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PubMed1987

The use of a quantitative assay in endotoxin testing.

By utilizing Limulus Amebocyte Lysate (LAL) and a chromogenic peptide substrate it is now possible to determine endotoxin concentrations quantitatively down to 10 EU/L (1 pg/mL) in a two stage assay. The optimal reaction conditions found for the two stages of the method, the endotoxin reaction with LAL and the measurement of the activation with the chromogenic substrate, are briefly described. The properties of the final kit reagents have also been investigated and the results are included. Finally, the usefulness of the present method is demonstrated by results obtained from testing therapeutical products as well as clinical plasmas. Some factors which may be critical in the performance of the assay are discussed.

Animals↗
PubMedDate not supplied

Collaborative study of a control standard endotoxin using the Limulus amebocyte (gel-clot) test.

A collaborative study was undertaken to determine the potency in endotoxin Units (EU) and International Units (IU) of a control standard endotoxin, LIF-1. Five laboratories from the Swedish Pharmaceutical Industry participated in the study. As reference preparations, two official standards, USP reference standard endotoxin, EC-5 (expressed in EU) and WHOs international standard endotoxin (expressed in IU), were used. The study was performed using the Limulus Amebocyte Lysate (LAL), gel-clot test. The test protocol included dilutions of the endotoxin in steps of 1:1.25 instead of the conventional 1:2 step dilution method. This gave more precise and standardized results. The content of one vial of LIF-1 (CSE) was calculated to be 22 EU and 16 IU which indicates that 1 EU (USP) corresponds to 0.7 IU (WHO).

Drug Contamination↗
PubMed1985

Vitamin B12-binding proteins of the horseshoe crab Limulus polyphemus.

Vitamin B12-binding proteins were detected in the body fluids and/or tissues of horseshoe crabs (Limulus polyphemus), clams and sponges. Among the biological specimens tested the Limulus plasma was especially rich in free B12-binding proteins. Gel filtration experiments revealed that Limulus plasma contains two classes of B12-binding proteins. One class of proteins, molecular weight in excess of 100,000, bind B12 preferentially with affinity constant of 5 X 10(11)M-1. The second type of proteins, molecular weights around 50,000, bind B12 with specificity approaching that of mammalian intrinsic factors. The binding constant of these proteins for B12 is around 10(11)M-1.

Animals↗
PubMed1983

Detection of endotoxin in antibiotic solutions with Limulus amoebocyte lysate.

Twenty-eight antibiotics were tested with the Limulus amoebocyte lysate assay to determine their non-inhibitory concentrations (NICs). The Limulus amoebocyte lysate assay was found to be a valid test for most of the antibiotics tested; the NICs were found to be greater than the minimum valid test concentrations. Borderline results were obtained with cefamandole nafate and neomycin sulfate. Polymyxin B and colistimethate contained too much endotoxin to permit determination of NICs. The NIC of tetracycline hydrochloride was dependent on the initial concentration of antibiotic. This dependence was most likely caused by the amount of base required to adjust the pH before testing.

Animals↗
PubMed2001

Endotoxin-neutralizing capacity of serum from cardiac surgical patients.

OBJECTIVE: To determine if endotoxin core antibody (EndoCAb) from the serum of cardiac surgical patients neutralizes endotoxin in an ex vivo biologic assay. DESIGN: Prospective blinded cohort study. SETTING: Academic medical center. PARTICIPANTS: Patients (n = 203) undergoing cardiac surgery. INTERVENTIONS: Sera were obtained from patients preoperatively. MEASUREMENTS AND MAIN RESULTS: EndoCAb levels were determined by enzyme-linked immunosorbent assay. Sera were incubated for 15 minutes at 37 degrees C with varying concentrations of endotoxin from a clinically relevant bacterium (Escherichia coli serotype O18), then tested for the presence of endotoxin activity using the validated Limulus amebocyte lysate assay. Median (interquartile range) IgM and IgG EndoCAb levels were 118 median units (range, 31 to 259 median units) and 208 median units (range, 108 to 401 medium units). Increasing levels of IgM EndoCAb were associated with increased neutralization of endotoxin (p < 0.0001). Increasing levels of IgG EndoCAb were associated with increased neutralization of endotoxin (p < 0.0001). An additive effect of IgM and IgG EndoCAb levels on endotoxin neutralization was observed without evidence of synergistic or plateau effects. EndoCAb levels did not completely predict serum neutralization capacity. CONCLUSION: Anti-EndoCAbs of both classes (IgM and IgG) were able to neutralize lipopolysaccharide from a clinically relevant bacterium in an ex vivo model. Neither Igm nor IgG appeared to be more capable of neutralization in this model. These antibodies did not completely predict neutralization capacity; other endogenous factors in human serum must be capable of lipopolysaccharide neutralization.

Antibodies, Bacterial↗
PubMed2004

Hypoxemic reperfusion after 120 mins of intestinal ischemia attenuates the histopathologic and inflammatory response.

OBJECTIVE: It has been suggested that reactive oxygen species play a pivotal role in the initial organ-tissue injury during reperfusion, eliciting inflammatory reaction and multiple organ failure. It was investigated if hypoxemic reperfusion attenuates tissue injury and inflammatory response. DESIGN: Randomized animal study. SETTING: Medical school laboratory. SUBJECTS: Twenty-five male pigs weighing 25-28 kg. INTERVENTIONS: Pigs were subjected to 120 mins of intestinal ischemia by clamping the superior mesenteric artery. Upon declamping, the animals were randomly assigned to receive either hypoxemic reperfusion (HR group, n = 9) reperfused with a Pao2 = 30-35 or normoxemic reperfusion (control group, n = 16) reperfused with a Pao2 = 100 mm Hg for 120 mins. Fluids without inotropes were given to combat circulatory shock during reperfusion. MEASUREMENTS AND MAIN RESULTS: Portal blood and intestinal and lung biopsies were collected at baseline, end of ischemia, and end of reperfusion. Histopathologic changes were scored, and interleukin-1beta, qualitative Limulus amebocyte, lysate test, and Pao2/Fio2 were measured. Eight of 16 animals of the control group and seven of nine of the HR group survived (p = .22). At the end of reperfusion, the intestinal (p = .004) and lung (p = .028) pathologic scores were lower in the HR group compared with controls. The only significant difference in concentration of interleukin-1beta in the portal blood between the two animal groups occurred 120 mins after reperfusion (p = .006). The number of HR animals with a positive Limulus test was significantly smaller compared with controls at 60 (p = .041) and 120 (p = .07) mins of reperfusion. During the period of ischemia, the Pao2/Fio2 decreased similarly in the control and HR group, whereas after 120 mins of reperfusion the rate was significantly higher in the HR group. CONCLUSIONS: Hypoxemic reperfusion represents an intervention that may attenuate the triggering of multifactorial cascade and organ tissue injury.

Animals↗
PubMed1988

Assay of pyrogenic contamination in pharmaceuticals by cytokine release from monocytes.

In 1984 it was reported that a batch of human recombinant growth hormone which had passed the rabbit pyrogen test and a Limulus amoebocyte lysate (LAL) test was pyrogenic in man (1). Further, it was shown that the growth hormone caused human monocytes to release leukocyte pyrogen which evoked fevers in rabbits (1). Leukocyte pyrogen is believed to comprise of a mixture of (at least) 2 lymphokines with potent pyrogenic and inflammatory activities, interleukin-1 (IL-1) and Tumour Necrosis Factor (TNF), which are synthesized and released by activated peripheral blood monocytes. The development at NIBSC of sensitive and specific bioassays and immunoassays of IL-1 (2) and TNF (3) have permitted a new type of pyrogen test based on the in vitro release by human monocytes of the lymphokines IL-1 and TNF. Studies are underway to evaluate the suitability of monocytic cell lines, rather than human monocytes, since these require a time-consuming and labour intensive preparation. Investigations are in progress to evaluate the suitability of the above "monocyte test" for pyrogen to replace or complement the rabbit pyrogen test which is routinely applied to biological medicines. The new method has already proved useful in detecting pyrogenic contamination of a large volume parenteral medicinal product which passed the rabbit pyrogen test but caused adverse reactions in patients.

Cells, Cultured↗
PubMed1984

[Endotoxins in gastroenterology--a medico-historical sketch].

First descriptions of effects of intestinal bacterial endotoxins date from the middle of the 19th century (P. L. Panum et al.). The antitoxic function of the liver has been investigated by I. P. Pawlow in 1893. At the turn of the last century the theory of "auto-intoxication" (C. Bouchard, I. I. Metschnikoff et al.) was well known, but there were also first systematic studies of the facultatively pathogenic intestinal bacteria (T. Escherich, H. Tissier, J. Strasburger). In the twenties of our century V. van der Reis and L. Bogendoerfer worked out important fundamentals of human gastrointestinal microecology. Endotoxins as component of cellular wall of gram-negative bacteria are found by A. Boivin et al., J. W. Walker et al. First applicable proof for the detection of endotoxins was the pyrogen test with rabbits. The Limulus amoebocyte lysate test (J. Levin and F. B. Bang) has been employed as a more simple, rapid and sensitive method and was introduced in gastroenterology in a larger extent. Connections between endotoxinaemia and liver diseases, effects of endotoxins on gastrointestinal mucosa and on the course of shock are subjects of actual investigations.

Endotoxins↗
PubMed1996

Inhibition of mouse liver cytochrome P-450 by gram-negative bacteria lipopolysaccharides.

The ability of bacterial endotoxins, of different origin, to modify the level of mouse liver cytochrome P-450 was investigated. Endotoxins, (lipopolysaccharides, LPSs) were isolated from Proteus, Escherichia, Salmonella, Bacteroides and Coxiella strains. The most potent inhibitor of cytochrome P-450 activity was S. typhi 0101 LPS, which at a dose of 1 microgram/mouse reduced the cytochrome P-450 activity to 59%. E. coli O55:B5, S. typhimurium, P. mirabilis O3, and C. burnetii LPSs, at dose 10 micrograms/mouse, decrease cytochrome P-450 level from 56 to 69%. B. ovatus LPS significantly suppressed the expression of cytochrome P-450 only at the highest dose used-100 micrograms/mouse. The comparison of inhibitory activity of P. mirabilis complete, S and R types of LPSs indicate that lipid A portion of LPSs are sufficient to decrease the cytochrome P-450 level. However, the core oligosaccharide of LPS significantly enhance that inhibition. The isolated O-specific polysaccharide part of P. mirabilis O3 LPS did not decrease cytochrome P-450 level. The comparison of biological activity of Proteus LPSs, tested by chromogenic Limulus amebocyte lysate (LAL) test, demonstrated the enhancement effect of O-polysaccharide part of tested LPSs.

Animals↗
PubMed1999

A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing.

Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS) and Staphylococcus aureus was comparable to that of MNC. Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6. The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay. As expected, S. aureus and C. albicans did not show any LAL activity. A. niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells. In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S. aureus and S. typhimurium, but the MM6 assay could not detect A. niger, C. albicans and Influenza virus.

Biological Assay↗
PubMed1986

Endotoxin-like activity associated with Lyme disease Borrelia.

The newly recognized spirochete, Borrelia burgdorferi, the causative agent of Lyme Disease, has been examined for endotoxin-like activities as measured by the standard Farmacopea Ufficiale della Republica Italiana rabbit fever test and the Limulus amoebocyte lysate assay. The suspension of heat-killed microorganism caused a febrile response at a dose of 1 X 10(8) bacteria pro kilo. Similar results were obtained in the Limulus assay where the heat-killed spirochetes stimulated formation of solid clot until the concentration of 1 X 10(5) per ml. Both in pyrogen test and in Limulus assay heat-killed Escherichia coli exhibited a higher degree of potency. These results show that LD-Borrelia possess endotoxin-like activities which could help in understanding the pathogenesis of the clinical symptomatology of the disease.

Animals↗
PubMed1982

Endotoxemia and hyperbilirubinemia in the neonate.

One hundred twenty-five neonates with varying serum bilirubin levels were tested for endotoxin by the limulus amebocyte lysate (LAL) test. Neither infection nor hemolytic disease was a contributing factor to the bilirubin levels. As the serum bilirubin level rose, positive LAL tests increased in frequency until the LAL test attained 100% at a level of 13 mg/dL. Bilirubin at different concentrations did not elicit positive LAL tests in plasma or normal saline in vitro. The LAL test was positive in urine obtained by suprapubic aspiration in 50% of neonates with positive serum LAL tests. Although bilirubin and endotoxin are cleared independently by hepatic cells with different functions, a striking relationship is evident between the endotoxin and bilirubin levels in the neonate. Immaturity of physiological liver functions in the neonate plays an important role. The LAL test cannot be used as an indicator of Gram-negative sepsis in neonates with unconjugated hyperbilirubinemia.

Bilirubin↗