Validation of Two Kinetic Assays for the Quantification of Endotoxin in Human Serum

Background: There is an emerging evidence of the role of the microbiome in neurological diseases. Endotoxin is a component of gram-negative bacteria and thought to be one of the possible signals between the gut microbiota and the immune system. Previous studies explored the blood levels of endotoxin using an endpoint chromogenic assay.Methods: We validated and compared the analytical performance of two kinetic assays for the quantification of endotoxin in serum: (1) the Limulus Amebocyte Lysate (LAL) Kinetic-QCL assay and (2) the turbidimetric LAL Pyrogent-5000 assay. We used the best-performing validated assay to measure the endotoxin level in 20 patients with multiple sclerosis (MS) and eight healthy controls.Results: The Pyrogent-5000 and QCL assay achieved similar performance in regard to spike recovery and linear dilution; however, the Pyrogent-5000 had a better signal to noise in the calibrator curve. By using the Pyrogent-5000 assay, we found that serum samples from MS patients and healthy controls have a similar level of endotoxin; hence, we did not find evidence to support a penetration of endotoxin in the blood of MS patients. Our findings do not exclude a role of endotoxin in mediating signals from the gut microbiota in MS patients directly at the gut–blood barrier where numerous antigen-presenting cells are actively sensing metabolites and bacterial products.

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

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.

Development of a Gold Nanoparticle-Based Colorimetric Sensor for Water for Injection At-Line Impurity Testing

The best-known rapid test using gold nanoparticles (AuNPs) is the human chorionic gonadotropin pregnancy test. AuNPs are a powerful tool in point-of-care testing because of their flexibility, modifiability, and visibility. Here, we report a method to detect impurities for at-line process control in water-for-injection (WFI) manufacturing through the example of endotoxins. If a distinct concentration of these amphipathic molecules, originated from gram-negative bacteria, enters the human body, it will result in septic shock, followed by organ failure and possibly death. Every fluid given parenterally is subject to strict regulatory requirements and therefore endotoxin testing. Through use of traditional methods like the limulus amebocyte lysate (LAL) test, it takes more than 2 h to complete. With the presented method, one-fifth of the sample volume is sufficient compared with the LAL test. Once the assay components have been mixed, the result can be interpreted visually within 2 min without the use of further instruments.

Metode Monocyte Activation Test (MAT) dan Recombinant Factor C (rFC) sebagai Alternatif Metode Pengujian Pirogen bagi Perusahaan Farmasi di Indonesia

Peningkatan kualitas produk industri farmasi mendorong tersedianya uji yang akurat dan efisien untuk mendeteksi adanya pirogen. Endotoksin merupakan salah satu dari pirogen yang umumnya ditemukan sebagai kontaminan pada produk obat. Hingga saat ini pengujian pirogen yang digunakan secara umum di Indonesia adalah metode Rabbit Pyrogen Test (RPT) dan Limulus Amebocyte Lysate (LAL). Namun, kedua metode ini memiliki kekurangan yang hampir sama, yaitu penggunaan hewan hidup sebagai bahan uji. Untuk alasan ini, saat ini sedang dikembangkan metode in vitro yang lebih menguntungkan untuk uji pirogen, yaitu dengan Recombinant Factor C (rFC) dan Monocyte Activation Test (MAT). Tujuan dari review ini adalah memberikan pemaparan mengenai kelebihan dan kekurangan metode uji pirogen dan endotoksin yang sesuai dengan kebutuhan konsumen, serta kajian penggunaan metode alternatif pada industri farmasi di Indonesia. Diharapkan dengan pemaparan dan kajian yang diberikan dapat digunakan sebagai pertimbangan penggunaan metode uji pirogen dan endotoksin yang lebih efektif dan efisien.

The Long-Term Effect of Bleeding for Limulus Amebocyte Lysate on Annual Survival and Recapture of Tagged Horseshoe Crabs

In the U.S., 525,000 horseshoe crabs (Limulus polyphemus) per year have been captured during 2013–2017, brought to biomedical facilities, and bled to produce Limulus amebocyte lysate (LAL), then mostly released to the area of capture. The Atlantic States Marine Fisheries Commission estimates short-term bleeding-induced mortality to be 15% (4% to 30%), resulting in mortality of approximately 78,750 horseshoe crabs annually in recent years comprising a minor portion (<13%) of the up to one million annual coastwide landings dominated by harvest for bait. However, the long-term effect of bleeding for LAL on annual survival and spawning behavior is unknown; thus, results from short-term studies alone might underestimate bleeding effects at the population level. To address this knowledge gap, we analyzed data from the U.S. Fish and Wildlife horseshoe crab tagging database to estimate the differences in survival and recapture rates of bled and not bled horseshoe crabs tagged in the same years and geographic area. Contrary to expectation, survival was not lower for bled crabs compared to unbled crabs. Differences varied, but survival estimates tended to be higher for bled crabs than for unbled crabs. However, biomedical culling and selection for younger or healthier animals could have resulted in biomedically tagged individuals representing a healthier subset of the overall population with subsequent higher survival. Furthermore, the tagging analysis revealed a post-bleeding reduction in capture probability, which could indicate decreased spawning activity, evident in males more than females. Continued tagging of bled and unbled crabs in the same geographic area while recording age class and sex will contribute to the further resolution of LAL production’s effect on horseshoe crab populations.

Interference of Gold Nanoparticles with In vitro Endotoxin Detection Assays

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.