Author: Eagle Biosciences

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Products for Host Cell Protein Detection!

by Eagle Biosciences

Eagle Biosciences is excited to highlight our product series for Host Cell Protein Detection!

Host cell proteins (HCPs) are a major class of impurities produced during biotherapeutic manufacturing. They must be removed from the final drug product to both assure patient safety and maintain drug efficacy. Our wide range of Host Cell Protein Detection Kits are easy to use and highly sensitive.

What are HCPs and why must they be removed from biologic drugs?

HCPs are proteins produced or encoded by the host organisms used to produce recombinant therapeutic proteins. Genetic engineering allows the host organism cells to be transformed to produce a protein of interest. During the recombinant protein production, host cells also coproduce proteins related to the normal cell functions such structural proteins, as well as proteins required for normal cellular growth and function, and vary in both number and concentration depending on the chosen host species and the manufacturing process being used. In general, apart from the therapeutic protein of interest, all endogenous proteins co-expressed by the host cells are called host-cell proteins.

Why must HCPs be removed from biologic drugs?

HCPs must be removed from the final biotherapeutic product to avoid adverse effects. Almost all HCPs carry safety risks as foreign proteins due to the potential to elicit immune response in humans (e.g, cytokine storm). In addition, some HCPs can also act to enhance the immune response to a drug product. Certain HCPs can also affect drug product stability and efficacy if not adequately removed or inactivated.

How are HCPs detected?

ELISAs are widely used for detecting HCPs, where they are generally configured in a sandwich assay format for improved specificity. In this scenario, a microplate-bound antibody is used for analyte capture, then a second analyte-specific antibody (that binds a different epitope on the target molecule) is added to enable detection. By incorporating a reference standard (e.g., a purified protein) into the assay design, it is possible to quantify the analyte of interest and confirm that its concentration meets regulatory requirements. Advantages of ELISA are that it is sensitive and compatible with high sample throughput – key considerations for biopharmaceutical manufacturing.

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Technical Spotlight: iLite Assay Ready Cells

by Eagle Biosciences

iLite Assay Ready Cells

iLite® Assay Ready Cells are developed by our partners at Svar Life Science. The cell based solutions are based on the iLite technology, a cleverly engineered cell-based assay system with a dual reporter gene readout. They offer the ease of use and robustness of a Ligand Binding Assay and can be developed for virtually any pharmaceutical target and allows an easy, rapid and accurate test format for measurement and quantification of drug activity and immunogenicity.

Key Benefits

Assay Ready Format:
iLite cells are delivered as Assay Ready Cells, and stored at -80°C. There is no need for cell culturing and continuous maintenance of cells – just thaw and dilute before use in the assay.

Normalization Gene:
A second reporter gene used as an internal control will compensate for differences in cell number. In addition, it can be used to compensate for serum matrix effects, other complex matrices or if luciferase is sensitive to the compound you are analyzing.

Sensitivity and Specificity:
The sensitivity of iLite cells is enhanced through up-regulation of key components, such as the receptor and certain signaling pathway proteins. The up-regulation of receptors also confers a higher specificity, and this is enhanced through the use of chimeric transcription factors and synthetic reporter gene promoters.

These elements are used as a lock and key to transcription of the reporter gene – only the chimeric transcription factor can bind to the synthetic promoter, and endogenous transcription factors are thereby unable to trigger expression of the reporter gene. In this way, pathway cross-talk can be effectively minimized.

Precision:
The Assay Ready Format reduces assay variability in comparison with cells in culture. Traditionally, cell-based assays are known for their high variability, with %CV often over 25%. Robustness assays of iLite assays have shown that repeatability and intermediate precision are in the range of 4-11% CV, and sample accuracy between 92-107%.

Find all of the iLite products offered on the iLite Assay Ready Cells product page.

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Biomarker Spotlight: FGF-23

by Eagle Biosciences


Fibroblast Growth Factor 23 (FGF-23) is a hormone primarily produced by osteocytes and osteoblasts in bones, playing a key role in regulating phosphate and vitamin D metabolism. It lowers serum phosphate levels by reducing phosphate reabsorption in the kidneys and suppresses the activation of vitamin D, thereby decreasing intestinal phosphate and calcium absorption. FGF-23 requires the co-receptor Klotho to bind to fibroblast growth factor receptors (FGFRs) and exert its effects, primarily in the kidneys. Dysregulation of FGF-23 is associated with several pathological conditions, making it a valuable biomarker for assessing phosphate balance and mineral metabolism.


image credit: https://www.kidney-international.org/

Why Measure FGF-23?

Measuring FGF-23 is essential for advancing research on phosphate metabolism, bone biology, and endocrine regulation. It provides insights into the mechanisms underlying phosphate homeostasis and the complex interplay between FGF-23, Klotho, and fibroblast growth factor receptors. FGF-23 is also a valuable marker for studying how mineral metabolism affects systemic processes, including cardiovascular function and aging. In experimental models, FGF-23 measurements help elucidate the effects of genetic mutations, dietary phosphate intake, and novel therapeutic interventions on phosphate regulation. Additionally, FGF-23 serves as a biomarker to explore the broader effects of mineral dysregulation on inflammation, oxidative stress, and metabolic disorders, enhancing our understanding of its role in health and disease.

Related Products:

*Use Promo Code: FGF25 to save 25% on your order!

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MedFrontier Intact FGF23 Assay Featured in a Recent Publication!

by Eagle Biosciences

We’re thrilled to announce that the MedFrontier Intact FGF23 Assay was featured in a recent publication! Details are available via the abstract and full-text access provided below.

Abstract

Multiple myeloma commonly manifests with symptoms arising from the involvement of various organs, particularly the bone and kidneys. In this report, we detail the case of a 44-year-old man who was diagnosed with multiple myeloma associated with reduced bone density. He exhibited clinical findings of osteomalacia due to Fanconi syndrome (characterized clinically by bone pain and proximal weakness and biochemically by elevated serum alkaline phosphatase, hypophosphatemia, hypouricemia, and glucosuria). With phosphate replacement, there was a notable improvement in bone pain, osteomalacia, and bone mineral density. Nevertheless, the patient continued to experience renal wasting of phosphate, uric acid, and glucose despite achieving remission from multiple myeloma for nearly 2 years. Our case highlights several important clinical features of myeloma-associated Fanconi syndrome, including the need to recognize this complication to appropriately treat the underlying bone disease while avoiding osteoclast inhibitors and the long-term persistence of the proximal renal tubulopathy despite achieving remission from myeloma and correction of osteomalacia.

Alireza Zomorodian, Naim M Maalouf, Long-term Evolution of Hypophosphatemia and Osteomalacia in a Patient With Multiple Myeloma, JCEM Case Reports, Volume 2, Issue 7, July 2024, luae137, https://doi.org/10.1210/jcemcr/luae137

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Matriks Biotek ELISA Kits Now Available at Eagle Biosciences!

by Eagle Biosciences

Eagle Biosciences, Inc. is thrilled to announce a distribution partnership with Matriks Biotek, a leader in the development of ELISA kits for therapeutic drug monitoring!

About Matriks Biotek

Matriks Biotek was founded in 2002 by Prof. Haluk Ataoglu MD, PhD (Microbiology & Immunology). Their core business values include science, innovation, reliability, perfection and customer satisfaction. They were the first R&D company to globally commercialize ELISA kits for monitoring biological drugs, providing reliable tools for numerous research applications. With an offering of over 90 validated ELISA kits covering 36 biological drugs, Matriks Biotek’s product line includes quantitative free drug detection kits, qualitative anti-drug antibody detection kits, total antibody detection kits!

Key performance features of Matriks Biotek ELISA Kits:

  • Reliable Results: High recovery rates (85-115%) for robust results
  • Convenience: Minimal sample volume requirements (10-25 µl), even for small samples like mouse serum
  • Speed: Rapid processing times (70-140 minutes) for efficient workflows
  • Versatility: Broad sample compatibility (human, mouse, rat, and monkey serum or plasma)
  • Dynamic Range: Optimized for Cmax – Cmin (trough) values

Check out the full catalog, or reach out to us with any questions — we’d love to hear from you!

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Bovine IgM ELISA Employed in a Recent Publication!

by Eagle Biosciences

A recent publication employed the Bovine IgM ELISA Assay Kit to help measure the effects of commercial-scale heat treatments on milk proteins! This research provides key data on protein retention and functionality after processing. Check out the details and access the full findings below.

Abstract

Two pasteurization steps are often used in the preparation of whey protein concentrate (WPC) before evaporation into a dry product. The Pasteurized Milk Ordinance (PMO) in the United States requires that raw bovine milk be pasteurized using a process that meets minimum heat treatment requirements to achieve reductions in pertinent microorganisms. In addition, WPC produced from USDA-approved plants must comply with CFR Subpart B §58.809, which dictates that all fluid whey used in the manufacture of dry whey products shall be pasteurized before being condensed. These heat treatments are effective at inactivating the most thermally resistant bacterium, such as Coxiella burnetii; however, they can also alter milk proteins—inducing denaturation, aggregation and reduced bioactivity. Though the impact of thermal treatments on whey proteins has been examined, the specific influence of 2 high-temperature-short-time (HTST) pasteurization steps on the retention of proteins in WPC remains unknown. This study aimed to investigate the effect of commercial-scale HTST pasteurization of both raw milk and the resulting sweet whey on the products’ overall protein profile. Three distinct batches of raw milk (RM) and corresponding pasteurized milk (PM), the resulting whey (RW) and pasteurized whey (PW) produced at commercial scale were analyzed. Assessments of denaturation were conducted through solubility testing at pH 4.6 and hydrophobicity evaluation via anilinonaphthalene-1-sulfonic acid assay (ANS). Additionally, enzyme-linked immunosorbent assay (ELISA), PAGE (PAGE) and liquid chromatography tandem mass spectroscopy (LC-MS/MS) were employed to compare the retention of key bioactive proteins before and after each HTST pasteurization step. The percentage of soluble whey protein decreased from RM to PM and from RW to PW, but no significant differences were observed via hydrophobicity assay. ELISA revealed a significant reduction in key bioactive proteins, such as lactoferrin, immunoglobulin A and immunoglobulin M, but not immunoglobulin G, after HTST pasteurization of RM and RW. PAGE and LC-MS/MS revealed a significant decrease in the retention of lactoferrin and key milk fat globular membrane proteins, such as xanthine dehydrogenase oxidase/xanthine oxidase, lactadherin and fatty acid binding protein. Additionally, xanthine oxidase activity was significantly reduced after HTST pasteurization of milk and whey. This research helps to identify the limitations of the current processing techniques used in the dairy industry and could lead to innovation in improving the retention of bioactive proteins.

Haas, Joanna et al. Effects of high temperature short time (HTST) pasteurization on milk and whey during commercial whey protein concentrate production. Journal of Dairy Science, Volume 0, Issue 0. DOI: 10.3168/jds.2024-25493

If you have any questions about this kit or any of our other offerings, contact us here.

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Niacin Microtiter Plate Assay Utilized in a Recent Study!

by Eagle Biosciences

The Niacin Microtiter Plate Assay Kit was utilized in a recent study! Researchers developed a novel approach to study microglia, key immune cells in the CNS, under different conditions. Read on for a summary of this groundbreaking research!

Summary

Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis—and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.

Wogram, E et al. Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes. Immunity. 57(9)p:2216-2231.e11 doi:10.1016/j.immuni.2024.07.019

If you have any questions about the Niacin Microtiter Plate Assay Kit or any of our other offerings, contact us here.

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Aldosterone ELISA Utilized in a Recent Study!

by Eagle Biosciences

The Aldosterone ELISA Assay Kit was utilized in a recent study! The study looked at how blood flow in the kidneys and biomarker levels relate to kidney and heart problems in children experiencing shock. Access the abstract and full text below.

Abstract

Importance: Pediatric acute kidney injury (AKI) is a prevalent and morbid complication of shock. Its pathogenesis and early identification remain elusive.

Objectives: We aim to determine whether renal blood flow (RBF) measurements by point-of-care ultrasound (POCUS) and renin-angiotensin-aldosterone system (RAAS) hormones in pediatric shock associate with vasoactive requirements and AKI.

Design, Setting, and Participants: This is a single-center prospective, noninterventional observational cohort study in one tertiary PICU in North American from 2020 to 2022 that enrolled children younger than 18 years with shock without preexisting end-stage renal disease.

Main Outcomes and Measures: RBF was measured by POCUS on hospital days 1 and 3 and plasma RAAS hormone levels were measured on day 1. The primary outcome was the presence of AKI by Kidney Disease Improving Global Outcomes criteria at first ultrasound with key secondary outcomes of creatinine, blood urea nitrogen (BUN), Vasoactive-Inotrope Score (VIS), and norepinephrine equivalent dosing (NED) 48 hours after first ultrasound.

Results: Fifty patients were recruited (20 with AKI, mean age 10.5 yr, 48% female). POCUS RBF showed lower qualitative blood flow (power Doppler ultrasound [PDU] score) and higher regional vascular resistance (renal resistive index [RRI]) in children with AKI (p = 0.017 and p = 0.0007). Renin and aldosterone levels were higher in the AKI cohort (p = 0.003 and p = 0.007). Admission RRI and PDU associated with higher day 3 VIS and NED after adjusting for age, day 1 VIS, and RAAS hormones. Admission renin associated with higher day 3 creatinine and BUN after adjusting for age, day 1 VIS, and the ultrasound parameters.

Conclusions and Relevance: In pediatric shock, kidney blood flow was abnormal and renin and aldosterone were elevated in those with AKI. Kidney blood flow abnormalities are independently associated with future cardiovascular dysfunction; renin elevations are independently associated with future kidney dysfunction. Kidney blood flow by POCUS may identify children who will have persistent as opposed to resolving AKI. RAAS perturbations may drive AKI in pediatric shock.

Fisler, Grace et al. Kidney Blood Flow and Renin-Angiotensin-Aldosterone System Measurements Associated With Kidney and Cardiovascular Dysfunction in Pediatric Shock. Critical Care Explorations 6(8):p e1134, August 2024. DOI: 10.1097

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FGF23 C-Terminal ELISA Employed in a Recent Study

by Eagle Biosciences

In a recent study, researchers employed the FGF23 C-Terminal ELISA Kit from Biomedica Immunoassays to explore the link between dietary phosphorus and key health markers among Puerto Rican adults. Dive into the findings to learn how phosphorus intake from various foods might impact health!

Abstract

Phosphorus (P) additives may be deleterious for health. We measured the P content of key foods, and associations of P intake with biomarkers in the Boston Puerto Rican Health Study (BPRHS). Direct chemical analysis of 92 foods was done with the molybdenum blue spectrophotometric method and inductively coupled plasma mass spectrometry (ICP-MS). A novel algorithm was used to determine bioavailable, natural, and added P. We estimated P intakes from foods in 1323 participants, aged 45–75 y, and associations of these with serum P, fibroblast growth factor 23 (FGF23), parathyroid hormone (PTH), and Klotho. Relationships between intakes and status markers were assessed with Pearson’s correlations and t-tests. Our food analyses generally support P values in the USDA nutrient database, with the exceptions of American and cheddar cheese, which had more P than in the database. Women had higher added P intake than men, and younger participants had higher added P than those older. Total P intake tended to be positively associated with serum P and klotho, and inversely associated with PTH, but relationships were not strong. Puerto Rican adults have high intake of additive P. Culturally sensitive interventions that highlight dietary quality are needed.

O.J. Akinlawon, et al. Phosphorous intake in foods and phosphorus status markers in circulation in the Boston Puerto Rican Health Study, Journal of Food Composition and Analysis, Volume 136, 2024, 106681, ISSN 0889-1575, https://doi.org/10.1016/j.jfca.2024.106681.

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Calprotectin ELISA Utilized in a Recent Publication!

by Eagle Biosciences

Researchers recently utilized our Calprotectin ELISA Assay Kit in an innovative study examining a new treatment for cystic fibrosis (CF)! The study focused on how this treatment influenced both the intestinal microbiome and inflammation, alongside important clinical markers in children with CF. Check out the abstract and access the full text of the study below.

Abstract

The intestinal microbiome influences growth and disease progression in children with cystic fibrosis (CF). Elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA), the newest pharmaceutical modulator for CF, restores the function of the pathogenic mutated CF transmembrane conductance regulator (CFTR) channel. We performed a single-center longitudinal analysis of the effect of ELX/TEZ/IVA on the intestinal microbiome, intestinal inflammation, and clinical parameters in children with CF. Following ELX/TEZ/IVA, children with CF had significant improvements in body mass index and percent predicted forced expiratory volume in one second, and required fewer antibiotics for respiratory infections. Intestinal microbiome diversity increased following ELX/TEZ/IVA coupled with a decrease in the intestinal carriage of Staphylococcus aureus, the predominant respiratory pathogen in children with CF. There was a reduced abundance of microbiome-encoded antibiotic resistance genes. Microbial pathways for aerobic respiration were reduced after ELX/TEZ/IVA. The abundance of microbial acid tolerance genes was reduced, indicating microbial adaptation to increased CFTR function. In all, this study represents the first comprehensive analysis of the intestinal microbiome in children with CF receiving ELX/TEZ/IVA.

Reasoner SA, et al. 2024. Longitudinal profiling of the intestinal microbiome in children with cystic fibrosis treated with elexacaftor-tezacaftor-ivacaftor. mBio 15:e01935-23.https://doi.org/10.1128/mbio.01935-23

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