Modified micromethod of the Limulus amoebocyte lysate assay for endotoxin.

A Limulus amoebocyte lysate microtechnique performed in petrolatum wells on a microscope slide is described. Injection of a dye solution in ethanol directly into the wells leads to an unambiguous interpretation of the results. Twelve samples can be tested on a single slide, and compact storing of the samples is possible.     

Purification and properties of the clotting enzyme from limulus lysate

The clotting enzyme from Limulus lysate which is involved in the gelation reaction of lysate with endotoxin has been purified and some of its properties determined. It was isolated from endotoxin-treated lysate and purified by gel filtration, ion exchange chromatography, and disc gel electrophoresis. Reaction of clotting enzyme with lysate clottable protein produces a clot or gel such as occurs with the gelation of lysate by endotoxin. Purified clotting enzyme has an approximate molecular weight of 84,000 (subunit MW 43,000), is isoelectric at pH ca. 5.5, trypsin-like, heat labile and pH sensitive.     

Use of gas chromatography-mass spectrometry versus the limulus amebocyte lysate test for the determination of airborne endotoxin in confined swine buildings

Airborne endotoxin (lipopolysaccharide, LPS) in filter samples collected in three different swine confinement buildings was determined by using the Limulus amebocyte lysate test and by applying gas chromatography-mass spectrometry (GC-MS) to analyze 3-hydroxy fatty acids (3-OH-FAs). The amounts of LPS as shown by GC-MS were 30–50 times larger than the amounts detected by the Limulus test. GC-MS revealed that 21% of the LPS collected on cellulose acetate filters and 26% on polycarbonate filters remained on the filters after buffer extraction. Better correlation with the Limulus test and the 3-OH-FA measurements was achieved when considering the sum of 3-OH C12:0 and 3-OH C14:0 rather than the sum of all of the detected 3-OH-FAs (i.e. those with 12–18-carbon chains), indicating that the bioactivity of the LPS was dependent upon the relative distribution of the 3-OH-FAs. Linear regression analysis between air concentrations of dust and endotoxin/LPS gaveR ²values that varied from 0.407 to 0.739. The air concentrations of LPS were lower in the uninsulated swine house (a green house with an alternative housing system) than in the two insulated buildings (conventional housing systems; one designed as a climate chamber, i.e. climatic parameters could be controlled), whereas the opposite was found for the concentrations of LPS in airborne dust from the three buildings. The numbers of viable bacteria and fungi were highest in the uninsulated swine house. Moreover the relative distribution of 3-OH-FAs in that building differed from the distribution in the insulated buildings, which reflects differences in the microflora, probably mainly due to differences between the housing systems and the design of the buildings.     

Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries.

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.     

Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.     

Comparison of limulus amebocyte lysates and correlation with the United States Pharmacopeial pyrogen test.

Six limulus amebocyte lysate (LAL) preparations obtained from five different suppliers were evaluated for sensitivity, dependability, cost, convenience of use, and correlation with the United States Pharmacopeial (USP) rabbit pyrogen test method. Endotoxins from various gram-negative microorganisms were used for the evaluation. Major differences among the LAL preparations lie in the area of sensitivity. Differences, up to 100-fold, exist in the sensitivity of the various LAL preparations to the same endotoxin. The LAL tests in general were 3 to 300 times more sensitive than was the USP rabbit pyrogen test method. The LAL and the USP rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. However, large discrepancies in correlation were found when partially degraded endotoxins were compared. One LAL preparation responded to both intact and degraded endotoxin, whereas others responded only to intact endotoxin; the latter closely correlated with the febrile response of the rabbit. Therefore, proper selection of an LAL preparation is important for its application in clinical, pharmaceutical, public health, and environmental areas.     

Comparison of limulus amebocyte lysates and correlation with the United States Pharmacopeial pyrogen test.

Six limulus amebocyte lysate (LAL) preparations obtained from five different suppliers were evaluated for sensitivity, dependability, cost, convenience of use, and correlation with the United States Pharmacopeial (USP) rabbit pyrogen test method. Endotoxins from various gram-negative microorganisms were used for the evaluation. Major differences among the LAL preparations lie in the area of sensitivity. Differences, up to 100-fold, exist in the sensitivity of the various LAL preparations to the same endotoxin. The LAL tests in general were 3 to 300 times more sensitive than was the USP rabbit pyrogen test method. The LAL and the USP rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. However, large discrepancies in correlation were found when partially degraded endotoxins were compared. One LAL preparation responded to both intact and degraded endotoxin, whereas others responded only to intact endotoxin; the latter closely correlated with the febrile response of the rabbit. Therefore, proper selection of an LAL preparation is important for its application in clinical, pharmaceutical, public health, and environmental areas.     

显色基质鲎试剂法在人血白蛋白热原检测中的应用

为了对显色基质法测定人血白蛋白中的内毒素含量方法进行探讨。以内毒素,鲎试剂及显色基质,在一定的条件下反应释放出对硝基苯胺(PNA),溶液呈现黄色,于波长545nm处比色读数,其产色深浅与内毒素浓度呈线性关系,从而定量测定出检品中内毒素含量。结果表明标准曲线的线性相关系数r≥0.98,人血白蛋白经3.3倍稀释后无干扰作用。显色基质法与家兔法比较,有灵敏、快速、能定量、重复性好的特点,可用于人血白蛋白内毒素含量的测定。     

Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.     

A new micromethod for mechano-chemical research.

A new micromethod to study activity of enzymes in crystalline and amorphous solid samples subjected to tension is described. Both static (up to 200%) and dynamic (with an amplitude up to 50% and frequency of 10(-3)-50 Hz) deformation may be applied to the sample during the activity measurement. Strain-induced changes in activity of thin (3-10 microns) needle-like strips (0.2-0.5 microns long), cut out of the microtome sections of cross-linked crystals of pancreatic carboxypeptidase A is measured in a droplet of substrate solution (with volume of 10(-6)-10(-9) l) with a pH-microelectrode technique.     

Statistical procedure for evaluating the sensitivity of Limulus amoebocyte lysate by using a reference lysate.

A study was designed to estimate variability of the Limulus amoebocyte lysate test by comparing a reference lysate against itself. Three technicians performed parallel tests, i.e., titrated side by side, the contents of two vials of reference lysate on 4 different days using 24 vials of the United States reference lysate and 12 vials of the United States reference endotoxin. Each parallel test was replicated three times. From the sensitivity endpoints, ratios were calculated for each parallel test. These ratios were converted to the logarithm for estimating variability among technicians and among vials of endotoxin. By using the overall variability of log ratios, a statistical procedure was developed to evaluate the sensitivity of each lot of licensed lysate submitted to the Bureau of Biologics for release.     

Improved microtechnique for endotoxin assay by the Limulus amebocyte lysate test

A simple microtechnique for the endotoxin assay with Limulus amebocyte lysate is described. Mixtures of 1 μl of reagent with 1 μl of test sample are incubated in 5-μl capillaries which are subsequently dipped into a dye solution. In the absence of a firm gel the dye enters the capillaries whereas a firm gel, formed in the presence of endotoxin, prevents the stain from entering the capillaries. The technique offers a great saving of reagent; unequivocal distinction between positive and negative results, a good reproducibility, and a sensitivity equal to or higher than those in previously reported techniques could be demonstrated.     

Statistical procedure for evaluating the sensitivity of Limulus amoebocyte lysate by using a reference lysate.

A study was designed to estimate variability of the Limulus amoebocyte lysate test by comparing a reference lysate against itself. Three technicians performed parallel tests, i.e., titrated side by side, the contents of two vials of reference lysate on 4 different days using 24 vials of the United States reference lysate and 12 vials of the United States reference endotoxin. Each parallel test was replicated three times. From the sensitivity endpoints, ratios were calculated for each parallel test. These ratios were converted to the logarithm for estimating variability among technicians and among vials of endotoxin. By using the overall variability of log ratios, a statistical procedure was developed to evaluate the sensitivity of each lot of licensed lysate submitted to the Bureau of Biologics for release.     

Biological activity of lipopolysaccharides from clinical Bacteroides fragilis strains isolated in Poland determined in reaction with limulus amoebocyte lysate

The aim of this study was to determine a biological activity of lipopolysaccharides (LPS) from clinical Bacterioides fragilis strains isolated in Poland by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides were extracted from nine clinical B. fragilis strains by the procedure of Westphal and Jann (1965). Crude LPS preparations were purified with ultracentrifugation. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit). Tests were performed according to the recommendations of the producer (Charles River Endosafe Ltd., USA). E. coli O55:B5 LPS and LPS preparations from reference B. fragilis strains were applied to compare the results of examinations. Activities of endotoxins from clinical B. fragilis strains isolated in Poland determined in reaction with Limulus amoebocyte lysate were differentiated. Among endotoxins of clinical B. fragilis strains the most active was the preparation from strain cultured in the case of pancreatic ulcer (B. fragilis 80/81 LPS). Lipopolysaccharides of examined B. fragilis strains were less active in BET test than E. coli O55:B5 LPS.     

Automation of the Limulus amoebocyte lysate test by using the Abbott MS-2 microbiology system.

A rapid, automated method for the performance of the Limulus amoebocyte lysate endotoxin assay has been developed by using the Abbott MS-2 Microbiology System. This instrument automatically determines sequential changes in the optical density of up to 176 samples at 1- or 5-min increments during a 1-h assay period. Graphic representation of optical density changes can be viewed on a cathode-ray tube or reproduced by using a hard-copy printer. Limulus amoebocyte lysate preparations that were obtained from different commercial producers and that had similar endotoxin sensitivities by the conventional gelation method varied somewhat in reactivity when determinations were based upon rate changes in optical density. Lysates from Associates of Cape Cod, Difco Laboratories, and M. A. Bioproducts were the most readily adaptable to the MS-2 System. Use of the MS-2 system increased the sensitivity of these preparations from 60- to 250-fold, and as little as 1 pg/ml was detected. Adaptation of the MS-2 instrument for this purpose provides an objective, reproducible, automated method for the performance of Limulus amoebocyte lysate tests on a variety of fluids.     

A limulus amoebocyte lysate activating activity (LAL activity) that lacks biological activities of endotoxin found in biological products

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS-binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IRA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.     

Studies on the sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assays.

The sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assay were studied by means of artificially contaminated parenterals. Various gram-negative and gram-positive bacterial strains were used as was one strain of the yeast Candida albicans. The numbers of organisms needed to elicit positive responses in distilled water and normal saline were recorded and compared. The sensitivity and specificity of the Limulus amebocyte lysate assay for the detection of bacterial endotoxin from gram-negative bacteria were demonstrated. Variable results were recorded with gram-positive bacteria and Candida albicans.     

Comparison of direct serial dilution and most-probable-number methods for determining endotoxins in meats by the Limulus amoebocyte lysate test.

When the endotoxin content of ground beef was determined by direct serial dilution and by three-tube most-probable-number methods, the results were not significantly different, although the latter method provided more specific values for individual samples.     

Comparison of the Limulus amoebocyte lysate test with plate counts and chemical analyses for assessment of the quality of lean fish.

The quality of lean fish was assessed simply and rapidly with Limulus amoebocyte lysate. The endotoxin levels agreed with aerobic plate counts and chemical indices of spoilage. Correlation between level of endotoxin and level of total volatile bases was found to be highly significant (r = 0.8579; P less than 0.001).