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PubMed1976

Sensitivity of the Limulus test and inhibitory factors in the radiopharmaceuticals.

The basic sensitivity of the Limulus test and the inhibitory factors were examined in 21 radiopharmaceuticals commonly used in Japan. The sensitivity of the Limulus test using pre-gel was found to be 1 mug/ml for Escherichia coli endotoxin. This sensitivity is about ten times that of the rabbit test adopted by USP and JP. The Limulus test was applicable, with full sensitivity and without inhibitory reaction, for the evaluation of 99mTcO4-, 99mTc-albumin, 99mTc-MAA, 99,Tc-Sn-colloid, 131I-Hippuran, Na131I, Na251CrO49, 67Ga citrate, and 57Co-bleomycin as commercially supplied. On the other hand, with 111In-DTPA, 99mTc-phytate, 99mTc-pyrophosphate, 99mTc-DTPA, 131I-polyvinylpyrrolidone (PVP), 59FeCl3, Na232PO4, 198Au colloid, and selenomethionine (Se-75), the pH required adjustment to avoid inhibition of the gelation reaction. Benzyl alcohol showed an inhibitory effect on the gelation reaction at concentrations of more than 1%. Iodine-131-Bromsulphalein (BSP) and 131I-rose bengal showed intense inhibition of the gelation reaction.

Arthropods↗
PubMed1976

Measurement of endotoxin. II. Comparison of reactivities measured by radioimmunoassay and with the Limulus test.

Various endotoxins and the extracts of gram-positive bacteria were measured immunologically by radioimmunoassay and also biologically by the Limulus test. The minimum amount of endotoxin detectable with the Limulus test was in the range from 1ng/ml to 1 mug/ml, with the lysate of sensitivity, 100 ng/ml [E. coli O111: B4 (B) lipopolysaccharide]. On the other hand, by the radioimmunoassay they were estimated in the range o- 0.3 to 10 times of dry weight. Endotoxin-like activity was detected in the ether extracts of gram-positive bacteria at a minimum concentration between 1 mug/ml and 100 mug/ml with the Limulus test. However, most of them were estimated by the radioimmunoassay to be under 1/50 of dry weight. Various substances such as thrombin, thromboplastin, polyinosinic-polycytidylic acid, polyadenylic-polyuridylic acid, carrageenan and human colonic mucosal antigen had cross reactivities of various degrees in the minimum concentration from 10 mug/ml to 10 mg/ml. Compounds such as thrombin and thromboplastin cross-reacting in the Limulus test were scarcely measured by the radioimmunoassay except for polynucleotides. From this study, it has become clear that the radioimmunoassay method is quite specific and accurate for quantitative measurements of endotoxin.

Animals↗
PubMed1979

[Determination of endotoxemia by means of the Limulus test in patients with septicemia (author's transl)].

The Limulus test was applied to determine bacterial endotoxemia in 42 patients suspected of having septicemia. Hemocultures were practiced simultaneously. A comparative study is made of the two presently most commonly recommended tests for neutralizing the factors inhibiting the endotoxin-amebocyte reaction. According to our results the dilution of plasma in isotonic saline solution is preferable; we obtained a sensitivity of determination of 0.0001 micrograms of E. coli endotoxin per ml of plasm. Seventeen cases of bacteriemia were diagnosed, only one of which with sepsis due to Serratia marcescens responded positively to the limulus test. The test was positive in three of the remaining 25 patients. Two of them had liver failure. The basic role that liver failure plays in the high level of endotoxin in the blood is discussed. The prognostic value of this test and the low correlation between bacteremia and endotoxemia are also commented on

Endotoxins↗
PubMed1999

Application of the classic Limulus test and the quantitative kinetic chromogenic LAL method for evaluation of endotoxin concentration in indoor air.

The classic (gel-clot procedure) Limulus test (CLT) and the quantitative kinetic chromogenic LAL method (KQCL) used for the evaluation of bacterial endotoxin concentration in the indoor air of dwellings were compared. The scientific procedure included analyses of 40 air samples supplemented by the analysis of 20 sample duplicates (selected at random) which were taken during the fall season from 10 flats located in 3 towns of the Upper Silesian region (southern Poland). The particulate aerosol probes were sampled by Harvard impactor and Casella sampler. The same samples were analyzed in the Netherlands using the quantitative kinetic chromogenic LAL method, and in Poland using the classic Limulus test. Comparison of both methods revealed that the quantitative kinetic chromogenic LAL method was more precise, with better reproducibility (the coefficient of variation between analyses of the main probe and its duplicate was over two times smaller in the KQCL method than in the CLT method), fully automated in the phase of analysis and data reading, and faster and more effective than the classic Limulus test. Nevertheless, on the basis of the obtained results, the usefulness of the classic Limulus method for assessment of the degree of pollution of indoor air with bacterial endotoxin seems to be confirmed as in the majority of examined samples (21 out 40) the results obtained by both methods were of the same order of magnitude, and in the remaining 19 samples did exceed one order of magnitude. Thus, the data received by means of the classic Limulus test may be regarded as acceptable.

Air Microbiology↗
PubMed1989

[Rapid diagnostic methods for fungal infections--using chromogenic limulus tests and enzymatic measurement of D-arabinitol].

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.

Humans↗
PubMed1992

[Limulus test (factor G) and polysaccharides from fungus].

Picogram quantifies per ml of endotoxin showed a positive limulus test. The concentration more than ten pg/ml of curdlan ((1-3)-beta-D-glucan) was also positive to a conventional limulus test, which contains a factor G. One nanogram per ml of CM-curdlan was positive, but higher concentration of 10 pg/ml of CM-curdlan decreased its optical density of the conventional limulus test. More than one hundred ng/ml of mannan from Saccharomyces cerevisiae did not react with factor G. However, a culture supernatant of Saccharomyces cerevisiae activated factor G. This result suggested that beta-glucan activated factor G. In addition, culture supernatant of Aspergillus fumigatus, Rhodotorula rubra, Candida parapsilosis, Candida tropicalis, Candida krusei also activated factor G, but Cryptococcus neoformans did not activate it.

Antimicrobial Cationic Peptides↗
PubMed1995

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.

Adult↗
PubMed1991

[Investigation of limulus test during and after cardiopulmonary bypass. Part II. Effect of washing of the bypass circuit].

Previously we reported the presence of Limulus positive substance (LPS) in blood and urine for several days following cardiopulmonary bypass (CPB), and foreign particles in the primining fluids. The purpose of this study was to evaluate the effect of washing of the CPB circuit on appearance of LPS and to define the nature of it. Semi-quantitative Limulus test was performed in patients using washed CPB circuit (n = 54) and without washing (n = 41). LPS in the blood were negative in all patients before CPB. LPSs in the priming fluids did not disappear after washing, however the amounts of it in the blood were significantly lower than those without washing. Especially at second postoperative day. Limulus test were positive in 10% of patients with washed CPB circuit and 60% of those without it (p less than 0.01), and all the urine samples of patients with washed CPB circuit were negative to Limulus test at 1st postoperative day. The number of foreign particles decreased in parallel with the smaller pore size of the filter. Since reactions of Pregell to ethylene oxide gas, ethylene chlorohydrin and ethylene glycol were negative, ethylene oxides did not seem to be the LPS. These results indicate the superiority of employment of washed CPB circuit with micropore filter. However, further exploration is still mandatory to elucidate the LPSs.

Adolescent↗
PubMed1977

Studies on a simple Limulus test, a slide method.

A simple micromethod of Limulus test was studied. Briefly, this method is as follows. A 20 microliter aliquot of Pregel lysate solution and the same amount of sample are dropped on the same place on the silicone coated slide glasg and mixed well. Then it is incubated at 37 degrees C for 30 minutes in a moisture chamber. After the incubation, the vibration of the droplet surface is observed. When the vibration is no longer observed, it is judged as positive reaction, and when the vibration is still observed as before the incubation, it is judged as negative reaction. Compared with the conventional tube method, this method has advantages as below: 1) the judgement of the result is relatively objective, 2) shorter incubation period is enough for the reaction to take place and 3) smaller amount of Limulus lysate is enough for each sample. Besides these merits, the detection limit of this method is not inferior to that of the tube method.

Limulus Test↗
PubMed2004

[The role of bacterial translocation and endotoxemia in pathogenesis of obturation ileus, caused by colorectal carcinoma. Limulus test--a method for quick diagnostics of endotoxemia].

Colorectal carcinoma is one of most common diseases in human body. Often it is presented for the first time by its complicated forms--obturation, perforation, bleeding etc. Most common is the obturation of the large bowel. His clinic is caused not only by intoxication of carcinoma, but by massive flow of endotoxin in systemic circulation. In this publication we are trying to explain changes in human body and we offer a test for quick and early diagnosis--Limulus test.

Bacterial Translocation↗
PubMed1981

Application of a Limulus test device in rapid evaluation of gonococcal and nongonococcal urethritis in males.

A test device incorporating Limulus amoebocyte lysate (Mallinckrodt, Inc., St. Louis, Mo.) was developed for the rapid, presumptive diagnosis of gonococcal and nongonococcal disease in males. The device, which was evaluated in 550 men with exudative urethritis, consisted of a specimen collection syringe, a dilution reservoir containing 10 ml of pyrogen-free water, and a Limulus amoebocyte lysate single-test vial. After specimen collection, the syringe was affixed to the dilution reservoir for rapid, accurate dilution of the clinical sample. Contamination of the specimen and potential biohazards to the user were prevented. The diluted sample was then transferred (via the collection syringe) to the lysate test vial for assay of endotoxin. Various incubation times at 37 degrees C were also studied in an additional 301 male patients, and time was reduced from the standard 60 to 30 min while still retaining equivalent predictability of culture results (P less than 0.05). Of the 550 males evaluated with the test device, 366 had positive cultures for Neisseria gonorrhoeae, and 184 were negative. A sensitivity of 99.2% and a specificity of 96.7% were obtained with the test device. Overall ability to predict culture results was 98.4%. Gram-stain sensitivity and specificity were 96.4% and 99.5%, respectively, with an overall accuracy of 97.5%. There were no statistical differences between the Limulus amoebocyte lysate test and Gram stain in predicting cultures (P less than 0.05). Thus, use of the Limulus amoebocyte lysate test device would enable the private physician to make an accurate, presumptive diagnosis of gonococcal and nongonococcal disease in males with exudative urethritis within 30 min without the need of a microscope and to initiate proper therapy during the patient's initial evaluation.

Diagnosis, Differential↗
PubMed1982

Use of the Limulus test to determine the hygienic status of milk products as characterized by levels of gram-negative bacterial lipopolysaccharide present.

The Limulus test was used to investigate the occurrence of Gram-negative bacterial lipopolysaccharide (LPS) in milk and dairy products. This test specifically detects the extremely heat-resistant LPS, and so determines the total accumulated quantity of LPS in a product whether it has been heat-treated or not. A positive correlation between number of psychrotrophic bacteria and amount of LPS in fresh farm milk was found (r = 0.63). Fresh farm milk and freshly produced market milk contained on average less than 1 ng LPS/ml. The Limulus test is considered highly suitable for use in the dairy industry, being a method which will characterize quickly and specifically the hygienic level of a product by the extent of the cumulative contamination by Gram-negative bacteria.

Animals↗
PubMed1994

[Limulus test (factor G pathway) positive substance during cardiopulmonary bypass].

Limulus cascade reaction is found to be frequently activated by the plasma in the patients undergoing cardiopulmonary bypass (CPB). Recently, it is considered that this phenomenon occurs by factor G pathway reactive activity (GPRA) rather than endotoxin that activates factor C pathway. In the present report, we investigated circulating endotoxin and GPRA in 32 patients undergoing CPB. Plasma was pretreated by the New PCA method, and assay was performed by two limulus tests; Toxicolor (reactive with endotoxin and GPRA) and Endospecy (endotoxin-specific). Thirty-two patients were classified into four groups by clinical diagnosis, Group A; dissecting aneurysm of the aorta (6 patients), Group B; ischemic heart disease (9 patients), Group C; heart valve disease (8 patients), Group D; congenital heart disease and myxoma in left atrium (9 patients). GPRA in Ringer's lactate hardly increased during previous circulation before operation. The highest plasma GPRA concentration in all patients was observed at the end of CPB, 415 +/- 342pg/ml, but plasma endotoxin concentration was within normal range. At the end of CPB, plasma GPRA concentrations were 761 +/- 180pg/ml in Group A, 646 +/- 341pg/ml in Group B, 230 +/- 86pg/ml in Group C, and 113 +/- 65pg/ml in Group D. GPRA increase ratio (GPRA increase value/CPB time) of Groups A and B was significantly higher (p < 0.05) than that of Groups C and D. It was suggested that the origin of plasma GPRA during CPB was not the CPB circuit but the tissue of the patients.

Adolescent↗
PubMed1982

The Limulus test of the cerebrospinal fluid in the diagnosis of disc hernia and its possible correlation with the auto-immune pathogenesis of root pain.

The writers examined by means of the Limulus test 130 samples of cerebrospinal fluid and eighteen samples of serum obtained from subjects affected by extremely varied neurological conditions as well as by lumbar and sciatic pain of disc origin. They demonstrated that the Limulus test was positive in cases of lumbar and sciatic pain in which clinical, myelographic and surgical findings gave positive results. It is therefore probable that there is correlation between positivity of the test and an auto-immune pathogenesis of root pain in disc hernia.

Autoimmune Diseases↗
PubMed1974

Assessment of limulus test for detecting endotoxaemia.

The occurrence of endotoxaemia in 46 surgical patients has been investigated using a sensitive preparation of limulus lysate. Altogether 23 patients with local and systemic Gram-negative infections were shown to have endotoxaemia. An overt infection is not a prerequisite for a positive limulus test result, since endotoxin can be absorbed via the peritoneal cavity in patients with "chemical" peritonitis. Over 70% of the tests performed during episodes of Gram-negative infection or peritonitis gave positive results. In 23 patients without clinical evidence of infection or peritonitis only 15% of the limulus test results were positive. The implications of these findings and the use of the test in clinical practice are discussed.

Adult↗