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PubMed2002

Rapid and simple estimation of microbiological quality of raw milk using chromogenic Limulus amoebocyte lysate endpoint assay.

A rapid chromogenic Limulus amoebocyte lysate (LAL) endpoint assay for the enumeration of total mesophilic microbial loads and coliforms was investigated as a means to assess the microbiological quality of raw milk. For experiment 1, raw milk samples (n = 25) were stored in a refrigerator (2 +/- 2 degrees C) and then analyzed at regular intervals (1, 5, 10, and 15 days). For experiment 2, fresh raw milk samples (n = 50) were tested to determine the utility of the LAL assay for fresh raw milk. The sample was diluted threefold in a 96-well microtiter plate with pyrogen-free water and assayed with a chromogenic LAL kit to find a final reaction point. The LAL results were compared with standard plate counts (SPC) and coliform counts determined by conventional plating methods. The results of the LAL assay were strongly correlated to conventional SPC (r2 = 0.93; n = 100) and were highly correlated to coliforms (r2 = 0.74; n = 100). A highly significant linear relationship (r2 = 0.82; n = 50) was also observed between the predicted SPC based on the LAL value and the actual SPC. The results of LAL testing were classified into one of seven contamination groups. The data set for SPC was effectively differentiated using the LAL technique (P < 0.01). The chromogenic LAL assay was found to be a rapid (within 16 min) and simple (not requiring specific instruments) method for monitoring microbial levels in raw milk. This method may be successfully implemented to rapidly determine highly microbial contaminated raw milk (> 3.0 log10 CFU/ml of SPC).

Animals↗
PubMed1984

Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system.

The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.

Automation↗
PubMedDate not supplied

Automation and computerization of chromogenic LAL assay method for bacterial endotoxin using 96-well microtiter plate.

A high degree of automation was achieved for a chromogenic substrate Limulus amebocyte lysate (LAL) assay method for bacterial endotoxin using a Cetus Pro/Group Liquid Handler and 96-well microtiter plates. A Titertek Multiskan Reader was interfaced with an IBM PC using a LOTUS MEASURE software to capture optical density values of samples in a LOTUS 1-2-3 spreadsheet. A password protected, menu-driven macro programmed in LOTUS 1-2-3 automates the calculation, evaluation of assay parameters, documentation, and generation of a formatted three-page report suitable as a primary record. All assay operations, including testing 19 samples against a four-point standard curve in replicates of four each, the calculation of results, and generation of a report, are completed in less than 40 minutes. The relative standard deviation (RSD) of the assay is approximately 7%, which compares favorably with the robot automated system.

Automation↗
PubMedDate not supplied

Diagnostic value of effluent endotoxin level in gram-negative peritonitis in CAPD patients.

OBJECTIVE: To evaluate the diagnostic usefulness of effluent endotoxin by Limulus amoebocyte lysate (LAL) assay in gram-negative peritonitis patients on continuous ambulatory peritoneal dialysis (CAPD) therapy. DESIGN: Prospective study with patients serving as their own controls. Standard microbiologic work up and endotoxin analysis of effluents (night dwell) were done during the pre- and posttreatment phases. SETTING: Specimens from three teaching hospitals were processed and tested at a common center. Patients were left for treatment at their respective centers without intervention. PATIENTS: 32 clinical peritonitis and 40 infection-free CAPD patients were studied. RESULTS: 75% (n = 24) of cultures were positive: 41.6% (n = 10) gram-negative and 58.4% (n = 14) gram-positive bacteria. Effluents of pre- and posttreated gram-negative cultures had endotoxin levels of 1.53 +/- 0.169 and 0.214 +/- 0.085 endotoxin units (EU)/mL, respectively (p < 0.0001); pre- and posttreated gram-positive levels of 0.102 +/- 0.06 and 0.122 +/- 0.052 EU/mL, respectively (p > 0.05); pre- and posttreated culture-negative peritonitis levels of 0.110 +/- 6.025 and 0.087 +/- 0.031 EU/mL, respectively (p > 0.05); peritonitis-free effluents contained 0.117 +/- 0.079 EU/mL. The diagnostic specificity and the sensitivity of LAL assay were 100% and 98.2% respectively. CONCLUSION: Where initial microbiological work-up cannot demonstrate a pathogen, effluent endotoxin determined by quantitative LAL assay is a useful marker for diagnosis and management, within safe time limits, of gram-negative peritonitis in CAPD patients.

Adult↗
PubMed1982

The detection of endotoxin by in vitro production of endogenous pyrogen: comparison with limulus amebocyte lysate gelation.

The sensitivities of leukocyte endogenous pyrogen (EP) production and limulus amebocyte lysate (LAL) gelation to endotoxin from E. coli (minimum i.v. pyrogenic dose 4 ng/kg in rabbits) were determined. Concentrations of 0.5-1.0 ng/ml could be detected by LAL. The minimum endotoxin concentrations which generated detectable EP from 2 X 10(6) monocytes was 10-fold lower (0.05-0.1 ng/ml). At an endotoxin concentration of 0.4 ng/ml the minimum number of monocytes required for detectable EP production was 5 X 10(5). It is concluded that the LAL gelation test cannot safely be used to exclude significant endotoxin contamination in a cellular system where EP production is being measured. The same conclusion applies even more forcibly to the in vitro production of lymphocyte activating factor (LAF, interleukin-1), since it appears that LAF and EP are identical and sub-pyrogenic amounts of EP are easily detectable in the LAF assay.

Animals↗
PubMed2000

Native and heterologous neuropeptides are cardioactive in Drosophila melanogaster.

Nine neuropeptides isolated from Drosophila melanogaster and five neuropeptides, previously isolated from the CNS of Limulus with antisera to FMRFamide-related peptides, were tested for their effects on the myogenic heart of Drosophila melanogaster. Of the native peptides, TDVDHVFLRF-NH(2) (Dromyosuppressin), DPKQDFMRFamide, and PDNFMRFamide significantly slowed the heart. Of the Limulus peptides, DEGHKMLYFamide (LP1) increased heart rate significantly, GHSLLHFamide (LP2) and PDHHMMYFamide (LP3) decreased the heart's rate, while DHGNMLYFamide (LP4) and GGRSPSLRLRFamide (LP5) had no effect at the concentrations we employed. Dromyosuppressin, DPKQDFMRFamide, and PDNFMRFamide from Drosophila, and LP2 and LP3 from Limulus, which belong to a novel group of peptides structurally unrelated to FMRFamide, are among only a very few substances from within the general group of neuropeptides and neurohormones known to slow the heart of Drosophila, and as such offer an important tool for investigating the molecular mechanisms underlying the control of the pacemaker.

Journal Article↗
PubMed1995

A comparative study of the accurate measurement of endotoxin in liposome-encapsulated hemoglobin.

We have examined three different methods of endotoxin determination utilizing the Limulus Amebocyte Lysate (LAL) assay to accurately determine endotoxin levels in Liposome Encapsulated Hemoglobin (LEH), 1) the gel-clot method, 2) chromogenic spectroscopic-based LAL, and 3) the turbidimetric method which determines endotoxin levels in solutions based on the time needed to reach a specific degree of turbidity. Both the chromogenic and turbidimetric methods require significant dilution of the LEH preparation before accurate measurement can be made. We have tested the levels of endotoxin in LEH solutions using these methods and measured LEH, liposome, and hemoglobin samples spiked with known amounts of endotoxin. A comparison of the three methods shows that the absolute value of endotoxin measured in LEH by the three methods can vary significantly. However, within any one assay the spiked amount of endotoxin in the sample can be accurately measured. The accuracy of these methods may also be complicated by the binding of endotoxin to LEH. This was evident by mixing free endotoxin with LEH followed by centrifugation to separate the LEH. Biological activity of endotoxin bound to LEH was measured by exposure to RAW264.7 followed by the expression of tumor necrosis factor.

Chromogenic Compounds↗
PubMed1995

[History of the LAL-test: validation and regulatory acceptance]

Bacterial endotoxins gram-negative bacteria are the most relevant substances inducing hyperthermia in humans (pyrogens). Since endotoxins may contaminate pharmaceutical preparations during production process, purity is assured by monitoring the increase in body temperature of rabbits exposed to such preparations. This bioassay can be replaced by the LAL-test in which the clotting reaction of blood cells of the horseshoe crab limulus polyphemus is measured after contact with bacterial endotoxins. Although this reaction is significantly more sensitive to endotoxins than hyperthermia in rabbits, the LAL-test had to undergo 25-30 years of validation to achieve regulatory acceptance. Although endotoxin induced blood clotting in Limulus is quite similar to the same reaction in humans, acceptance of the assay as an alternative to testing in rabbits was delayed, since chemicals present in pharmaceutical preparations may interfere with the LAL clotting reaction. In addition, fever can be induced also by substances other than endotoxins. Therefore it has to be proven for each new preparation that the LAL-test can replace the rabbit pyrogen test in a case by case validation according to guidelines, as e.g. the German guideline, which was released in 1993. During the past decade some laboratories of the pharmaceutical industry in Germany were able to replace 95% of the rabbit pyrogen tests by the LAL-test without increasing the risk for humans.

Journal Article↗
PubMed2000

Concordance of endotoxemia with gram-negative bacteremia. A meta-analysis using receiver operating characteristic curves.

OBJECTIVE: To apply meta-analysis to compare the concordance between the results of 2 types of limulus amebocyte lysate (LAL) assay, gelation (GLAL) and chromogenic (CLAL), with the detection of gram-negative bacteremia in patients with suspected bacteremia. DESIGN: Meta-analysis using receiver operating characteristic-based analytical method. DATA SOURCES: MEDLINE literature search and manual reviews of article bibliographies together with direct approaches to authors of potentially eligible studies. STUDY SELECTION: The studies that were selected had all included at least 10 patients, of whom at least 2 patients were diagnosed with gram-negative bacteremia, and all had data available for extraction into a contingency table format. RESULTS: Fifty-six studies (28 GLAL and 28 CLAL studies) met the inclusion criteria. Studies were stratified by type of test (GLAL vs CLAL). Each analysis was repeated with smaller studies excluded. There was no difference between the 2 types of LAL assays. Among the CLAL studies, there was no difference between studies that did versus those that did not use the sepsis syndrome criteria as a basis for patient inclusion. Among 45 studies for which data on the proportion of non-Enterobacteriaceae were available, there was a trend toward higher concordance as this proportion increased. CONCLUSIONS: The concordance between the LAL test and the detection of gram-negative bacteremia in patients with suspected bacteremia is no higher with the CLAL assay than with the original GLAL version. However, the concordance is higher among studies with a higher proportion of non-Enterobacteriaceae among the gram-negative bacteremia isolates.

Endotoxemia↗
PubMed1989

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin.

A new reagent for the chromogenic Limulus amebocyte lysate (LAL) assay is described. LAL was formulated for optimal performance in either an endpoint procedure or a kinetic procedure with the chromogenic substrate, buffer, and LAL components colyophilized as a single reagent. The kinetic chromogenic method required an incubating microplate reader coupled to a computer for collection and analysis of data. The kinetic method had a longer incubation time than the endpoint method and spanned a range of over 3 orders of magnitude compared with the 1-order-of-magnitude range of the endpoint assay. The kinetic method was less subject to operator error, since readings were continuous and automatic. The endpoint test was more operator intensive, requiring both addition of acetic acid to stop the reaction and transfer of the sample to the reading device. A single-step chromogenic reagent was also prepared without lyophilization by mixing reconstituted gel clot LAL with a buffer and a chromogenic substrate. The reagent prepared in this manner performed as well as the colyophilized agent.

Animals↗
PubMed1991

Quantitative Limulus lysate assay for endotoxin and the effect of plasma.

The effects of plasma and chromogenic substrate on the kinetics of the endotoxin-activated Limulus amoebocyte lysate (LAL) assay were determined. A linear correlation was observed between the rate of development of turbidity (optical density 405) with the LAL reagent and the concentration of endotoxin over a four log ten-fold range. Like chromogenic substrate, the addition of dilution and heat treated plasma to the reaction resulted in an increase in optical density proportional to the concentration of plasma present. The presence of the treated plasma also resulted in an accelerated increase in optical density with comparable results when testing plasma at different concentrations and, additionally, serum. This accelerated increase in optical density may not be recognised in assays that monitor the progress of the reaction at a single time point and may confound assays of plasma samples that use chromogenic substrate. Plasma obtained from endotoxin sensitive and resistant strains of mice showed similar effects. The use of kinetic methodology means that a quantitative assay for endotoxin in plasma can be achieved, its variability comparable with that seen with semiquantitative serial dilution but with greater economy of the LAL reagent.

Chromogenic Compounds↗
PubMed1995

Detection of (1-3)-beta-D-glucan in a rat model of aspergillosis.

The G test containing factor G, fractioned from the Limulus lysate, was used to detect (1-3)-beta-D-glucan in a rat model of aspergillosis. Aspergillus fumigatus strain MF-13, 1 x 10(4) conidia, were inoculated transtracheally into rats treated with cortisone acetate (100 mg/kg) and fed a low-protein (8%) diet. Increased serum (1-3)-beta-D-glucan was found on the sixth day after inoculation in concentrations of 370 +/- 178 pg/ml (mean +/- SD) in untreated controls, and 154 +/- 43 pg/ml in rats treated with 0.5 mg/kg of amphotericin B. On day 11 (1-3)-beta-D-glucan concentrations were 2,590 +/- 2,940 pg/ml and 448 +/- 442 pg/ml, respectively. The elevation in levels of (1-3)-beta-D-glucan increased in correlation with the elevation of galactomannan antigen titers; (1-3)-beta-D-glucan is thus measurable during experimental aspergillosis in rats.

Amphotericin B↗
PubMed1986

A rapid sensitive monoclonal assay for lipid A in solution.

Lipid A is the toxic component of bacterial endotoxins (LPS) and LPS has been thought to be clinically important in Gram-negative bacterial sepsis, as well as in non-bacteremic states where endotoxemia of enteric origin may be deleterious. The presently accepted method of detecting both LPS and its common lipid A moiety is the Limulus lysate amebocyte assay (LAL), but this test cross-reacts with non-bacterial antigens and serum contains natural inhibitors to the reaction. As an extension of previous work using a polyclonal antibody, we have developed a rapid and sensitive monoclonal antibody assay for lipid A. This 3-step inhibition ELISA is reproducible in aqueous solutions and capable of detecting less than 10 pg/ml of this shared toxic endotoxin component. The assay does not detect intact LPS but acid hydrolysis releases the active lipid A for reaction. While already valuable in detecting lipid A in biological solutions, the presence of naturally occurring anti-lipid A immunoglobulins in serum interfere with the reaction and cause false positives in this inhibition assay. Clinical usefulness of the assay will depend on removal of these antibodies from serum prior to testing.

Antibodies, Monoclonal↗
PubMed2000

Pyrogenic reactions associated with single daily dosing of intravenous gentamicin.

OBJECTIVE: To identify risk factors associated with an unexpected outbreak of pyrogenic reactions (PR) following intravenous gentamicin. DESIGN: We conducted two cohort studies. PRs were defined as chills, rigors, or shaking within 3 hours after initiating the gentamicin infusion during the preepidemic (December 1, 1997-January 15, 1998) or epidemic (May 1-June 15, 1998) periods. We tested gentamicin vials for endotoxin using the limulus amebocyte lysate assay. SETTING: Inpatient services of a large community hospital in Los Angeles, California. RESULTS: During the epidemic period, 22 (15%) of 152 patients developed documented PRs following intravenous gentamicin. PRs were more likely among patients receiving single daily dosing (SDD) than multiple daily dosing gentamicin (20/73 [27%] vs. 2/79 [3%]; relative risk, 10.8; 95% confidence interval, 2.6 44.7). Laboratory analysis of gentamicin vials found endotoxin levels that were higher among Fujisawa-brand gentamicin (implicated brand) than gentamicin used after the outbreak terminated (non-implicated brand). Although endotoxin levels in the vials did not exceed US Pharmacopeia limits (1.7 endotoxin units/mg gentamicin), the use of SDD gentamicin may place patients at greater risk of receiving doses of endotoxin above the threshold for PRs in humans. CONCLUSIONS: Reassessment of the acceptable amounts of endotoxin in gentamicin and other parenteral products should be considered when dosing intervals used in clinical practice change.

Adolescent↗
PubMed1992

Mechanisms of amplification, deactivation, and noise reduction in invertebrate photoreceptors.

In this review we have discussed the problem of deactivation at both the rhodopsin and G protein levels. Of particular interest is the novel observation that rhodopsin deactivation can be modulated by light. This modulation is likely to play an important role in light adaptation by reducing the gain of transduction. One interesting possibility is that this modulation involves the phosphorylation of an arrestin-like molecule, but this remains to be tested. One of the experimental advantages of Limulus photoreceptors is the large size of the single photon responses and the fact that even single G proteins produce a detectable response. This made possible the observation that nonhydrolyzable GTP analogues produce discrete transient events rather than the step-like events that would be predicted by previous models. This observation led us to a new view of how enzyme deactivation is coupled to GTP hydrolysis on G protein. According to this view, enzymes are activated by G protein, but can be deactivated by processes that are not dependent on G protein or the hydrolysis of GTP. We have conducted several types of experiments, including some on the vertebrate rod system, that strongly support this hypothesis. A second major theme of this review is transduction noise. The available biochemical evidence suggests that both G protein and G protein-activated enzymes are likely to become spontaneously active and generate undesirable noise. Our measurements indicate, however, that this noise is orders of magnitude smaller than would be predicted by simple models, suggesting that special mechanisms must exist for suppressing this noise. We have proposed a specific mechanism by which enzymes regulated allosterically by multiple subunits could act as coincidence detectors to reduce transduction noise. Finally, there is the fundamental question of which second messengers have a direct role in invertebrate phototransduction. After Fesenko et al. (1985) showed that the light-dependent conductance in vertebrate rods was modulated by cGMP and not by Ca2+, there was rapid progress in understanding the vertebrate photoreceptor transduction mechanism. Now that it has been established that invertebrate light-dependent channels are regulated by cGMP and not by Ca2+, we can expect rapid progress in understanding invertebrate phototransduction. A key question that needs to be answered is whether the InsP3-Ca2+ pathway somehow triggers changes in cGMP or whether there is an altogether different pathway by which cGMP metabolizing enzymes are affected by light.

Adaptation, Physiological↗
PubMed1987

Quantitative measurement of endotoxin in canine plasma using the new endotoxin-specific chromogenic test.

Endospecy (a lyophilized mixture of factor G-free limulus coagulation enzymes and chromogenic substrate, Boc-Leu-Gly-Arg-pNA) coupled with modified perchloric acid (PCA) pretreatment was carried out for the quantitative measurement of endotoxin in canine plasma. The endotoxin recovery from normal canine plasma was 99.9 +/- 7.7% (n = 20). The full recovery of endotoxin illustrated the applicability of the modified PCA pretreatment to the Endospecy in removal of interfering factors in a canine plasma. The normal canine plasma endotoxin level was less than 3.0 pg.ml-1 when Escherichia coli 0111:B4 endotoxin was used as a reference. The canine plasma endotoxin levels were markedly high(1-40 ng.ml-1) at 5 min after intravenous administration of 25 micrograms.Kg-1 (total 150-200 micrograms).

Animals↗
PubMed1981

Preparation and characterization of detoxified lipopolysaccharide-protein conjugates.

Alkaline treatment of Pseudomonas aeruginosa type 5 lipopolysaccharide (LPS) resulted in reduced toxicity as measured by both the Limulus amoebocyte assay and the rabbit pyrogenicity test. Chemical analysis of the deacylated LPS (D-LPS) revealed that ester-linked fatty acids were removed while the amide-linked fatty acids remained intact. The neutral and amino sugar compositions for native LPS and D-LPS were identical within experimental error. Antigenic determinants for complement-dependent human opsonic antibody were retained under these deacylation conditions. To enhance its immunogenicity, D-LPS was covalently coupled to Pseudomonas pili and the 1,4-diaminobutyl derivatives of Pseudomonas exotoxin A and tetanus toxoid. Quantitative amino sugar analyses revealed that 2.6 and 3.2 mol of D-LPS were covalently bound to aminobutyl Pseudomonas exotoxin A and aminobutyl tetanus toxoid, respectively. Gel electrophoresis data indicated at least 1 mol of D-LPS covalently bound per pilus subunit protein. Initial immunologic data indicated that antibody against D-LPS could be induced when the D-LPS is covalently attached to protein carriers.

Amino Acids↗
PubMedDate not supplied

Enhancing effect of non-ionic surfactant on the inactivation of lipopolysaccharide by steam-heat treatment I.

Polyoxyethylene (20) sorbitan mono-fatty acid esters strongly enhanced the inactivation of lipopolysaccharide (LPS) by steam-heat treatment at 121 degrees C, as assayed by using the Limulus amebocyte lysate (LAL) and the pyrogen test. In an aqueous solution containing 0.1% surfactant, the decrease of LPS (1 microgram/ml) from E. coli 055:B5 at 121 degrees C followed first-order kinetics. Based on the LAL assay, 0.1% surfactant was essential to achieve 3-log cycle reduction of LPS with concomitant loss of pyrogenicity by steam-heat treatment for 20 min at 121 degrees C. Steam-heat treatment for 20 min at 121 degrees C in the absence of surfactant was insufficient to achieve depyrogenation. Polyoxyethylene (9) lauryl ether and decaglycerin mono-laurate similarly enhanced depyrogenation by steam-heat treatment. The effects of all the surfactants were concentration-dependent for all of the six kinds of LPS examined.

Feasibility Studies↗