Chromogenic Limulus Test Research Articles

Indoor airborne endotoxin assessment in homes of Paris newborn babies

The aims of this study were first to assess airborne endotoxin levels in the dwellings of 162 newborns living in Paris twice during a 1-year period, and second, to identify predictors for endotoxin concentrations using questionnaire data in relation to housing factors and living conditions. Air samples were collected on a glass fiber filter in polystyrene filter holders, using a pump at a flow rate of 3.5 l/min for 24 h placed in the main room of the home. Endotoxin levels were measured using a chromogenic kinetic Limulus Amoebocyte Lysate test. Geometric means (geometric standard deviation) of airborne endotoxin levels at two different visits were respectively 0.509 (4.289) EU/m3 and 0.557 (3.029) EU/m3. Airborne endotoxin levels were significantly increased: (i) in cold season (P = 0.024), with (ii) the presence of visible cockroaches in the previous 12 months at home (P < 0.001), (iii) increased number of inhabitants per square meter (P = 0.012), (iv) the high frequency of cleaning with the floor cloths (P = 0.0014), and (v) the low frequency of vacuuming (P = 0.0045). This study provided for the first time airborne endotoxin levels issued from repeated measurements in Paris dwellings. PRACTICAL IMPLICATIONS This analysis contributed to identify a few factors that determined indoor airborne endotoxin levels. However, the predictive model including housing factors and living conditions poorly estimated endotoxin levels. Consequently, multiple samples and longer sampling periods might improve the estimate of long-term airborne endotoxin exposure especially its variability, in cohort studies.

Exposure to house dust endotoxin and allergic sensitization in adults.

It has been suggested that exposure to elevated levels of endotoxin decreases the risk of allergic sensitization. To examine the associations between current exposure to bacterial endotoxin in house dust and allergic sensitization in adults. In 1995-1996, we conducted a nested case-control study following a cross-sectional study performed within the European Community Respiratory Health Survey (ECRHS). Data of 350 adults aged 25-50 years was analysed. Allergic sensitization was assessed by measurement of specific immunoglobulin E (IgE) against several inhalant allergens. Living room floor dust samples were taken. The endotoxin content was quantified using a chromogenic kinetic Limulus amoebocyte lysate test. Multiple logistic regression analysis showed a negative association between exposure to house dust endotoxin and severe allergic sensitization. Odds ratios (95% CI) adjusted for place of residence, gender, age, and 'caseness' were 0.80 (0.64-1.00) for sensitization to >/=1 allergen and 0.72 (0.56, 0.92) for sensitization to >/=2 allergens using 3.5 kU/l as a cut-off value for sensitization. With regard to single allergens, the protective effect of endotoxin was strongest for pollen sensitization [aOR (95% CI) = 0.74 (0.58, 0.93)]. Our results indicate that current exposure to higher levels of house dust endotoxin might be associated with a decreased odds of allergic sensitization in adults.

Levels and predictors of endotoxin in mattress dust samples from East and West German homes.

East-West differences in prevalence of asthma and allergies were suggested to be associated with lifestyle factors. To describe endotoxin levels in mattress dust samples from East and West German homes collected approximately 10 years after reunification. To identify factors that may account for an East-West difference. Dust was collected from mattresses of 2157 infants and 2108 mothers living in Leipzig (former East Germany) and Munich (West Germany). Endotoxin was measured using a chromogenic kinetic Limulus amoebocyte lysate test. Data on bedding, dwelling, and housing characteristics, and occupants' behavior were collected using a self-administered questionnaire. Endotoxin levels were significantly higher in Leipzig compared with Munich for the infants' mattresses but not for the mothers' mattresses. Apart from this, predictors for the mothers' and the infants' mattresses were very similar. Pet-ownership and contact with animals outside the home were most influential. Endotoxin levels in mattress dust were highest in summer and increased with the number of persons living in the household. The overall percentage of variability in endotoxin levels explained by these factors was low. Endotoxin levels were associated with lifestyle factors discussed within the framework of the hygiene hypothesis. None of these factors explains the difference in infant's mattress dust endotoxin between Leipzig and Munich or could be used as a surrogate for endotoxin. Endotoxin levels in mattress dust are associated with a number of factors discussed in the framework of the hygiene hypothesis; among other things they are associated with pet-ownership, contact with pets and number of persons living in the home. However, none of these factors and not even a combination of factors explains the variability of endotoxin levels between homes.

Abnormal accumulation in lipopolysaccharide in biliary epithelial cells of rats with self-filling blind loop

Primary sclerosing cholangitis (PSC) is known to be frequently associated with inflammatory bowel diseases. In a rat with self-filling blind loop (SFBL), a proposed animal model for PSC, hepatobiliary inflammation has previously been demonstrated. In this study, we assessed the involvement of lipopolysaccharide (LPS), a bacterial endotoxin, in the pathogenesis of hepatobiliary inflammation of the SFBL model. The hepatic localization of LPS was examined by immunohistochemistry using an anti-lipid A antibody. The portal blood concentration of LPS was measured by an endotoxin-specific chromogenic Limulus test (Endospecy test). LPS was localized in the biliary epithelial cells (BECs) of rats with SFBL, and the portal blood concentration of LPS was significantly higher than that of sham-operated rats. Development of hepatobiliary inflammation, peribiliary fibrosis, and injury to the intestinal mucosa were histologically confirmed. Constriction in the biliary trees was radiologically demonstrated. These findings suggested that abnormal accumulation of LPS, which may be derived from portal blood, in BECs was involved in the pathogenesis of hepatobiliary inflammation with intestinal injury.

Biological characterization of endotoxins released from antibiotic-treated Pseudomonas aeruginosa and Escherichia coli.

The supernatants taken from Pseudomonas aeruginosa and Escherichia coli cultures in human sera or chemically defined M9 medium in the presence of ceftazidime (CAZ) contained high levels of endotoxin, while those taken from the same cultures in the presence of imipenem (IPM) yielded a very low level of endotoxin. The biological activities of endotoxin in the supernatants were compared with those of phenol water-extracted lipopolysaccharide (LPS). The endotoxin released from the organisms as a result of CAZ treatment (CAZ-released endotoxin) contained a large amount of protein. The protein, however, lacked endotoxic activity, since the endotoxin did not show any in vivo toxic effects in LPS-hyporesponsive C3H/HeJ mice sensitized with D-(+)-galactosamine (GalN) or any activation of C3H/HeJ mouse macrophages in vitro. The activities of CAZ- and IPM-released endotoxin (as assessed by a chromogenic Limulus test) were fundamentally the same as those of P. aeruginosa LPS, since their regression lines were parallel. The CAZ-released endotoxin was similar to purified LPS with respect to the following biological activities in LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice: lethal toxicity in GalN-sensitized mice, in vitro induction of tumor necrosis factor- and NO production by macrophages, and mitogen-activated protein kinase activation in macrophages. The macrophage activation by CAZ-released endotoxin as well as LPS was mainly dependent on the presence of serum factor and CD14 antigen. Polymyxin B blocked the activity. These findings indicate that the endotoxic activity of CAZ-released endotoxin is due primarily to LPS (lipid A).

Measurement of different types of endotoxin in murine plasma by pretreatment with alkaline reagent and kinetic chromogenic Limulus test

Using a combined pretreatment methodology employing an alkaline reagent and a kinetic chromogenic Limulus test using Endospecy®, different types of endotoxins in murine plasma were able to be measured quantitatively. The detection of smooth (S)-form Salmonella abortus equi LPS (S-LPS) was approximately twice as sensitive as Helicobacter pylori LPS in aqueous solution. S-LPS added to whole mouse blood was recovered quantitatively from plasma, but the rough (R)-form S. minnesota LPS was not. The rates of clearance of S-LPS from the circulation following intravenous injection were shown to be different among BALB/c, C3H/HeN and B10Sn mice. Infection of mice with Salmonella typhimurium 3 days earlier resulted in accelerated clearance of subsequently injected LPS from the circulation. Significantly higher amounts of free non-microbe-associated LPS were detected in the blood of mice 1 h after infection with Escherichia coli O111:B4 followed by immediate treatment with ceftazidime, in comparison with non-antibiotic-treated but infected mice.

Application of a new perchloric acid treatment method to measure endotoxin in both amniotic fluid and cord blood by an endotoxin-specific chromogenic Limulus test in intra-amniotic infection.

Endotoxin in both amniotic fluid and cord blood was measured to detect intra-amniotic fetal infection. Both amniotic fluid and cord blood plasma were pretreated by a perchloric acid treatment, and the endotoxin level was measured by Endospecy test. Cut off values for endotoxin in amniotic fluid and cord blood were 8.5 pg/mL and 7.6 pg/mL, respectively. Escherichia coli intra-amniotic infection caused respiratory distress syndrome (RDS)-mimicking pneumonia. Abnormally high values of endotoxin in both amniotic fluid and cord blood were detected. Intra-amniotic infection caused by Gram-positive bacteria (group B streptococci, Enterococcus fecalis) was shown to be endotoxin negative in both amniotic fluid and cord blood. In cases of negative amniotic fluid culture, measurement of the value of endotoxin in the amniotic fluid is useful in identifying intra-amniotic fetal infection.

Application of a new perchloric acid treatment method to measure endotoxin by an endotoxin‐specific chromogenic Limulus test in neonatal septicemia

The endotoxin in blood was measured to establish both the cut-off value and to detect Gram-negative septicemia. We employed a new perchloric acid treatment method using an endotoxin-specific chromogenic Limulus test (Endospecy test). The cut-off value of endotoxin in blood was 11.2 pg/mL. All cases of septicemia (n = 7) showed high values of endotoxin. Three cases were Group B streptococci, and two cases were Escherichia coli. The others were showed to be negative in blood cultures. The paired values of endotoxin titers during a 48 h interval were useful to evaluate the effectiveness of antibiotics.

Clinical and experimental studies of the limulus test after digestive surgery.

A chromogenic limulus test (Toxicolor test) revealed a transiently increased blood endotoxin level in some patients after digestive surgery. However, most showed no clinical signs of endotoxemia. The Endospecy test of these specimens, which reacts specifically with endotoxin, did not show a high value, while the Gluspecy test, which reacts specifically with (1-->3)-beta-D-glucan (glucan), did. These findings thus suggested that the gauze used for surgery contains a large amount of glucan. The intraperitoneal injection of this gauze-saline into rats results in a phenomenon similar to that observed in clinical studies. These findings suggested the involvement of substances other than endotoxin such as glucan in the postoperative positive-Toxicolor phenomenon. In addition, the Toxicolor test also showed a higher value in patients with liver cirrhosis than in those without. In an experimental study in rats, the liver cirrhosis group also showed a significantly high value. These results therefore suggest that the glucan clearance mechanism is impaired in the reticuloendothelial system in liver damage such as cirrhosis.

Suppression by methylprednisolone of augmented plasma endotoxin-like activity and interleukin-6 during cardiopulmonary bypass

It has been reported that plasma endotoxin, measured by Limulus amoebocyte lysate assays, markedly increased during extracorporeal circulation (ECC). Therefore, this study was undertaken to see if pretreatment with methylprednisolone 30 mg kg-1 modifies the endotoxaemia and associated increases in plasma cytokines in 17 patients undergoing cardiac surgery requiring ECC. We found that methylprednisolone suppressed significantly the ECC-induced increases in plasma endotoxin, measured by a conventional Limulus amoebocyte lysate assay (Toxicolor), and interleukin-6. Plasma concentrations of endotoxin, measured by a highly specific chromogenic Limulus test (Endospecy test), tumour necrosis factor-alpha and interleukin-1 beta did not increase significantly in either the control or methylprednisolone groups.

Diagnostic and prognostic significance of plasma endotoxin determination in febrile patients with haematological malignancies

We evaluated the clinical utility of a new endotoxin-specific chromogenic limulus test in febrile patients with haematological malignancies. The specificity is assured by the removal of factor G, which is sensitive to (1→3)-β- d-glucan, from horseshoe crab amoebocyte lysate. The sensitivity and specificity of the test to systemic gram-negative bacterial infections were 69.7 and 96.3%, respectively. Meanwhile, gram-negative bacteria grew in only 39.7% of endotoxaemic samples. Thus, it seems appropriate to consider gram-negative bacteraemia and endotoxaemia as different entities. Endotoxaemia was significantly associated with septic shock and infectious death, especially in patients with neutropenia. The new test, the results of which are available within 3 h, should help physicians to recognise this ominous sign early and to initiate a prompt countermeasure to endotoxaemia.

A new sensitive microplate assay of plasma endotoxin.

We have developed a microplate method for determining endotoxin in platelet-rich plasma-using Endospecy, an endotoxin-specific chromogenic Limulus test reagent. Nonspecific activators and inhibitors of the test were eliminated by exposing samples (5 microliters) to the alkali reagent consisting of KOH, CaCl2, Triton X-100, ethyleniminepolymer and N,N-bis(2-hydroxyethyl)glycine. The recoveries of various endotoxins were almost complete and not enhanced by dilution. The dose-response curve was linear over endotoxin concentrations of 2-400 pg/ml with good precision (C.V. less than 5.0%). Normal human plasmas (n = 30) contained less than 5.0 pg/ml of endotoxin in reference to that of Escherichia coli 0111: B4. All plasma samples with high concentration of endotoxin by a conventional method showed high values by the microplate assay as well. Since it does not require centrifugation, the new treatment allows the whole reactions to proceed on the same microplate. This permits us to apply the Limulus test to an automated assay system, making plasma endotoxin determination simpler and more rapid than a conventional test tube method.

A new sensitive method for determining endotoxin in whole blood

We developed a microplate method for determining endotoxin in whole blood with Endospecy, an endotoxin-specific chromogenic limulus test reagent. The factors in blood that would interfere with the test were successfully removed by exposing samples to 0.66 mol/l HNO 3 containing 0.25% Triton X-100. Recoveries of various endotoxins spiked into whole blood of humans and experimental animals were almost complete, and were not enhanced by sample dilution. Normal endotoxin concentration in human whole blood was less than 10 pg/ml in reference to Escherichia coli 0111 : B4 endotoxin. Measurements on paired whole blood and platelet-rich plasma (PRP) samples from 50 normal and 132 diseased subjects showed a good correlation ( r = 0.901, P < 0.01). This microplate whole blood method has advantages over the conventional PRP method in that it requires less time, less amount of sample and limulus reagent, and less risk of contamination during the procedure.

Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system

A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10 −7-10 −11 g ml −1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10 −7-10 −11 g ml −1.

Endotoxemia in Patients on Hemodialysis

We examined endotoxins and limulus amebocyte lysate-reactive material (LAL-RM) in serum from 87 patients on hemodialysis, using the conventional chromogenic limulus test (CCLT) and a new specific endotoxin assay with factor G-free LAL (endotoxin-specific test: EST). All patients with regenerated cellulose dialyzers had increased CCLT values, whereas the EST values were not higher than in controls. This discrepancy can be explained by the LAL-RM which is cellulose-derived and cross-reacts with LAL via factor G. Using EST for measurements of endotoxin, 6 patients out of 87 had pathological endotoxemia and all of these patients were associated with either cirrhosis, infection, or malignancy. Some patients who had no complications showed fever before or during dialysis, but they did not have high endotoxin levels. EST is useful for the diagnosis of endotoxemia in patients on hemodialysis because of the presence of LAL-RM in serum. Endotoxemia is rare in patients on hemodialysis, if they are not associated with infection, cirrhosis, or other complications.

Perioperative change of plasma endotoxin levels in early infants

As a preliminary study to elucidate the relationship of endotoxemia to postoperative morbidity, the plasma endotoxin levels in 64 surgical neonates were quantitated by the chromogenic limulus test (Toxicolor test; Seikagaku Kogyo, Tokyo, Japan). The preoperative levels of plasma endotoxin were 64 +/- 59 pg/mL in the group of infants with perforated peritonitis (n = 9), 63 +/- 51 pg/mL in the group of infants with gastroschisis (n = 7), and 15 +/- 16 pg/mL in the group of infants with ileus (n = 28), while the mean level was 6 +/- 5 pg/mL in the remaining 20 surgical neonates who had no signs of ileus or peritonitis. In the serial determination of plasma endotoxin in 28 neonates, the levels on the first postoperative day increased significantly compared with the preoperative levels (16 +/- 18 pg/mL to 46 +/- 25 pg/mL, P less than .01). They decreased gradually to 8 +/- 5 pg/mL within a week in 15 neonates who had no postoperative complications. However, in 13 neonates who had postoperative complications such as wound infection or postoperative ileus, the postoperative levels of plasma endotoxin increased to a much higher level and remained there. In this article the relationship of clinical endotoxemia to postoperative thrombocytopenia and hyperbilirubinemia is analyzed, and the usefulness of evaluating endotoxemia in surgical neonates is discussed.

False-positive result in Limulus test caused by Limulus amebocyte lysate-reactive material in immunoglobulin products

Limulus amebocyte lysate (LAL)-reactive material other than endotoxin was detected in the plasma and urine of patients after intravenous immunoglobulin therapy. Thirty-seven vials of six different immunoglobulin products were analyzed for the LAL-reactive material by combined use of a conventional chromogenic Limulus test and a chromogenic endotoxin-specific test. The amount of LAL-reactive material in reconstituted immunoglobulin solutions ranged from a mean (standard deviation) of 10.2 (2.1) to 2,448.1 (988.9) pg/ml, and there were statistically significant differences among the six brands. The levels of LAL-reactive material in plasma increased in proportion to the amounts contained in the immunoglobulin products administered. The material accumulated in the blood with repeated administration. Urinary excretion of the material was less than 5% of the total amount administered. Such material seems to be derived from the cellulose-based membranes used during preparation of the blood products. Thus, interpretation of Limulus test results of patients receiving immunoglobulin therapy requires special consideration.

Endotoxin levels in milk and plasma of mastitis-affected cows measured with a chromogenic limulus test

A chromagenic limulus test (“Toxicolor”) was applied to cow's milk and plasma after treatment with perchloric acid to remove interfering factors. The endotoxin levels in normal cow's milk and plasma were all <10 pg ml −1. In acute mastitis, the milk endotoxin level averaged (1.1 ± 0.7) × 10 3pg ml −1 in the cases where Gram-negative bacteria were isolated, while the plasma endotoxin concentration was normal. The endotoxin levels in the quarters infected with Gram-positive bacteria were all normal, both in milk and plasma. In gangrenous mastitis due to Gram-negative bacteria, the endotoxin concentration was very high in both milk [(9.3 ± 5.3) × 10 6pg ml −1] and plasma (85.2 ± 68.2 pg ml −1). In similar cases due to Gram-positive bacteria, endotoxin levels were all normal, both in milk and plasma, resembling the acute mastitis due to Gram-positive bacteria. The test was considered suitable for the diagnosis of mastitis due to Gram-negative organisms and the levels of endotoxin detected would aid in assessing the prognosis.

Determination of the endotoxin content of egg products using a miniaturized chromogenic Limulus test

A chromogenic Limulus amoebocyte lysate assay was applied to monitor endotoxin concentration in egg products. Analysis of differently contaminated whole egg probes revealed a strong correlation of endotoxin concentration to total bacterial count 6 x 10(4) cfu x ng-1, where cfu = colony-forming unit) as well as to number of Enterobacteriaceae (1 ng/7 x 10(2) cfu). Similar relations were also found for egg white and egg yolk probes. A significant influence of heat pretreatment of egg probes (65 degrees C, 60 min) on endotoxin detection could be excluded. Up to a concentration of 10 mg x ml-1 endotoxin-free whole egg material did not interfere with the test system. A miniaturized version of the chromogenic Limulus test, which can be carried out in microtiter plates, is described.

リムルステスト及びD-arabinitol測定による深在性真菌症の迅速診断法

Conventional chromogenic limulus test (toxicolor test) reacts with glucan of bakers yeast as well as endotoxin, because toxicolor test contains factor G which is sensitive to glucan and polysaccharide of fungus. A new endotoxin specific limulus test (endospecy test) was developed to improve reaction which responded only to endotoxin. The toxicolor test showed the positive reaction to the culture media of candida albicans 7N strain in RPMI 1640 medium, but the endospecy test did not. D-arabinitol, measured by fluometric enzymatic method, also was positive to culture media. These results suggested that a combination of the toxicolor and the endospecy test and enzymatic measurement of D-arabinitol could be applied to rapid diagnostic methods of fungal infection.