[en] Brief item

[en] The feasibility of endotoxins test of radiopharmaceuticals with limulus agent and the approach to take off the inhibition/enhancement effect of radiopharmaceuticals on limulus agent have been studied. Results of the test for 8 radiopharmaceuticals have been given

[en] Full text: In recent years, several (¹⁸F) labeled radiopharmaceuticals other than 2-(¹⁸F)fluoro-2-deoxy-D-glucose have become increasingly important for molecular imaging studies in oncology, and a rapid BET assay is essential to quantify the bacterial endotoxin that may be present in such radiopharmaceutical preparations, prior to use in patients. The radiochemical synthesis of (¹⁸F) labelled radiopharmaceuticals like 3'-Deoxy-3'-(¹⁸F)fluorothymidine ((¹⁸F)FLT), (¹⁸F) fluoroazomycinarabinoside ((¹⁸F)FAZA) etc is based on use of SEP-PAK cartridge for isolation and purification instead of HPLC purification and requires 15% ethanolic water as elution solvent. BET assay for these radiopharmaceuticals can be routinely performed by Portable Test System (PTS)kinetic reader obtained from Endosafe Inc. The assay is based on kinetic chromogenic method and is known to be inhibited by presence of ethanol. Since all the above mentioned radiopharmaceuticals preparations contains ethanol, the aim of our study was to detect at what ppm/percentage level of ethanol, in final formulation, inhibition in BET assay is observed. The ppm/percentage levels of ethanol in final radiopharmaceutical formulations were detected by gas chromatography. In the present study, the BET Assay for (¹⁸F)FDG and (¹⁸F)FDG and (¹⁸F)FLT was performed at six different dilutions i.e 1:500, 1:200, 1:100, 1:50, 1:20 and 1:10 and all these dilutions were less than the Maximum Valid dilution (MVD). (¹⁸F) FDG was chosen as control since the elution solvent for it is 100% water and for all the dilutions mentioned above, the spike recovery was between 50-200%. The CV of negative and positive control samples were found to be 0.0% and less than 10% respectively. However, in case of (¹⁸F)FLT, for 1:20 and 1:10 dilutions, the spike recovery was below 50% but coefficient variance of negative and positive control sample were same as (¹⁸F)FDG. These results indicate inhibition at 1:20 and 1:10 dilution due to presence of ethanol in (¹⁸F)FLT, while at 1:200 dilution, BET assay of (¹⁸F)FLT by kinetic chromogenic method showed consistent result satisfying all the parameters. Thus our result show that, for radiopharmaceuticals, containing upto 15% ethanol, the bacterial endotoxin quantification can be satisfactorily done with the kinetic chromogenic BET assay using the PTS kinetic reader and the problem of inhibition by ethanol can be avoided if 1:200 dilution of the radiopharmaceutical is used in the assay

[en] The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation. (author)

[en] Various endotoxins and the ether extracts of grampositive bacteria were measured immunologically by radioimmunoassay and also biologically by the Limulus test. The minimum amount of endotoxin detectable with the Limulus test was in the range from 1 ng/ml to 1 μg/ml, with the lysate of sensitivity, 100 ng ml [E. coli 0111: B4(B) lipopolysaccharide]. On the other hand, by the radioimmunoassay they were estimated in the range of 0.3 to 10 times of dry weight. Endotoxin-like activity was detected in the ether extracts of grampositive bacteria at a minimum concentration between 1 μg/ml and 100 μg/ml with the Limulus test. However, most of them were estimated by the radioimmunoassay to be under 1/50 of dry weight. Various substances such as thrombin, thromboplastin, polynosinic-polycytidylic acid, polyadenylic-polyuridylic acid, carrageenan and human colonic mucosal antigen had cross reactivities of various degrees in the minimum concentration from 10 μg/ml to 10 mg/ml. Compounds such as thrombin and thromboplastin cross-reacting in the Limulus test were scarcely measured by the radioimmunoassay except for polynucleotides. From this study, it has become clear that the radioimmunoassay method is quite specific and accurate for quantitative measurements of endotoxin. (auth.)

[en] The limulus amoebocyte lysate (LAL) is commonly used for bacterial endotoxin assay. There are several methods of the LAL test ranging from the semi-quantitative to the fully-automated. Gel-clot, kinetic turbidometric, and chromagenic are three different methods available. The gel-clot assay is a simple qualitative method and best used for low-volume laboratories. The morphology of physical gel-clot is often difficult to examine due to the delicate nature of the system as it is thermosensitive and therefore has not been studied in detail. In recent years there have been significant advances in computer aided imaging. These have resulted in a significant increase in the methods available for the detection and quantify the presence of bacterial endotoxin in the laboratory. One of the key tools in applying physics-based models to gel-clot image processing has been the analysis of color histograms. Physics-based models of reflection predict that the shape of the histogram is related not only to the illumination color and object color, but also to such non-color properties such as surface roughness and imaging geometry. However the relationship is complex and cannot be solved analytically. Therefore we have developed a method for estimating these properties by interpolating between histograms that come from images of known scene properties. We present tests of our algorithm on simulated data for analyzing color histograms that yields estimates of gel-clot, phase angle between the camera and light source, and illumination intensity. These three scene parameters are related to three histogram measurements. Our method for estimating gel-clot properties is very fast, and requires only a single color image. (Author)

[en] Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of the innate immune system, yet mechanisms of their action and in particular the role of glycans remain elusive. Efficient non-invasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here we report a new method for labeling LPS and other lipoglycans with luminescent quantum dots. The labeling is achieved by partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS-Qdot conjugates is demonstrated by the labeling of mouse monocytes. This simple method should find broad applicability in studies concerned with visualization of LPS biodistribution and identification of LPS binding agents.

[en] The authors modified the method of Braude of labelling of endotoxins with ⁵¹Cr. A higher uptake of the isotope by endotoxin was obtained (98.4%) which has a favourable effect on the accuracy of measurements with labelled endotoxins. (author)

[en] The present work is to study the effect of toxins (δ-endotoxins) extracted from strains of Bacillus thuringiensis isolated from the mud on the fly Sabkhat Dejoumi Ceratitis capitata, a pest of citrus and fruit trees. Among 51 isolated tested, 15 showed a very significant insecticidal activity, characterized by mortality rates exceeding 80 pour cent. These mortality rates are caused by endotoxins of Bt revealed variability between them. The preliminary results of this study encourage us towards the characterization of the insecticidal activity produced by strains of Bt for large scale application.

[en] Several methods for the extraction of endotoxin or lipopolysaccharide from Gram negative bacteria have been described. However, the product is often contaminated with nucleic acids or proteins in a proportion depending on the extraction method used. Molecular and immunological studies require further purification of the raw LPS. We present here, a simple method for the purification of raw LPS obtained by the standard hot phenol-water procedure using size exclusion chromatography in Sepharose CL-6B. We demonstrated that the using of DNAse and RNAse treatment of the sample before the chromatographic step is necessary to abrogate the nucleic acid contamination in the LPS fraction. The spectrophotometric properties of the pure LPS were verified, supporting the immediate online detection of the LPS and oligonucleotides fractions spectrophotometrically at 206 nm. The mobile phase used (NaCl 0.2 M) do not absorb at 206 nm while maintains the LPS aggregates and therefore, allows the separation of the LPS fraction from the oligoribonucleotide and desoxioligoribonucleotide fractions. The yield of pure LPS was around 98%. Chemical and biological characterizations were conducted in order to assess the feasibility of the procedure developed. (Author)