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PubMed1993

Beta-D-glucan concentrations detected by Toxicolor and Endospecy tests in the urine of patients with urinary fungal infections.

Beta-D-glucan is an essential component of the cell wall of fungi. We measured its concentration in the urine of patients with funguria using the chromogenic endotoxin assay kits, Toxicolor and Endospecy. These assay systems use the same Limulus coagulation enzymes. Since the Endospecy test detects endotoxin but not factor G, which is activated by beta-D-glucan, the beta-D-glucan concentration can be calculated by subtracting the Endospecy value from the Toxicolor value. Concentrations of beta-D-glucan were found to be significantly higher in urine samples from patients with funguria (> or = 10(3) colony-forming units/ml) than in non-infected samples.

Bacterial Infections↗
PubMed2004

Method for estimation of toxic endotoxin in inactivated salmonella vaccine in D-galactosamine-sensitized mice.

We developed a method to estimate the content of the toxic endotoxin in inactivated Salmonella vaccine in D-galactosamine-sensitized mice. Ten-fold serially diluted vaccines were injected intraperitoneally into D-galactosamine-sensitized mice. Lethality in the mice was judged 3 days after the injection. The best result was obtained when C3H/HeN mice were used for the test. Correlation was observed between the endotoxin content measured by Limulus amoebocyte lysate assay and the LD50 in the mouse safety test (r=0.81). These results suggested that this test could be applied to the estimation of endotoxin content in inactivated vaccines of Salmonella.

Animals↗
PubMed1985

Biological activities of chemically synthesized partial structure analogues of lipid A.

Analogues of the nonreducing sugar part of lipid A were chemically synthesized and tested for biological activities such as Limulus amebocyte lysate gelation, interferon- and tumor necrosis factor-induction, lethal toxicity in galactosamine-sensitized mice, and pyrogenicity. A 4-O-monophosphorylglucosamine derivative possessing 2-N-3-tetradecanoyl-oxytetradecanoyl and 3-O-tetradecanoyl groups (GLA-27) exhibited all activities tested except for pyrogenicity. Alteration of the acyl substituents or dephosphorylation as well as acylation or phosphorylation of the 6-OH caused most activities of GLA-27 to diminish or disappear altogether. On the other hand, the biological activities expressed by GLA-27 were not significantly affected even when the glucosamine backbone was changed to 1-deoxy type, epimer type at C-3 (allose form), or 3-amino type. These results indicate that the acyl substituents and the phosphorylation positions rather than the backbone structures in these partial structure analogues of lipid A affect the expression of biological activities of endotoxin. The results also clearly indicate that some biological activities of endotoxin can be expressed separately from pyrogenicity.

Animals↗
PubMed2002

Detection of pyrogens in intravenous IgG preparations.

Five different intravenous IgG (i.v. IgG) preparations were assessed for their capacity to modify the pyrogenic response to bacterial lipopolysaccharide (LPS) of rabbits under the conditions of a pharmacopoeal test. Four of the five preparations were found to mitigate the reaction rendering the result "non-pyrogenic" with an LPS dose proved pyrogenic when administered in saline or in albumin. Bacterial LPS was found readily detectable by a simple Limulus amoebocyte lysate (LAL) gelation test. Four of six brands of i.v. IgG were found reactive in the test under conditions adjusted to detect the FDA limit. The reaction obtained upon addition of standard LPS to the negative preparations supported the validity of the assay. The LAL reactivity of two of the reactive preparations was inhibited by laminarin, a compound known to inhibit Limulus lysate gelation by beta-D-glucan, but not by Polymyxin B. Specific detection of bacterial endotoxins in i.v. IgG solutions requires inhibition of the beta-D-glucan pathway of the Limulus lysate coagulation. Using an appropriate inhibitor, the LAL gelation test is suitable to detect a potential endotoxin contamination in i.v. IgG which might have not been unravelled by the in vivo test for pyrogens.

Animals↗
PubMedDate not supplied

Bacterial endotoxin testing: a report on the methods, background, data, and regulatory history of extraction recovery efficiency.

Since the mid-1970s the Limulus Amebocyte Lysate (LAL) assay has been used to test medical devices for bacterial endotoxins. The Association for the Advancement of Medical Instrumentation (AAMI) recently published a standard designated ANSI/AAMI ST 72: 2002, Bacterial Endotoxins--Test methodologies, routine monitoring, and alternatives to batch testing, which addresses LAL testing and associated issues. In order to perform the bacterial endotoxins test (BET), the test article must be extracted in an aqueous medium, with the extract being used as the test solution. In the early years of testing, and periodically throughout LAL test history, questions have arisen about validation of the extraction efficiency of endotoxins from medical devices. The AAMI Microbiological Methods Committee appointed a Task Group to thoroughly research the issue of extraction efficiency and to recommend whether validation of extraction efficiency is necessary for LAL testing of medical devices.

Advisory Committees↗
PubMed1987

Infection and labor: the detection of endotoxin in amniotic fluid.

Intra-amniotic infections are frequently caused by gram-negative organisms. Lipopolysaccharide, or endotoxin, is a component of the cell wall of these bacteria. The Limulus amebocyte lysate assay is the most sensitive test clinically available for the detection of endotoxin in biologic samples. The diagnostic value of the Limulus amebocyte lysate assay test for the detection of intra-amniotic infection has been examined and compared with that of the Gram stain. The Gram stain had a 60.8% sensitivity and 9.6% false positive rate. The Limulus amebocyte lysate assay had a 69% sensitivity and a 4.8% false positive rate. When both tests were used in combination, the sensitivity improved to 95.6%, whereas the false positive rate was 14.3%. The Limulus amebocyte lysate assay test is a rapid, sensitive, inexpensive, and simple test for the detection of gram-negative intra-amniotic infections.

Amniotic Fluid↗
PubMed1991

Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media.

A procedure for the purification of Neisseria meningitidis lipopolysaccharide (LPS) from outer membrane vesicles (OMV) in spent growth media was developed. Five different LPS strains of group A N. meningitidis were grown in tryptic soy broth with vigorous aeration for 36-48 h, and centrifuged to collect both cells and supernatants. The amount of LPS in the OMV in the supernatants was higher or at least equal to that in the cells. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate to dissociate LPS from OMV. The LPS was then separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 column in 1% sodium deoxycholate, and precipitated from the column fractions in 70% ethanol. In addition, LPS was also extracted from cells with hot phenol-water, ultracentrifuged once after treatment with ribonuclease, and purified on Sephacryl S-300. When compared with an improved phenol-water extraction method, the LPS obtained from either OMV or cells by the above methods gave a 40-180% increase in yield. The LPS also had much higher activities in limulus amebocyte lysate assay, rabbit pyrogenic test, and enzyme-linked immunosorbent assay. The LPS purified from cells and from OMV were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Antigen-Antibody Reactions↗
PubMed1988

A procedure for the efficient incorporation of wild-type lipopolysaccharide into liposomes for use in immunological studies.

Previous studies on the mechanism of action of lipopolysaccharides (LPS) on macrophages have used wild-type lipopolysaccharide (wt-LPS) containing liposomes. In these studies the endotoxin was incorporated into liposomes by suspending the wt-LPS in the buffer used to rehydrate the lipid. Using this approach (buffer method), we observed that less than 10% of Salmonella minnesota smooth LPS is incorporated into multilamellar vesicles (MLV). If the non-incorporated material is not effectively separated from the liposomal form, erroneous conclusions on the mechanism of action of LPS can be drawn. Prolonged sonication of the wt-LPS-MLV suspension resulted in almost complete incorporation of the LPS into the resulting small unilamellar vesicles (SUV). In order to prepare MLV, we briefly soniated the buffer preparation, dehydrated the resulting smaller vesicles and then rehydrated the mixture (dry method). This procedure resulted in almost complete incorporation of the wt-LPS into MLV. The ability of wt-LPS in MLV prepared by the dry method to activate macrophages or trigger gelation of Limulus amoebocyte lysate was reduced by 100-1000-fold compared to the non-incorporated wt-LPS. This indicates that at least 99% of the wt-LPS is incorporated in MLV made by the dry method.

Animals↗
PubMed2000

The detection and interpretation of endotoxaemia.

A considerable body of evidence has accumulated that implicates endotoxin in the pathogenesis of the sepsis syndrome. This has raised interest in the possibility of measuring endotoxin as a surrogate marker of Gram negative infection, particularly since conventional microbiological tests have an inevitable delay. The Limulus amoebocyte lysate (LAL) assay has been used most widely to measure endotoxin in clinical samples. However, there are several important limitations that need to be borne in mind. These include the dangers of contamination, lack of precision and accuracy, and both false positive and false negative results. Endotoxaemia is present in the blood of about 30% of patients with bacteraemia, but endotoxaemia does not predict either Gram negative bacteraemia or Gram negative infection, nor does it predict survival from sepsis. There is some correlation with severity of sepsis, but the level of precision is poor. At the present time, there is no place for routine endotoxin testing in clinical practice. In particular, the positive predictive value of the test is insufficiently high to be of clinical use. It may be that the LAL assay, or one of the newer developments, may be more useful in excluding Gram negative infection, but that remains to be shown.

Endotoxemia↗
PubMed1988

[The growth and lipopolysaccharide production of Escherichia coli and Pseudomonas aeruginosa].

The course of growth and LPS production of two strains of type cultures of Escherichia coli (ATCC 11229, ATCC 25922), one E. coli mutant strain P400 and one type strain of Pseudomonas aeruginosa (ATCC 15442), grown partly by repeated cultures in BHI and partly also in minimal medium or in 1:10 diluted PC broth in a gyratory shaker (60 rpm) at 30 degrees C, was monitored respectively by counting the cfu and by simultaneous determination of LPS by means of the three miniaturized LAL-tests, i.e. the capillary test, the "Mini" endotoxin test and the Coatest endotoxin method. All three tests yielded generally comparable and reproducible results. The LPS content for a defined number of cfu was virtually in the same order of magnitude in all cases, regardless of the nutrient content of the culture medium. The quantity of LPS was relatively high in the initial phases of growth but then decreased significantly to constant levels in the stationary phase. There was a remarkable increase in the yield of LPS in the mid- and late stages of the exponential phase in the three strains, in contrast to the mutant in which the LPS content declined continuously. A possible explanation for this variation could be due to the fact that specific cell membrane proteins, which are lacking in the mutant, react differently with the LPS and thus with the Limulus Amoebocyte lysate. When the LAL tests are used for the rapid determination of the gram negative bacterial load of in particular perishable fresh foods, in which generally bacterial cells are at different stages of the exponential growth phase, then it is necessary to standardize each method specifically both for the product and for the storage conditions.

Colony Count, Microbial↗
PubMed1983

Rapid evaluation of gonococcal and nongonococcal urethritis in men with Limulus amoebocyte lysate and a chromogenic substrate.

A chromogenic substrate was used with Limulus amoebocyte lysate (LAL) and compared by parallel testing with the traditional gelation LAL method for the rapid evaluation of exudative urethritis in 125 male patients. Of these patients, 67 had positive cultures for Neisseria gonorrhoeae and 58 were negative. The corresponding prevalence of gonococcal urethritis was 53.6%. For assay, diluted urethral samples and chromogenic substrate were added directly to single-test LAL vials, and objective color endpoint determinations were made visually after a 10-min incubation period at 37 degrees C. Sensitivity and specificity were 98.5% and 93.1%, respectively, with an overall accuracy in predicting culture results of 96.0%. The predictive value of a positive LAL test was 94.3% in our patient population; in a population with a prevalence of gonococcal urethritis of only 10%, the predictive value would be 61.3%. Results were not statistically different from those obtained by the 30-min gelation LAL method or by Gram-stained smears read by experienced microscopists (P greater than 0.05). Unlike the delicate gel, the color endpoint was not prone to accidental mechanical disruption during incubation or reading. Thus, use of a chromogenic substrate greatly improved the utility and speed of the LAL assay for evaluating men with exudative urethritis while not affecting the accuracy of the test.

Chromogenic Compounds↗
PubMed1987

Endotoxin inactivation by the humoral components in the tolerant rat serum.

A rapid inactivation of endotoxin has shown to occur following its incubation in serum obtained from endotoxin-tolerant rats with the aid of the limulus amebocyte lysate (LAL) assay. The tolerant rat had large quantities of lipopolysaccharide inhibitor (LPSI) activity, which does not appear to be complement. Heating tolerant rat serum for 60 min at 56 degrees C or the addition of lead acetate to the tolerant serum both resulted in the loss of LPSI activity. This paper focuses on the most unique properties of LPSI, namely it's alteration of activity after heating or the addition of lead acetate, compared with those properties of inhibitors for endotoxin which have been previously demonstrated by a number of investigators.

Animals↗
PubMed2002

Plasma endotoxin level of healthy donors.

The plasma level of endotoxin was determined in 116 healthy blood donors. After a routine physical and laboratory investigations the endotoxin level was determined with Limulus amebocyte lysate assay (LAL-test) by the chromogenic kinetic method of Bio-Whittaker Co. (USA). Its sensitivity was 0.005-50 EU/ml. The plasma level of endotoxin in most of the healthy donors was less than 1 EU/ml (in the range of 0.01-1.0 EU/ml), but always measurable. The average +/- S.D. was 0.128 +/- 0.215 EU/ml. Because of the high standard deviation and high range of values, the data were distributed into two groups with the means of 0.05 +/- 0.022 EU/ml and 0.294 +/- 0.186 EU/ml. The difference between the groups was significant (p < 0.001). In conclusion, endotoxin can be measured in plasma of healthy individuals.

Animals↗
PubMed2006

Testing the role of calmodulin in the excitation of Limulus photoreceptors.

The phototransduction cascade in Limulus ventral photoreceptors involves multiple second messengers, including Ca(2+) and cGMP. Light-induced Ca(2+) release from intracellular stores is an intermediate step, but the subsequent Ca(2+)-activated reaction remains to be determined. The possibility that Ca(2+)/calmodulin (Ca(2+)/CaM) might be involved is suggested by the high calmodulin content of the transducing lobe. To test whether CaM can excite the transduction cascade we injected a 25 microM Ca(2+)/CaM solution. This produced a rapid, brief depolarization similar to that produced by light, suggesting a role for CaM in the cascade. However, an important caveat is that Ca(2+) dissociating from the Ca(2+)/CaM complex might excite this process. Several control experiments argue against, but do not entirely eliminate this possibility. To test whether endogenous CaM has a function in excitation, trifluoperazine was pressure injected into the rhabdomeric region. The response to brief flashes was not affected, but the response to steady illumination was transiently attenuated by each injection. We conclude that calmodulin should be considered a candidate to couple intermediate and late stages of the transduction cascade.

Animals↗
PubMed1985

Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.

Endotoxins↗
PubMed1989

The International Standard for Endotoxin: evaluation in an international collaborative study.

An ampouled preparation of bacterial endotoxin, coded 84/650, was evaluated in 35 laboratories in 12 countries for its suitability to serve as the International Standard for Endotoxin. The ampouled preparation was calibrated in terms of the USA National Standard, EC5, in Limulus Amoebocyte Lysate gelation, turbidimetric and chromogenic tests and in rabbit pyrogen tests. On the basis of the results reported here, with the agreement of the participants in the study, and with the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 84/650 was established in 1986 as the International Standard for Endotoxin for Limulus Gelation Tests with an assigned unitage of 14,000 IU of endotoxin per ampoule.

Animals↗
PubMed1985

Spontaneous endotoxinemia in premature infants: correlations with oral feeding and bowel dysfunction.

Infants admitted to a tertiary care nursery were tested serially to determine the frequency and epidemiology of spontaneous endotoxinemia, a phenomenon suggested by previous studies. Plasma and stools were tested for endotoxin-like activity (ELA) using a Limulus amoebocyte lysate method and results were correlated with clinical data. We detected ELA in plasma of 28 of 47 infants (60%) tested throughout their hospital stay: only two of 58 separate episodes could be attributed to infection. Endotoxinemia was not consistently associated with classical signs of fever, shock, and jaundice. Prior to oral feeding, little or no ELA was detected in stools and endotoxinemia was ascertained in only six of 45 infants (13%). With feeding, fecal ELA concentrations rose sharply, and endotoxinemia was detected in 56% of remaining infants (p less than 0.001). Bowel disease predisposed to endotoxinemia: 16 of 20 infants (80%) with necrotizing entercolitis or difficult establishment on feeding were affected, compared to five of 17 infants (29%) without such problems (p less than 0.01). Fecal ELA concentrations were not abnormally elevated in those with bowel disease. We conclude that endotoxinemia occurs commonly in immature infants as their fecal flora develops with feeding but the amount of circulating endotoxin required for injury and the patterns this takes require further investigation.

Endotoxins↗