Limulus Amebocyte Lysate Reaction Research Articles

Highly Sensitive Electrochemical Endotoxin Sensor Based on Redox Cycling Using an Interdigitated Array Electrode Device

The Limulus amebocyte lysate (LAL) reaction-based assay, the most commonly used endotoxin detection method, requires a skilled technician. In this study, to develop an easy-to-use and highly sensitive endotoxin sensor, we created an electrochemical endotoxin sensor by using an interdigitated array electrode (IDAE) device with advantages of amplifiable signals via redox cycling and portability. We added Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as an electrochemical substrate for an LAL reaction and detected p-aminophenol (pAP) released from LGR-pAP as a product of an endotoxin-induced LAL reaction via an IDAE device. The IDAE device showed a great redox cycling efficiency of 79.8%, and a 4.79-fold signal amplification rate. Then, we confirmed that pAP was detectable in the presence of LGR-pAP through chronoamperometry with the potential of the anode stepped from -0.3 to 0.5 V vs. Ag/AgCl while the cathode was biased at -0.3 V vs. Ag/AgCl. Then, we performed an endotoxin assay by using the IDAE device. Our endotoxin sensor detected as low as 0.7 and 1.0 endotoxin unit/L after the LAL reaction for 1 h and 45 min, respectively, and these data were within the cut-off value for ultrapure dialysis fluid. Therefore, our highly sensitive endotoxin sensor is useful for ensuring medical safety.

Electrochemical Immunoassay with Dual-Signal Amplification for Redox Cycling within a Nanoscale Gap

Here, we report a highly sensitive and easy-to-use electrochemical immunoassay using a dual-signal amplification strategy of redox cycling within a nanoscale gap and the Limulus amebocyte lysate (LAL) cascade reaction. After the antigen–antibody reaction using an endotoxin-labeled antibody, the LAL reaction was induced by endotoxin as the first signal amplification. Subsequently, p-aminophenol (pAP) liberated from peptide-conjugated pAP (Boc-Leu-Gly-Arg-pAP; LGR-pAP), at the last step of the LAL reaction, was detected with redox cycling within a nanoscale gap using a fabricated device as the second signal amplification. First, an electrochemical immunoassay for goat immunoglobulin G (IgG), used as a model analyte, was performed with an endotoxin-labeled antibody using a 96-well plate. The results indicated that the endotoxin-labeled antibody was usable for immunoassays. However, a high background was observed. To decrease the background and increase the efficiency of the reactions, magnetic beads in an endotoxin-free test tube were used for the immunosorbent assay. After optimization of the condition, goat IgG was successfully detected with single-signal amplification of the cascade reaction at concentrations as low as 10 pg/mL (67 fM) using a Au disc electrode. Finally, an immunoassay for goat IgG on magnetic beads using a redox cycling-inducing device was performed. With the dual-signal amplification strategies of redox cycling within a nanoscale gap and LAL reactions, the detection limit was 70 fg/mL (470 aM). Our highly sensitive and easy-to-use immunoassay strategy provides a platform for clinical diagnosis to detect early-stage infections.

A highly sensitive endotoxin sensor based on redox cycling in a nanocavity.

We report a highly sensitive and rapid electrochemical method for the detection of endotoxin, based on a Limulus amebocyte lysate (LAL) assay using redox cycling at a pair of electrodes in a nanocavity for electrochemical signal amplification. We have previously developed Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as a substrate for the amperometric LAL assay, and in this work, Z-Leu-Gly-Arg-aminomethylferrocene (LGR-AMF) was newly prepared. They were examined as substrates for a LAL-based endotoxin assay using a nanocavity device. During the last step of the endotoxin-induced LAL cascade reaction, pAP or AMF is generated from the substrate, which can be detected electrochemically with efficient signal amplification by redox cycling between the two electrodes in the nanocavity. A device with a 190 nm-high nanocavity was fabricated by photolithography. With the fabricated device in model assay solutions prepared by mixing LGR-pAP and pAP, we demonstrated that pAP could be quantitatively detected from the difference in oxidation potentials between LGR-pAP and pAP. For LGR-AMF and AMF, a difference in the formal potential of 0.1 V was obtained which was considered to be insufficient to distinguish AMF from LGR-AMF. However, we showed for the first time that analytes such as AMF can be detected by differences in diffusion coefficients between the analyte and coexisting molecules (such as LGR-AMF) using a device with high redox-cycling efficiency. Next, the endotoxin assay was performed using the fabricated nanocavity device. Using this method, endotoxin was detected at concentrations as low as 0.2 and 0.5 EU L-1 after LAL reaction times of 1 h and 30 min, respectively, using the LGR-pAP substrate. However, the endotoxin assay using LGR-AMF was not successful because the clotting enzyme did not react with LGR-AMF. This problem might be solved by further design of the substrate. Our nanocavity device represents an effective platform for the simple and rapid detection of endotoxin with high sensitivity.

Multi-institutional study of a newly developed bioluminescent endotoxin measurement method based on the limulus amebocyte lysate reaction

Multi-institutional study of a newly developed bioluminescent endotoxin measurement method based on the limulus amebocyte lysate reaction

Detection of endotoxin using a photonic crystal nanolaser

Fast and reliable detection of endotoxin (ET) in medical equipment and pharmaceutical products is an essential precursor to clinical treatment. In this study, we demonstrate the use of shifts in wavelength of photonic crystal nanolasers for sensing the Limulus amebocyte lysate reaction, which is a standard method for detecting ET. From working curves of wavelength shift vs ET concentration, whose correlation factors were as high as 98%, we detected a required concentration of 0.001 EU/ml within 33 min and detected a low concentration of 0.0001 EU/ml.

Electrochemical sensor with substitutional stripping voltammetry for highly sensitive endotoxin assay.

We have developed a novel method for detection of endotoxin with extra-high sensitivity by using substitutional stripping voltammetry (SSV). In this method, a p-aminophenol (pAP) conjugated peptide (Boc-Leu-Gly-Arg-pAP; LGR-pAP) was used as a substrate for a protease, which is activated at the last step of the endotoxin-induced Limulus amebocyte lysate (LAL) cascade reaction. Extra-highly sensitive detection of pAP liberated by the endotoxin-induced LAL reaction was successfully realized with SSV, based on the accumulation of an amperometric signal owing to exchange of the oxidation current of pAP generated at an electrode in a reaction cell with silver deposition on another electrode in a deposition cell. This reaction is driven by the difference in the redox potential between pAP/quinoneimine and silver/silver ion. The amount of the deposited silver is quantified by anodic stripping voltammetry (ASV). This SSV-based endotoxin assay was performed with a chip device comprising two cells, each of which was connected via a liquid junction made of Vycor® glass. The reaction cell and the deposition cell contained a standard endotoxin sample with LAL regents containing LGR-pAP and AgNO3 solution, respectively. After the cells were electrically connected for 60 min, ASV was conducted in the deposition cell to quantify the total electrical charge derived by the oxidation of free pAP in the reaction cell. The ASV signal increased with the increase of the endotoxin concentration in the sample solution in the range of 0.5-1000 EU L(-1).

Saline and buffers minimize the action of interfering factors in the bacterial endotoxins test

The bacterial endotoxins test (BET) is the most sensitive assay for measuring endotoxin levels in solution. However, it is difficult to quantify endotoxin levels in some solutions because unknown interfering factors may inhibit or enhance the Limulus amebocyte lysate (LAL) coagulation reaction. We investigated the mechanisms of this interference and found that interference can be reduced or totally suppressed by preparing sample solutions in saline, Dulbecco’s phosphate-buffered saline (D-PBS), N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), or 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffers. We examined the inhibitory effect on the interfering action of various reagents. The reagents examined were classified into two groups: a weak interference and a strong interference group. The interference of the strong interference group was suppressed by adding endotoxin and the test factors to LAL individually. Endotoxin peaks analyzed by gel-filtration HPLC disappeared in the presence of interfering factors. When buffers were used to prepare sample solutions instead of water, endotoxin peaks were maintained and interference with LAL reaction was suppressed. These results indicate that for the strong interference group, interference of the LAL reaction was a direct consequence of interfering factors binding to endotoxin. This alters endotoxin complexation, but this effect may be suppressed by preparing solutions in saline or other buffers instead of in water.

Early detection of the Limulus amebocyte lysate reaction evoked by endotoxins

The gelation of Limulus amebocyte lysate (LAL) evoked by bacterial endotoxins can be detected earlier than with usual methods by using laser scattering photometry to recognize the formation of small particles of clotted enzyme produced when the reaction mixture is agitated. The appearance of these small particles means that the influence of endotoxins has stimulated activation of the clotting enzyme across the LAL cascade, and the timing of their appearance is related to endotoxin concentration. This new method can be used for quick and sensitive endotoxin assay. The average endotoxin level of healthy volunteers was assayed to be 0.0738 pg/ml [0.0312–0.3445 pg/ml] ( n = 11) within 70 min from the start of the assay.

Detection of anti-lipopolysaccharide antibodies to Vibrio cholerae O1 and O139 using a novel microtiter limulus amebocyte lysate (LAL) assay

This paper describes a new assay for measuring antibodies to lipopolysaccharide (LPS) of Vibrio cholerae using blocking of the limulus amebocyte lysate (LAL) reaction in a microtiter plate. When V. cholerae LPS was coated onto a microtiter plate, and then LAL reagent was added, a typical gel reaction occurred. However, when the LPS-coated plates were first incubated with serum containing anti-cholera antibodies, the gel reaction did not occur. Blocking of the gel reaction was serotype specific, in that anti-O1 serum blocked the O1 LAL reaction but not the O139 LAL reaction, and anti-O139 serum neutralized the O139 reaction but not the O1 reaction. Preliminary data suggested that the LAL titers were comparable to the vibriocidal titers but that titers using the LAL assay may have been slightly higher. This study showed that antibodies to V. cholerae blocked the LAL reaction and suggested that the LAL blocking assay could be applicable for determining antibodies to other endotoxin-containing bacteria and microorganisms.

Lipopolysaccharide (LPS) Neutralizing Peptides Reveal a Lipid A Binding Site of LPS Binding Protein

Endotoxic shock follows a cascade of events initiated by release of lipopolysaccharide during infection with Gram-negative organisms. Two overlapping 15-mer peptides were identified, corresponding to residues 91-108 of human lipopolysaccharide binding protein that specifically bound the lipid A moiety of lipopolysaccharide with high affinity. The peptides inhibited binding of lipopolysaccharide to lipopolysaccharide binding protein, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of tumor necrosis factor alpha following lipopolysaccharide challenge both in vitro and in vivo. These results suggest lipopolysaccharide binding protein residues 91-108 form at least part of the lipopolysaccharide binding site. Moreover, derivatives of lipopolysaccharide binding protein residues 91-108 might modulate lipopolysaccharide toxicity in the clinical setting.

Functional domains of recombinant bactericidal/permeability increasing protein (rBPI23).

The 23-kDa recombinant amino-terminal bactericidal/permeability increasing protein fragment (rBPI23) has all of the antibacterial and antiendotoxin properties of the holoprotein. In the current studies, we have identified multiple active domains within rBPI23 with chemical and proteolytic cleavage fragments and with synthetic overlapping peptides. We also demonstrate a novel, high affinity heparin binding property for rBPI23, in addition to its established bactericidal and lipopolysaccharide binding properties. Cleavage fragments and synthetic, overlapping peptides of rBPI23 were analyzed for inhibition of the lipopolysaccharide-induced Limulus amebocyte lysate reaction, for bactericidal activity, and for heparin binding. Three separate, active domains were identified in amino acid regions 17-45, 65-99, and 142-169. A single synthetic peptide (85-99) was bactericidal. These results indicate that rBPI23 is comprised of three separate functional domains which contribute to the high affinity interaction of rBPI23 with Gram-negative bacteria. The individual activity of each domain and the cooperative interaction among domains provide the basis for developing rBPI23 analogues with increased biologic efficacy.

Extraction of bacterial lipopolysaccharides from soil samples and their determination using the Limulus amebocyte lysate reaction

Lipopolysaccharides were extracted from soil using an aqueous phenol procedure and determined by using the Limulus amebocyte lysate coagulation reaction. When heat-killed Escherichia coli cells were added to soil at a concentration of organisms expected to occur in subsurface material, there was a 10% recovery of the added cellular lipopolysaccharides (LPS); because of the sensitivity of the Limulus amebocyte lysate (LAL) reaction this recovery is sufficient for the method to be a rapid (4 h) estimate of the Gram-negative bacteria present in soil samples. By using a kinetic assay LPS could be measured over a concentration range of five orders of magnitude, which is important with environmental samples.

Limulus amebocyte lysate reaction with streptococcal pyrogenic exotoxin.

Three immunologically distinct pyrogenic exotoxins derived from group A streptococci were found to cause coagulation of limulus amebocyte lysate. These exotoxins were shown to be several thousand times less active for lysate coagulation than gram-negative endotoxin.