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Fluorometric detection typically relies on the use of an antibody that has been labeled with a fluorophore. Time-resolved fluorescence (TRF) is very similar to standard fluorometric detection. But TRF relies on the use of very specific fluorescent molecules, called lanthanide chelate labels, which allow detection of the emitted light to take place after excitation has occurred. The most commonly used lanthanide chelate label is the europium ion (Eu3+). It emits light over a longer period after excitation (microseconds as opposed to nanoseconds). This helps reduce background noise by delaying the start of the measurement until after the background signal has decayed. The measurement of assay signal using non-visible light spectra can increase dynamic range and is not limited to the same extent by saturation.

AxisPharm Europium conjugation technology can be applied to microplate-based assay or immunochromatographic assay. Our technology can provides significantly higher sensitivity in comparison with other particle-based assays. please feel free to contact us for more details.

Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassays. This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones. ELISA test kits are widely used for the in vitro quantitative measurement of different disease biomarkers in human serum, urine or stool samples. The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result, based on different signal detection methods, like Colorimetric ELISA, Chemiluminescent ELISA, Electrochemiluminescence, Immuno-PCR, and so on. AxisPharm helps our clients develop customized ELISA assays, including direct, indirect, sandwich and competitive assays with fast turnaround time. Our professional team is ready to help you with high-quality and cost-effective testing services to make your project a success. please feel free to contact us for more details.

Difference Between ELISPOT and ELISA

October 18, 2022 Posted by Axispharm

With the wide application of ELISA technology in the medical and biological fields, new breakthroughs have been made in the detection of various cytokines and antibodies in vitro. When studying the mechanism of immune response, enzyme-linked immunosorbent assay (ELISA) was used to detect free cytokines (CK) or antibodies in body fluids. Metabolized or combined with target organs, but cannot accurately reflect the level of antibodies and CK in vivo. In the 1980s, based on the basic principle of ELISA technology, researchers established a solid-phase enzyme-linked immunospot technique (ELISPOT) for detecting specific antibody-secreting cells and CK-secreting cells in vitro.

Main difference between ELISPOT and ELISA:

The ELISPOT method is derived from ELISA and breaks through the traditional ELISA method. It is an extension and new development of quantitative ELISA technology. Both detect cytokines or other soluble proteins produced by cells, and their biggest differences are:

1. ELISA measures the absorbance on a microplate reader through a color reaction, and compares it with the standard curve to obtain a quantitative total amount of soluble protein.

2. ELISPOT shows clearly distinguishable spots on the corresponding positions where the soluble protein is secreted by cells through the color reaction. The spots can be manually counted directly under the microscope or counted by the ELISPOT analysis system. One spot represents one spot. Active cells to calculate the frequency of cells secreting the protein or cytokine. (Some studies measure not only the amount of cytokine produced, but also the frequency of cells secreting the cytokine)

3. Because it is a single-cell level detection, ELISPOT is more sensitive than ELISA and limiting dilution methods, and can detect 1 cell secreting this protein from 200,000-300,000 cells.

4. The capture antibody is a high-affinity, high-specificity, low-endotoxin monoclonal antibody produced by Swedish high-tech biological company MABTECH, BD, R&D. When researchers activate cells with stimulators, they will not affect the secretion of cytokines by activated cells.

Detection principle

Cells are stimulated to locally produce cytokines, which are captured by specific monoclonal antibodies. After cell dissociation, the captured cytokines bind to biotin-labeled secondary antibodies, which in turn bind to alkaline phosphatase-labeled avidin. After incubation with BCIP/NBT substrate, “purple” spots appeared on the PVDF plate, indicating that the cells produced cytokines, and the results were obtained after the spots were analyzed by the ELISPOT enzyme-linked spot analysis system.

Application field

Prediction of rejection in transplantation, vaccine development, Th1/Th2 analysis, autoimmune disease research, tumor research, allergic disease research, infectious disease research, epitope mapping analysis, screening of compounds and drug immunological responses, etc.

CHEMISTRY CONTRACT RESEARCH SERVICES

Custom Synthesis – Pharmaceutical Intermediates, ADC Linkers, Bio-labeling Reagent, Fluorescent Dye Probes

As a biotechnology company conveniently located in San Diego, AxisPharm offers the biotech communite a range of services that include ADC linker custom synthesis, long term FTE/flexible FTE, and CRO tech transfer services.

Our highly experienced chemists are equipped with PhD level degrees and substantial experience in the pharmaceutical industry. We utilize state-of-the-art instruments for synthesis, purification and analysis in order to best serve the scientific community, promising a swift, successful result that will exceed your expectations.

We tailor each project to our clients‘ obligations and preferences by using existing routes or designing/developing robust approaches in order to fulfill our customers’ demand.

AxisPharm provides long term and flexible FTE services to best help our clients tackle synthetic challeges and lead optimizations.

At AxisPharm, we conduct pilot scale synthesis validation and provide detailed technology write ups in order to ensure a smooth technology transfer.

Classification and application of fluorescent dyes

August 16, 2023 Posted by Axispharm

Fluorescent dyes refer to substances that absorb light waves of a certain wavelength and emit light waves with a wavelength greater than that of the absorbed light. Most of them are compounds containing a benzene ring or a heterocyclic ring with a conjugated double bond. Fluorescent dyes can be used alone or combined into composite fluorescent dyes. Under the microscope, it functions by absorbing light at a given wavelength and re-emitting it at a longer wavelength. This fluorescence produces different colors that can be visualized and analyzed.

The composite fluorescent dye is a fluorescent dye synthesized by fluorescence resonance energy transfer technology, which is composed of a donor and an acceptor fluorescent substance molecules that are very close in distance and can transfer energy between each other. The complex dye is excited at the excitation wavelength of the acceptor molecule and emits a photon at the emission wavelength of the donor molecule. The development of fluorescent dyes is very rapid, and the fluorescent dyes developed for scientific research and clinical applications have basically covered the entire spectral range from ultraviolet to visible light and infrared.

Fluorescent dyes are commonly used for fluorescent labeling of various biomolecules (antibodies, peptides, various proteins, etc.) for monitoring drug delivery to target tissues, imaging, and other processes.

Fluorescent dye classification:

1. Fluorescein dyes, including fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. and their analogs. This is a class of compounds with more benzene rings. The most widely used is FITC (as shown in the picture of FITC-labeled tissue fluorescence), which is excited by an argon ion laser at 488 nm and emits blue-green fluorescence at 525 nm. FITC can bind to various antibody proteins and stably exhibits blue-green fluorescence in alkaline solution.

2. Rhodamine dyes, including red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. TRITC emits yellow fluorescence at 570 nm when excited at 550 nm.

3. Cy series cyanine dyes, cyanine dyes usually consist of two heterocyclic ring systems, including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, cy7 nhs ester , cy3b nhs ester and their analogs.

4. Alexa series dyes, which are series of fluorescent dyes developed by Molecular Probes. Its excitation light and emission light spectrum cover most of the visible light and part of the infrared spectral region, and it is widely used. Features high brightness, stability, instrument compatibility, multiple colors, pH insensitivity, and water solubility. Includes Alexa Fluor 350, 405, 430, alexa fluor 488 maleimide, 532, 546, 555, 568, 594, 610, 633, alexa 647 nhs ester, 680, 700, 750. At present, Alexa series dyes are widely used in the market and gradually replace traditional fluorescent dyes. For example, Alexa Fluor 488 can replace FITC and Cy2; Alexa Fluor 555 can replace Cy3 and TAMRA; Alexa Fluor 633 can replace APC, Cy5, etc.

5. APDye Fluors, APDye Fluors is the equivalent fluorescent dye of alexa fluors developed by axispharm. APDye Fluors series fluorescent dyes are bright in color, optically stable, very good in light stability of dyeing brightness, and very competitive in price. It can be applied to the labeling and localization of tissues, cells and biomolecules in biomedical research. Its excitation and emission spectra cover most of the visible and part of the infrared spectral region and are suitable for most fluorescence microscopes.

6. Protein dyes, including phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc. Most of them are proteins found in cyanobacteria. Such fluorescent dyes can be coupled with Cy series cyanine dyes to form complex dyes for antibody labeling. For example, PE-Cy3/Cy5/Cy7, APC-Cy7, PerCP-Cy5.5, etc. are commonly used in the market.

Scientific research application

The earliest application of dyes in biochemistry was to directly stain sections and then visualize them. With the continuous development of biotechnology, computer technology and fluorescence spectrometry technology, many dyes, especially fluorescent dyes, have been widely used in cell detection, tumor gene protein analysis, toxic analysis, clinical medical diagnosis and so on.

Due to its high sensitivity and convenient operation, fluorescent dyes have gradually replaced radioisotopes as detection markers, and are widely used in fluorescent immunology, fluorescent probes, and cell staining. Including specific DNA staining, for chromosome analysis, cell cycle, apoptosis and other related research. Numerous nucleic acid dyes are also useful counterstains in multicolor staining systems as background controls, labeling nuclei so that the spatial relationships of intracellular structures can be seen at a glance.

Immunoassay

Fluorescence-labeled monoclonal antibody technology expands infinite application space for flow cytometry in the study of cell membranes and various functional antigens in cells, tumor gene proteins and other fields. Fluorescent probes can be covalently bound to monoclonal antibodies via protein cross-linkers. The most commonly used dyes for immunofluorescence labeling are fluorescein isothiocyanate (FITC), phycoerythrin (PE) and AlexaFluor series dyes.

Nucleic acid amplification testing

Nucleic acid fluorescent dyes stain the nucleus and quantitatively measure the fluorescence intensity of the cell, so that the content of DNA and RNA in the nucleus can be determined, and the cell cycle and cell proliferation can be analyzed. There are a variety of fluorescent dyes that can stain DNA or RNA in cells. Commonly used DNA dyes include propidium iodide (PI), DAPI, Hoechst 33342, etc., and RNA dyes include thiazole orange, acridine orange, etc.

Ideal fluorescent dyes generally have the following characteristics:

1. It has high photon yield and high signal intensity;

2. Strong absorption of excitation light, reducing background signal;

3. The distance between the excitation spectrum and the emission spectrum is large to reduce the interference of background signals;

4. It is easy to combine with the labeled antigen, antibody or other biological substances without affecting the specificity of the labeled substance;

5. Good stability, not easily affected by light, temperature, pH, specimen anticoagulant and fixative.

Chemiluminescence immunoassay (CLIA) is an analytical method that combines a chemuluminescence system with an immune reaction therefore has the advantages of high sensitivity, wide linearity range of detection, for high throughput, low analyte concentration, time sensitive testing and isolation. The assay format is similar to enzyme-linked immunoassays, usually based on heterogeneous assays where antibodies or antigens are immobilized on a solid phase but one of the components is conjugated with a chemiluminescent label. The benefits of CLIAs can be enhanced using magnetic beads as a solid platform which improves the separation of the un-bound reagents and reduces the interferences using a magnetic field. CLIAs have been widely used in the fields of clinical diagnosis, life science, environmental monitoring, food safety and drug analysis. Professional team and scientists in AxisPharm have developed both magnetic beads or 96-well plates based CLIA test kits, with highly sensitive, flexibility and easy-to-use format.

If you are interested in our chemiluminescent immunoassay based kits development, please feel free to contact us for more details.

In the development of small molecule drugs, it is necessary to evaluate the bioavailability of the compound as early as possible, so as to lay the foundation for subsequent development. In addition to using experimental animals to evaluate bioavailability, various in vitro analysis methods have been developed to evaluate the ability of compounds to penetrate the intestinal wall. These in vitro experiments are not only lower in cost but also higher in throughput and shorter experimental time. Among them, Caco-2 monolayer cell permeability analysis assay has become one of the standards in vitro methods.

CaCO-2 cells are derived from human colon cancer and have the typical characteristics of intestinal epithelial cells, such as the formation of a polarized monolayer columnar epithelium, and densely differentiated microvilli on the top surface of the cells neatly arranged to form a striated border and intercellular connection.

The determination of Caco-2 cell permeability not only helps to determine intestinal permeability but also can identify specific transporters or efflux proteins and intestinal phase II drug-metabolizing enzymes. Therefore, this Caco-2 permeability assay provides valuable information for drug development in an effective and reproducible manner, which is not available in non-biological models, and even in many other biological models.

The Efflux Ratio can be calculated by measuring the transport from A-B and B-A in two different directions, so as to determine whether a molecule has active efflux.

Caco-2 Permeability Assay Protocol

Compound required

10 mM DMSO 100µL or 2 mg

Compound Test Concentration

10µM

Cell Culture

21 days

Replicates

N=2

Incubation Conditions

Incubation in CO2 incubator at 37ºC for 90 min

Positive control compound

digoxin （or other compounds）

Integrity of the monolayer

TEER>200 Ω•cm2

Analysis method

LC-MS／MS

Data delivery

Papp, Efflux Ratio, %Recovery

Data calculation

Papp = CRecx VRec/ (A x t x C0) Efflux Ratio = Papp (B→A) /Papp (A→B) Recovery = [(Vrec x Crec) + (Vd x Cd)] / (Vd x C0)

Bioconjugation Service at AxisPharm

Bioconjugation is the process of linking molecules/polymers or biomolecules/biopolymers together by covalent bonds. Conjugation can enhance drug efficacy by improving pharmacokinetic and pharmacodynamic properties and by reducing plasma protein binding. For instance, introducing polyethylene glycol (PEG) or polysaccharide reduces toxicity and improves solubility and stability.

AxisPharm offer our clients with affordable and high quality custom bioconjugation service that covers:

Early de-risking and optimization

Process development and optimization

Formulation development

Analytical method development

Drug substance manufacture

Drug product manufacture

Payload and linker manufacturing

Our bio conjugation service delivers affordable and high-quality custom bioconjugation service, using combinations of payloads, linkers, and conjugation methods.

Antibody-Drug Conjugates (ADCs)

Antibody-Oligonucleotide Conjugates (AOCs)

Protein-drug Conjugation

Fluorophore Conjugation (products: fluorescent dyes)

Biotin conjugation (products: biotin peg)

Polymer–drug conjugation (products: polymer peg)

Polymer-drug-target ligand conjugation

PEGylation and cross-linking

Cysteine-based conjugation

Lysine-based conjugation

Thio-engineered antibody

Carbohydrate-based conjugation

Unnatural amino acids-based conjugation (product: amino peg)

Analytical – AxisPharm has the analytical tools and experience that are critical for this field. We are equipped with sophisticated mass spectrometers for small and large molecules to analyze and quantify protein, antibody and their conjugates. We routinely use high resolution mass spectrometer on real time analysis of drug-antibody ratio for bioconjugation synthesis, process optimization and quality control. Please click for real time analysis case study.

Synthetic Chemistry and Biology Expertise – AxisPharm has a large collection of ADC linker, such as PEG linker, Peptide Linker, Sugar Liner, Dye Probe in stock and has extensive experience in synthesis, purification and characterization of both small and large molecules to support bioconjugation R&D.