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PubMed1996

The use of human monocytoid lines as indicators of endotoxin.

We wish to develop an in vitro test system for pyrogenic substances. A major source of pyrogen activity is endotoxin. Here we describe a highly sensitive endotoxin-monitoring system based on cytokine measurement in human cell lines of myelomonocytoid origin. The following measures were taken to develop an endotoxin monitoring system of high sensitivity. (i) Mono Mac 6 (MM6) and THP-1 cells, which both represent advanced stages of myelomonocytic development, were better suited as endotoxin indicators than the more immature U-937 line. (ii) In order to enhance cell surface expression of CD14, a major lipopolysaccharide (LPS) receptor, cells were pretreated for 2 days with calcitriol. (iii) The use of fetal calf serum (FCS) without detectable endotoxin traces was essential for maintaining a high LPS sensitivity. (iv) Selected subclones of either THP-1 or MM6 were significantly more sensitive LPS indicators than bulk cultures from which the clones originated. (v) Based on stimulation indices, a commercial tumor necrosis factor-alpha (TNF-alpha) immunoassay proved to be a more sensitive LPS indicator than other cytokine assays or the expression of procoagulant activity/tissue factor. Thus we were able to eliminate the disadvantage of previous cell line-based systems (i.e., low sensitivity) without loss of reproducibility which is seen when using fresh blood, monocytes or monocyte-derived macrophages. High endotoxin sensitivity is a prerequisite for a test system specifically indicating pyrogen activity, because it permits the testing of substances at higher dilutions, thereby minimizing nonspecific interference.

Calcitriol↗
PubMed1985

Evaluation of robot automated chromogenic substrate LAL endotoxin assay method for pharmaceutical products testing.

The robot automated chromogenic substrate LAL assay method was evaluated for endotoxin testing using three lots each of 12 pharmaceutical products. As many as 216 assays, including automated standard curve construction and sample preparation, can be performed in a single day of unattended operation. The method is linear (r greater than .99) in the range of 0 to 0.2 EU/ml. The precision of the method determined by assaying a lot of calcium gluconate for four days was 6%, 10%, and 10% for within an assay block, between assay blocks, and between assay days, respectively. Recovery of endotoxin when spiked into products ranged from 81% to 110% and was within the statistical variation (2 sigma limit) of the method. The endotoxin levels detected in a biological raw material by the chromogenic substrate assay method correlated well with that of the gel-clot LAL assay method. The endotoxin content of the majority of the pharmaceutical products tested was well below the sensitivity of both the chromogenic substrate and the gel clot LAL assay methods.

Automation↗
PubMed1983

The processing and collaborative assay of a reference endotoxin.

A preparation of Escherichia coli bacterial endotoxin, the latest of successive lots drawn from bulk material which has been studied in laboratory tests and in animals and humans for suitability as a reference endotoxin, has been filled and lyophilized in a large number of vials. Details of its characterization, including stability studies, are given. A collaborative assay was conducted by 14 laboratories using gelation end-points with Limulus amebocyte lysates. Approximate continuity of the unit of potency with the existing national unit was achieved. The lot was made from the single final bulk but had to be freeze-dried in five sublimators. An assessment was therefore made for possible heterogeneity. The results indicate that the lot can be used as a large homogeneous quantity. The advantages of using it widely as a standard for endotoxins are discussed.

Animals↗
PubMed1984

Biological activities of Eikenella corrodens outer membrane and lipopolysaccharide.

Highly purified preparations of the outer membrane and lipopolysaccharide (LPS) of Eikenella corrodens strain ATCC 23834 and the outer membrane fraction (OMF) of strain 470 were tested in in vitro biological assays. The OMFs of both strains were found to be mitogenic for BDF and C3H/HeJ murine splenocytes. The E. corrodens LPS was mitogenic for BDF spleen cells; however, doses of LPS as high as 50 micrograms/ml failed to stimulate C3H/HeJ cells. When incubated with T-lymphocyte-depleted C3H/HeJ splenocytes, the strain 23834 OMF demonstrated significant mitogenic activity, indicating that the OMF is a B-cell mitogen by a mechanism other than that elicited by conventional LPS. The E. corrodens 23834 OMF and LPS were stimulators of bone resorption when tested in organ cultures of fetal rat long bones. In contrast, the strain 470 OMF was only weakly stimulatory. Both OMFs and LPSs demonstrated "endotoxic" activity, since as little as 0.062 micrograms of E. corrodens LPS and 0.015 micrograms of the OMFs induced gelation in the Limulus amebocyte clotting assay. Thus, despite having a "nonclassical" LPS biochemistry, the E. corrodens LPS elicits classical endotoxic activities. These results also indicate that the surface structures of E. corrodens have significant biological activities as measured in vitro. The expression of such activities in vivo may play an important role in the pathogenesis of periodontitis as well as other E. corrodens infections.

Animals↗
PubMed1986

Bacterial endotoxin retention by inline intravenous filters.

Filters used in i.v. administration sets were tested for their ability to retain bacterial endotoxins for up to 96 hours of continuous infusion. Inline filters composed of cellulose ester, polyacrylate, polypropylene, polyethylene, or Posidyne Nylon 66 were used during continuous infusion of 5% dextrose injection at 83 mL/hr. One milliliter of inoculum containing 10(8) Escherichia coli was injected through a port upstream from the filter. A bacterial filter was used to monitor the sterility of effluent from the inline filters. The effluent was tested with limulus amebocyte lysate (LAL) that could detect endotoxin concentrations greater than 50 pg/mL. A control solution was monitored for viability of the bacteria throughout the course of the study, and positive endotoxin controls were used to confirm the sensitivity of the LAL. Samples of effluent were tested at 0, 4, 19, 24, 48, 72, and 96 hours. Effluent from all filters was sterile throughout the study. LAL assay indicated that only the effluent from filters containing Posidyne Nylon 66 was free of endotoxins for 96 hours. Effluent from the other filters contained endotoxins immediately after injection of the E. coli. Of the inline filters tested, only the one composed of Posidyne Nylon 66 was able to retain E. coli endotoxin for 96 hours. Further study is needed with E. coli and other microorganisms that are likely contaminants of i.v. infusions.

Drug Contamination↗
PubMed2000

Evaluation of the applicability of the bacterial endotoxin test to antibiotic products.

Fundamental conditions for applying the bacterial endotoxin test to antibiotic products were investigated so as not to affect the level of regulation by the rabbit pyrogen test. According to accuracy evaluation of test methods, the kinetic-turbidimetric and kinetic-colourimetric assays were shown to allow more accurate measurement and, therefore, more sensitive detection of interference to the bacterial endotoxin test than the gel-clot method. In total, 102 antibiotic products were evaluated on their interfering effect to show that the antibiotics could be categorized into three groups depending on intensity of the interference. Although the test was shown to be applicable even to the group showing the strongest interference, it was assumed to be crucial to use appropriate reagents and an accurate test method for avoiding approval of a pyrogenic product. Accordingly, lists of antibiotics are presented to provide limits of concentration for eliminating interference and endotoxin limits for approval to facilitate effective bacterial endotoxin tests.

Animals↗
PubMed2006

beta-Glucans in standardized allergen extracts.

BACKGROUND: Allergen extracts contain variable quantities of bacterial endotoxin. Recent studies have suggested that (1-->3)-beta-D-glucans (beta-glucans), also microbial cell wall components, may have adjuvant properties that could affect allergen immunotherapy. OBJECTIVE: To determine the quantities of beta-glucans in standardized allergen extracts. MATERIALS AND METHODS: Ninety-four lots of 13 standardized allergen extracts were tested for beta-glucan content by Glucatell assay, and for endotoxin content by a specific, chromogenic formulation of the Limulus amebocyte lysate test. RESULTS: Standardized allergen extracts contain variable quantities of endotoxins and beta-glucans. As in our previous work, endotoxin activity was greatest in cat pelt and Dermatophagoides farinae, and least in the pollens. There was no correlation between endotoxin and beta-glucan levels (r = 0.1887; P = 0.07). beta-Glucan content was highest for grass pollen (median content, 10.6 ng/ml; range, 0.4-41.8 ng/ml), ragweed pollen (32.9 ng/ml; range, 6.5-41.2 ng/ml), and cat pelt (25.5 ng/ml; range, 16.7-41.1 ng/ml), and lowest for cat hair (4.9 ng/ml; range, 1.2-10.3 ng/ml), D. farinae (1.2 ng/ml; range, 0.4-5.2 ng/ml) and Dermatophagoides pteronyssinus (1.8 ng/ml; range, 0.4-6.7 ng/ml). CONCLUSIONS: beta-Glucans are present in standardized allergen extracts. The effects of these quantities of beta-glucans on allergen immunotherapy and allergen skin testing require further study.

Allergens↗
PubMed1984

Synthesis and quality assurance of [11C]alpha-aminoisobutyric acid (AIB), a potential radiotracer for imaging and amino acid transport studies in normal and malignant tissues.

Carbon-11 labeled alpha-aminoisobutyric acid (AIB), a synthetic amino acid, was prepared by the modified Bucherer-Strecker amino acid synthesis from acetone, ammonium carbonate and [11C]KCN in the presence of carrier KCN. This method results in the labeling of AIB in the carboxyl group. The label is stable in this position because AIB is not a metabolized after cellular uptake. AIB is rapidly accumulated in viable cells including malignant cells. Since it is a non-metabolized amino acid, AIB offers the possibility of studying amino acid transport in vivo without interference by radiolabeled metabolic products. Radiochemical yields of [11C]AIB of 35-60% have been obtained in 70-80 min with radiopurities greater than 99%. Carrier added syntheses gave 15-25 mCi of [11C]AIB with specific activities of 0.3 Ci/mmol. Our quality control program which insures that [11C]AIB is suitable for imaging studies in patients with cancer includes HPLC analyses of product identity and purity, apyrogenecity and isotonicity assays, and a sensitive test for cyanide.

Aminobutyrates↗
PubMed1988

The influence of lipopolysaccharide content on the apparent B cell stimulating activity of anti-mu preparations.

Anti-mu preparations differ greatly in their ability to stimulate mouse B cells to incorporate tritiated thymidine (TdR). We have found that these differences may be due in part to different levels of lipopolysaccharide (LPS) content. In this report we show that LPS concentrations as low as 0.025 ng/ml stimulate the proliferation of T-depleted (C57BL/6 X DBA/2)F1 (B6D2F1) spleen cells, provided that 5 X 10(-5) M 2-mercaptoethanol is also present. Each of six commercial anti-mu preparations tested for LPS content contained more than this amount. We describe a technique that uses polymyxin B-agarose to remove nanogram quantities of LPS from anti-mu preparations. In B6D2F1 B cells, LPS-depleted anti-mu preparations induced much more uniform tritiated thymidine incorporation than did non-depleted preparations; but there was little difference between the two preparations when tested on B cells from C3H/HeJ (LPS hyporesponsive) mice.

Absorption↗
PubMedDate not supplied

Variability in the LAL test: comparison of three kinetic methods for the testing of pharmaceutical products.

Data presented in this study indicate that there is considerable variability in spike recoveries between three different kinetic LAL methods as used for the testing of a variety of parenteral products. However, successful spike recoveries were observed for each of the thirteen products under test on at least one of the three kinetic methods used, suggesting that problems caused by spike recovery variability may be easily controlled in the laboratory by flexibility in the choice of method.

Limulus Test↗
PubMed1992

Isolation and biological activities of endotoxin from Leptospira interrogans.

Endotoxins extracted with ethylenediaminetetraacetate (EDTA) from Leptospira interrogans serovars icterohaemorrhagiae and canicola and Leptospira biflexa serovar patoc were tested for various biological activities characteristic of endotoxins. The presence of lipopolysaccharide biological activity was demonstrated by the Limulus amoebocyte lysate test, pyrogenicity in rabbits, complement interaction inhibiting the erythrocyte lysis, and chicken-embryo lethality. The lipopolysaccharides did not induce the local Shwartzman reaction. The lipopolysaccharides of serovars icterohaemorrhagiae and canicola were immunogenic in rabbits and were cytotoxic to chicken-embryo fibroblasts.

Animals↗
PubMedDate not supplied

Current practices in endotoxin and pyrogen testing in biotechnology. The Quality Assurance/Quality Control Task Group. Parenteral Drug Association.

This article presents the results of a nationwide survey of the biotechnology industry regarding endotoxin and pyrogen testing and control. It identifies procedures and methods being used by biotechnology companies, and firms working with biotechnology products, in the testing for and detection of endotoxin and other pyrogenic substances. The review attempts to identify areas of commonality and standardization within the industry and includes topics for discussion at the end of the survey results.

Biotechnology↗
PubMed1978

Detection of lipopolysaccharide in suspected bacteriuric urine using a carbocyanine dye.

Currently practiced methods for the detection of gram negative bacteriuria require culturing and overnight incubation. Such an approach to bacteriuria detection is unacceptable for any screening program which requires rapid presumptive evidence of infection. In this study, the lipopolysaccharide-dependent formation of a unique dye absorption spectra of the cationic carbocyanine dye, 1-ethyl-2-[3-(1-ethylnaphtho[1,2d]-thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2d]-thiazolium bromide, was used to detect bacteriuria caused by gram negative organisms in a hospitalized population. In an evaluation of 168 first morning and randomly collected suspected bacteriuric urines, the dye test detected 66% of the loop plate positive urines with false positive and false negative values of 28% and 34%, respectively. However, 37% of the false positive results occurred in urines containing less than 10(5) gram negative bacteria/ml and an additional 24% of the false positives were seen for patients currently receiving antibiotic treatment. Urine specimens were also evaluated using the limulus lysate assay for lipopolysaccharide.

Bacteriuria↗
PubMed1984

Naturally occurring pasteurellosis in laboratory rabbits: chemical and serological studies of whole cells and lipopolysaccharides of Pasteurella multocida.

Whole cells and lipopolysaccharides (LPS) of 10 isolates of Pasteurella multocida from laboratory rabbits were subjected to chemical and serological analysis. LPS of most of these isolates possessed pyrogenic potency comparable to LPS from Salmonella minnesota 9700, although their average ketodeoxyoctonate content was only 18% of that of salmonella. A gel diffusion precipitin test for somatic antigens extracted in a formal-saline solution demonstrated several isolates with three to four somatic antigens, with some variation in the major somatic type from one test to another. Conversely, the use of LPS as antigen in the gel diffusion precipitin test (i) eliminated cross-reactivity with reference antisera and (ii) often resulted in the organism being typed as serotype 12 even when the type 12 antigen was a minor antigen in the formal-saline extracts. Antisera from specific pathogen-free rabbits immunized with either whole cells or LPS of two isolates were tested against whole cells of LPS of the 10 isolates by enzyme immunoassay and indirect hemagglutination. Both whole cells and LPS of one of the isolates (isolate 2) were serologically specific, whereas those of the other isolate (isolate 1) were moderately to strongly cross-reactive with other isolates. The data indicate that although LPS is the major antigen responsible for typing based on the gel diffusion precipitin test, substances other than LPS (probably capsular polysaccharide) are responsible for the type specificity that forms the basis for the A, B, D, or E classification of this organism.

Animal Diseases↗