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PubMed1975

Use of limulus assay to compare the biological activity of peptidoglycan and endotoxin.

The gelation of a lysate prepared from the amebocytes of Limulus polyphemus, the horseshoe crab, has been used to detect endotoxin-like material in clinical studies and in "in vitro" experiments. The investigation of blood samples from 54 hospitalized patients suspected of having endotoxemia, revealed a positive limulus test in 14. Infections due to gram-positive organisms were not associated with positive assays. These results were in agreement with the observation that living gram-positive microorganisms - in contrast to gram-negative bacteria - did not initiate lysate gelation when studied "in vitro". Only very high concentrations of peptidoglycan, isolated from the cell walls of various gram-positive bacteria, induced the reaction. Therefore, these findings support the view, that the limulus test is relatively specific for the detection of gram-negative bacterial endotoxin and endotoxemia.

Animals↗
PubMedDate not supplied

[Research of the soluble microbial substances in organic fluids for the rapid diagnosis of some infections and particularly of bacterial meningitis (author's transl)].

A number of immunological and non-immunological techniques have been recently used to detect soluble microbial substances in body fluids of patients with acute meningitis, bacteremia, and lobar pneumonia. By the immunological methods capsular highly polymerized polisaccharide group- or type-specific antigens of the most common C. N. S. pathogens (N. meningitidis A, B, and C; Str. pneumoniae, H. influenzae type b, E. coli K1, mucoid Pseudomonas, Cryptococcus neoformans) can be detected and quantitated in spinal fluids, sera, urine and other fluids specimens from meningitic patients. Capsular type-specific antigens from pneumococcus, and likely from H. influenzae as well, can be detected in sputum from patients with lower respiratory infection. Among the various techniques, the radioimmunoassay appears as the most sensitive one, but high diagnostic sensitivity can be also achieved by using the latex agglutination, haemoagglutination inhibition and coagglutination tests. Counterimmunoelectrophoresis, however, is still the far most used technique for determining soluble microbial antigens, albeit its sensitivity is significantly less than the one of the above mentioned methods. High specificity and some advantages in serotyping the causal organisms are probably the main reasons of such preferential employment. Among the non-immunological techniques the evaluation of lactate and lactic dehydrogenase has been used by some Author for differentiating between bacterial and non bacterial meningitis, and the limulus test for detecting Gram-negative bacterial endotoxins with a high degree of sensitivity and specificity. Finally, the liquid gas chromatography has been evaluated in detection of some organic products (microbial?), such as acids, amines, neutral compounds, in spinal fluid, allowing the differential diagnosis between bacterial, tuberculous, viral, and cryptococcal meningitis. In the present review sensitivity, specificity, and other properties of each test alone and in comparison with the conventional microbiological methods (Gram and culture) are evaluated and the biological and pathogenic role and significance of the soluble microbial antigens and endotoxin are discussed.

Agglutination Tests↗
PubMedDate not supplied

Testing medical disposables using the Limulus Amoebocyte Lysate (LAL) test.

Plastic, single-use devices intended for the administration of drugs or for the removal or transfer of body fluids must be free of pyrogenic contaminants. Unfortunately, very little information is available regarding tests to detect such contaminants. For this reason, many manufacturers of medical disposables must rely on their own discretion when they devise methods for extracting these substances from their products. This article discusses the use and value of the Limulus Amoebocyte Lysate (LAL) test for estimating the concentration and extraction of bacterial endotoxins in disposable medical devices. When bioburden-control procedures are applied to products manufactured to GMP requirements, batch testing of products for pyrogenicity may not be essential.

Animals↗
PubMed1980

Circulating immune complexes in cystic fibrosis.

Recurrent respiratory infections associated with "mucoid" Pseudomonas aeruginosa characterize the advanced stages of cystic fibrosis. To determine if chronic antigenic stimulation is associated with circulating immune complexes (CIC), we assayed the sera of 20 hospitalized patients using the technique of precipitation with 4% polyethylene glycol. Elevated CIC levels, defined by > 310 micrograms IgG per ml, were found in 18 of 20 patients, (range, 350 to 3200 micrograms/ml). Serum, supernatant, and resuspended precipitates were assayed for hemagglutinating antibodies against pseudomonas lipopolysaccharide (LPS or endotoxin) and exotoxin A antigens. Both serum anti-LPS (range, 1:64 to 1:2048) and antitoxin titers (range, 1:64 to 1:16, 384) were markedly elevated and higher than titers in supernatants and resuspended precipitates, indicating antibody excess. "Enrichment" ratios for antibodies present in CIC were calculated by proportion of titer to immunoglobulin in the precipitated complex relative to these values in serum. Mean enrichment ratios of 13.1 and 13.9 were obtained for LPS antibody before and after 2 mercaptoethanol reduction, but the mean enrichment ratio for antitoxin was only 2.07. Serially diluted supernatants and precipitates were boiled for 1 hr and tested for endotoxin-like activity by the limulus test. At > 1:8 dilutions, precipitates were positive, and supernatants were negative. These findings indicate that CIC's are common in advanced cystic fibrosis, and analysis of the precipitated complexes demonstrates significant (> 13-fold) enrichment of antibodies against LPS but not exotoxin antigens, as well as endotoxin-like activity in boiled precipitates.

Antibodies↗
PubMedDate not supplied

The effect of refrigeration and mixing on detection of endotoxin in parenteral drugs using the Limulus Amebocyte Lysate (LAL) test.

Prior to testing for the presence of bacterial endotoxin, parenteral products are handled and stored in a variety of ways. Two incidents, detected by the U.S. Food and Drug Administration, revealed that differences in product handling and storage may have played a role in causing analytical discrepancies in the testing of identical samples. The testing procedure was the USP Bacterial Endotoxin test using Limulus Amebocyte Lysate (LAL) reagent. Consequently, an evaluation was made at the two principal factors that contributed to the suspected analytical anomaly. The factors were sample storage and the degree of agitation prior to sample analysis. Additional variables such as bacterial growth medium and adsorption potential of endotoxin by rubber stoppers were also evaluated. It was found that neither the medium employed to grow the E. coli endotoxin nor the storage temperature of the spiked solutions were problematic. However, it was shown that 20-40% of the spiked endotoxin was lost due to non-agitation of solution in vials in which the solution was in contact with the rubber stoppers. A suggested remedy for this problem is to store intact product containers in an upright position and to establish a uniform mixing procedure prior to endotoxin assay.

Endotoxins↗
PubMed1982

Pertussis antigens--screening models on toxicity.

From the same batch of B. pertussis bacteria two types of DPT-vaccines were produced after harvest of the inactivated organisms by centrifugation or acid precipitation. The first vaccine contained whole pertussis bacteria and the second an extracted antigen complex. In a screening programme with reliable animal tests, such as a modified mouse-weight-gain assay, mouse leukocytosis test, histamine sensitization in mice, allergic encephalitis in rats, and the limulus test it could be demonstrated that the vaccine with whole inactivated bacteria was more toxic than that with extracted antigens. Cell material harvested by centrifugation showed a lower rate of toxicity than that containing acid-precipitated pertussis organisms.

Animals↗
PubMed1999

Chemical and immunochemical characterization of limulus factor G-activating substance of Candida spp.

The limulus test is a well-established method for the diagnosis of both gram (-) sepsis and invasive fungal infection. To diagnose deep-seated fungal infections, a (1-->3)-beta-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. It is suggested that the limulus reactive substance was released from the fungi to the blood, however, its chemical properties were not precisely examined in detail because of the limited quantity available. In this study, we used chemically defined liquid medium to culture Candida spp. and collected the water soluble fraction, CAWS. The yield of CAWS was circa 100 mg/l, independent of the strain of Candida. CAWS reacted with limulus factor G (Fungitec G test MK) at concentrations as low as 100 ng/ml. Limulus factor G reactivity of CAWS was sensitive to (1-->3)-beta-glucanase, zymolyase and was, at least in part, bound to ConA-agarose. The ConA-bound fraction also reacted with anti-beta-glucan antibody. CAWS is mainly composed of mannan and (1-->6)-beta-glucan, in addition to protein, assessed by 1H-NMR spectroscopy. CAWS also reacted with typing sera of Candida spp., specific for cell wall mannan. Chemical, immunochemical and biochemical analyses of CAWS strongly suggested that the limulus factor G-activating substance was a mannan-beta-glucan complex, present within the architecture of the yeast cell wall.

Candida↗
PubMed1990

Endotoxin measurement: aerosol sampling and application of a new Limulus method.

A new Limulus test was designed specifically for measuring endotoxin in environmental aerosols. The new Limulus method has a detection limit for airborne endotoxin of 2.2 pg (NP-1 activity)/m3 and can precisely quantitate aerosols containing 14 or more pg/m3 from samples representing 6.5 m3 of air. Aerosols in the range 100 to 500 pg/m3 were measured with 95% confidence of +/- 32% and an aerosol of 10 pg/m3 with 95% confidence of +/- 50%. Qualitative information about airborne endotoxin was also obtained from the assay. A wide variety of filter media were found to inactivate lipopolysaccharide in solution. This implies that airborne endotoxin can be measured only relative to the conditions of a particular study and that comparison of endotoxin aerosol measurements made under different circumstances are invalid. The research and policy implications of this observation are discussed. The need for improved collection and extraction methods notwithstanding, it was proposed that a buffered, parallel-line Limulus assay method be adopted as the standard method for measuring environmental endotoxin. The kinetic-turbidimetric Limulus assay with resistant-parallel-line estimates (KLARE), rate response method, should be considered a prime candidate for the standard method because of its precision, sensitivity, resistance to interference by pH, internal validation of estimates, and ability to provide qualitative as well as quantitative information about airborne endotoxin.

Aerosols↗
PubMed1989

A modified Limulus amebocyte lysate test with increased sensitivity for detection of bacterial endotoxin.

We have developed a simple modification of the chromogenic Limulus amebocyte lysate test that increases the sensitivity for the detection of bacterial endotoxins. In this assay, free paranitroaniline, cleaved from synthetic chromogenic substrates by proteases that were generated by Limulus lysate after incubation with endotoxin, was then derived. Derivation was with p-dimethylaminocinnamaldehyde in the presence of strong acid, forming a stable Schiff base end product with much greater molar absorbancy than the parent chromogen. Conditions (times and temperatures of incubations, concentrations of reagents) for the augmented chromogenic procedure were optimized. A ten-fold or greater increase in sensitivity for bacterial endotoxin was obtained with the modified assay as compared with the standard chromogenic Limulus test, with unequivocal detection of endotoxin concentrations of less than 100 pg/ml. The greater sensitivity of this modified Limulus test increases its usefulness for a wide range of research applications and clinical investigations.

Colorimetry↗
PubMed1979

Stem cell migration induced by erythropoietin or haemolytic anaemia: the effects of actinomycin and endotoxin contamination of erythropoietin preparations.

The injection of erythropoietin or the induction of anaemia with phenylhydrazine leads to changes in murine pluripotent and granulocyte-macrophage stem cells indicating migration from marrow to spleen. In order to evaluate the interrelationship between erythroid differentiation and stem cell migration we have selectively suppressed erythroid differentiation with actinomycin D. Anaemia or EP injection resulted in stem cell changes consistent with migration; actinomycin blocked these changes in anaemic but not EP injected mice while blocking erythropoiesis in both groups. The erythropoietin contained from 0.01 to 1000 microgram/ml of endotoxin as defined by the limulus test; it decreased marrow erythropoiesis and stimulated marrow granulopoiesis. Adsorption of the erythropoietin preparation with limulus lysate removed endotoxin without decreasing erythropoietin activity. Adsorbed erythropoietin stimulated erythropoiesis and not granulopoiesis, and stem cell changes induced by its administration were largely blocked by actinomycin, suggesting that endotoxin in the non-adsorbed erythropoietin caused the actinomycin resistant stem cell changes. The observation that actinomycin blocks both erythroid differentiation and stem cell migration suggests that these two physiologic events are closely linked. The effects of injected erythropoietin on murine haemopoietic stem cells may, to a significant extent, be secondary to the presence of endotoxin in the erythropoietin preparations.

Anemia, Hemolytic↗
PubMed1987

Use of the Limulus amebocyte lysate test as a cost-effective screen for gram-negative agents of meningitis.

A retrospective review of data from two institutions demonstrated that the Limulus amebocyte lysate test was a simple and cost-effective means to screen cerebrospinal fluids (CSF) for Gram-negative agents of meningitis. Results of either gelation Limulus amebocyte lysate (LAL) or chromogenic Limulus amebocyte lysate (CLAL) tests on 1504 CSF were evaluated along with results of antigen detection tests [ADT; either latex agglutination (LA) or counterimmunoelectrophoresis (CIE)], cultures, and Gram-stains. All 127 CSF positive for Haemophilus or Neisseria by culture and/or ADT were also positive by Limulus test (100%). The sensitivities of Gram stain examination, culture, LA, and CIE for these two pathogens were 81%, 91%, 86%, and 63%, respectively. The Limulus test on lumbar CSF was positive in 67% of cases with other Gram-negative bacillary meningitis, in 33% of cases with Gram-negative bacillary abscess or ventriculitis, in none of the cases with Gram-positive, mycobacterial, treponemal, fungal, or viral meningitis, and in 1% of cases with either normal or otherwise negative bacteriologic findings. Overall, sensitivity and specificity of the Limulus test were 97% and 99%, respectively. The cost of either version of the Limulus test was less than 15% of the cost of ADT batteries for Haemophilus and Neisseria. Based on its sensitivity for Haemophilus and Neisseria in CSF, we propose its use as a cost-effective screen to minimize the need for the more expensive ADT batteries.

Adult↗
PubMed1993

[The quantitative assay of (1-->3)-beta-D-glucan in culture medium of Candida albicans using G-test].

It has been difficult to diagnose the deep-seated fungal infection. Limulus test which originally has been developed to detect endotoxin in blood is also activated by (1-->3)-beta-D-glucan, the cell wall component of the fungi. Factor G in limulus lysate is activated by (1-->3)-beta-D-glucan and not by endotoxin. The quantitative assay of (1-->3)-beta-D-glucan is possible by the G-test using factor G. (1-->3)-beta-D-glucan in RPMI culture medium of Candida albicans was periodically measured using G-test and the effect of antifungal drug or neutrophil to the changes of (1-->3)-beta-D-glucan in the culture medium was studied. Increase in the level of (1-->3)-beta-D-glucan was in parallel with the growth of Candida albicans. G-test may be applied to the clinical diagnosis of fungal infection.

Candida albicans↗
PubMed1978

Survey for positive Limulus amoebocyte lysate test in plasma from humans and common research animals.

A survey for positive Limulus amoebocyte lysate tests was conducted on apparently healthy humans, mongrel dogs, rats, mice, rabbits, and squirrel monkeys. Only mongrel dog (45.8%) and human (32.8%) plasma samples gave positive tests. In dogs, a significant correlation between positive Limulus amoebocyte lysate tests and the presence of intestinal parasites was found. Positives found in human plasma samples were thought to be due to the presence of background levels of endotoxin or some possible mimicker substance found in the plasma after chloroform extraction. It was concluded that there was a need to distinguish between these positive Limulus tests and those which represent significant endotoxemia.

Animals↗
PubMed1983

A rapid method to produce a sensitive Limulus amoebocyte lysate (LAL). I. Evaluation of inter and intra batch differences in LAL and hemolymph from Limulus polyphemus.

An improved method for preparing Limulus amoebocyte lysate (LAL) is described and compared with two other well-known procedures. In five Limuli, three different bleeding procedures and three different cell rupture methods were studied. Increased sensitivity of LAL was accomplished by avoidance of the anticoagulant N-ethylmaleimid (NEM) in the bleeding procedure, optimal methods for mechanical cell rupture and use of pyrogen free conditions. Reaction of LAL with as little as 10(-15) gram Lipopolysaccharide (LPS) per ml was generally obtained. To evaluate a possible explanation for the differences in reactivity of LAL between different Limuli, found in one experiment, six Limuli were bled, and the pH and the concentration of protein, Ca2+, Mg2+, Cu2+, Na+ and K+ were determined in LAL and cell-free hemolymph. Inter and intra batch variations were found in LAL, but there was no correlation between the sensitivity of LAL and the content of the above mentioned constituents in LAL and cell-free hemolymph. Experiments with use of NEM in the bleeding procedure and addition of NEM to homogenized cells in different concentrations, showed that NEM inhibits the reactivity of LAL to LPS. It is concluded that the modified method of producing LAL by bleeding without NEM and by using optimal methods for mechanical cell rupture is quick, simple and produces a very sensitive reagent for the Limulus test.

Animals↗