 12.6%, respectively. The recoveries of recombinant hirudin was 89.8% to 100.7%. The limit of quantification (LOQ) was 3.12 ng/ml. The concentration-time curve of the recombinant hirudin in the plasma could be explained as a two-compartment model. Pharmacokinetic parameters, including the half-life of distribution phase (t_1/2 α), the half-life of elimination phase (t_1/2 β), volume of apparent distribution (Vd), and area under the concentration-time curve from zero to infinite time (AUC_0–t) were 7.59 min, 46.99 min, 0.17 L/kg, and 204.5 mg/L/min, respectively, as determined by chromogenic substrate assay; 6.41 min, 47.28 min, 1.24 L/kg, and 575.18 mg/L/min, respectively, as determined by ELISA; 3.69 min, 701.90 min, 0.04 L/kg, and 4189 mg/L/min, respectively as determined by RA; and 4.57 min, 724.9 min, 0.09 L/kg, and 2329 mg/L/min, respectively, as determined by TCA-RA.

Conclusions

The chromogenic substrate assay on the concentration dynamics of the recombinant hirudin in the plasma is a specific, sensitive, and accurate analytical method for pharmacokinetic studies. Moreover, the pharmacokinetic parameters determined by the chromogenic substrate assay and ELISA are congruent except for AUC.     

Bacterial bioluminescence as a bioassay for mycotoxins.

The use of bacterial bioluminescence as a toxicological assay for mycotoxins was tested with rubratoxin B, zearalenone, penicillic acid, citrinin, ochratoxin A, PR-toxin, aflatoxin B1, and patulin. The concentrations of mycotoxins causing 50% light reduction (EC50) in Photobacterium phosphoreum were determined immediately and at 5 h after reconstitution of the bacteria from a freeze-dried state. Generally, less toxins were required to obtain an EC50 at 5 h. The effects of the above mycotoxins on bioluminescence were determined after 5, 10, 15, and 20 min of incubation with the bacterial suspensions. The concentration of rubratoxin B necessary to elicit an EC50 increased with time, whereas the concentration of citrinin, penicillic acid, patulin, and PR-toxin necessary decreased with time. There was very little change in the concentration of zearalenone, aflatoxin B1, and ochratoxin A required to elicit an EC50 with time. The bacterial bioluminescence assay was most sensitive to patulin and least sensitive to rubratoxin B.     

Rapid alterations of serum oxidant and antioxidant status with the intravenous administration of n-6 polyunsaturated fatty acids

In an attempt to achieve the safe intravenous administration of two n-6 polyunsaturated fatty acids (PUFAs), gamma-linolenic acid (GLA) and arachidonic acid (AA), and to study the subsequent changes on the total oxidant and antioxidant status, various steadily increasing doses of each acid were injected intravenously at different infusion times in 28 male rabbits. Blood samples were collected at 15-min time intervals by the hepatic veins and from the carotid artery; oxidant status was determined by the thiobarbiturate assay and total antioxidant status (TAS) was assessed by a colorimetric assay. Both n-6 PUFAs were administered with safety at a dose of 25 mg/kg within 10 min accompanied by an increase of malonodialdehyde concentrations in the hepatic veins and in the carotid artery 30-45 min, respectively, after the end of the infusion of GLA and/or AA. Similar changes did not occur in red cell membranes after the infusion of AA. TAS presented reciprocal changes to malonodialdehyde production; the main consumption of TAS was observed in all samples 30-60 min after the end of the infusion of n-6 PUFAs. The above-mentioned rapid alterations occurring in both serum oxidant and antioxidant status after GLA might have a future clinical therapeutic significance in conditions like cancer and disseminated infectious diseases.     

Electrochemical determination of arylsulfatase activity using high-performance liquid chromatography

A highly sensitive assay for arylsulfatase A was developed using high-performance liquid chromatography with electrochemical detection. The retention time of the enzymatic product, p-nitrocatechol, on a reverse phase column was approximately 2.0 min. The assay was able to detect 0.43 pmol p-nitrocatechol in a 20-microliter ethanol extract of the reaction mixture. The coefficients of variation for seven determinations of the intra- and interassays were 9 and 8%, respectively. The levels of arylsulfatase A activity in human saliva samples and leukocyte and platelet lysates determined by this assay were within 6 and 11%, respectively, of the levels determined by a spectrophotometric assay. The high-performance liquid chromatography assay may have utility in measuring arylsulfatase A activity in biological samples with low activity or specific activity or in samples with compounds which interfere with the spectrophotometric assay.     

Determination in plasma of angiotensin-converting enzyme inhibitor by inhibitor-binding assay

A method of determining a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite (RS-5139) in plasma by inhibitor-binding assay has been developed using high-performance liquid chromatography. The assay is based on the principle that the amount of inhibitor bound to the enzyme is inversely related to the amount of hippuric acid liberated on hydrolysis from the artificial substrate (hippuryl- -hystidyl- -leucine). Plasma was heated at 60°C for 15 min, to inactivate endogenous enzyme, and preincubated with rabbit-lung angiotensin-converting enzyme at 37°C for 3 min. The artificial substrate (5.75 mg/ml in pH 8.3 phosphate buffer containing sodium chloride) was added to the resulting solution, and the mixture was incubated for 30 min. The reaction was terminated by the addition of 2 M hydrochloric acid. The hippuric acid liberated on hydrolysis was extracted with ethyl acetate and determined by reversed-phase chromatography using methylparaben as an internal standard. The total concentration of the inhibitor and its metabolite were determined by this method after de-esterification by rat-plasma esterase. The standard curve was obtained by the regression analysis of log concentration against logit response. The within-day and day-to-day precision were satisfactory. The proposed method is simple, rapid and sensitive enough to determine angiotensin-converting enzyme inhibitor in plasma.     

Non-thermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro

Cultured human diploid fibroblasts and cultured rat granulosa cells were exposed to intermittent and continuous radiofrequency electromagnetic fields (RF-EMF) used in mobile phones, with different specific absorption rates (SAR) and different mobile-phone modulations. DNA strand breaks were determined by means of the alkaline and neutral comet assay. RF-EMF exposure (1800 MHz; SAR 1.2 or 2 W/kg; different modulations; during 4, 16 and 24h; intermittent 5 min on/10 min off or continuous wave) induced DNA single- and double-strand breaks. Effects occurred after 16 h exposure in both cell types and after different mobile-phone modulations. The intermittent exposure showed a stronger effect in the comet assay than continuous exposure. Therefore we conclude that the induced DNA damage cannot be based on thermal effects.     

The effect of extraction,storage, and analysis techniques on the measurement of airborne endotoxin from a large dairy

The objective of this study was to fill in additional knowledge gaps with respect to the extraction, storage, and analysis of airborne endotoxin, with a specific focus on samples from a dairy production facility. We utilized polycarbonate filters to collect total airborne endotoxins, sonication as the extraction technique, and 0.05% Tween 20 in pyrogen-free water (PFW) as the extraction solution. Endotoxin concentrations were determined via the Limulus amebocyte lysate (LAL) assay. The endotoxin concentrations in extracts after 15 and 30 min of filter sonication were similar, while the concentration in 60 min extracts was about twofold lower. Rapidly vortexing samples for up to 15 min after sonication did not increase the endotoxin concentration. However, concentrations were 13 and 26% lower in extracts that were centrifuged at 1,000 and 10,000g for up to 15 min, respectively. Field samples and endotoxin standard were also sonicated in glass or polypropylene tubes for up to 120 min. Regardless of the extraction vessel, a decrease in endotoxin concentration occurred when sonicated for >30 min. Samples and endotoxin standard subjected to 12 freeze–thaw cycles at −20°C only showed a slight but not significant decrease in endotoxin concentration. Our results also demonstrate the importance of simultaneously adding LAL reagent to 96-well plates before initiating the LAL assay.     

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5′-monophosphoric acid (AMP) in a buffer of required pH, 5′-nucleotidase was inhibited with Ni²⁺ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultra-violet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.     

Repair kinetics of gamma-ray induced DNA damage determined by the single cell gel electrophoresis assay in murine leukocytes in vivo

The repair kinetics of the gamma rays induced DNA damage was determined in murine peripheral blood leukocytes in vivo by the comet assay. Mice were exposed to 1.0 Gy of gamma rays in a 137Cs source and samples of peripheral blood were taken from their tails at different times. The repair was evaluated per mice in separate experiments by measuring the proportion of cells with tail (comets) in each sample. An average of nearly 80% of comets was obtained at the initial time after the exposure; 2 min later the frequency decreased to 45% and continued diminishing to 22% at 15 min. This evidences the presence of a rapid repair mechanism. For a period of 25 to 40 min after exposure there was a slight but consistent increase of comets from 22 to 38% followed by a second reduction, which could be due to a late repair process that causes strand breaks and then joined them. In summary our results indicated that this system seems to be appropriate for the study of the repair capacity of cells following exposure to ionizing radiation.     

Gamma-aminobutyric acid (GABA) in the rat brain: re-evaluation of sampling procedures and the post-mortem increase

—GABA levels determined after the brain was removed, then frozen, were found to be generally compatible to levels after the brain was frozen in situ provided removal and freezing were effected in less than 60 s. Optimal GABA values were realized when the brain was removed and frozen in liquid nitrogen within 30 s of death. Beginning at 60 s post-mortem, GABA levels increased until the 4th min with the greatest rate occurring between 60 and 120 s at 30 μg/g/min. When frozen brains were dissected into regions by a partial thawing technique, post-mortem increases were not found to occur. Microwave irradiation, investigated as a potential sampling technique for GABA assay, showed considerable variability between samples and was rejected as a practical alternative at this time.     

Ribonuclease: a spectrophotometric assay using acridine orange--RNA complex

Bovine pancreatic RNase can be accurately and sensitively assayed with RNA-acridine orange complex (1:1, wt/wt) as substrate. The enzyme is optimally assayed in 0.05 m sodium acetate buffer, pH 5.0, at 37°C with a RNA concentration of 0.300 mg/ml. The acridine orange released after 30 min incubation can be determined, after filtration, with a simple visible light spectrophotometer at 492 nm. The increase in absorbance at 492 nm was linear for final RNase concentrations of 0.05–0.6 μg/ml. The assay is applicable to crude cell-free extracts, but RNase T₁ was inactive in this assay.     

Measurement of glycinamide ribonucleotide synthetase activity in intact human fibroblasts and Chinese hamster ovary cells

An intact cell assay system based on Tween 80 permeabilization was used to investigate glycinamide ribonucleotide (GAR) synthetase activity in human fibroblasts and Chinese hamster ovary cells. Optimal conditions for the assay of the enzyme were determined with regards to ATP, MgCl2, NH4Cl and ribose-5'-phosphate concentrations as well as pH. Using the optimal assay conditions, the Vmax values as determined by Lineweaver-Burke double reciprocal plots were found to be 5.19 nmol GAR formed/5 X 10(5) cells/30 min for the fibroblasts and 13.4 nmol GAR formed/5 X 10(5) cells/30 min for the Chinese hamster ovary cells.     

Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography

A non-radiochemical assay procedure for CTP synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays. The assay was linear with time and protein concentration, although at low protein concentration a lag phase was observed. An amount of 2×10⁶ cells was already sufficient to determine the specific activity of CTP synthetase in HL-60 cells, lymphocytes and in lymphoblasts obtained from pediatric patients suffering from acute lymphoblastic leukemia.     

Heat-Stable Enterotoxin Produced by Yersinia enterocolitica Isolated from Patients

Twenty-three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15-fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 μg of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C × 20 min or 100 C × 60 min). These observations indicate that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat-stable enterotoxin of Escherichia coli. However its physicochemical properties seem to be different from those of E. coli.     

Effect of short-term culture of blood lymphocytes in vitro on the ability of Fcgamma-receptors to bind IgG1 and IgG2 and mediation of antibody-dependent cytotoxicity

The comparative study incubation influence at 37 degrees C on the lymphocyte Fc gamma-receptor (Fc gamma R) binding ability to IgG1 and IgG2 and on the antibody-dependent cell mediated cytotoxicity (ADCC) was performed. By means of the fluorometric binding assay it was determined that during 30 min of incubation the binding ability of Fc gamma R decreased while after 180 min of incubation it increased. Upon comparing both IgG1 and IgG2 binding abilities to lymphocytes no statistically important changes were noticed (p < 0.05). The changes in the binding ability of Fc gamma R for IgG1 and IgG2 had a similar pattern to the changes.