Author: Eagle Biosciences

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New Complement Assays from Svar Life Science!

by Eagle Biosciences

We are excited to bring you the newest addition to our line of Complement Assays, two new Factor P assays – one quantitative and one functional. Developed and Manufactured by Svar Life Science, these kits are designed to work optimally together. These assays are ideal for assisting in the development of next-generation complement therapies!

In addition to measuring the properdin (Factor P) concentration and determining its functionality, the Factor P assays can be easily adapted for more in-depth studies, granting a closer look at the amplification loop of the complement system.

About Factor P

Factor P (Complement P, Properdin, FP) is a positive regulator and an initiator of the alternative pathway (AP) for complement activation. It binds surface-bound C3 and C5 convertases and stabilizes them to amplify the activation cascade1. Factor P binding increases the half-life of the convertase complex approximately 10-fold2. It is suggested, however debated, that Factor P also can initiate complement activation by binding for example cell surfaces or certain biological substrates, recruiting C3b or C3(H2O) and Factor B and thus initiate the AP pathway1,3. Factor P opposes the negative regulation of Factor H that enhances the dissociation of C3b and Bb and mediates Factor I cleavage of C3b to the inactive iC3b3 (Figure 1).


Figure 1: Factor P as stabilizer and initiator of the alternative complement pathway (left) and the opposing negative regulation by Factor H3 (right).

Factor P is not produced in hepatocytes as most complement proteins, but instead by several cell types including monocytes, macrophages, T-cells and granulocytes. It is likely that transient increased concentration of Factor P enhances the AP upon local stimuli4. For example, neutrophils have Factor P-containing granules that are secreted upon stimulation and can enhance the platelet-granulocyte aggregate formation1. In plasma, Factor P is present in a concentration of approximately 4-25 µg/mL5. Factor P is an elongated 53 kDa glycoprotein that oligomerizes in vivo to dimers, trimers or tetramers (P2, P3 and P4) in a ratio of 26:54:20 (P2:P3:P4) in head to tail structures5,6. Mutations, deficiencies, protein levels as well as protein deposits of Factor P are connected to diseases and disorders summarized by Chen et al 3. Deficiencies generally increase the susceptibility for meningococcal disease and other infectious diseases7. Altered serum levels have been associated with for example C3 glomerulopathy, Lupus Nephritis, sepsis and chronic heart failure and IgA nephropathy8.

References

  1. Blatt, A. Z.; Pathan, S.; Ferreira, V. P. Properdin: A Tightly Regulated Critical Inflammatory Modulator. Immunol Rev 2016, 274 (1), 172–190. https://doi.org/10.1111/imr.12466.
  2. Fearon, D. T.; Austen, K. F. Properdin: Binding to C3b and Stabilization of the C3b-Dependent C3 Convertase. J Exp Med 1975, 142 (4), 856–863.
  3. Chen, J. Y.; Cortes, C.; Ferreira, V. P. Properdin: A Multifaceted Molecule Involved in Inflammation and Diseases. Mol. Immunol. 2018, 102, 58–72. https://doi.org/10.1016/j.molimm.2018.05.018.
  4. Cortes, C.; Ohtola, J. A.; Saggu, G.; Ferreira, V. P. Local Release of Properdin in the Cellular
    Microenvironment: Role in Pattern Recognition and Amplification of the Alternative Pathway of Complement. Front Immunol 2013, 3. https://doi.org/10.3389/fimmu.2012.00412.
  5. Pangburn, M. K. Analysis of the Natural Polymeric Forms of Human Properdin and Their Functions in Complement Activation. The Journal of Immunology 1989, 142 (1), 202–207.
  6. Smith, C. A.; Pangburn, M. K.; Vogel, C. W.; Müller-Eberhard, H. J. Molecular Architecture of Human Properdin, a Positive Regulator of the Alternative Pathway of Complement. J. Biol. Chem. 1984, 259 (7), 4582–4588.
  7. Fijen, C. A. P.; van den Bogaard, R.; Schipper, M. Properdin Defciency: Molecular Basis and Disease Association. Molecular Immunology 1999, 36, 863–867.
  8. Michels, M. A. H. M.; Volokhina, E. B.; van de Kar, N. C. A. J.; van den Heuvel, L. P. W. J. The Role of Properdin in Complement-Mediated Renal Diseases: A New Player in Complement-Inhibiting Therapy? Pediatr Nephrol 2019, 34 (8), 1349–1367. https://doi.org/10.1007/s00467-018-4042-z.

If you have questions about these new items, or any of our other offerings, contact us here.

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Rat NT-proBNP Product Spotlight

by Eagle Biosciences


We are excited to bring you the new Rat NT-proBNP ELISA Assay Kit! This product has been developed, validated, and manufactured by Biomedica in Austria. This assay extends our robust line of kits specific to cardiac biomarkers. Learn more below!

Biomarker Background

BNP is mainly expressed by the ventricular myocardium in response to volume overload and increased filling pressure. It is synthesized and secreted by cardiomyocytes. Mature BNP consists of 108 amino acids (proBNP or BNP-108). ProBNP is cleaved during secretion in a 1:1 ratio resulting in physiologically active BNP-32 and the biologically inactive 76 amino acid NT-proBNP. NT-proBNP (1-76) has greater plasma stability and a much longer biological half-life (90-120 minutes) than BNP, being considered as the preferred laboratory marker. BNP has a key role in cardiovascular homeostasis with biological actions including natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion. A high concentration of BNP in the bloodstream is indicative of heart failure.

Rat NT-proBNP ELISA Assay Kit Features

  • Low Sample Volume Required – 10 µL/well
  • Controls Included
  • Sample Values Provided

Related Products

NT-proBNP ELISA Assay Kit
NT-proCNP ELISA Assay Kit
NT-proANP ELISA Assay Kit
BNP Fragment ELISA Kit

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

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Anti-Human C4d Antibody Utilized in Recent Publication

by Eagle Biosciences

Liver Transplant: antibody-mediated rejection monitored by C4d

Liver transplantation has become a routine treatment for children with end stage liver failure. Antibody-mediated rejection of the transplant can be monitored by C4d. This recent study utilized the Anti-Human C4d Antibody from Biomedica. Check out the abstract below.

Abstract

Liver transplantation has become a routine treatment for children with end stage liver failure. Recently, the long term survival of pediatric patients after liver transplantation has improved, with a life expectancy much longer than that of adult recipients, but also with longer exposition of the graft to various injuries, including immunological, inflammatory and others. Biochemical tests, although important, do not always reflect graft injury. The aim of our study was to analyze the histopathology of the graft in late protocol biopsies and correlate it with the clinical and biochemical status of these patients. We analyzed 61 protocol liver biopsies taken from 61 patients. Biopsies were taken 9.03-17.09 years (mean 12.68, median 11.74 years) after transplantation. Liver specimens were examined particularly for the presence and stage of liver fibrosis, inflammation, steatosis, and acute or chronic cellular and humoral rejection. We did not find any abnormalities in 26 (42.6%) liver specimens. None of the patients had signs of cellular or antibody mediated rejection or chronic rejection. In 23 liver biopsies (37.7%), we found non-specific lymphoid infiltrates. Another problem was fibrosis (equal to or more than three on the Ishak scale)-we found it in 17 patients, including seven liver specimens (11.5%) with severe fibrosis (Ishak 5-6). Conclusions: Various pathomorphological abnormalities were found in more than half of patients with a median 11.74 years post-transplant follow-up. Most of them presented normal laboratory liver tests at the same time, suggesting a slow subclinical process leading to pathomorphological abnormalities. No single factor for the development of these abnormalities was found, but our study supports the need for protocol liver biopsies even in patients with normal/almost normal biochemical liver tests.

Markiewicz-Kijewska M, Szymańska S, Pyzlak M, Kaliciński P, Teisseyre J, Kowalski A, Jankowska I, Czubkowski P, Ismail H. Liver Histopathology in Late Protocol Biopsies after Pediatric Liver Transplantation. Children (Basel). 2021 Aug 1;8(8):671. doi: 10.3390/children8080671. PMID: 34438562; PMCID: PMC8392008.

Anti-Human C4d Antibody Features

  • Widely cited for ICH
  • For kidney, heart, liver and other transplants

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

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Assay Ready Cells Product Spotlight

by Eagle Biosciences

The value of assay-ready cells in providing biologically relevant data for robust therapeutic development.

One of our suppliers, Svar Life Science, was recently featured in an editorial Select Science! This exclusive interview was with Dr. Peter Betz Wolff of the Analytical Development department at AGC Biologics. Dr. Wolff works to implement and develop a range of cell-based assays to meet specific client needs. He shares how his team recommends the use of assay-ready cells to provide the valuable biologically relevant data required for robust pharmaceutical development.

“Compared to non-cell-based approaches, the biggest advantage with cell-based systems is that they are a biologically relevant system, as they include data concerning internal signal cascades.” says Dr. Peter Betz Wolff. “This is of considerable interest to the authorities when reviewing potential new biologics coming to market.”

Check out the full article here.

iLite® Assay Ready Cells are developed by our partners at Svar Life Science. Their iLite technology is based upon a reporter gene assay format, modified and adapted for applications during the whole drug development cycle as well as for monitoring of biological drugs. These cell lines can be developed for any biopharmaceutical target and assays for drug potency, i.e. drug activity, and neutralizing antibodies (NAbs) can easily be set-up using the same cell line. The Assay Ready cells are genetically engineered to be used with a reporter gene assay technique for detection the the drug potency and the NAbs.

Check out our full portfolio of iLite Assay Ready Cells here.

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Microscopy Product Spotlight: Stencell

by Eagle Biosciences

What is Stencell intended for?

Stencell is used notably for wound-healing assay & immunocytochemistry experiments. It is very helpful when you are working with super expensive reagents or very rare cell lines, and when you want to test various experimental hypothesis.

Stencell for wound-healing experiments

Stencell Designs

Each order is composed of 2 sheets of 50 identical stencils (=100 stencils). Upon receipt of your purchase order, you will be able to choose either the same design for your 2 sheets, or 2 different designs for your 2 sheets (1 per sheet of 50).

    • Solo: 1 circular well – Diam. 12 mm
    • Quartet: 4 circular wells – Diam. 3 mm
    • Nonet: 9 circular wells – Diam. 3 mm
    • Presto: 2 oblong wells spaced by 0.35 mm
    • Allegro: 2 oblong wells spaced by 0.62 mm

    Features

    1. Design your PDMS – Our design process offers total freedom to turn your idea into a PDMS.
    2. Stack and Play – Compose flow channels and chambers, stacking different designs of Stencell.
    3. Stick it to a variety of culture labware – So far, it has been successfully stuck onto the following: glass slides, plastic dishes, Transwell(R) inserts, Polyacrylamide gels.
    4. Remove it – Stencell is not glued. It can then be removed to trigger cell migration, switch from flow chamber to open window. (Very useful for wound healing experiments)
    5. It is compatible with imaging – These thin sheets of silicone are fully transparent, without autofluorescence.
    6. Parallelized and standardized experiments – One multiwell Stencell can perform several experimental conditions.
    7. It saves samples and reagents – Only a few microliters are required.

    Key Conditions of Success

    • When you remove the inner elements of your Stencell, use 2 tweezers: 1 to remove and the other 1 to press close to the junction in order to avoid breaking the Stencell. Alternatively, you can cut the junctions with a scalpel.
    • When you put the Stencell on top of your culture substrate, softly remove the bubbles and stick the Stencell, patting it with tweezers.
    • Depending on the substrate hydrophobicity, the droplet might not completely fill the well. You can:
      • Either fill the well with the standard volume. Then, help the droplets stick to the Stencell by connecting the liquid next to the Stencell walls using the pipette tip.
      • Or, you may add an excess volume of liquid (e.g. 30 μL) and remove it afterwards (e.g. 10 μL).
    • But: do not overfill (e.g. > 30 μL) the wells, otherwise two droplets may merge.
    • When you put your substrate and cells in the incubator. Wait for 1 to 2 hours until cells spread and fully occupy the windows space. If you need to wait longer, be sure that the droplets do not completely evaporate.

    For more information about this product or any others from the Microscopy line, contact us here.

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Microscopy Product Spotlight: Stampwell

by Eagle Biosciences

What is it Stampwell intended for?

Stampwell is highly helpful if you have tons of 3D samples to image. The V-shape Stampwell is used to image them (2 different sizes). And the Rectangular Stampwell is designed to image Zebrafish or Medaka embryos. It avoids spending hours to locate your sample in the dish. It prevents them from slipping from one well to another when you move your dish from the incubator to the microscope. And it allows to image and observe how your biological samples grow over several weeks.

3 Shapes of wells to choose freely and best fit your experiments:

  • V Shape: 7*6 pins in V-shape – Diameter 26mm – Exists in 2 different sizes: 300 or 500u
  • Rectangular:7*5 rectangular – Diameter 26mm
  • “My Shape”: design your wells, adapted to your samples and your experimental needs!

Features of Stampwell

  1. Cross-platform – Compatible with most 35 mm dishes and 6-well plates.
  2. Reusable – Use your stamp dozens of times.
  3. Medium Culture Conditioning – U Shape: the design of a groove increases the culture medium conditioning and cell aggregates survival.
  4. Long-term imaging over weeks with V-shape and Rectangular – the sample just lays on the bottom of the well and can freely grow within it.
  5. Safe transport of 3D objects – V-shape and Rectangular keep your samples at the bottom of the wells, even when the dish is upside down.
  6. Automatized multi-position image acquisition V-shape and Rectangular – provide a constraint-free but stable and parallelized immobilization of the samples.
  7. Customize your shape – My-Shape is your own design and number of wells. Contact us for details.

For more information about this product or any others from the Microscopy line, contact us here.

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Microscopy Product Spotlight: Everspark

by Eagle Biosciences

What is Everspark intended for?

Everspark is a hassle-free buffer used to decouple the preparation of your samples and their imaging. Use it if you need to prepare your samples in your lab and image them in your core facility 3 to 4 weeks later. Everspark is also compatible with imaging that uses red, far-red and green dyes (eg. AF488). Different colors can be used either at the same time or one after the other.

Features

  1. High stability over time for repeated measurements – One mounted sample is stable over 3 to 4 weeks and can be imaged multiple times
  2. Multicolor (including green) – Compatible with green, red, and far red dyes (JF646, JF549, AF647, DL550, CF568, DL650, CF680, SulfoCy5 and mEos2)
  3. Ready to Use – Can be used directly from the vial
  4. Up to 6 months performance – Individualized packaging for optimal longevity
  5. Techniques – PALM, dSTORM, HILO, TIRF pattern
  6. Customizable – Choose the MEA concentration, pH, optical index, or even additives

Use Method:

  • Rinse the coverslip with sterile water
  • Discard water in excess
  • Prepare sealant: 1 volume of sealant catalyst + 1 volume of sealant basis in a micro-dish – do not mix
  • Open the Everspark tube and pipette right away the desired volume onto a Wilco glass bottom dish
  • Flip the coverslips face towards the slide on the cavity filled with Everspark without making bubbles
  • Absorb excess of Everspark buffer on the sides with a kimwipe
  • Mix sealant
  • Seal quickly with the reconstituted sealant
  • Wait for the sealant to polymerize on the slide before moving the slide
  • Discard the open tube of Everspark buffer (cannot be used for another slide, once already opened)
  • Gently clean the coverslip with 70% ethanol
  • Perform regular dSTORM acquisition (best to wait a couple of hours)
  • Repeat dSTORM acquisition 24 hours up to 3 weeks on the same slide providing no air bubbles were present
  • Store the slides at 4°C and in the dark before repeating dSTORM acquisition on the same slide (up to 3 weeks)

For more information about this product or any others from the Microscopy line, contact us here.

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DKK-1 levels predict disease outcomes in Covid-19

by Eagle Biosciences

Circulating DKK-1 levels predict disease outcomes and mirror metabolic adaptations in patients with Covid-19

Individuals with low serum levels of DKK-1 (Dickkopf-1) are twice as likely to die from Covid-19 than those with high levels according to new research published by Nikolai Jaschke and colleagues in the Journal of Clinical Endocrinology & Metabolism.

The researchers found that circulating DKK1 levels vary in humans and change as a function of time during SARS-CoV-2 infection. The infection promotes metabolic adaptations that resembles fasting, which are mirrored by circulating DKK1 levels. DKK-1 levels predict disease outcomes in Covid-19 individuals.

The results of the study suggest a potential use of measuring circulating DKK1 as an indicator of disease severity in COVID-19 patients.

Abstract

Context and aims: Coronavirus disease 19 (COVID-19) trajectories show high interindividual variability, ranging from asymptomatic manifestations to fatal outcomes, the latter of which may be fueled by immunometabolic maladaptation of the host. Reliable identification of patients who are at risk of severe disease remains challenging. We hypothesized that serum concentrations of Dickkopf1 (DKK1) indicate disease outcomes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals.

Methods: We recruited hospitalized patients with PCR-confirmed SARS-CoV-2 infection and included 80 individuals for whom blood samples from 2 independent time points were available. DKK1 serum concentrations were measured by ELISA in paired samples. Clinical data were extracted from patient charts and correlated with DKK1 levels. Publicly available datasets were screened for changes in cellular DKK1 expression on SARS-CoV-2 infection. Plasma metabolites were profiled by nuclear magnetic resonance spectroscopy in an unbiased fashion and correlated with DKK1 data. Kaplan-Meier and Cox regression analysis were used to investigate the prognostic value of DKK1 levels in the context of COVID-19.

Results: We report that serum levels of DKK1 predict disease outcomes in patients with COVID-19. Circulating DKK1 concentrations are characterized by high interindividual variability and change as a function of time during SARS-CoV-2 infection, which is linked to platelet counts. We further find that the metabolic signature associated with SARS-CoV-2 infection resembles fasting metabolism and is mirrored by circulating DKK1 abundance. Patients with low DKK1 levels are twice as likely to die from COVID-19 than those with high levels, and DKK1 predicts mortality independent of markers of inflammation, renal function, and platelet numbers.

Conclusion: Our study suggests a potential clinical use of circulating DKK1 as a predictor of disease outcomes in patients with COVID-19. These results require validation in additional cohorts.

Check out the full article here.

Circulating DKK-1 levels were measured with the DKK-1 ELISA from Biomedica

  • Small sample volume – 20µl serum/well
  • Reliable –international validation guidelines
  • Easy – direct measurement
  • Widely cited in +170 publications

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

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Microscopy Product Spotlight: Chitozen

by Eagle Biosciences

What is Chitozen intended for?

Chitozen is used to image bacteria both still and alive under the microscope, or it can be used to perform long-term imaging of bacteria. It can also be used if you want to change the growth condition (e.g. antibiotics, chemicals, inhibitors) during the experiment and directly observe, in real-time, the bacteria new comportment under the microscope.

E.coli monolayers on Chitosan

For what bacteria is Chitozen used for?

Chitozen has been tested with the following bacteria: E. coli, Vibrio cholerae, Myxococcus xanthus, Mycobacterium smegmatis, Bacillus subtilis, Pseudomonas aeruginosa and Pseudomonas fluorescens. More bacteria being currently tested.

Growth of E.coli on Chitozen

Simplified Protocol

Features

  1. Full compatibility with most of your conditions of experiments – Size: the coverslip dimension (25x75mm) is compatible with the most common available sticky slides and microscope stages; it is also compatible with advanced microfluidic techniques, nanolithography
  2. 6 independent channels – Either perform up to 6 experiments at the same time or use 1 channel one day, and the others later
  3. Ready and fast – Assemble it within 2 minutes, use it the same day as its prepared
  4. Long lasting – A bench-stable surface coated with chitosan, the most efficient way to immobilize your bacteria on a microscope coverslip; storable for 2 months once assembled

For more information about this product or any others from the Microscopy line, contact us here.

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Microscopy Product Spotlight: Actiflash

by Eagle Biosciences

What is Actiflash intended for?

Actiflash is used to convert your inducible ERT model into a photo-inducible one. Use it if you want to control transcription (using Gal4-UAS) or induce recombination (using Cre-lox) in space and/or time for in-vivo cell tracking experiments and more. Actiflash is for research use only

Features

    1. Wide applicative scope – Technology capitalizing on the versatile use of Tamoxifen-OH for controlling functions of multiple types of proteins.
    2. Simple conditioning – Caged Cyclofen-OH is cell-permeant and can be added either in the external medium or directly injected for conditioning.
    3. Excellent chemical stability – Caged Cyclofen-OH does not generate any basal activation of protein function and it benefits from an excellent temporal resolution upon uncaging.
    4. Favorable wavelength ranges for uncaging – Uncaging requires either UV-A light or a strong IR laser. Visible light is inactive, which facilitates the experiments with biological samples.
    5. Photochemical stability – Caged Cyclofen-OH liberates Cyclofen-OH, which is photostable in contrast to Tamoxifen-OH encountering photodegradation under illumination.

Method of Use:

Calibration of the Actiflash concentration
It is advised to first establish the extent of phenotype sought for as a function of the Tamoxifen-OH concentration. Then the concentration of Actiflash used for sample conditioning is fixed at Tamoxifen-OH concentration causing 100% of the desired phenotype (in general 3-5 μM in cultured cells and zebrafish embryos).

Conditioning protocol
Incubate your samples in a serum-free medium for 90 mins, away from light.

Photoactivation
Illumination of Actiflash may be performed with UV (325-425nm range) light or multiphoton excitation (at 750 and 1064 nm with two- and three-photon excitation, respectively) to release Cyclofen-OH. You can use either benchtop UV lamps or light sources installed on microscopes.

The calibration of the photoactivation
The objective is to provide enough photons to exhaust the conversion of the Actiflash but without generating detrimental side-effects on the biological sample. Simply analyze the phenotype recovery with decreasing illumination duration. Then determine the shortest illumination duration leading to 100% uncaging of Actiflash.

5 Key Conditions of Success

  1. Actiflash is provided as a powder. Resuspend it with DMSO (warning: no water).
  2. Only unfreeze the aliquots you need for your experiment.
  3. Carefully calibrate the photoactivation of Actiflash (concentration for conditioning, power of the light source, geometry and duration of illumination). Then always work under the same conditions.
  4. Use a serum-free medium during the incubation step of the cell lines.
  5. In the dark, just before photoactivation, wash your samples (unnecessary for two-photon laser experiments).

For more information about this product or any others from the Microscopy line, contact us here.