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PubMed1985

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.

Animals↗
PubMed1989

Effect of some penicillins on the sensitivity of limulus amoebocyte lysate test.

A group of penicillins have been tested for their effect on the sensitivity of limulus amebocyte lysate test (LAL). Cloxacillin, cephalothin and cefuroxime inhibited the gel formation at concentrations above 2 mg mL-1, while ampicillin, methicillin and mecillinam showed no inhibitory effects upto concentrations of 10 mg mL-1. For benzylepenicillin the maximum non-inhibitory concentration was 10,000 units mL-1. However, the dilutions required to overcome inhibition were within the limits of maximum valid dilutions computed for each product showing that LAL test is valid for these products at their non-inhibitory concentrations.

Animals↗
PubMed1979

Limulus amebocyte lysate testing of nomral serum albumin (human) in the United States since 1975.

In order to determine the reactivity of Limulus Amebocyte Lysate to albumin products, over 1500 lots of 25% Normal Serum (Human) produced by 12 U.S. licensed manufacturers were tested over the past 4 years. Representative samples of 5% Normal Serum Albumin (Human) and Purified Protein Fraction (human) were also tested with Limulus Amebocyte Lysate. Problems associated with apparent false negative reactions are discussed. Model test protocols are presented that might be useful in determining the degree of false positivity of albumin products. These models could be adopted by blood fractionators to gather validation data to support substitution of the Limulus Amebocyte Lysate test for the rabbit pyrogen test.

Animals↗
PubMed1995

Application of the Limulus amoebocyte lysate test as an indicator of microbial contamination in pork carcasses.

The Limulus amoebocyte lysate (LAL) test, a simple and rapid method, was applied for the evaluation of bacterial contamination of carcasses in a slaughterhouse. Twenty-five pork carcasses were examined for bacterial contamination, and we evaluated the correlation between the LAL test and the methods usually employed for detecting indicator organisms of total viable cells, coliforms or Staphylococcus aureus. The reciprocal of the highest dilution found to be positive was used as the LAL index. A high correlation was observed between the LAL index and the number of coliforms, but not between the index and the number of other organisms. The LAL test was accomplished within 2 hr after sampling, and thus may serve as a useful tool for the substitute indicator of coliforms among bacterial contamination in carcasses.

Animals↗
PubMed1984

Immunodiagnosis of bacterial meningitis.

The quest for the search of rapid, cheap, easy to perform and highly sensitive and specific tests has resulted into the introduction and application of various new tests for the diagnosis of bacterial meningitis. Most of these tests are based on immunological principles, viz. counter-immunoelectrophoresis, latex agglutination test, co-agglutination, radio-immunoassay, haemagglutination inhibition as well as study of immune profile of cerebrospinal fluid. Apart from these certain non-immunological tests viz. Limulus amoebecyte lysate test, gas liquid chromatography, nitroblue tetrazolium dye reduction test have also been evaluated to make the laboratory diagnosis of this important clinical entity rapid and more reliable. These tests have been discussed and their current status presented.

Counterimmunoelectrophoresis↗
PubMedDate not supplied

The importance of the quality of water in Limulus Amebocyte Lysate tests.

Commercial pharmaceutical waters were evaluated regarding their interference in the chromogenic Limulus Amebocyte Lysate (LAL) test. Two of the four tested waters strongly inhibited the U.S. Reference Standard Endotoxin, while a Control Standard Endotoxin was less affected. The inhibitory effect could be mimiced by adding trace amounts of certain metal ions to a non-interfering water. In conclusion, the water used in the LAL test must be chosen with great care.

Endotoxins↗
PubMed2000

[The neutralizing effect of histidine-rich-polypeptides on LPS from suspected periodontal pathogens].

OBJECTIVE: Human parotid histidine-rich-polypeptides (HRPs) are a family of lowmolecular-weight, cationic polypeptides. HRP-1, HRP-3 and HRP-5 comprise 85%-90% of the total HRPs and are called major HRPs. There were many researches indicating the antimicrobial activities of HRPs. Recently, HRPs were reported to have an inhibitory action on the lipopolysaccharide (LPS) of E. coli, with HRP-5 being the most effective one among the three major HRPs. Since the LPS of oral gram-negative bacteria is thought to be one of the important etiological factors during the development of periodontal diseases, our experiment was aimed to investigate the neutralizing effect of human parotid HRP-5 on the LPS of anaerobic suspected periodontal pathogens, which have different chemical structures and biological activities compared with LPS of aerobic E. coli. METHODS: By using of preparative acid urea polyacrylamide gel electrophoresis (AU-PAGE), HRP-5 was purified from parotid saliva collected from healthy adults. Two stains of suspected periodontal pathogens, Porphyromonas gingivalis (P.g, 47-A) and Fusobacterium nuceatum (F. n, separated from subgingival plaque of a patient with adult periodontitis) were mass cultured. After harvested in the midlogarithmic phase, they were washed and lyophilized. The LPS of dried bacteria were extracted by the modified Westphal hot phenol-water procedures and purified by the enzyme digestion plus ultracentrifugation. Limulus test was applied to test the neutralizing effect of HRP-5 on the LPS-induced gelation of Limulus amoebocyte lysate. In brief, the standard LPS of E. coli, or extracted LPS of P. g or F. n, was preincubated with HRP-5 in a tube separately in room temperature for 10 minutes. Then the reagent of Limulus amoebocyte lysate was added in all the tubes, continued the incubation in 37 degrees C for one hour. After that, the gelation level of every tube was observed. RESULTS: LPS extracted from P. g and F. n both showed good purity and strong activities to induce gelation of Limulus amoebocyte lysate. The gelation induced by LPS (1 ng/ml) of these two anaerobic suspected periodontal pathogens were weakly inhibited by HRP-5 (10 micrograms/ml), similar to that observed with standard LPS of E. coli. To get a complete neutralizing effect on LPS, it may be important to increase the concentration of HRP-5. CONCLUSION: It appeared that HRPs could neutralize the endotoxic properties of LPS of suspected periodontal pathogens, therefore may contribute to periodontal health. The present investigation further confirmed that HRPs are important components of the host non-immune defense system.

Escherichia coli↗
PubMed1977

Detection of endotoxins in human blood and plasma. An improved in-vitro pyrogen test.

We describe an improved in-vitro procedure for detection of endotoxin in human blood and plasma by use of Limulus amoebocyte lysate. Increasing concentrations of Escherichia coli endotoxin added to a constant amount of the lysate cause a proportional increase in protein precipitated by the endotoxin. By measuring the amount of protein precipitated, it was possible to determine the equivalent E. coli endotoxin concentration in unknown samples, when samples were run with E. coli endotoxin standards and negative controls. The E. coli endotoxin, present in human whole blood and platelet-rich plasma, failed to react with the lysate. However, the concentration of endotoxin in whole blood and platelet-rich plasma could be measured with this Limulus test after lysing the platelets to release the endotoxin and subsequently removing the inhibitory proteins by chloroform precipitation. With this procedure it was possible accurately and repeatedly to determine E. coli equivalent endotoxin concentrations as low as 195 ng per liter of whole blood or 49 ng per liter of platelet-rich plasma.

Analysis of Variance↗
PubMed1981

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.

Autoanalysis↗
PubMed1973

Rapid detection of gram-negative bacteriuria by use of the Limulus endotoxin assay.

The Limulus in vitro endotoxin assay was evaluated as a possible method for the prompt detection of significant gram-negative bacteriuria in children. This assay is capable of detecting endotoxin associated with intact cell walls of viable gram-negative bacteria as well as free endotoxin. Quantitative results are obtained following a 1-h incubation of Limulus lysate and 10-fold dilutions of otherwise untreated urine. A standard curve of Limulus activity and viable cell counts of Escherichia coli and Klebsiella pneumoniae in urine demonstrated that a positive Limulus reaction at a dilution of 1:100 or 1:1,000 indicated a colony count of at least 100,000 bacteria/ml. A positive Limulus reaction only from undiluted urine or at a dilution of 1:10 indicated less than 100,000 cells/ml. These experimental observations were confirmed by comparing the Limulus test with quantitative plate counts on 209 urine specimens from a mixed pediatric population. These results indicate that the Limulus assay is a simple, accurate method for rapid presumptive detection of gram-negative bacteriuria in patients where an immediate diagnosis is needed. This test would also seem promising for screening large patient populations for bacteriuria or for monitoring the effectiveness of treatment of urinary tract infections.

Adolescent↗
PubMed1975

Opsonizing antibodies, host factors, and the limulus assay for endotoxin.

The role of endotoxin in the pathophysiology of human gram-negative rod bacteremia is controversial. Gelation of lysates prepared from the amebocytes of Limulus polyphemus is a highly sensitive means for quantifying endotoxin-like activity in vitro, but variable results have been reported in several clinical studies. We performed limulus tests on plasmas obtained simultaneously with culture-positive blood from 68 patients with gram-negative bacteremia and related results to heat-stable opsonizing activity against the autologous infection strain. Overall, limulus positivity was 52% in this series. Positive tests were observed with 78% of plasmas with opsonic titers is less than or greater to 1:20 but with only 27% of plasmas whose titers were is greater than or equal to 1:80 (P less than 0.005). There was a strong association of positive tests with leukopenia. thrombocytopenia, and more severe underlying disease. In vitro study showed a 10-fold reduction in the sensitivity of the gelation test by antibodies against the endotoxin used and an additional 10-fold reduction when the test system included phagocytic cells.

Antibodies, Bacterial↗
PubMed1981

Limulus amebocyte lysate test for endotoxemia: investigations with a femtogram sensitive spectrophotometric assay.

The question of specificity of Limulus amebocyte lysate (LAL) test in the diagnosis of endotoxemia has been a limiting factor of its clinical application. Using a femtogram-sensitive spectrophotometric LAL assay 35 of 36 septic postoperative patients showed an excellent correlation (almost 100%) between positive LAL tests and culture-proven gram-negative bacteremia. Twenty patients of this group demonstrated a significant correlation (p < 0.05) between elevated total white blood cell counts and Escherichia coli Endotoxin equivalents (EcEe). All 22 liver cirrhotic patients with potentially enteric endotoxemia yielded LAL positive reactions (100%) but 9 of 22 correlated significantly as to leucocytosis and elevated EcEe (2 p < 0.01). A significant correlation between EcEe and leucocyte counts was found for all neonates and all parturients respectively (2 p < 0.001), 2 p < 0.01). In vitro tests showed that leucocytes gave positive LAL tests. Supernates of a Ficoll sedimented crude leucocyte preparations, induced LAL positivity in a dose dependent manner, while all reagents per se used in the preparation, were negative. This study suggests that heat-labile factors from leucocytes apart from endotoxin are responsible for positive LAL reactions.

Adult↗
PubMed1995

Acute toxicity and depression of phagocytosis in vivo by liposomes: influence of lysophosphatidylcholine.

Small unilamellar phospholipid vesicles (liposomes), intended as drug carriers, have recently been demonstrated to reversibly depress phagocytic activity in rats when injected in a single high dose (2g of lipid per kg body weight) as revealed by the carbon clearance test. Depression of the phagocytic function was found to vary widely depending on the lipid used [M. Brandl et al., Pharm. Pharmacol. Lett., 4 (1) 1-4, 1994]. This study has now been extended in two directions: Firstly, liposomes made of the same type of lipid but different batches of raw material were compared in terms of their influence on phagocytosis as well as for their contents of impurities. The test revealed great variability of RES suppression between different batches of hydrogenated soy PC, whereas the reproducibility of the carbon clearance test was satisfactory with liposomes made of a single batch of raw material. Thin layer chromatographic analyses of the used phosphatidylcholines (PCs) and limulus tests on lipopolysaccharides revealed lysophosphatidylcholine (lysoPC) as the only impurity which showed parallels with the observed differences in phagocytosis. Secondly by "spiking" phosphatidylcholine with increasing amounts of lysoPC the latter could be proven to enhance RES depression by liposomes in a dose-dependent manner. At the same time a strong and dose-limiting increase in acute toxicity of PC vesicles was observed with increasing contents of lysoPC. However, in cholesterol-containing vesicles lysoPC-spiking did not significantly alter their behaviour, for lysoPC contents of up to 10%. Only PC/cholesterol-vesicles containing lysoPC contents as high as 15% provoked enhanced RES depression and toxicity compared to lysoPC-free vesicles. LysoPC and cholesterol in liposomes are known to play a destabilizing and stabilizing role respectively within liposomal bilayers which might influence recognition and uptake of vesicles by macrophages and thus modulation of phagocytosis.

Animals↗