[Studies on Limulus test: preparation of Limulus test reagent from Taiwan horseshoe crab].
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Animals↗
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[Pyrogen test and Limulus test--on the relation between the 2 tests].
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Animals↗
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.
Adult↗
[Pyrogen testing in vitro using the Limulus test].
The applicability of the Limulus test for the pyrogen test was checked in comparison to the pyrogen test in rabbits. In 6 out of 24 lots of raw materials and drugs pyrogens could be detected by means of the pyrogen test in rabbits. 2 of these 6 lots showed positive reaction in the Limulus test, there were no false positive results. Testing 7 bacterial strains in modified quantity of germs the Limulus test turned out to be more sensitive than the pyrogen test in rabbits. The application of this in vitro test as a complement to the pyrogen test in rabbits for a certain kind of problems is discussed.
Animals↗
Determination of endotoxins in sugar with the Limulus test.
The Limulus amebocyte lysate test has been used for determination of pyrogens in sugar of different qualities. All the samples of domestic white sugar and beet raw sugar produced in Sweden during 1976 had a very low content of endotoxins, less than 10 ng/g of sugar. Imported cane raw sugar was, however, highly contaminated. The highest value obtained corresponds to about 100 mg of Escherichia coli endotoxin per g of raw sugar. Such crude sugar cannot, even after refining, be used for medical purposes. Instead, Swedish beet sugar is used as the raw material for production of invert sugar solutions for parenteral administration. The amount of endotoxin in this sugar is less than 1 ng/g.
Carbohydrates↗
[Investigation of Limulus test during and after cardiopulmonary bypass--analysis of Limulus positive substance and particles in the cardiopulmonary bypass circuit].
Patients undergoing heart surgery with cardiopulmonary bypass (CPB) frequently have positive Limulus test without any remarkable signs of endotoxemia. CPB circuit could be one of the possible causes of this phenomenon, but the exact mechanism has not been studied. Accordingly, a prospective study was made in 41 patients undergoing heart surgery with CPB. Limulus test were performed on the samples of priming fluids of CPB including the plasma and urine of those patients during and after surgery. Besides the Limulus test, the CPB fluids were analysed by the false positive test. Microscopic examinations were carried out on the priming fluids before and after using a 40 microns filter during recirculation without washing the CPB circuit and the fluid from a 4 microns filter been washed by 2000 ml of Martose-10 in order to find out the presence of any foreign particles in the CPB circuit. The size and the number of particles were measured by coulter counter. The studies revealed the following facts, 1) Limulus test; most of the samples from priming fluids (31/35), plasma circulating the CPB (36/36), urine during CPB (22/25) and plasma (34/35) and urine (21/30) just after the surgery revealed positive. However, only 4 samples from plasma at 3POD and 2 samples from urine at 2POD revealed positive, 2) false positive test of the samples from priming fluids was negative meaning the Limulus positive substances are not endotoxin, 3) under microscopic analysis and coulter counter, there were about 40 particles over 50 microns per ml of priming fluids before washing the CPB circuit.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Detection of endotoxin in biological products by the limulus test.
The limulus amebocyte lysate (LAL) test is established as a beneficial quality assurance measure for the parenteral drug industry because of its sensitivity, specificity, and simplicity. Limulus amebocyte lysate reacts with various forms of endotoxin to form an opaque gel under acceptable conditions of pH, temperature, and ionic content. Although certain materials and conditions may alter the lysate-endotoxin reaction, the test is not significantly limited by inhibition or non-specific activation. Many U.S. drug firms apply the LAL test generally for monitoring production water and other ingredients, for an in-process control, and as a supplemental end product test for pyrogenic contamination. Specific applications are made for bacterial and viral vaccines, antineoplastic agents, radiopharmaceuticals and drugs designed for intrathecal injection. Efforts to standardize LAL test technique and lysate potency continue.
Animals↗
Endotoxin levels in milk and plasma of mastitis-affected cows measured with a chromogenic limulus test.
A chromogenic 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 less than 10 pg ml-1. In acute mastitis, the milk endotoxin level averaged (1.1 +/- 0.7) X 10(3) pg 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) X 10(6) pg 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.
Animals↗
[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.
Animals↗
Combination of conventional and endotoxin-specific limulus tests for measurement of polysaccharides in sera of rabbits with experimental systemic candidiasis.
Factor G, the coagulation enzyme from limulus amebocytes, is activated by (1-->3)-beta-D-glucan but not by endotoxin. The endotoxin-specific limulus test, which is devoid of factor G, reacts only with endotoxin. However, the conventional limulus test which includes factors G and C, reacts with not only endotoxin but also with (1-->3)-beta-D-glucan. In this study, the culture supernatant of Candida albicans in RPMI medium activated the conventional limulus test, but not the endotoxin-specific limulus test. All five rabbits inoculated intravenously with Candida albicans (1.0 x 10(7) CFU/rabbit) showed increased levels of reactivity with the conventional limulus test, whereas no elevation in the levels of the endotoxin-specific limulus test was observed in the sera of any infected rabbits. Only one serum sample from rabbit No. 5 on day 8 showed a positive Cand-tectest. The difference in the titers of the two limulus tests was suggestive of a diagnosis of Candida infection.
Amino Acid Sequence↗
Perchloric acid treatment and use of chromogenic substrate in the Limulus test: application to veterinary diagnosis.
The Limulus test was carried out for endotoxin determination using a synthetic chromogenic substrate after perchloric acid(PCA) treatment to remove inhibiting substances in the blood. PCA treatment completely removed non-specific amidolytic activity and other factors which interfered with the Limulus test. The recovery of added endotoxin was about 100% irrespective of animal species. Endotoxin levels in PCA-treated blood plasma of healthy domestic animals were invariably below 1 pg ml-1. Many of the cows with gangrenous mastitis, from which Gram-negative rods were isolated, showed high endotoxin levels ranging from 22.8 to 138.0 pg ml-1. Endotoxin was also detected in the blood of cows affected with salmonellosis, colibacillosis, Haemophilus somnus infection and urethritis associated with Proteus.
Animals↗
Establishment of a new perchloric acid treatment method to allow determination of the total endotoxin content in human plasma by the limulus test and clinical application.
We established a new method of plasma treatment for the removal of interfering factors in the plasma to allow detection of endotoxin by limulus test. The limulus test used was an endotoxin-specific chromogenic test, the Endospecy test. Perchloric acid (PCA) treatment and centrifugation (PCA method) is usually used to remove interfering factors from plasma, with the precipitate being discarded and the supernatant used to detect endotoxin. As the solubilized precipitates of endotoxin-spiked plasma and some patient plasma were found to contain the Endospecy activity, we have devised a new method assaying endotoxin in both the supernatant and precipitate. This study confirmed that the solubilized precipitate of endotoxin-spiked plasma had Endospecy activity and found that the precipitate had other endotoxin activities, such as lethality in galactosamine-sensitized mice and pyrogenicity in rabbits. We also confirmed that interfering factors were completely removed from plasma samples by this new method. The endotoxin level after the new PCA method was found to be about 8 times higher than that determined after PCA treatment and the new PCA method surpasses the conventional PCA method with regard to the positive rate of endotoxin contents in clinical samples. These results indicate that the new PCA method is superior to the PCA method as a plasma pretreatment method for limulus test.
Amidohydrolases↗
Solubilization of limulus test reactive material(s) from Candida cells by murine phagocytes.
Solubilization of limulus test reactive materials from Candida was examined in the presence or absence of phagocytic cells. Solubilized limulus test reactive materials (LTRM) were detected in culture supernatant, and hot water and sodium hydroxide extracts of the acetone dried cells of Candida parapsilosis. Suspensions of Candida cells also reacted with limulus test, and LTRM were released from the acetone dried cells by serum treatment. After treatment of the acetone dried cells with polymorphonuclear leucocytes (PMN) or macrophages (M phi), a significant amount of LTRM was solubilized. Significant amounts of LTRM were also released by PMN during treatment of live and growing C. parapsilosis. The reactivity of LTRM was completely inhibited by the addition of excess amount of purified (1----3)-beta-D-glucan, suggesting LTRM from Candida cells as described above would contain (1----3)-beta-D-glucan. These results suggested that LTRM during fungal infection would come from the extracellular water soluble polysaccharide fraction as well as the insoluble cell wall fraction solubilized by the action of phagocytes.
Animals↗
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↗
[The Limulus test in studies of pyrogenicity. Applications in radiopharmacy].
Preparation, standardization and use of the Limulus test, in relation to the official Rabbit test. Applications of the Limulus test to radiopharmaceuticals.
Animals↗
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↗
[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↗
[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↗