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PubMed1977

Limulus test, parenteral drugs and biological products: an approach.

The authors report their experience with the LAL test for endotoxin assay. They have examined parenteral drugs, blood products and biological substances employed in immunology. The results were compared with rabbit pyrogen tests. All the data suggest that if correctly performed, the LAL test is a simple, rapid and sensitive test. This assay appears to be very useful for materials employed in immunology in order to test the possible contamination with bacterial endotoxin, since the interference between endotoxin and immunologic response is well known.

Animals↗
PubMed1989

Quantitative assay of lentinan in human blood with the limulus colorimetric test.

A conventional limulus test detects not only endotoxin but also beta (1----3) glucan. Therefore, using a quantitative limulus test (the limulus colorimetric test) we studied the pharmacokinetics of lentinan, an antitumor beta (1----3) glucan, in the blood of 10 health volunteers and three patients with advanced gastric cancer. The calibration curve of lentinan in the human plasma was linear in the range of 0 to 100 ng/ml. When incubated with human plasma at 37 degrees C in vitro, lentinan had the recovery of almost 100% as compared to the initial concentration even after 60-min incubation, indicating the stability of lentinan in human plasma. When 1 mg of lentinan was intravenously administered over a 2 hr period, lentinan concentration reached the maximum levels (50-80 ng/ml) at the end of the drip infusion and decreased gradually thereafter. In the near future, the more appropriate modes of lentinan administration will be determined by further investigation of its kinetics in the human body.

Colorimetry↗
PubMed2001

[18F-FDG injections produced by a solid phase 18F-fluorination (FDG MicroLab): effects of 18F-FDG and the components on endotoxin and sterility tests].

Effects of 18F-FDG and components of the injections on endotoxin tests (Limulus tests) and sterility tests (Blood culture system) were determined with 18F-FDG injections produced by a solid phase 18F-fluorination (FDG MicroLab, GE). 18F-FDG injections with endotoxins shortened the time for gelling (turbidimetry), compared with that of the control (saline). Blood culture systems inoculated with 18F-FDG injections and microorganisms showed positive results within 72 h of incubation for every species of microorganisms used in the present study (Bacillus subtilis, Candida albicans, Clostridium sporogenes, Micrococcus luteus). These results were quite similar to those for the control samples inoculated with saline and the microorganisms. Consequently, 18F-FDG and the components of the injections produced by the present methods may not significantly affect the endotoxin tests and sterility tests.

Bacillus subtilis↗
PubMed1991

Endotoxin liberation by strains of N. meningitidis isolated from patients and healthy carriers.

The main objective of this study was to assess whether the capacity of Neisseria meningitidis to release endotoxin depends upon the type of strain or upon bacterial mass. Endotoxin release was studied in 32 strains isolated from patients with meningococcal infections and in 49 from asymptomatic carriers, using a quantitative test (limulus test with a chromogenic substrate). The results show that the strains from patients release significantly higher amounts of endotoxin than strains from carriers regardless of serogroup and isolation site. No correlation was found between stage of bacterial growth and the amount of endotoxin liberated. These findings suggest that endotoxin liberation is a characteristic of certain strains of N. meningitidis and is not determined simply by bacterial mass.

Adult↗
PubMed1998

Factor G pathway reactive activity (GPRA) after surgery for abdominal aortic aneurysm.

BACKGROUND: Following major surgery, detection of endotoxaemia using the Toxicolor (Limulus) test has been reported. In addition to endotoxins, this test detects a reactant from human tissue, factor G pathway reactive activity (GPRA). We measured endotoxin and GPRA in 10 patients during and after elective surgery for abdominal aortic aneurysm. Additionally, we measured phosphokinase (CPK) and GPRA levels in the muscle of 10 patients during abdominal aortic aneurysm surgery and 6 during open laparotomy for other causes. METHODS: Samples were taken from the arterial lines prior to surgery, before cross-clamping of the aorta, immediately after and 1, 3, 6, and 18 hours after declamping. Muscle specimens were taken from the rectus abdominus and homogenized in many levels. Endotoxin concentrations were measured with the Endotoxin-Specific test. GPRA concentrations were determined by subtracting the values from the Endotoxin-Specific test from those given by the Toxicolor test (Limulus assay with achromogenic substrate). CPK was also measured. RESULTS: Endotoxin did not significantly increase during or after surgery, but GPRA was elevated. GPRA in muscle correlated significantly with CPK in muscle. CONCLUSIONS: We conclude that GPRA was elevated after surgery, while endotoxin did not increase significantly. The GPRA probably originated from human muscle tissue.

Aged↗
PubMed1982

Rapid evaluation of female patients exposed to gonorrhea by use of the Limulus lysate test.

The Limulus amoebocyte lysate (LAL) assay was used to evaluate 115 females who were named as sexual contacts by men with culture-proven gonorrhea. These patients were treated for gonorrhea before laboratory confirmation, as recommended by the Centers for Disease Control, because of the lack of rapid screening tests and the serious consequences of undetected infection. For the LAL assay, endocervical samples were collected with depyrogenated cotton-tipped swabs, and the swabs were placed in 10 ml of diluent to assay for endotoxin; the negative predictive value of the LAL assay at this dilution was 100%. Incubation was carried out at 37 degrees C for 30 min; positive or negative results were indicated by gelation or lack of gelation, respectively. Lysate sensitivity was 0.3 ng/ml, with an Escherichia coli endotoxin standard. Single endocervical cultures and the LAL assay were both positive in 71 patients, but the Gram stain was positive in only 36 (50.7%) of these cases. For the 44 culture-negative cases, the LAL assay was negative in 21 (47.7%). Thus, the LAL assay was able to selectively exclude approximately half of the culture-negative gonorrhea contacts and would have spared these patients inappropriate therapy and contact tracing, without excluding culture-positive gonorrhea cases.

Female↗
PubMed1985

The history of the development of the Limulus amebocyte lysate test.

The Limulus Test for bacterial endotoxins was developed as the result of investigations of the effects of Gram-negative bacteria on blood coagulation in the horseshoe crab Limulus polyphemus. Analysis of the factors required for blood coagulation (all of which are contained within Limulus amebocytes) has revealed marked similarities between blood coagulation in the horseshoe crab and mammals. The Limulus Test (LT) is the most sensitive test for bacterial endotoxins now available and correlates with a wide variety of other assays. An increasing number of applications of the LT have been described, including detection of endotoxins in pharmaceutical preparations, food, radioisotopes, water supplies, and medical devices, as well as in various body fluids such as blood, cerebrospinal fluid, and urine. The "basic research" that unexpectedly led to the discovery and subsequent development of the Limulus Test provides an example of the value of biomedical investigations directed at understanding pathophysiologic phenomena and demonstrates the importance of studies of comparative physiology.

Animals↗
PubMed1982

Determination of endotoxin using fluorescent probe.

Bacterial endotoxin induces gel-formation of amebocyte lysate. This gelation test, the limulus test, was first described by Levin and Bang [1]. The limulus test has been widely used for the detection of endotoxin, not only in fundamental bacteriological research but also in clinical diseases [2]. Although the technique of the limulus test is simple, the endotoxin level can not be determined quantitatively. We developed a method to determine the endotoxin in physiological saline solution and glucose solutions using a fluorescent probe, fluorescamine.

Endotoxins↗
PubMed1979

Rapid presumptive diagnosis of gonococcal urethritis in men by the limulus lysate test.

In an evaluation of the limulus assay as a method for detecting endotoxin in urethral exudates, positive results of urethral samples at a 1/200 dilution were obtained from 73 out of 73 patients with culture-positive gonococcal urethritis while negative results were obtained from 26 out of 27 patients with cuture-negative urethral specimens. A specimen from one patient, which gave negative results on Gram stain and culture, gave positive results to the limulus test. The overall accuracy of the limulus test for predicting culture results was 99% (p less than 0.001). Thus, in preliminary studies of otherwise healthy men, the results of the limulus assay correlated with those of biological methods for diagnosing urethral gonorrhoea; the test may, therefore, be of use in identifying cases of nongonococcal urethritis.

Adult↗
PubMed1989

[Rapid diagnosis of gram-negative bacteriuria with endotoxin-specific chromogenic test].

Limulus amebocyte lysate test is a simple, yet sensitive laboratory method for detecting endotoxin. Although the specificity of the test was questioned, we recently solved this problem by removing a (1----3)-beta-D-glucan-sensitive factor G from the lysate (Endospecy, Seikagaku Kogyo Co., Ltd, Tokyo). In this study, we have established a method for the determination of endotoxin urine in using Endospecy. An aliquot of 0.2 ml of Endospecy dissolved in 4.4 ml of 0.1 mol/l Tris-HCl buffer (pH 8.0) was added to 5 microliter of urine sample. The mixture was incubated at 37 degrees C for 30 min. After diazo-coupling, absorbance was measured at 545 nm. The standard curve using Escherichia coli 0111: B4 endotoxin showed a good linearity. The addition-recovery test of various endotoxins showed nearly 100% recovery. Normal urine contained less than 60 pg/ml of endotoxin. Urine from patients with Gram-negative bacteriuria showed definitely high values, whereas urines infected either with Gram-positive bacteria or with fungi all showed nearly normal values. This method will be useful as a rapid and reliable test for the diagnosis of urinary tract infection with Gram-negative bacteria.

Endotoxins↗
PubMed1980

Comparison of plasma extraction techniques in preparation of samples for endotoxin testing by the Limulus amoebocyte lysate test.

Due to the presence of inhibitory and possible mimicking substances in plasma difficulties have occurred in the use of the Limulus amoebocyte lysate test. Currently, there are a variety of extraction techniques discussed in the literature which are used to remove these interfering substances, but there is little information comparing these techniques. Five such procedures were compared in their ability to provide an extracted plasma sample in which low levels of endotoxin could be detected by the Limulus amoebocyte lysate test. Results indicated that some procedures adversely affected endotoxin detection. The dilution + heating extraction method was found to be as effective as the widely used chloroform extraction method. Comparison of Limulus amoebocyte lysate test results from healthy human plasma samples extracted by these two methods indicated that lysate type and not extraction procedure was associated with previously reported questionable positive tests. Thus, ambiguities associated with Limulus amoebocyte lysate tests of plasma samples may be due not only to extraction method but also the lysate type employed.

Endotoxins↗
PubMed1978

Lack of specificity of the limulus lysate test in the diagnosis of pyogenic arthritis.

The diagnosis of pyogenic arthritis may be difficult to confirm since culture results are sometimes negative. This study attempted to evaluate the utility of the limulus lysate assay for the early detection of pyogenic arthritis due to gram-negative organisms. Seven-one specimens of synovial fluid from 46 patients were evaluated for reactivity in the limulus test, pyrogenic responses in rabbits, total white blood cell count, total neutrophil count, total red blood cell count, and protein and glucose concentrations. All patients with culture-proven septic arthritis or presumptive septic arthritis had joint fluid specimens that yielded a positive result in the limulus assay (12 patients). However, 52.9% of patients (18 of 34) who had a nonseptic cause for their joint effusion also had a joint fluid specimen that was positive in the limulus test. A positive limulus test result showed a significant correlation with an elevated total white blood cell count (P less than 0.0005), an elevated absolute neutrophil count (P less than 0.0005), and a decreased concentration of glucose (P less than 0.005) in synovial fluid, and the production of fever in rabbits after injection of synovial fluid (P less than 0.05). Thus, this study suggests that a positive result in the limulus test on joint fluid is nonspecific for a septic process, but a negative result would be evidence against it.

Arthritis, Infectious↗
PubMed1994

An improved sandwich ELISA method for the determination of immunoreactive schizophyllan (SPG).

As it is important to determine the optimal serum concentration of schizophyllan (SPG) when it is used as an anti-cancer drug, we devised a solid-phase ELISA. We also developed a sandwich ELISA using murine anti-SPG monoclonal antibody as the first antibody and rabbit anti-SPG serum as the second antibody in order to improve the detection sensitivity. This assay was able to determine SPG concentrations over 1.0 ng/ml and the absorbance at 490 nm was directly proportional to the SPG concentration. SPG in rabbit serum, obtained after intravenous and intramuscular injection (SPG; 10 mg/kg), was determined by this sandwich ELISA. Furthermore, the sensitivity of this ELISA method was compared with that of the Limulus test. The Limulus test is able to detect SPG in physiological saline (pH 6.3) at concentrations greater than 1.0 microgram/ml, but the sensitivity increased when SPG was dissolved in alkaline solution (pH 12.0), enabling SPG to be measured almost down to 1.0 ng/ml. These data suggest that our sandwich ELISA may be used for the measurement of SPG in blood or tissue.

Animals↗
PubMed2001

[Replacement of the pyrogen-test as batch control test for the biological substances aprotinin and urokinase].

In the Pharmeuropa Vol. 10, No. 4, December 1998, the European Pharmacopoeia Commission published an appeal to replace the pyrogen test by the limulus test for a total of 10 biological substances. These substances included the active ingredients aprotinin and urokinase, contained in our sales products Antagosan and Actosolv. After successful validation of the limulus test, we calculated endotoxin limit concentrations for these and submitted these to the European Department for Quality of Medicines (EDQM). We expect - not least on the basis of the data submitted by ourselves - that the pyrogen test will be replaced in future as a quality control test by the limulus test in testing aprotinin and urokinase compounds for the presence of pyrogenic substances. This leads to a reduction in the number of rabbits used as opposed to the current pyrogen test for testing the stated compounds.

Animal Testing Alternatives↗
PubMed1975

Limulus lysate test for gram-negative bacterial meningitis. Bedside application.

The limulus lysate on cerebrospinal fluid was evaluated in 335 infants and children as a method for the rapid diagnosis of Gram-negative bacterial meningitis. Positive limulus tests were obtained within one hour in 33 of 34 cases of Hemophilus influenzae meningitis; four additional patients with Gram-negative meningitis also showed positive limulus lysate tests. Conversely, 13 patients with Gram-positive bacterial meningitis all yielded negative limulus assays. All 48 cases of aseptic meningitis and 236 children with no meningitis showed negative limulus assays. Antibiotic therapy prior to hospitalization did not vitiate the validity of the test. A bedside adaptation of the limulus test, performed by house officers and medical students, showed approximately 98% agreement with the laboratory assay.

Arachnida↗