Pyrogens, including endotoxins and non-endotoxins, are key factors that impact the safety of parenterally administered drugs as contaminants. Currently, traditional pyrogen detection methods primarily rely on the Rabbit Pyrogen Test (RPT) and the Limulus Amebocyte Lysate (LAL) assay, both of which are derived from animal-based systems. However, both methods possess certain limitations. The RPT demonstrates lower reproducibility and a higher false-positive rate compared to the LAL assay; in contrast, the LAL test is limited to detecting endotoxins from Gram-negative organisms. The Monocyte Activation Test (MAT), which is based on monocytes, has emerged as the most promising alternative to traditional pyrogen detection methods. However, the MAT is operationally complex, time-consuming, and exhibits significant individual variability; it typically requires a substantial volume of human blood, thereby considerably limiting its practical application. To overcome these limitations, we developed a rapid pyrogen detection method based on monocytes derived from human embryonic stem cells (hESCs), integrated with a luciferase reporter system. By leveraging the stability and pluripotency of hESCs, we can obtain numerous hESC-derived monocytes (hESC-Mono) through directed invitro differentiation. These hESC-Mono exhibit batch-to-batch consistency and closely resemble peripheral blood monocytes in functionality. We have demonstrated that hESC-Mono possess sensitive reporting capabilities for at least three types of pyrogens: lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria; lipoteichoic acid (LTA), a key constituent of the cell wall of Gram-positive bacteria; and fungal Zymosan. Furthermore, we have confirmed that hESC-Mono maintain stable expression of Toll-like receptors, which are among the primary determinants of the reliable detection of pyrogens. Consequently, we have successfully developed and validated a novel pyrogen detection method based on the integration of an invitro hPSC-directed differentiation system with a luciferase-based reporter assay, thereby offering a promising alternative to traditional pyrogen detection methods.

For more than 40 years, the Limulus Amebocyte Lysate (LAL) test has served as the standard method for bacterial endotoxin Testing (BET). The critical proteins required for endotoxin detection in LAL assays are sourced from amoebocytes-blood cells extracted from horseshoe crabs.Animal-free reagents have been developed to support conservation efforts and the 3Rs (Replacement, Reduction, and Refinement). One such alternative is the recombinant cascade reagent (rCR), which contains three recombinant proteins that replicate the natural enzymatic cascade found in horseshoe crab amoebocytes to detect bacterial endotoxins.This study assessed the equivalency of rCR to FDA-licensed LAL reagents using 563 pharmaceutically relevant samples. Of these, 134 samples were contaminated with natural environmental endotoxin (NEE), allowing for a direct statistical comparison.Results showed that the rCR assays were equivalent in performance to FDA-licensed LAL assays, detecting endotoxin at similar levels under real-world conditions. Equivalency was demonstrated using methods consistent with those outlined in compendial guidance for bacterial endotoxin testing.

Gram-negative bacteria feature an asymmetric outer membrane, composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) or lipooligosaccharides (LOS) in the outer leaflet, which underpins virulence and antibiotic resistance. Conventional LPS assays like the Limulus amebocyte lysate test face ethical and practical challenges, motivating the development of aptamer-based detection. Here, we probe the interaction of the LA27 DNA aptamer (Kd≈46nM) with biomimetic membranes incorporating LOS/LPS from Akkermansia muciniphila, Flavobacterium sp. Root935, and Paenalcaligenes hominis strains. Asymmetric supported lipid bilayers (SLBs) and symmetric large unilamellar vesicles (LUVs) were characterized by neutron reflectometry (NR), small-angle neutron scattering (SANS), and dynamic light scattering (DLS). NR confirmed preserved bilayer asymmetry and revealed that LA27 deeply penetrates Akkermansia LOS bilayers, evidenced by increased core hydration and structural disruption, whereas interactions with Flavobacterium LOS are moderate, and those with Paenalcaligenes LPS are confined to surface adhesion due to extensive LPS O-antigen chains. SANS and DLS of LUVs corroborated these modes: Akkermansia vesicles showed negligible changes in lamellar thickness and hydrodynamic radius, consistent with insertion, while those containing Flavobacterium LOS and Paenalcaligenes LPS exhibited significant increases in both size-related metrics, indicative of surface binding for those latter cases. These results elucidate how polysaccharide composition and supramolecular architecture of LOS/LPS govern aptamer affinity and specificity, providing a rigorous framework for nano-bio interface studies and laying the groundwork for selective, aptamer-based endotoxin sensing platforms.

Carcinoscorpius and Tachypleus lysates assay for detecting endotoxin in milk and groundwater: Toward reducing reliance on Limulus amebocyte lysate.

Pyrogens, which include endotoxin and non-endotoxin pyrogens (NEPs), act on immune cells in the bloodstream, causing various effects such as fever and endotoxic shock. The limulus amebocyte lysate test, a commonly used endotoxin test in the manufacturing of pharmaceuticals and medical devices, can detect endotoxin but not NEPs. The monocyte activation test (MAT), which uses monocytes, is a testing method included in the European Pharmacopoeia (EP 11.5; 07/2024:20630) that can detect NEPs. The MAT detects the cellular response following activation of Toll-like receptors (TLRs) by pyrogens; released cytokines, such as IL-6, are often the targets of detection. This cytokine release is regulated by the transcription factor NF-κB. In this study, we investigated whether it is possible to detect pyrogens with an NF-κB reporter gene-expressing cell line, using the NOMO-1 cell line as a model monocyte-like line. This study demonstrates that the reporter gene-expressing cells can detect 0.0125 EU/mL lipopolysaccharide (LPS) after 3 hours of incubation, and a stable calibration curve for LPS quantification can be created. Moreover, these cells can detect agonists for TLR1–9 in a concentration-dependent manner. Pharmaceuticals, including blood products and antibody drugs, were used in LPS recovery tests to confirm that they do not interfere with LPS detection. This study demonstrates that NF-κB reporter cells facilitate a simpler, more concise MAT, eliminating the complexity associated with enzyme-linked immunosorbent assays. Moreover, using the NOMO-1 cell line allows for the detection of a wider range of NEPs compared with using existing reporter gene-expressing cell lines.

Pyrogens, which include endotoxin and non-endotoxin pyrogens (NEPs), act on immune cells in the bloodstream, causing various effects such as fever and endotoxic shock. The limulus amebocyte lysate test, a commonly used endotoxin test in the manufacturing of pharmaceuticals and medical devices, can detect endotoxin but not NEPs. The monocyte activation test (MAT), which uses monocytes, is a testing method included in the European Pharmacopoeia (EP 11.5; 07/2024:20630) that can detect NEPs. The MAT detects the cellular response following activation of Toll-like receptors (TLRs) by pyrogens; released cytokines, such as IL-6, are often the targets of detection. This cytokine release is regulated by the transcription factor NF-κB. In this study, we investigated whether it is possible to detect pyrogens with an NF-κB reporter gene-expressing cell line, using the NOMO-1 cell line as a model monocyte-like line. This study demonstrates that the reporter gene-expressing cells can detect 0.0125 EU/mL lipopolysaccharide (LPS) after 3 hours of incubation, and a stable calibration curve for LPS quantification can be created. Moreover, these cells can detect agonists for TLR1-9 in a concentration-dependent manner. Pharmaceuticals, including blood products and antibody drugs, were used in LPS recovery tests to confirm that they do not interfere with LPS detection. This study demonstrates that NF-κB reporter cells facilitate a simpler, more concise MAT, eliminating the complexity associated with enzyme-linked immunosorbent assays. Moreover, using the NOMO-1 cell line allows for the detection of a wider range of NEPs compared with using existing reporter gene-expressing cell lines.

Background and Objectives:The secretion of outer membrane vesicles (OMVs) is a universal event among bacteria. In this study, we characterized OMVs from pathogenic and non-pathogenic strains of Escherichia coli and assessed the effect of pathogenic OMVs on NLR signaling pathways.Materials and Methods:OMVs were extracted by differential centrifugation and characterized by scanning electron microscopy (SEM), SDS-PAGE, Limulus amebocyte lysate (LAL) test, and nucleic acid extraction. Then, the Caco-2 cells were treated with the pathogenic OMVs to evaluate their effect on NLR signaling pathways.Results:SEM showed that pathogenic and non-pathogenic strains produced OMVs in the range of 9–72.9 and 45–270 nm, respectively. The SDS-PAGE revealed that both OMVs had protein bands ranging from 25 to 100 kDa. The LAL test displayed that the concentration of LPS was 2.368 and 0.055 EU/ml in pathogenic and non-pathogenic OMVs, respectively. The evaluation of nucleic acid contents showed no significant difference between both types of OMVs. The assessment of pathogenic OMVs’ effect on NLR genes demonstrated that the expression level was changed in some genes.Conclusion:The characterization of OMVs showed that both strains of E. coli secrete OMVs in different sizes and contents. Besides, it was revealed that OMVs can regulate gene expression.

Endotoxin detection is paramount for monitoring bacterial contamination in food, pharmaceuticals, and clinical diagnostics. The limulus amebocyte lysate (LAL) test, which relies on horseshoe crab blood, has long been the gold standard for endotoxin detection. However, the widespread adoption of this method is constrained by ethical concerns and the high costs associated with harvesting endangered species. Although nanozyme-based colorimetric methods present a more cost-effective and straightforward alternative, their application is limited by suboptimal selectivity and sensitivity. In this study, we report the synthesis and rigorous characterization of the bimetallic PCN-242 (Fe2Co) metal-organic framework (MOF), synthesized using 2-amino terephthalic acid and a pre-synthesized [Fe2Co(μ3-O)(CH3COO)6] cluster. Steady-state kinetic analyses revealed that PCN-242 (Fe2Co) MOF exhibits a significantly higher affinity for hydrogen peroxide (H2O2) compared to horseradish peroxidase (HRP) and other iron-based MOFs. The development of a PCN-242 (Fe2Co)-based colorimetric sensor demonstrated a low limit of detection (LOD) of 1.36 μg mL-1 for endotoxins, with excellent selectivity and reproducibility, thereby enabling effective detection of bacterial endotoxins. Recognizing the potential of the PCN-242 (Fe2Co) MOF beyond endotoxin detection, we explored its utility in glucose biosensing. Moreover, incorporating glucose oxidase (GOx) into the PCN-242 (Fe2Co) MOF framework further enhanced its peroxidase-like catalytic activity. This integration enabled sensitive glucose detection, achieving LODs of 4.24 μM for glucose and 2.2 μM for H2O2 within a linear range of 1 to 150 μM. The dual functionality of PCN-242 (Fe2Co) MOF as a peroxidase mimic and biosensor platform highlights its potential for advanced catalytic and diagnostic applications, offering a versatile and ethical alternative to conventional methods.

ObjectiveThe purpose of this study was to evaluate the effects of different endodontic treatments – chelation and antimicrobial photodynamic therapy (aPDT), on the reduction of endotoxin levels in root canals.MethodsEighty human single-rooted teeth had their crowns sectioned, and their root canals were subsequently prepared. All samples and materials were sterilized using Cobalt-60 irradiation. Subsequently, 10 µL of fresh lipopolysaccharide (LPS) was inoculated into the root canals and incubated for 3 days. On the fourth day, experimental treatments were applied to the root canals according to the groups (n=10): [1] pyrogen-free water, [2] 0.005% methylene blue, [3] diode LASER and [4] 0.005% methylene blue + diode LASER (PDT), [5] 2.5% sodium hypochlorite, [6] 17% trisodium ethylenediaminetetraacetic acid (EDTA), [7] 10% tetrasodium EDTA, [8] 18% etidronate (HEBP). The exposure time for each solution and light irradiation was 5 minutes. The samples collected after treatment were analyzed using the Limulus Amebocyte Lysate test to quantify endotoxins. The data obtained were subjected to Kruskal-Wallis analysis followed by Dunn’s test with a significance level of 5%.ResultsAll treatments demonstrated efficacy in reducing endotoxin levels in root canals compared to the use of pyrogen-free water (control). A statistically significant reduction was observed in the groups treated with 17% EDTA and 18% HEBP compared to the control group.ConclusionShort-term application (5 minutes) of 17% EDTA and 18% HEBP chelating solutions significantly reduced LPS in root canals and may be effective adjuncts in endodontic therapy.

Abstract. Acute alcoholic pancreatitis (AAP) represents a significant cause of hospitalizations in surgical departments, with severe cases leading to multi-organ failure and high mortality rates, up to 30%. Effective management of AAP requires accurate patient condition assessment, which directly influences treatment strategies, including fluid resuscitation, pain relief, nutritional support, and antibiotic use. A key factor in AAP outcomes is the development of hypovolemia, caused by fluid loss into the peritoneal and retroperitoneal spaces, exacerbating systemic and local hypoperfusion, tissue ischemia, and pancreatic necrosis. Proper fluid therapy is essential to prevent both hypovolemia-related complications and fluid overload, which may lead to pleural effusion, cerebral edema, and cardiovascular dysfunction. Current guidelines for fluid therapy in AAP focus on maintaining specific physiological parameters such as mean arterial pressure, heart rate, central venous pressure, urine output, and hematocrit levels. However, the use of non-invasive methods like bioimpedance analysis, which measures tissue resistance to determine fluid distribution, may offer more precise insights into the patient's fluid status, enabling better management of infusion therapy. The aim to optimize fluid management in patients with moderate to severe AAP and reduce infectious complications through the use of bioimpedance analysis. Materials and methods. A total of 110 patients were enrolled, including 101 men and 9 women. The patients were divided into two groups: one treated with standard fluid therapy using Ringer's lactate solution and the other receiving Sterofundin ISO solution, with infusion volumes adjusted according to bioimpedance measurements. Laboratory tests, including procalcitonin levels and the Limulus amebocyte lysate (LAL) test, were used to monitor inflammatory markers and endotoxin levels, while bioimpedance measurements were conducted on days 1, 3, 5, 8, 11, and 14 to dynamically assess fluid balance. The results showed that patients treated with Sterofundin ISO and bioimpedance-guided fluid management experienced a significant reduction in extracellular fluid volume deviations and infectious complications compared to those receiving standard treatment. In the second group, the incidence of infectious complications in patients with moderate AAP decreased from 35.9% to 21.62%, and in severe cases, from 81.3% to 44.45%.. Key words: acute alcoholic pancreatitis, LAL-test, bioimpedance measurement, infusion, infectious complications

Veterinary autogenous vaccines, similar to all injectable pharmaceutical products, must be tested to assess endotoxin concentrations. The Limulus Amebocyte Lysate Test (LAL test) is widely used in in vitro quality control assays for endotoxin detection, although it presents some ethical issues related to the production of reagents and is also characterized by a low specificity due to other contaminants that can activate the reaction. For all these reasons, a new recombinant factor C LAL test was developed. In this study, we described the comparison between two LAL test methods for in vitro quality control of veterinary autogenous vaccines, with the aim of evaluating the most suitable method and establishing an endotoxin concentration range for two different matrices. Two hundred batches of two different vaccine matrices were tested using the kinetic chromogenic LAL test and recombinant factor C endotoxin detection assay commercial kits. Statistical analysis conducted after the validation of the recombinant factor C test exhibited a statistically significant correlation between the two methods and for both vaccine matrices, suggesting that the animal-free assay can be used as a routine quality control test for veterinary autogenous vaccines.

Highly sensitive and rapid optical fiber bio-sensor for endotoxin detection using anti-resonance effect

Acute pancreatitis (AP) is an inflammatory condition of the pancreas, characterized by acute, intense abdominal pain. AP accounts for 20-30% of hospitalizations due to abdominal symptoms. The incidence of AP is increasing globally by 2-5% annually, ranging from 3.4 to 73.4 cases per 100,000 people. A critical concern in AAP is the potential development of infectious complications, which may require surgical intervention and increase mortality rates. Effective management and monitoring of these complications are essential. Research Justification. The diagnosis of AAP in clinical practice commonly relies on serum levels of α-amylase, lipase, C-reactive protein, and leukocyte counts. However, these markers have limitations and do not fully reflect the level of intoxication. The accuracy of C-reactive protein as a marker is not well established and is used mainly as a prognostic indicator. Serum α-amylase levels tend to decrease sharply after the fifth day of illness, regardless of the patient's condition. Lipase levels in diabetic patients can exceed reference values even without pathology, necessitating adjustments for AAP diagnosis. These criteria are most effective during the enzymatic phase of AAP (days 1-5). To predict the course of AAP and assess the adequacy of therapy, as well as to determine the need for surgical intervention, serum procalcitonin levels are commonly used. However, some studies question the diagnostic accuracy of procalcitonin, particularly regarding its correlation with organ failure, infectious complications, and patient mortality. Materials and Methods: This study involved 55 AAP patients, 51 men (92.7%) and 4 women (7.3%), with an average age of 49.49±9.87 years. Patients were categorized based on the severity of their condition: moderate (39 patients, 70.9%) and severe (16 patients, 29.1%). The control group consisted of 10 healthy individuals (9 men, 90.0%; 1 woman, 10.0%; average age 45.22±7.55 years). AAP was classified according to the Atlanta classification of acute pancreatitis. The study included patients with moderate to severe AAP who were hospitalized within 48 hours of symptom onset. All patients received treatment according to clinical guidelines, including intensive fluid therapy with Ringer's lactate. The Limulus Amebocyte Lysate (LAL) test and procalcitonin levels were measured on days 5, 8, 11, and 14 of hospitalization. For suspected infected necrosis, patients underwent contrast-enhanced computed tomography (CT) on day 14, with fine-needle aspiration biopsy or surgical intervention if necessary. Results. On day 5, LAL test values in the moderate and severe AAP groups were within reference ranges, and procalcitonin levels did not exceed 0.5 ng/ml. By day 14, a significant increase in LAL test values was noted in both groups, with a corresponding rise in procalcitonin levels. The ROC analysis for LAL test values on day 14 in the moderate AAP group showed an area under the curve (AUC) of 0.966, with 92.86% sensitivity and 96% specificity. For procalcitonin, the AUC was 0.723, with 71.43% sensitivity and 92% specificity. Conclusion. The LAL test is a sensitive and specific marker for detecting endotoxemia and monitoring the progression of AAP, particularly in predicting the development of infectious complications. The study demonstrates that LAL test values significantly correlate with the severity of AAP and may provide better diagnostic accuracy than procalcitonin, especially in the later stages of the disease.

Limitations of Limulus amebocyte lysate test for endotoxin control in raw materials for liposomal nanoformulations.

Bacterial cellulose is a unique biomaterial produced by various species of bacteria that offers a range of potential applications in the biomedical field. To provide a cost-effective alternative to soft-tissue implants used in cavity infills, remodeling, and subdermal wound healing, in vitro cytotoxicity and in vivo biocompatibility of native bacterial cellulose were investigated. Cytotoxicity was assessed using a metabolic assay on Swiss 3T3 fibroblasts and INS-1832/13 rat insulinoma. Results showed no cytotoxicity, whether the cells were seeded over or under the bacterial cellulose scaffolds. Biocompatibility was performed on Sprague-Dawley rats (males and females, 8 weeks old) by implanting bacterial cellulose membranes subcutaneously for 1 or 12 weeks. The explanted scaffolds were then sliced and stained with hematoxylin and eosin for histological characterization. The first series of results revealed acute and chronic inflammation persisting over 12 weeks. Examination of the explants indicated a high number of granulocytes within the periphery of the bacterial cellulose, suggesting the presence of endotoxins within the membrane, confirmed by a Limulus amebocyte lysate test. This discovery motivated the development of non-pyrogenic bacterial cellulose scaffolds. Following this, a second series of animal experiments was done, in which materials were implanted for 1 or 2 weeks. The results revealed mild inflammation 1 week after implantation, which then diminished to minimal inflammation after 2 weeks. Altogether, this study highlights that unmodified, purified native bacterial cellulose membranes may be used as a cost-effective biomedical device provided that proper endotoxin clearance is achieved.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

Dietary soy protein and soy isoflavones have anti-inflammatory properties. Previously, we reported that feeding soy protein concentrate diet (SPC) with low or high isoflavone (LIF or HIF) to young (seven-week-old) obese (fa/fa) Zucker rats inhibits lipopolysaccharide (LPS) translocation and decreases liver inflammation compared to a casein control (CAS) diet. The current study investigated whether SPC-LIF and SPC-HIF diets would reduce liver inflammation in adult obese Zucker rats fed a CAS diet. A total of 21 six-week-old male obese (fa/fa) Zucker rats were given CAS diet for 8 weeks to develop obesity then randomly assigned to CAS, SPC-LIF, or SPC-HIF (seven rats/group) diet for an additional 10 weeks. The expression of LPS-translocation, inflammation, and intestinal permeability markers were quantified by qPCR in liver, visceral adipose tissue (VAT), and colon. LPS concentration was determined in both the colon content and fecal samples by a Limulus amebocyte lysate (LAL) test. SPC-LIF and SPC-HIF diets significantly decreased liver LPS-binding protein (LBP) expression compared to CAS diet (p < 0.01 and p < 0.05, respectively). SPC-HIF diet also significantly decreased liver MCP-1 and TNF-α expression (p < 0.05) and had a trend to decrease liver iNOS expression (p = 0.06). In the colon, SPC-HIF diet significantly increased LBP expression compared to CAS diet (p < 0.05). When samples from all three groups were combined, there was a negative correlation between colon LBP expression and liver LBP expression (p = 0.046). SPC diets did not alter the expression of intestinal permeability markers (i.e., occludin, claudin 3, and zonula occludens-1) in the colon or inflammation markers (i.e., TNF-α and iNOS) in VAT or the colon. LPS levels in the colon content did not differ between any groups. Fecal LPS levels were significantly higher in the SPC-LIF and SPC-HIF groups compared to the CAS group (p < 0.01). In conclusion, SPC, particularly SPC with HIF, reduces liver LBP expression and inflammation makers (i.e., TNF-α and MCP-1 expression) in adult obese Zucker rats, likely by reducing LPS translocation.

Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products

Creating an effective and safe vaccine is critical to fighting the coronavirus infection successfully. Several types of COVID-19 vaccines exist, including inactivated, live attenuated, recombinant, synthetic peptide, virus-like particle-based, DNA and mRNA-based, and sub-unit vaccines containing purified immunogenic viral proteins. However, the scale and speed at which COVID-19 is spreading demonstrate a global public demand for an effective prophylaxis that must be supplied more. The developed products promise a bright future for SARS-CoV-2 prevention; however, evidence of safety and immunogenicity is mandatory before any vaccine can be produced. In this paper, we report on the results of our work examining the safety, toxicity, immunizing dose choice, and immunogenicity of QazCoVac-P, a Kazakhstan-made sub-unit vaccine for COVID-19. First, we looked into the product's safety profile by assessing its pyrogenicity in vaccinated rabbit models and using the LAL (limulus amebocyte lysate) test. We examined the vaccine's acute and sub-chronic toxicity on BALB/c mice and rats. The vaccine did not cause clinically significant toxicity-related changes or symptoms in our toxicity experiments. Finally, we performed a double immunization of mice, ferrets, Syrian hamsters, and rhesus macaques (Macaca mulatta). We used ELISA to measure antibody titers with the maximum mean geometric titer of antibodies in the animals' blood sera totaling approximately 8 log2. The results of this and other studies warrant recommending the QazCoVac-P vaccine for clinical trials.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.