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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↗
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↗
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↗
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↗
PubMed1979

Dry up method as a revised Limulus test with a new technique for gelatin inhibitor removing.

To detect endotoxins, Limulus test, especially its tube method, is recently used most widely. But this method has shortcomings considerably, for example, the lack of the objectivity on the judgement, the necessity of long handling time and the requirement of relatively large amount of Limulus lysate. To revise these shortcomings we established a new method. In our method, sample and Limulus lysate are mixed on a silicone coated slide glass and incubated at 37 degrees C for 30 min, then heated to dry up for the judgement. In samples which contain protein, the pretreatments with (NH4)2SO4, dilution and boiling are performed to remove gelation inhibitor. It was proved that this method could be applied to such samples as physiological saline, plasma, urine, transudate, exudate, cerebrospinal fluid (CSF) and suspension of Escherichia coli (E. coli). This method has advantages in its (1) objectivity of the judgement because of the clear difference of the dry up patterns between positive reaction and negative, (2) shortness of the handling time (results can be obtained within 2 hr from sampling), (3) requirement of little amount of sample and of Limulus lysate (a fifth volume of sample and a tenth volume of lysate are needed compared with the conventional method) and (4) sensitivity (0.1 or 0.5 ng/ml of lipopolysaccharide (LPS)).

Adult↗
PubMed1979

Measurement of endotoxins with the limulus test in burned patients.

High titres of endotoxin as measured by the Limulus test were usually found in burned patients who had raised body temperatures, and were colonized with gram-negative bacteria; also some infected patients showed raised endotoxin without a raised temperature. Patients vaccinated with an antipseudomonas vaccine rarely showed endotoxin in their plasma but occasional plasma samples from vaccinated patients had a high titre of endotoxin which appeared unrelated to infection or to a raised temperature in the patient.

Adolescent↗
PubMed1979

Calibration of the limulus test for blood products.

The Protein Fractionation Centre has been using the Limulus Amoebocyte Lysate test for detecting the presence of pyrogens for some five years. All tests on final plasma products have been carried out in parallel with the EP rabbit pyrogen test. Good correlation between both tests has been obtained with products such as distilled water and crystalloid solutions. However, it is more difficult to correlate results when dealing with plasma proteins. Each product appears to react differently. Some normally pass the limulus test undiluted while others, such as immune globulins and albuminoid products, have to be diluted to various concentrations before they pass. A preliminary statistical analysis has been carried out on the results of pyrogen testing on Stable Plasma Protein Solution, firstly to ascertain that a significant correlation does exist between the LAL test and the rabbit test, and secondly to try to obtain "cut off" values for the limulus test which compare well with the official EP temperature rises in the rabbit test.

Animals↗