Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Limulus Test”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources
PubMed2004

Endotoxin testing in inhalation grade lactose-a useful quality parameter?

Alpha-lactose monohydrate is the standard carrier used for dry powder inhalation drug products. The physico-chemical characteristics of this carrier material need to be monitored and specified carefully in order to guarantee functionality of the powder formulation. But also microbiological acceptance criteria need to be considered during development and routine testing. In this study, the endotoxin content of 19 batches of alpha-lactose monohydrate provided by two different manufacturers was determined with a semi-quantitative LAL assay. The endotoxin content was found to be less than 18 endotoxin units (EU)/g lactose in all cases and less than 3 EU/g in most cases. Assuming that the typical lactose has an endotoxin content of 10 EU/g and that a patient inhales six times daily 25 mg of lactose and that the total amount of lactose reaches the lung, this translates to an endotoxin exposure of 1.5 EU per day. On the other hand, the proposal for endotoxins in air limits the endotoxin concentration to 50 EU/m3 which corresponds to approximately 3333 EU inhaled endotoxins a day during normal breathing (breathing at rest conditions). The maximum endotoxin exposure by dry powder inhalations is thus two log steps lower than the recommended acceptable daily intake.

Administration, Inhalation↗
PubMed2005

Synthesis of lipid A and its analogues for investigation of the structural basis for their bioactivity.

As a step to elucidate the structural requirements for the endotoxic and antagonistic activity of lipid A derivatives, we have focused, in the present study, on the effects of the acyl moieties and acidic groups at the 1- and 4'- positions. We synthesized a new analogue corresponding to Rubrivivax gelatinosus lipid A, which has a characteristic symmetrical distribution of acyl groups on the two glucosamine residues with shorter acyl groups (decanoyl groups [C(10)] and lauryl groups [C(12)]) than Escherichia coli lipid A. Carboxymethyl analogues in which one of the phosphates was replaced with a carboxymethyl group were also synthesized with different distribution of acyl groups. Biological tests revealed that the distribution of acyl groups strongly affected the bioactivity. The synthetic Ru. gelatinosus type lipid A showed potent antagonistic activity against LPS, whereas its 1-O-carboxymethyl analogue showed weak endotoxic activity. These results demonstrated that when the lipid A has shorter (C(10), C(12)) hexa-acyl groups, the bioactivity of lipid A is easily affected with small structural difference, such as the difference of acidic group or the distribution of acyl groups, and the bioactivity changes from endotoxic to agonistic or vice versa at this structural boundary for the bioactivity. We also designed, based on molecular mechanics calculations, and synthesized lipid A analogues possessing acidic amino acid residues in place of the non-reducing end phosphorylated glucosamine. Definite switching of the endotoxic or antagonistic activity was also observed depending on the difference of the acidic groups (phosphoric acid or carboxylic acid) in the lipid A analogues.

Endotoxins↗
PubMed1986

Structure-activity relationship of lipid A: comparison of biological activities of natural and synthetic lipid A's with different fatty acid compositions.

To investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-15-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.

Animals↗
PubMed1992

Bovine colostrum fraction as a serum substitute for the cultivation of mouse hybridomas.

Fractions of bovine colostrum were prepared and their ability to support the growth of mouse-mouse hybridomas in culture was tested. Whey was prepared from defatted colostrum by removal of casein using acid precipitation. An ultrafiltrate was obtained from cleared whey by filtration through membranes with a nominal molecular mass cut-off of 100,000 Da. Colostrum ultrafiltrate contained 1.16 milligrams protein, 0.24 milligrams immunoglobulin G (IgG) and less than 0.24 EU (endotoxin unit)/ml endotoxins. The effect of defatted colostrum, whey and ultrafiltrate as serum substitutes was examined by cultivation of hybridoma cells in minimal essential medium containing different concentrations of the supplements. Under optimal conditions in ultrafiltrate-supplemented medium, the maximal cell concentration was 35-40% of that obtained using 10% foetal bovine serum, and IgG production per cell was equal to that achieved using serum. In 1% defatted colostrum the maximum hybridoma concentration was about 30% of that in 10% serum, but at higher concentrations hybridoma growth was significantly reduced. The growth-promoting activity of whey was low. The results show that bovine colostrum ultrafiltrate provides a very attractive alternative to serum for production of monoclonal antibodies.

Animals↗
PubMed1989

Activation of mononuclear cells by aqueous extracts from hollow-fibre haemodialysers.

Monocytes from patients treated by long-term haemodialysis with cellulosic membranes can show functional signs of activation depending on the dialyser module. To elucidate the mechanism of this phenomenon, aqueous extracts of various hollow-fibre dialysers were tested for their ability to induce in-vitro interleukin-1 generation in peripheral blood mononuclear cells from healthy donors. Extracts were prepared by passing 0.5 litres of sterile endotoxin-free water from the dialysate to the blood compartment of the dialyser. Different dialysers containing cellulosic membranes, i.e. cuprophan, and hemophan, were studied. Extracts were dried by lyophilisation and resuspended in cell culture medium before incubation with peripheral blood mononuclear cells for 18 hours at 37 degrees C in a 5% CO2 atmosphere. Interleukin-1 was assayed by biological or radioimmunological methods. Extracts from steam-autoclaved, dry-stored, or gamma-sterilised, wet-stored cuprophan and hemophan modules resulted in interleukin-1 activity that did not differ from negative controls. By contrast, extracts from cuprophan caused significant interleukin-1 production when prepared from ethylene oxide-sterilised, dry-stored dialysers. This monokine-inducing activity could not be neutralised by the addition of polymyxin B and was heat unstable, indicating that the cell-activating stimulus was not endotoxin. Extensive rinsing of the module with water before extract preparation totally mitigated the in-vitro production of interleukin-1. Our results suggest that the extract-induced activation of peripheral blood mononuclear cells found with some dialysers containing membranes of cellulosic origin cannot be exclusively related to the membrane polymer, but depends on a number of other parameters such as sterilisation and storage mode of a given membrane.

Adjuvants, Immunologic↗
PubMed1982

Analytical methods for microbiology in the next decade.

As microbiologists develop and modify methods and turn to automated equipment to decrease analysis time, the precision and accuracy of the methods must be ensured through collaborative study. Rapid, validated methods are needed in many areas. Sterility testing and the use of biological indicators are among subjects of interest to FDA and industry. These groups are also studying particulate analysis. The limulus amebocyte lysate test is being considered for use in detecting endotoxins in drugs and medical devices, in diagnosing illness, and in assessing the microbiological quality of foods and water. Reliable methods are needed for detecting viruses in foods to assure a safe food supply. Studies are needed on the Vibrionaceae to better understand the mechanisms of invasiveness and host response. Methods must also be developed to study pathogens in the water and food supply.

Drug Contamination↗
PubMed1992

Bactericidal activity of beta-lactams and amikacin against Haemophilus influenzae: effect on endotoxin release.

Ampicillin or cefotaxime, alone or in combination with amikacin, were tested at levels achievable in CSF for bactericidal activity against eight clinical isolates of Haemophilus influenzae serotype b. Endotoxin release was determined by the limulus amoebocyte lysate test and by macrophage tumour necrosis factor production for each beta-lactam antibiotic, alone and in combination with amikacin. Accelerated killing was observed when amikacin was added to ampicillin or cefotaxime; however, the additional antibiotic-induced bacterial lysis observed after the addition of amikacin to beta-lactam antibiotics was not associated with an increase in endotoxin release.

Amikacin↗
PubMed1994

Assay of endotoxin in human plasma using immobilized histidine, Limulus amoebocyte lysate and chromogenic substrate.

The Limulus amoebocyte lysate test for endotoxin is inhibited or enhanced by many substances. It is particularly difficult to determine endotoxin in plasma. In order to overcome this problem, we have modified the specific endotoxin assay method by using a membrane filter unit, a chromogenic Limulus amoebocyte lysate reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This immobilized histidine method consists of the endotoxin adsorption step on immobilized histidine, the separation step, in which Limulus amoebocyte lysate-interfering substances are removed, and the Limulus amoebocyte lysate test. Preheating of plasma samples (40-fold dilution with distilled water, at 100 degrees C for 7.5 min) was necessary, and it was necessary to dilute the sample more than 100-fold for the adsorption step. Under these conditions, the fraction of endotoxin recovered from plasma by the immobilized histidine method was almost 1. Moreover, by increasing the sample volume and extending the Limulus amoebocyte lysate reaction time, the sensitivity could be increased. By using the immobilized histidine method, 50-200 units/l of endotoxin in plasma samples can be accurately assayed. The method was used for the determination of plasma endotoxin in rabbits.

Animals↗
PubMed1987

The Limulus amebocyte lysate assay. A rapid and sensitive method for diagnosing early gram-negative peritonitis in patients undergoing continuous ambulatory peritoneal dialysis.

The Limulus amebocyte lysate (LAL) assay was used in a blinded, prospective fashion to analyze peritoneal fluids from 35 consecutive patients undergoing continuous ambulatory peritoneal dialysis (CAPD), who presented with clinical peritonitis. The results were correlated with standard microbiologic culture results. The LAL assay was positive in all three patients with gram-negative peritonitis, was appropriately negative in 24 of 28 gram-positive infections (sensitivity, 100%; specificity, 86%) and was positive in two of five cases in which there was no microbiologic growth. One of the two patients in this last group yielded a gram-negative organism two days later. It was then demonstrated that therapeutic concentrations of a variety of antibiotics (cefazolin sodium, gentamicin sulfate, tobramycin sulfate, ticarcillin disodium, penicillin G potassium, vancomycin hydrochloride, metronidazole hydrochloride, piperacillin sodium, and trimethoprim/sulfamethoxazole) did not interfere with the LAL assay. Together, these data indicate that the LAL assay is useful for identifying patients at high risk for gram-negative peritonitis and for excluding from possible aminoglycoside exposure the majority of patients with peritonitis undergoing CAPD, most of whom will have gram-positive infections. Furthermore, lack of antibiotic interference allows the possibility of monitoring treatment efficacy.

Anti-Bacterial Agents↗
PubMed1978

Diagnostic limulus lysate assay for endophthalmitis and keratitis.

The limulus lysate assay is an inexpensive, reliable, and rapid means of detecting and presence of Gram-negative endotoxin. In all ten cases of experimentally induced Proteus endophthalmitis in rabbits, the assay was positive, and the assay was appropriately negative in all ten cases of Staphylococcal endophthalmitis, ten cases of Candida endophthalmitis, and ten cases of sterile endophthalmitis in rabbits. In a clinical assessment of keratitis, the assay of corneal scrapings was positive in 11 of 13 Gram-negative corneal ulcers. In a similar study of clinical endophthalmitis, both Gram-negative cases had a negative limulus assay, but two cases are insufficient to be conclusive. The assay may prove to be a useful adjunct both to standard diagnostic evaluations and in the rapid direction of appropriate therapy for these conditions.

Animals↗
PubMed1984

In vivo effect of polymyxin B on pertussis vaccine.

Pertussis vaccine contains lipopolysaccharide (endotoxin). Polymyxin B sulfate neutralizes endotoxin activity in vivo and in vitro from Enterobacteriaceae and Salmonella-derived endotoxin. In vitro, polymyxin B eliminates the endotoxin reaction of pertussis vaccine in the Limulus lysate test. In this study, platelet and WBC counts and antibody response were compared in rabbits given either pertussis vaccine alone or pertussis vaccine and polymyxin B intravenously. Pertussis vaccine-induced leukopenia and thrombocytopenia were eliminated in the polymyxin B group. The antibody titers in the animals receiving pertussis vaccine and polymyxin B were somewhat lower and rose more slowly. Since the toxicity of pertussis vaccine is related in part to endotoxin, we suggest that a clinical study using a combination of the vaccine with an endotoxin-neutralizing agent be done to assess both amelioration of side effects caused by the vaccine and any effect on immunogenesis.

Animals↗
PubMed2000

High-efficiency passive elution of bacterial lipopolysaccharides from polyacrylamide gels.

We recently described a method for recovering polyacrylamide-gel-separated bacterial lipopolysaccharides (LPS) based on the sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole followed by passive elution from 32 microm average size gel microparticles into water. With this procedure, the recovery of rough- or semismooth-type LPS after 3 h elution is about 70-80%, while that of smooth LPS is only about 10%. Here we evaluated whether a simple replacement of water with other eluents would enhance the passive diffusion of LPS. We found that solutions of the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and Triton X-100, or mixtures of the organic solvents acetonitrile and triethylamine and water, increased the recovery of a smooth LPS band from Vibrio cholerae O1 in a concentration-dependent manner. Furthermore, a quantitative recovery of rough or smooth LPS from V. cholerae O1, Escherichia coli O111:B4, E. coli K-235, or Serratia marcescens was feasible in 1% SDS or DOC after 3 h or in 5% triethylamine after only 2 min. A simple dilution of SDS or DOC or evaporation of triethylamine rendered the eluted LPS preparations compatible with biochemical activity determination, as tested by Limulus amebocyte lysate assay. Thus, this improved micropurification method may be a suitable interface between analytical gel electrophoresis and further characterization or use of LPS.

Detergents↗
PubMed2001

Chemical and biological evaluation of endotoxin contamination on natural rubber latex products.

Relationship between pyrogenicity and bacterial endotoxin contamination on latex products was demonstrated by chemical analysis and biological assays. In commercially available latex products' surveillance, water extracts prepared from one surgical glove and two silicone elastomer-coated Foley catheters sterilized by gamma-irradiation were obviously pyrogenic in rabbits. The induced fever was monophasic at low dose of the pyrogenic extracts and biphasic at high dose. These extracts exhibited limulus amebocyte lysate gelation activity, and induced inflammatory cytokine (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) production from MM6-CA8 human monocytoid cells. These biological properties, including pyrogenicity, completely disappeared by treating the pyrogenic extracts with endotoxin-adsorbent affinity column. Limulus amebocyte lysate activity and cytokine production from MM6-CA8 cells induced by the extracts were significantly decreased by endotoxin inhibitors, an active fragment peptide of an 18-kDa cationic antimicrobial protein and a synthetic lipid A B464 analogue. Furthermore, very small amounts of 2-keto-3-deoxyoctonate and 3-hydroxy fatty acid, which are common constituents of bacterial endotoxins, were detected by gas chromatography-mass spectrometry analysis of the pyrogenic extracts. These findings clearly showed that the pyrogenicity found in these latex products originated from endotoxins contaminating the products.

Animals↗
PubMed1982

Periodic neutropenia and monocytopenia.

A patient with periodic neutropenia exhibited simultaneous monocytopenia, and epinephrine infusion revealed no monocytes in the marginating pool during neutropenia. Lymphocytes, eosinophils, and platelets also fluctuated periodically, but serial bone marrow studies and epinephrine infusion data indicate these fluctuations could have represented epiphenomena rather than a more global form of periodic hematopoiesis. Bone marrow descriptions of most cases of periodic neutropenia have indicated a "maturation arrest" at the promyelocyte or myelocyte stage prior to development of neutropenia; peripheral blood monocytes are usually normal or fluctuate out of phase with neutrophils. In this present case, "maturation arrest" occurred at the myeloblast stage, and neutrophils and monocytes cycled together. Morphologically normal eosinophilopoiesis with a mean eosinophil to erythroid ratio in the marrow of 0.27 +/- 0.10 (SD) persisted despite a sustained disappearance of promyelocytes.

Agranulocytosis↗
PubMed2006

Recombinant factor C assay for measuring endotoxin in house dust: comparison with LAL, and (1 --> 3)-beta-D-glucans.

BACKGROUND: Measurement of exposure to environmental endotoxin is frequently performed using a Limulus amebocyte lysate (LAL) based assay. Recently, a new method has become available with similar sensitivity and potentially greater specificity using recombinant Factor C (rFC) from the horseshoe crab Carcinoscorpius rotundicauda. A preliminary study was carried out to determine the comparability of LAL and rFC in measuring endotoxins in house dust for large scale epidemiologic studies. METHODS: House dust samples were collected from family rooms by vacuuming 1 m2 of the center of the room. Sixty sieved house dust samples were assayed for endotoxin by LAL (Cambrex, KQCL lysate) and rFC (Pyrogene, Cambrex) and for (1 --> 3)-beta-D-glucans by ELISA. The resistant parallel line estimation was used for data analysis of LAL and rFC. A four-parameter logistic fit with inverse prediction was used to calculate (1 --> 3)-beta-D-glucan levels of the samples. RESULTS: The spike recovery was 113.63% (95% CI = 101.69, 125.57%) for LAL and 99.69% (95% CI = 90.14, 109.24%) for rFC assays. The LAL assay gave higher endotoxin estimates compared with rFC. The LAL and rFC estimates were highly correlated (r = 0.86, P < 0.0001). The difference between LAL and rFC endotoxin estimates correlated with the LAL estimates (r = 0.51, P < 0.0001). However, the difference was not correlated with (1 --> 3)-beta-D-glucans. CONCLUSION: LAL and rFC gave comparable results, hence either assay can be used for studies of endotoxin exposure. The current study shows that (1 --> 3)-beta-D-glucan is not a major factor interfering with endotoxin measurements in house dust using a Cambrex KQCL LAL preparation.

Air Microbiology↗
PubMed1989

Bile acids inhibit endotoxin-induced release of tumor necrosis factor by monocytes: an in vitro study.

Endotoxins play an important role in the pathogenesis of complications of surgery in obstructive jaundice. Preoperative treatment with orally administered deoxycholic acid prevented endotoxin-related complications, such as renal malfunction. Other bile acids, however, were less effective, and the mechanism of action is not known. Endotoxin toxicity is considered to be largely mediated by tumor necrosis factor/cachectin, a cytokine release by mononucler phagocytes. Therefore, we studied the influence of different bile acids on endotoxin-induced tumor necrosis factor production by monocytes in vitro. Bile acids inhibit tumor necrosis factor production through a direct inhibitory effect on the monocytes. Deoxycholic acid was the most effective, chenodeoxycholic acid was less effective and ursodeoxycholic acid was ineffective in the concentrations used. Bile acids did not inactivate endotoxin as measured in a chromogenic Limulus amebocyte lysate assay. The therapeutic effect of bile acids in obstructive jaundice can be explained by an inhibition of endotoxin-induced tumor necrosis factor release by mononuclear phagocytes.

Bile Acids and Salts↗
PubMed1995

Pyrogen reactions to human serum albumin during plasma exchange.

Reactions to human serum albumin (HSA) in therapeutic plasma exchange (TPE) are rare. Nevertheless, older literature describes possible adverse effects, including specific immune responses to albumin or other proteins, and reactions due to contaminating organisms or pyrogen. During an eight day period three patients in our unit had unusual reactions after infusion of 1.5-2 L of HSA. Patient 1 had trembling that persisted for 20 min. Patient 2 had shaking for 40 min despite calcium gluconate infusion, and fever to 100.8 degrees F. Patient 3 had severe rigors that subsided after 90 min when meperidine was finally given, and fever to 103.5 degrees F. Record reviews revealed that all three patients had received HSA from the same lot, and that only one other TPE patient had received HSA from that lot. Neither our pharmacy nor the manufacturer was aware of other reactions associated with that lot. Material from a bottle only partially infused to patient 3 was negative in culture and was negative for pyrogen when retested by the manufacturer. Nevertheless, because patients 1 and 2 had each had multiple previous uneventful TPEs and because all three patients tolerated subsequent TPEs without incident when another brand of HSA was used, we conclude that these patients had pyrogen reactions to the implicated HSA lot. This experience illustrates the value of cluster recognition in arousing suspicion of unusual reactions to HSA and the value of recorded lot numbers in pursuing such suspicions. Apheresis personnel should be aware of the potential for pyrogen reactions with HSA and should record lot numbers of all fluids infused during TPE.

Adult↗