Endotoxin recovery using limulus amebocyte lysate (LAL) assay

Biologicals ◽  
2016 ◽  
Vol 44 (5) ◽  
pp. 434-440 ◽  
Author(s):  
Jay S. Bolden ◽  
Rob E. Warburton ◽  
Robert Phelan ◽  
Marie Murphy ◽  
Kelly R. Smith ◽  
...  
Keyword(s):  
Limulus Amebocyte Lysate ◽  
Lal Assay

Endotoxins in Ophthalmic Viscosurgical Devices

2003 ◽  
Vol 13 (2) ◽  
pp. 176-184 ◽  
Author(s):  
H. Burkhard Dick ◽  
A.J. Augustin ◽  
T. Pakula ◽  
N. Pfeiffer
Keyword(s):  
Positive Sample ◽  
Gram Negative Bacteria ◽  
European Pharmacopoeia ◽  
Limulus Amebocyte Lysate ◽  
Dilution Series ◽  
Sample Extract ◽  
Immunological Reactions ◽  
Negative Controls ◽  
Lal Assay

Purpose To measure the endotoxin concentration (EC) of 25 commercially available, hyaluronic acid- and hydroxypropylmethylcellulose-based (HPMC) ophthalmic viscosurgical devices (OVDs). Methods The in vitro Limulus amebocyte lysate (LAL) assay, which indicates the presence of endotoxins originating from gram-negative bacteria, was used to determine the EC. The procedure was performed according to the European Pharmacopoeia/USP. EC including duplicate determinations, negative controls, dilution series with control standard endotoxin, dilution series with sample extract and positive sample control. Results 16 OVDs (Amvisc®, Amvisc® Plus, Biolon®, Coatel®, Healon®, Healon® GV, Healon®5, HPMC Ophtal® L, Microvisc®, Microvisc® Plus, Ocucoat®, Provisc®, Rayvisc®, Viscoat®, Visco Shield® 2%, Visko® 1.4%) had an EC under 1.2 endotoxin units/mL, five (Adatocel®, HPMC Ophtal® H, LA Gel®, Viscorneal®, Viscorneal® Plus) had an EC ≥ 1.2 and ≤ 24 EU/ml, and four (Biocorneal®, Dispasan® also named Ophthalin, Dispasan® Plus, Visko® 1%) had an EC of > 24 EU/ml. Discussion To avoid viscoelastic-related inflammatory or immunological reactions, the use of pure OVDs is recommended, especially for surgical procedures with an inherent possibility of leaving viscoelastic remnants in the eye (e.g., cataract surgery, viscocanalostomy or penetrating keratoplasty).

Concordance of EndotoxemiaWith Gram-Negative Bacteremia

2000 ◽  
Vol 124 (8) ◽  
pp. 1157-1164 ◽  
Author(s):  
James C. Hurley
Keyword(s):  
Contingency Table ◽  
Operating Characteristic ◽  
Meta Analysis ◽  
Sepsis Syndrome ◽  
Inclusion Criteria ◽  
Limulus Amebocyte Lysate ◽  
Gram Negative ◽  
Study Selection ◽  
Gram Negative Bacteremia ◽  
Lal Assay

Abstract 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.

scholarly journals Modification of sample processing for the Limulus amebocyte lysate assay enhances detection of inflammogenic endotoxin in intact bacteria and organic dust

2017 ◽  
Vol 23 (3) ◽  
pp. 307-318 ◽  
Author(s):  
Kimberly A Hoppe Parr ◽  
Suzana Hađina ◽  
Brita Kilburg-Basnyat ◽  
Yifang Wang ◽  
Dulce Chavez ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Specific Activity ◽  
Organic Dust ◽  
Limulus Amebocyte Lysate ◽  
E Coli ◽  
Pre Treatment ◽  
Dose Dependent ◽  
Lal Assay ◽  
Endotoxin Content ◽  
Limulus Amebocyte Lysate Assay

The pro-inflammatory potency and causal relationship with asthma of inhaled endotoxins have underscored the importance of accurately assessing the endotoxin content of organic dusts. The Limulus amebocyte lysate (LAL) assay has emerged as the preferred assay, but its ability to measure endotoxin in intact bacteria and organic dusts with similar sensitivity as purified endotoxin is unknown. We used metabolically radiolabeled Neisseria meningitidis and both rough and smooth Escherichia coli to compare dose-dependent activation in the LAL with purified endotoxin from these bacteria and shed outer membrane (OM) blebs. Labeled [14C]-3-OH-fatty acids were used to quantify the endotoxin content of the samples. Purified meningococcal and E. coli endotoxins and OM blebs displayed similar specific activity in the LAL assay to the purified LPS standard. In contrast, intact bacteria exhibited fivefold lower specific activity in the LAL assay but showed similar MD-2-dependent potency as purified endotoxin in inducing acute airway inflammation in mice. Pre-treatment of intact bacteria and organic dusts with 0.1 M Tris-HCl/10 mM EDTA increased by fivefold the release of endotoxin. These findings demonstrate that house dust and other organic dusts should be extracted with Tris/EDTA to more accurately assess the endotoxin content and pro-inflammatory potential of these environmental samples.

scholarly journals Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test

ALTEX ◽  
2021 ◽  
pp. 3-19
Author(s):  
Thomas Hartung
Keyword(s):  
Whole Blood ◽  
Implementation Process ◽  
Monocyte Activation ◽  
Limulus Amebocyte Lysate ◽  
25Th Anniversary ◽  
Safety Testing ◽  
Cell Therapies ◽  
Horseshoe Crabs ◽  
One Year ◽  
Lal Assay

The whole blood pyrogen test invented 25 years ago, and its variant based on cryo-preserved blood one year later, brought momentum into the field of pyrogen testing, which, despite the broad application of the Limulus amebocyte lysate (LAL) assay, aka bacterial endotoxin test (BET), consumed several hundred thousand rabbits per year world-wide. The resulting international validation and lengthy acceptance and implementation process of what are called now monocyte activation tests (MATs) finally is impacting on animal numbers – at least in Europe – reducing them by more than 70% and counting. The author sees no reason for continuing any regulatory rabbit testing for pyrogens except the lack of acceptance of MATs in some regions of the world. The availability of MATs has opened also the discussion about the shortcomings of LAL/BET, namely its restriction to Gram-negative pyrogens, non-reflection of the potency of these in humans, interference and masking by many products, and animal welfare concerns for horseshoe crabs. The obvious advantages of MATs in all these respects should lead to a shift from LAL/BET to MATs. We are starting to see this for vac­cines and medical devices, but other areas like safety testing of blood transfusions, cell therapies and nanomaterials, and the assessment of air-borne pyrogens still need to grasp the opportunity provided by MATs. While the different MATs can jointly serve these needs, the whole blood MAT has some advantages as discussed here.

Interference of Gold Nanoparticles with In vitro Endotoxin Detection Assays

2020 ◽  
Vol 16 (2) ◽  
pp. 204-213 ◽  
Author(s):  
Melissa A. Vetten ◽  
Mary Gulumian
Keyword(s):  
Gold Nanoparticles ◽  
Gold Nanoparticle ◽  
Surface Reactivity ◽  
In Vitro Assays ◽  
Limulus Amebocyte Lysate ◽  
Chromogenic Assay ◽  
Nanoparticle Suspensions ◽  
Endotoxin Contamination ◽  
Factor C

Background: Endotoxin-free engineered nanoparticle suspensions are imperative for their successful applications in the field of nanomedicine as well as in the investigations in their toxicity. Gold nanoparticles are known to interfere with various in vitro assays due to their optical properties and potential for surface reactivity. In vitro endotoxin testing assays are known to be susceptible to interference caused by the sample being tested. Objective: This study aimed to identify a preferred assay for the testing of endotoxin contamination in gold nanoparticle suspensions. Methods: The interference by gold nanoparticles on three assays namely, the commonly used limulus amebocyte lysate chromogenic assay, the limulus amebocyte lysate gel-clot method, and the less common recombinant Factor C (rFC) assay, was tested. Results: Possible interference could be observed with all three assays. The interference with the absorbance- based chromogenic assay could not be overcome by dilution; whilst the qualitative nature of the gel-clot assay excluded the possibility of distinguishing between a false positive result due to enhancement of the sensitivity of the assay, and genuine endotoxin contamination. However, interference with the rFC assay was easily overcome through dilution. Conclusion: The rFC assay is recommended as an option for endotoxin contamination detection in gold nanoparticle suspensions.

scholarly journals Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test

2021 ◽  
Vol 22 (4) ◽  
pp. 2135
Author(s):  
Takashi Kanno ◽  
Changmin Kim ◽  
Daisuke Yamanaka ◽  
Ken-ichi Ishibashi ◽  
Hiroshi Tanaka ◽  
...  
Keyword(s):  
Quantitative Methods ◽  
Japanese Cedar ◽  
False Positives ◽  
Affinity Column ◽  
Deep Mycosis ◽  
Specific Affinity ◽  
Japanese Cedar Pollen ◽  
Cedar Pollen ◽  
Positive Factor ◽  
Lal Assay

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.

scholarly journals Contamination of erythropoietin by endotoxin: in vivo and in vitro effects on murine erythropoiesis

Blood ◽  
1979 ◽  
Vol 54 (1) ◽  
pp. 146-158 ◽  
Author(s):  
KS Zuckerman ◽  
PJ Quesenberry ◽  
J Levin ◽  
R Sullivan
Keyword(s):  
Significant Inhibition ◽  
Erythropoietic Activity ◽  
Limulus Amebocyte Lysate ◽  
Endogenous Erythropoietin ◽  
Significant Change ◽  
In Vitro Effects ◽  
Stimulation Of

Abstract Endotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU- e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10(-5)-10(-3) mug of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.

scholarly journals Endotoxins in surgical instruments of hip arthroplasty

2016 ◽  
Vol 50 (3) ◽  
pp. 405-410 ◽  
Author(s):  
Vania Regina Goveia ◽  
Isabel Yovana Quispe Mendoza ◽  
Gilberto Lima Guimarães ◽  
Flavia Falci Ercole ◽  
Bráulio Roberto Gonçalves Marinho Couto ◽  
...  
Keyword(s):  
Femoral Head ◽  
Teaching Hospital ◽  
Hip Arthroplasty ◽  
Exploratory Study ◽  
Surgical Instruments ◽  
Gel Formation ◽  
Limulus Amebocyte Lysate ◽  
Positive Analysis ◽  
Hip Arthroplasties

Abstract OBJECTIVE To investigate endotoxins in sterilized surgical instruments used in hip arthroplasties. METHOD A descriptive exploratory study conducted in a public teaching hospital. Six types of surgical instruments were selected, namely: acetabulum rasp, femoral rasp, femoral head remover, chisel box, flexible bone reamer and femoral head test. The selection was based on the analysis of the difficulty in removing bone and blood residues during cleaning. The sample was made up of 60 surgical instruments, which were tested for endotoxins in three different stages. The EndosafeTM Gel-Clot LAL (Limulus Amebocyte Lysate method) was used. RESULT There was consistent gel formation with positive analysis in eight instruments, corresponding to 13.3%, being four femoral rasps and four bone reamers. CONCLUSION Endotoxins in quantity ≥0.125 UE/mL were detected in 13.3% of the instruments tested.

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