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PubMed1985

Interaction between limulus amoebocyte lysate and soluble antigens from Pseudomonas aeruginosa and Staphylococcus aureus studied by quantitative immunoelectrophoresis.

To investigate the interaction of Limulus amoebocyte lysate (LAL) with gram-negative bacteria, soluble antigens from sonicated Pseudomonas aeruginosa were studied by various crossed-immunoelectrophoresis methods before and after reaction with LAL. Of 64 possible, at least 7 antigens were affected, as indicated by precipitin pattern, after the reaction with LAL. The precipitates corresponding to lipopolysaccharide and Pseudomonas "common antigen" disappeared. This reaction was inhibited when LAL was pretreated with lipopolysaccharide or by heating. Several of the reacting antigens have been shown to cross-react with many other strains of both gram-negative and gram-positive bacteria. Soluble antigens from a protein A-deficient strain of Staphylococcus aureus were also studied. LAL reacted with at least four of these antigens, including the teichoic acid complex. It is concluded that LAL is highly reactive with lipopolysaccharide, but it can react with other antigens from gram-negative and gram-positive bacteria as well. It is suggested that LAL interacts with biologically important antigens from the bacterial membrane. It is proposed that the reactivity and specificity of LAL for various microbial antigens can be studied by immunoelectrophoretic techniques.

Antigens, Bacterial↗
PubMed1994

Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with monoclonal antibody against Limulus peptide C.

Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by the reaction of endotoxin with Limulus amebocyte lysate, was synthesized, and a monoclonal antibody against it was raised. A new microassay for endotoxin was developed, using this antibody in an enzyme-linked immunosorbent assay for generated peptide C-like immunoreactivity. A linear relationship between absorbance and endotoxin concentration was obtained. Control standard endotoxin in water could be detected to a level of 0.001 endotoxin unit per ml. The endotoxin levels in plasma samples from normal humans, rabbit, mice, and guinea pigs were generally found to be below the detection limit of 0.01 endotoxin unit per ml of plasma. The color and turbidity of specimens did not interfere with the assay. The consumption of Limulus amebocyte lysate in the assay was less than 5% of that in the gel-clot and chromogenic assays. With raw lysate, which was much more stable in solution than chloroform-treated lysate, the assay was still highly sensitive to endotoxin but was totally unresponsive to natural glucans. The monoclonal antibody cross-reacted with peptide C-like immunoreactivity generated in Tachypleus amebocyte lysate, which gave equal sensitivity in the endotoxin assay.

Animals↗
PubMed1994

Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.

Animals↗
PubMed1983

Limulus assay for bacterial endotoxin in synovial fluid.

The limulus assay for the detection of bacterial endotoxin has been applied to the study of synovial fluid. Three of 5 patients (60%) with culture-positive gonococcal arthritis had positive SF limulus assay results; as did 2 of 11 (18%) with presumptive evidence of gonococcal arthritis, 3 of 6 (50%) with nongonococcal infectious arthritis, and none of 47 patients with noninfectious arthritis. Endotoxin levels ranged from 0.25 to 128.0 ng/ml. As at present applied to synovial fluid the commercial limulus assay appears to be specific for the infectious process but apparently lacks sensitivity.

Arthritis↗
PubMed1979

Relationship between endotoxaemia and protein concentration of ascites in cirrhotic patients.

Endotoxaemia was investigated by the Limulus assay in 42 cirrhotic patients with ascites and in 33 without ascites. The incidence of endotoxaemia in the former group (59.5%) was significantly (P less than 0.05) higher than in the latter (36.4%). Correlation between endotoxaemia and specific gravity and concentrations of total protein, albumin, and globulin in ascitic fluid was studied in the group with ascites. The specific gravity of ascites in 25 patients with endotoxaemia was significantly greater than that in 17 patients without endotoxaemia (P less than 0.01). The concentration of total protein in patients with endotoxaemia (13.95 +/- 7.18 milligram, mean +/- SD) was nearly twice as high (P less than 0.01) as in patients without endotoxaemia (7.49 +/- 3.60 milligram). The protein content of those who showed reactions greater or equal to 2(+) in the Limulus assay (16.78 +/- 7.14 milligram) was a significantly (P less than 0.05) higher than in those with 1(+) reaction (11.26 +/- 6.33 milligram). Moreover, the concentration of albumin in patients with endotoxaemia (7.68 +/- 4.60 milligram) was more than twice that of the patients without endotoxaemia (3.39 +/- 1.58 milligram, P less than 0.01). On the other hand, globulin concentration in patients with endotoxaemia was 1.6 times that of patients without endotoxaemia (P less than 0.01). Similar differences were noted between endotoxaemic and non-endotoxaemic patients in the ascites-to-serum ratio in protein, albumin, and globulin. These results suggest that in liver cirrhosis endotoxaemia may cause an increase in protein concentrations in ascitic fluid, and that it may be a precipitating factor in the formation of ascites.

Adult↗
PubMed1982

Endotoxaemia and complement activation in acute pancreatitis in man.

Twenty-four patients who experienced 26 attacks of acute pancreatitis were studied. Endotoxaemia, as measured by the limulus lysate assay, was present in 13 of the attacks. Six out of seven patients with systemic complications of the disease had endotoxaemia. C3 catabolism was increased in all 26 attacks of pancreatitis, and a falling level of C3 during attacks of severe pancreatitis was associated with a fatal outcome. There was statistical evidence of more complement activation in serum samples taken when patients had positive limulus lysate tests than when endotoxin was not detected in their blood.

Acute Disease↗
PubMed1978

Purification of specific precipitinogen and extraction of endotoxin from Haemophilus influenzae.

After purifying a Haemophilus influenzae precipitinogen from endotoxic activity by means of ultracentrifugation, column chromatography (Sepharose 6B) and ion exchange chromatography (DEAE Sephadex A25) a fraction was obtained which still contained a specific precipitinogen that was virtually free of endotoxin. Furthermore, during the chromatographic procedures fractions with a high and a low molecular weight endotoxic activity were found. The limulus lysate test was more sensitive in the high molecular weight fractions and the LD50 in mice in the low molecular weight fractions with endotoxic activity.

Animals↗
PubMed1985

Effect of anticoagulants on the chromogenic Limulus lysate assay for endotoxin.

We have examined the effects of anticoagulants on the chromogenic Limulus lysate assay for endotoxin. The results indicate that both heparin and CPD Adenine 1 have a striking dose related inhibitory effect on the assay. At concentrations of heparin as low as about 30 U/ml there was a 90% reduction in detectable endotoxin. The inhibitory effect of up to 100 U/ml of heparin could be neutralised by the addition of protamine sulphate. Anticoagulants appear to be an additional factor which can adversely affect the Limulus chromogenic assay for endotoxin. This further emphasises the need for alternative approaches to the measurement of endotoxin.

Adenine↗
PubMed1992

Limulus amoebocyte lysate assay in the diagnosis of peritonitis in patients receiving continuous ambulatory peritoneal dialysis.

AIMS: To evaluate the Limulus amoebocyte lysate (LAL) assay for differentiating Gram positive from Gram negative peritonitis in patients receiving continuous ambulatory peritoneal dialysis (CAPD). METHODS: One hundred and six patients with suspected peritonitis were studied. LAL assay was performed by adding 0.1 ml of CAPD fluid to 0.1 ml of LAL reagent and incubating in a heating block for 60 minutes at 37 degrees C. The sensitivity of the reaction was determined by: (i) diluting endotoxin in distilled water and used (filter sterilised) peritoneal dialysis fluid; and (ii) diluting a broth culture of E coli used in peritoneal dialysis fluid. A positive LAL assay was defined as the constant stability of the clot through an inversion of 180 degrees. RESULTS: Compared with bacterial culture, the LAL assay had a sensitivity of 65% and a specificity of 98%. The sensitivity of microscopy compared with culture of Gram negative organisms was 76%; overall sensitivity of microscopy in comparison was 80%. CONCLUSIONS: The Gram stain was more sensitive than has previously been reported; the LAL assay was specific but insensitive for the diagnosis of CAPD peritonitis. There was a correlation between reduced leucocyte count and culture; this was reduced in cases from which Gram negative organisms had been isolated. It is recommended that laboratories evaluate their Gram stain procedure to improve its sensitivity because the LAL assay is not a satisfactory substitute.

Diagnosis, Differential↗
PubMed1976

Acute inhalation toxicity of cotton plant dusts.

The number of free lung-cells was studied in guinea-pigs after acute exposure to extracts of various cotton dusts. A good correlation was found between the increase in number of leucocytes in the airways and the number of Gram-negative bacteria in the different dusts. Experiments using the Shwartzmann reaction and the Limulus titration test demonstrated a relationship between the content of different endotoxins in the dusts and the pulmonary reaction. A model for the acute exposure effects after exposure to cotton dust is proposed.

Animals↗
PubMed1989

Endotoxin levels in farming: absence of symptoms despite high exposure levels.

The endotoxin concentration in air was measured in farms where 11 farmers had experienced febrile reactions or allergic alveolitis and in a random sample of farms with 17 symptomless farmers. Samples were obtained during normal dairy farming in eight reference farms (background samples) and in all farms during the handling of material which had probably caused symptoms or disease or, in reference farms, maximal spore exposure (worst case samples). In addition, parallel samplers were used in reference farms, one with a cyclone (5 microns cutoff) and one without, to measure the dust and endotoxin concentrations in the respirable fraction and total dust. The endotoxin worst case values varied from less than 0.01 to greater than 50 micrograms/m3 in symptom farms (median 6.4 micrograms/m3, geometric mean 2.2 micrograms/m3) and from less than 0.01 to greater than 50 micrograms/m3 in reference farms (median 42 micrograms/m3, geometric mean 29 micrograms/m3). This difference was not statistically significant. The background values in reference farms were 1.3 (median) and 0.4 (geometric mean) micrograms/m3. The differences between samples with and without cyclone and between background and worst case samples were statistically significant (p less than 0.02). About 75% of the activity was found in the non-respirable fraction. No correlation was found between exposure to endotoxin and symptoms in farmers. There were weak, but statistically significant, correlations between endotoxin concentrations and total spore count or dust concentrations. The surprisingly high endotoxin values in the respirable fraction of air from environments which apparently did not cause symptoms raises the concern that the Limulus amebocyte assay might be sensitive to other components in the dust rather than endotoxin.

Agricultural Workers' Diseases↗
PubMed1992

Rapid diagnosis of gram negative pneumonia by assay of endotoxin in bronchoalveolar lavage fluid.

BACKGROUND: Diagnosis of ventilator associated pneumonia can be made by quantitative cultures of bronchoalveolar lavage fluid or of protected specimen brushings, though cultures require 24-48 hours to provide results. In 80% of cases aerobic Gram negative bacteria are the cause. METHODS: A rapid diagnostic method of assessing the endotoxin content of lavage fluid by Limulus assay is described. Forty samples of lavage fluid were obtained from patients with multiple trauma requiring mechanical ventilation for a prolonged period. Pneumonia was diagnosed on the basis of clinical, radiological, and bacteriological findings, including quantitative cultures of lavage fluid. RESULTS: A relation was observed between the concentration of endotoxin in lavage fluid and the quantity of Gram negative bacteria. The median endotoxin content of lavage fluid in Gram negative bacterial pneumonia was 15 endotoxin units (EU)/ml; the range observed in individual patients was 6 to > 150 EU/ml. In patients with pneumonia due to Gram positive cocci and in non-infected patients the median endotoxin level was 0.17 (range < or = 0.06 to 2) EU/ml. An endotoxin level greater than or equal to 6 EU/ml distinguished patients with Gram negative bacterial pneumonia from colonised patients and from those with pneumonia due to Gram positive cocci. CONCLUSION: The measurement of endotoxin in lavage fluid is a rapid (less than two hours) and accurate diagnostic method. It should allow specific and early treatment of Gram negative bacterial pneumonia.

Bronchoalveolar Lavage Fluid↗
PubMed1978

Mitogenic and protective activity associated with a lipopolysaccharide from Coxiella burnetii.

A purified lipopolysaccharide (LPS) isolated from the rickettsial agent of Q fever, Coxiella burnetii phase 1, possesses some of the in vivo and in vitro biological properties previously only associated with bacterial endotoxins. The Coxiella LPS is mitogenic for guinea pig leukocytes, induces non-specific resistance in mice to virulent Candida albicans, causes dermal Schwartzman reactions, and is positive by the limulus lysate assay.

Animals↗