This document describes the development of an assay to detect anti-PEG antibodies using BioScale's Acoustic Membrane MicroParticle (AMMP) technology. The assay uses paramagnetic beads coated with biotinylated PEG to capture anti-PEG antibodies from serum samples. The bead-antibody complexes are then captured on an acoustic membrane coated with Protein A. Changes in mass on the membrane produce a signal proportional to the amount of anti-PEG antibodies present. Preliminary results suggest the assay can detect IgG anti-PEG antibodies at concentrations below 1000 ng/mL, which is more sensitive than other published methods. The AMMP technology provides a generic platform to detect antibodies against a variety of PEG molecules by modifying
Radioimmunoassay (RIA) is a sensitive biochemical test that uses the principle of competitive binding between labeled and unlabeled antigens/analytes for a limited number of antibody binding sites. RIA combines the specificity of antigen-antibody reactions with the sensitivity of radioactive measurements. It involves incubating a sample containing an unknown amount of antigen with a known amount of labeled antigen and antibody. The amount of labeled antigen bound to the antibody is inversely proportional to the concentration of unlabeled antigen in the sample. By comparing results to a standard curve of known concentrations, the amount of antigen in the unknown sample can be determined with picogram-level sensitivity. RIA has applications in measuring hormones, vitamins, drugs and tumor markers.
This document describes various tests used to evaluate analgesic agents and their mechanisms of action, including the hot plate test, tail clip method, and writhing test using mice. It also discusses the carrageenan-induced rat paw edema test and use of the Ehrlich ascites carcinoma model to study cancer cells. Parameters observed in developmental toxicity, fertility, and teratogenicity tests are outlined. Finally, the mechanisms and methods of genetic toxicology tests like the Ames test and mechanisms of mutation are summarized.
This document provides an overview of basic immunologic procedures used in labeled immunoassays. It describes key terminology like ligands, receptors, sandwich technique and different types of immunoassays including ELISA, RIA, IRMA, fluorescent and chemiluminescent assays. Radioimmunoassays use radioactive labels while enzyme immunoassays use enzymatic labels that are detected through reactions with substrates. The document also discusses various labeling techniques, assay formats (e.g. competitive, non-competitive, homogeneous, heterogeneous), applications and advantages/disadvantages of different immunoassay methods.
This document provides an overview of radioimmunoassay (RIA), a sensitive and specific immunological technique for detecting antigens. It discusses the theory, principle, requirements, and procedure of RIA. RIA involves reacting an antigen with an antibody that has been radiolabeled. A competitive binding reaction then occurs between the radiolabeled and non-radiolabeled antigens. Measurement of radioemission allows for sensitive detection. RIA has applications in detecting hormones, drugs, toxins, and viruses in biological samples. Though sensitive, it requires specialized equipment and handling of radioactive materials poses health risks.
This presentation explains about the principle and procedure involved in elisa method of immunoassay, development o f elisa , application advantages and disadvantages of elisa
Radioimmunoassay (RIA) is a sensitive technique for detecting small quantities of substances using the principle of competitive binding between labeled and unlabeled antigens or ligands to an antibody. RIA of digitalis involves using radioactively labeled digoxin to compete with digoxin in serum samples for binding sites on anti-digoxin antibodies. The bound and unbound fractions are then separated, and the radioactivity counted to quantify the concentration of unlabeled digoxin in the serum sample. RIA of digitalis is used to monitor digoxin levels in patients receiving the drug to treat heart conditions. RIA provides high sensitivity and specificity for detection of biological substances and is used in various fields including endocrinology, pharmac
This presentation explains about the Immunoassay ,radio immuno assay, definition, types, Principle , procedure, steps involved ,advantages ,disadvantages ,Application, RIA in insulin. RIA in Digitalis drug ligand etc....
Radioimmunoassay (RIA) is a sensitive biochemical test that uses the principle of competitive binding between labeled and unlabeled antigens/analytes for a limited number of antibody binding sites. RIA combines the specificity of antigen-antibody reactions with the sensitivity of radioactive measurements. It involves incubating a sample containing an unknown amount of antigen with a known amount of labeled antigen and antibody. The amount of labeled antigen bound to the antibody is inversely proportional to the concentration of unlabeled antigen in the sample. By comparing results to a standard curve of known concentrations, the amount of antigen in the unknown sample can be determined with picogram-level sensitivity. RIA has applications in measuring hormones, vitamins, drugs and tumor markers.
This document describes various tests used to evaluate analgesic agents and their mechanisms of action, including the hot plate test, tail clip method, and writhing test using mice. It also discusses the carrageenan-induced rat paw edema test and use of the Ehrlich ascites carcinoma model to study cancer cells. Parameters observed in developmental toxicity, fertility, and teratogenicity tests are outlined. Finally, the mechanisms and methods of genetic toxicology tests like the Ames test and mechanisms of mutation are summarized.
This document provides an overview of basic immunologic procedures used in labeled immunoassays. It describes key terminology like ligands, receptors, sandwich technique and different types of immunoassays including ELISA, RIA, IRMA, fluorescent and chemiluminescent assays. Radioimmunoassays use radioactive labels while enzyme immunoassays use enzymatic labels that are detected through reactions with substrates. The document also discusses various labeling techniques, assay formats (e.g. competitive, non-competitive, homogeneous, heterogeneous), applications and advantages/disadvantages of different immunoassay methods.
This document provides an overview of radioimmunoassay (RIA), a sensitive and specific immunological technique for detecting antigens. It discusses the theory, principle, requirements, and procedure of RIA. RIA involves reacting an antigen with an antibody that has been radiolabeled. A competitive binding reaction then occurs between the radiolabeled and non-radiolabeled antigens. Measurement of radioemission allows for sensitive detection. RIA has applications in detecting hormones, drugs, toxins, and viruses in biological samples. Though sensitive, it requires specialized equipment and handling of radioactive materials poses health risks.
This presentation explains about the principle and procedure involved in elisa method of immunoassay, development o f elisa , application advantages and disadvantages of elisa
Radioimmunoassay (RIA) is a sensitive technique for detecting small quantities of substances using the principle of competitive binding between labeled and unlabeled antigens or ligands to an antibody. RIA of digitalis involves using radioactively labeled digoxin to compete with digoxin in serum samples for binding sites on anti-digoxin antibodies. The bound and unbound fractions are then separated, and the radioactivity counted to quantify the concentration of unlabeled digoxin in the serum sample. RIA of digitalis is used to monitor digoxin levels in patients receiving the drug to treat heart conditions. RIA provides high sensitivity and specificity for detection of biological substances and is used in various fields including endocrinology, pharmac
This presentation explains about the Immunoassay ,radio immuno assay, definition, types, Principle , procedure, steps involved ,advantages ,disadvantages ,Application, RIA in insulin. RIA in Digitalis drug ligand etc....
Assignment on General principles of ImmunoassayDeepak Kumar
Assignment on General principles of immunoassay: theoretical basis and optimization of immunoassay, heterogeneous and homogenous immunoassay systems. Immunoassay methods evaluation; protocol outline, objectives and preparation. Immunoassay for digoxin and insulin
Radioimmunoassay is a binding assay that uses antibodies and radioactivity to measure the amount of bound and free antigen. Radioactively labeled antigen, called a tracer, competes with unlabeled antigen for binding sites on antibodies. The ratio of bound to free tracer is measured and used to determine the amount of unlabeled antigen present. Radioimmunoassay is versatile, fast, and sensitive but requires the use of radioactivity which is hazardous. It works on the principle that the distribution of antigen between bound and free fractions is determined by the association constant of the antibody-antigen interaction.
Immunoassays are chemical tests that use an immunological reaction to detect or quantify a specific substance in a blood or body fluid sample. They work by measuring the formation of antibody-antigen complexes. Immunoassays can be qualitative, detecting only presence or absence, or quantitative, measuring the actual amount present. Common uses include measuring hormones, drugs, proteins, and markers of diseases. Radioimmunoassays were an early type of immunoassay that used radioactive labels on antigens or antibodies to detect complexes via radiation measurement. While sensitive, radioimmunoassays require special handling due to the radioactive materials.
Radioimmunoassay (RIA) is a sensitive technique introduced in 1960 to detect hormone levels in blood using antibodies and radioactive tracers. It represented the first invitro assay that could detect hormone levels and revolutionized research and clinical practice. RIA uses a radioactive label on the antigen or antibody to quantify its binding to antibodies or antigens, respectively, through competition. It allows for the detection of minute quantities of substances and is widely used in clinical diagnostics and research.
This document discusses immunoassays and two common types - radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). RIA was developed in the 1950s and involves competition between labeled and unlabeled antigens for binding with antibodies. Radioactivity of the antigen-antibody complex is then measured. ELISA uses an enzyme like alkaline phosphate to visualize antigen-antibody reactions. It has two types - direct ELISA using a double antibody sandwich and indirect ELISA using direct antigen coating. Both assays are used to detect various antigens and antibodies and have applications in clinical diagnosis, food testing, and toxicology.
This document provides an overview of two immunoassay techniques: ELISA and RIA. ELISA (enzyme-linked immunosorbent assay) detects the presence of an antigen or antibody using an enzyme-linked secondary antibody that produces a colored product when reacted with a substrate. RIA (radioimmunoassay) uses a radiolabeled antigen or antibody to compete with unlabeled antigens in a sample, and measures radioactivity to determine antigen concentration. Both techniques rely on the specificity of the antigen-antibody reaction and can be used to detect various targets like hormones, drugs, and infectious diseases.
This document provides information about radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques. It discusses the principles, requirements, methodology, and applications of RIA. Key steps in RIA include radio label production, conjugate preparation, antibody production and characterization, and separation techniques. RIA has applications in pharmaceutical analysis and pharmacokinetic studies. ELISA can be used to detect antigens or antibodies and has advantages of sensitivity and accurate measurement of low analyte levels. Both techniques have widespread uses in fields like immunoassay, drug analysis, and HIV testing.
Non Specific Binding of Antibodies in Immunoassays Expedeon
Find out more about non-specific binding here: http://www.innovabiosciences.com/innova/non-specific-binding.html
How to Overcome all of your Problems with Secondary Antibodies
The latest Innova Biosciences webinar focuses on how to overcome the problems of using secondary antibodies. For instance, the use of secondary antibodies:
• Requires a series of incubations and wash steps that are both tedious and time consuming. It is amazing how many times people state how much they hate those wash steps!
• Can often be a source of non-specific staining within experiments which make data interpretation difficult or even impossible.
• Multi-colour analysis often results in cross species re-activity.
Secondary antibodies are generally used either because there are no directly labeled primary antibodies or to increase sensitivity. In this seminar, we will review:
• How labeling of your own antibodies overcomes the need for secondary antibodies.
• How easy it really is to label an antibody using Innova's 30 seconds hands-on antibody labeling kits and design your own unique research tools.
• Application data such as flow cytometry and western blotting generated using directly labeled antibodies
• And question the hypothesis of secondary vs. primary labeled antibodies.
Radioimmunoassay (RIA) is a sensitive technique that uses radioactively labeled molecules and antibodies to detect trace amounts of substances. It works by measuring the competition between a radioactive antigen and a non-radioactive antigen for binding to a limited number of antibodies. RIA has applications in measuring hormones, vitamins, drugs, and markers of infection or cancer. It provides high specificity and sensitivity, allowing detection of picogram quantities. However, it requires special handling of radioactive materials and trained personnel.
This study characterized structural modifications to hepatitis B surface antigen (HBsAg) particles that accompany the loss of immunogenicity induced by heating the particles to 60°C for 3 weeks. Electron microscopy and atomic force microscopy showed that heated particles aggregated into long chains and had altered surface topography compared to untreated spherical particles. Infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated heated particles had decreased alpha-helical content in surface proteins and chemical alterations to tryptophan residues and surface lipids. These structural changes, particularly to epitopes and the particle surface, explain the decreased immunogenicity observed in potency assays with antibodies. The results demonstrate the close relationship between HBsAg particle structure and surface integrity with its ability
1. Radioimmunoassay (RIA) is an immunoassay technique used to detect and quantify substances such as hormones, drugs, and proteins in body fluids using radioactive isotopes. It combines the specificity of antigen-antibody reactions with the sensitivity of radioactive measurements.
2. In RIA, a labeled antigen competes with an unlabeled antigen of interest in a sample for binding to an antibody. The amount of labeled antigen bound is inversely proportional to the amount of unlabeled antigen present.
3. Detection of the bound radioactive labels allows for highly sensitive quantification of the unlabeled antigen in the sample down to picogram levels. RIA is widely used in clinical diagnostics and research.
Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are immunoassay techniques that use antigen-antibody reactions to detect and measure substances like hormones, vitamins, drugs, and biomarkers. RIA uses radiolabeled antigens or antibodies while ELISA uses enzyme-labeled antigens or antibodies, avoiding the hazards of radioactivity. Both techniques are highly specific and sensitive, capable of detecting substances at the nanogram level. They have various applications like hormone level testing, therapeutic drug monitoring, and infectious disease diagnosis.
Immunoassay is a bioanalytical technique that quantifies an analyte using the interaction between an antigen and its corresponding antibody. Radioimmunoassay (RIA) is an immunoassay technique that utilizes radioactive isotopes, such as iodine-125, as labels. RIA was developed in 1959 and allowed for the first in vitro measurement of hormone levels in blood plasma. A key component of RIA is the standard curve, which is generated using known concentrations of unlabeled antigen and is used to quantify unknown samples. RIA provides high sensitivity and specificity but also involves risks associated with the use of radioactive materials.
Hormones, Proteins, etc. present in blood in minute concentration can be assayed by the recent advanced technique of “Enzyme Immuno Assay” without involving any disadvantage. The basic reaction is the interaction between an antibody and an antigen.
Radioimmunoassay (RIA) uses antibody-antigen binding and radioactivity to separate and quantify proteins. It revolutionized research and clinical practice in areas like blood banking and endocrinology. RIA was introduced in 1960 as an assay for insulin levels in plasma. Enzyme-linked immunosorbent assay (ELISA) is similar but uses an enzyme reaction instead of radioactivity, avoiding radiation hazards. ELISA can detect antigens or antibodies and is used to analyze hormones, vitamins, drugs, and diagnose infections. Both RIA and ELISA are highly sensitive and specific immunoassays used widely in research and clinical settings.
This gene encodes D-2hydroxyglutarate dehydrogenase, a mitochondrial enzyme belonging to the FAD-binding oxidoreductase/transferase type 4 family. This enzyme, which is most active in liver and kidney but also active in heart and brain, converts D-2-hydroxyglutarate to 2-ketoglutarate. Mutations in this gene are present in D-2-hydroxyglutaric aciduria, a rare recessive neurometabolic disorder causing developmental delay, epilepsy, hypotonia, and dysmorphic features.
To purchase this antibody, use the following link: http://www.stjohnslabs.com/d2hgdh-antibody-p-68883?filter_name=STJ27209
Radioimmunoassay is an assay technique that uses the binding of antigens and antibodies to measure concentrations of substances. It uses a radioactive tracer that competes with the antigen in a sample for binding to a limited number of antibodies. This allows quantification by measuring the bound versus unbound radioactive tracer. RIA has high sensitivity and specificity, and has revolutionized research and clinical practice in areas like endocrinology, pharmacology, and cancer detection.
ANTIMICROBIAL PEPTIDES & THEIR POTENTIAL TO COMBAT ANTIMICROBIAL RESISTANCE O...Kazimierz Murzyn
Presentation given during Cost AMiCI meeting in Tallinn Nov 2017
by Maria Olivia Pereira, Assistant Professor University of Minho Department of Biological Engineering Braga, Portugal, Professor in Biomedical Engineering Principal Investigator in the Biofilm Group
Radioimmunoassay allows for the measurement of wide range of materials of clinical and biological importance. This technique has a significant impact on medical diagnosis due to the ease with which the tests can be carried out, while assuring precision, specificity and sensitivity.
The radioimmunoassay technique, as the name implies, achieves sensitivity through the use of radionuclides and specificity that is uniquely associated with immunochemical reactions. It can detect substance from a range of Nano gram(ng) to Pico gram(pg).
This document provides information and advice for homeowners considering selling their house. It discusses reasons why selling in the fall makes sense, including strong buyer demand, less competition from other listings, and the potential for a quicker sale process before new mortgage regulations take effect. It also outlines factors to consider when choosing a real estate agent and pricing the house correctly to attract multiple offers. The rest of the document provides tips and statistics related to home prices, mortgage rates, and the risks of selling without an agent.
Martine PINVILLE, Secrétaire d’Etat chargée du Commerce, de l’Artisanat, de la Consommation et de l’Economie sociale et solidaire et Axelle LEMAIRE, Secrétaire d’Etat chargée du Numérique
saluent l’adoption par le Conseil national de la consommation (CNC) d’un avis portant sur les plateformes numériques collaboratives. Le CNC y formule plusieurs recommandations destinées à améliorer l’information des utilisateurs de ces sites.
Assignment on General principles of ImmunoassayDeepak Kumar
Assignment on General principles of immunoassay: theoretical basis and optimization of immunoassay, heterogeneous and homogenous immunoassay systems. Immunoassay methods evaluation; protocol outline, objectives and preparation. Immunoassay for digoxin and insulin
Radioimmunoassay is a binding assay that uses antibodies and radioactivity to measure the amount of bound and free antigen. Radioactively labeled antigen, called a tracer, competes with unlabeled antigen for binding sites on antibodies. The ratio of bound to free tracer is measured and used to determine the amount of unlabeled antigen present. Radioimmunoassay is versatile, fast, and sensitive but requires the use of radioactivity which is hazardous. It works on the principle that the distribution of antigen between bound and free fractions is determined by the association constant of the antibody-antigen interaction.
Immunoassays are chemical tests that use an immunological reaction to detect or quantify a specific substance in a blood or body fluid sample. They work by measuring the formation of antibody-antigen complexes. Immunoassays can be qualitative, detecting only presence or absence, or quantitative, measuring the actual amount present. Common uses include measuring hormones, drugs, proteins, and markers of diseases. Radioimmunoassays were an early type of immunoassay that used radioactive labels on antigens or antibodies to detect complexes via radiation measurement. While sensitive, radioimmunoassays require special handling due to the radioactive materials.
Radioimmunoassay (RIA) is a sensitive technique introduced in 1960 to detect hormone levels in blood using antibodies and radioactive tracers. It represented the first invitro assay that could detect hormone levels and revolutionized research and clinical practice. RIA uses a radioactive label on the antigen or antibody to quantify its binding to antibodies or antigens, respectively, through competition. It allows for the detection of minute quantities of substances and is widely used in clinical diagnostics and research.
This document discusses immunoassays and two common types - radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). RIA was developed in the 1950s and involves competition between labeled and unlabeled antigens for binding with antibodies. Radioactivity of the antigen-antibody complex is then measured. ELISA uses an enzyme like alkaline phosphate to visualize antigen-antibody reactions. It has two types - direct ELISA using a double antibody sandwich and indirect ELISA using direct antigen coating. Both assays are used to detect various antigens and antibodies and have applications in clinical diagnosis, food testing, and toxicology.
This document provides an overview of two immunoassay techniques: ELISA and RIA. ELISA (enzyme-linked immunosorbent assay) detects the presence of an antigen or antibody using an enzyme-linked secondary antibody that produces a colored product when reacted with a substrate. RIA (radioimmunoassay) uses a radiolabeled antigen or antibody to compete with unlabeled antigens in a sample, and measures radioactivity to determine antigen concentration. Both techniques rely on the specificity of the antigen-antibody reaction and can be used to detect various targets like hormones, drugs, and infectious diseases.
This document provides information about radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques. It discusses the principles, requirements, methodology, and applications of RIA. Key steps in RIA include radio label production, conjugate preparation, antibody production and characterization, and separation techniques. RIA has applications in pharmaceutical analysis and pharmacokinetic studies. ELISA can be used to detect antigens or antibodies and has advantages of sensitivity and accurate measurement of low analyte levels. Both techniques have widespread uses in fields like immunoassay, drug analysis, and HIV testing.
Non Specific Binding of Antibodies in Immunoassays Expedeon
Find out more about non-specific binding here: http://www.innovabiosciences.com/innova/non-specific-binding.html
How to Overcome all of your Problems with Secondary Antibodies
The latest Innova Biosciences webinar focuses on how to overcome the problems of using secondary antibodies. For instance, the use of secondary antibodies:
• Requires a series of incubations and wash steps that are both tedious and time consuming. It is amazing how many times people state how much they hate those wash steps!
• Can often be a source of non-specific staining within experiments which make data interpretation difficult or even impossible.
• Multi-colour analysis often results in cross species re-activity.
Secondary antibodies are generally used either because there are no directly labeled primary antibodies or to increase sensitivity. In this seminar, we will review:
• How labeling of your own antibodies overcomes the need for secondary antibodies.
• How easy it really is to label an antibody using Innova's 30 seconds hands-on antibody labeling kits and design your own unique research tools.
• Application data such as flow cytometry and western blotting generated using directly labeled antibodies
• And question the hypothesis of secondary vs. primary labeled antibodies.
Radioimmunoassay (RIA) is a sensitive technique that uses radioactively labeled molecules and antibodies to detect trace amounts of substances. It works by measuring the competition between a radioactive antigen and a non-radioactive antigen for binding to a limited number of antibodies. RIA has applications in measuring hormones, vitamins, drugs, and markers of infection or cancer. It provides high specificity and sensitivity, allowing detection of picogram quantities. However, it requires special handling of radioactive materials and trained personnel.
This study characterized structural modifications to hepatitis B surface antigen (HBsAg) particles that accompany the loss of immunogenicity induced by heating the particles to 60°C for 3 weeks. Electron microscopy and atomic force microscopy showed that heated particles aggregated into long chains and had altered surface topography compared to untreated spherical particles. Infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated heated particles had decreased alpha-helical content in surface proteins and chemical alterations to tryptophan residues and surface lipids. These structural changes, particularly to epitopes and the particle surface, explain the decreased immunogenicity observed in potency assays with antibodies. The results demonstrate the close relationship between HBsAg particle structure and surface integrity with its ability
1. Radioimmunoassay (RIA) is an immunoassay technique used to detect and quantify substances such as hormones, drugs, and proteins in body fluids using radioactive isotopes. It combines the specificity of antigen-antibody reactions with the sensitivity of radioactive measurements.
2. In RIA, a labeled antigen competes with an unlabeled antigen of interest in a sample for binding to an antibody. The amount of labeled antigen bound is inversely proportional to the amount of unlabeled antigen present.
3. Detection of the bound radioactive labels allows for highly sensitive quantification of the unlabeled antigen in the sample down to picogram levels. RIA is widely used in clinical diagnostics and research.
Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are immunoassay techniques that use antigen-antibody reactions to detect and measure substances like hormones, vitamins, drugs, and biomarkers. RIA uses radiolabeled antigens or antibodies while ELISA uses enzyme-labeled antigens or antibodies, avoiding the hazards of radioactivity. Both techniques are highly specific and sensitive, capable of detecting substances at the nanogram level. They have various applications like hormone level testing, therapeutic drug monitoring, and infectious disease diagnosis.
Immunoassay is a bioanalytical technique that quantifies an analyte using the interaction between an antigen and its corresponding antibody. Radioimmunoassay (RIA) is an immunoassay technique that utilizes radioactive isotopes, such as iodine-125, as labels. RIA was developed in 1959 and allowed for the first in vitro measurement of hormone levels in blood plasma. A key component of RIA is the standard curve, which is generated using known concentrations of unlabeled antigen and is used to quantify unknown samples. RIA provides high sensitivity and specificity but also involves risks associated with the use of radioactive materials.
Hormones, Proteins, etc. present in blood in minute concentration can be assayed by the recent advanced technique of “Enzyme Immuno Assay” without involving any disadvantage. The basic reaction is the interaction between an antibody and an antigen.
Radioimmunoassay (RIA) uses antibody-antigen binding and radioactivity to separate and quantify proteins. It revolutionized research and clinical practice in areas like blood banking and endocrinology. RIA was introduced in 1960 as an assay for insulin levels in plasma. Enzyme-linked immunosorbent assay (ELISA) is similar but uses an enzyme reaction instead of radioactivity, avoiding radiation hazards. ELISA can detect antigens or antibodies and is used to analyze hormones, vitamins, drugs, and diagnose infections. Both RIA and ELISA are highly sensitive and specific immunoassays used widely in research and clinical settings.
This gene encodes D-2hydroxyglutarate dehydrogenase, a mitochondrial enzyme belonging to the FAD-binding oxidoreductase/transferase type 4 family. This enzyme, which is most active in liver and kidney but also active in heart and brain, converts D-2-hydroxyglutarate to 2-ketoglutarate. Mutations in this gene are present in D-2-hydroxyglutaric aciduria, a rare recessive neurometabolic disorder causing developmental delay, epilepsy, hypotonia, and dysmorphic features.
To purchase this antibody, use the following link: http://www.stjohnslabs.com/d2hgdh-antibody-p-68883?filter_name=STJ27209
Radioimmunoassay is an assay technique that uses the binding of antigens and antibodies to measure concentrations of substances. It uses a radioactive tracer that competes with the antigen in a sample for binding to a limited number of antibodies. This allows quantification by measuring the bound versus unbound radioactive tracer. RIA has high sensitivity and specificity, and has revolutionized research and clinical practice in areas like endocrinology, pharmacology, and cancer detection.
ANTIMICROBIAL PEPTIDES & THEIR POTENTIAL TO COMBAT ANTIMICROBIAL RESISTANCE O...Kazimierz Murzyn
Presentation given during Cost AMiCI meeting in Tallinn Nov 2017
by Maria Olivia Pereira, Assistant Professor University of Minho Department of Biological Engineering Braga, Portugal, Professor in Biomedical Engineering Principal Investigator in the Biofilm Group
Radioimmunoassay allows for the measurement of wide range of materials of clinical and biological importance. This technique has a significant impact on medical diagnosis due to the ease with which the tests can be carried out, while assuring precision, specificity and sensitivity.
The radioimmunoassay technique, as the name implies, achieves sensitivity through the use of radionuclides and specificity that is uniquely associated with immunochemical reactions. It can detect substance from a range of Nano gram(ng) to Pico gram(pg).
This document provides information and advice for homeowners considering selling their house. It discusses reasons why selling in the fall makes sense, including strong buyer demand, less competition from other listings, and the potential for a quicker sale process before new mortgage regulations take effect. It also outlines factors to consider when choosing a real estate agent and pricing the house correctly to attract multiple offers. The rest of the document provides tips and statistics related to home prices, mortgage rates, and the risks of selling without an agent.
Martine PINVILLE, Secrétaire d’Etat chargée du Commerce, de l’Artisanat, de la Consommation et de l’Economie sociale et solidaire et Axelle LEMAIRE, Secrétaire d’Etat chargée du Numérique
saluent l’adoption par le Conseil national de la consommation (CNC) d’un avis portant sur les plateformes numériques collaboratives. Le CNC y formule plusieurs recommandations destinées à améliorer l’information des utilisateurs de ces sites.
El documento describe la lluvia de ideas o brainstorming, una herramienta de trabajo grupal que facilita el surgimiento de nuevas ideas sobre un tema o problema determinado. Genera muchas ideas en poco tiempo con pocos recursos, cuyos resultados pueden usarse inmediatamente o guardarse para otros proyectos. Requiere un facilitador experto para evitar que sea caótico e intimidante.
Yoga teacher training provides the skills needed to become a certified yoga instructor. The training typically involves 200 hours of instruction and allows graduates to add RYT (Registered Yoga Teacher) to their name. Online programs and coaching organizations offer yoga teacher training with flexible schedules at low costs. The training helps students improve their own yoga practice and develop the calmness and pain management skills needed to teach others effectively. Graduates will be prepared for career opportunities and feel physical changes within their own bodies from regular yoga practice.
Este documento resume los conceptos clave de los modelos VAR, cointegración y corrección de error. Explica que la cointegración permite realizar regresiones significativas entre variables integradas de orden 1 (I(1)), aunque no sean estacionarias. Define la cointegración como la relación estacionaria que surge de la combinación lineal de variables no estacionarias. Finalmente, presenta los pasos para especificar un modelo de cointegración empírico utilizando pruebas de raíz unitaria, causalidad de Granger y tests de cointegra
This document provides information about the seven continents and five oceans. It describes the geographic makeup and surrounding bodies of water for each continent: North America, South America, Europe, Asia, Africa, Australia, and Antarctica. It also gives details on the location and sizes of the five oceans: Pacific, Atlantic, Indian, Arctic, and Southern. The objectives are to name each continent and ocean, locate them on a map, and do identifying activities.
A miniaturized sandwich immunoassay platformQing Chen
This document describes a new miniaturized sandwich immunoassay platform (MSIP) for detecting protein-protein interactions (PPIs) in a high-throughput manner. The MSIP combines antibody microarray technology with co-immunoprecipitation methods to allow simple, rapid, and large-scale PPI detection using small amounts of cell lysate. Evaluation of the MSIP showed it could accurately identify both known interacting and non-interacting protein pairs. Compared to traditional resin-based co-immunoprecipitation, the MSIP has higher sensitivity and throughput while being simpler and more cost-effective. The MSIP is presented as an effective method for validating PPIs identified by other techniques like yeast two-hybrid screening
A visual chip-based coimmunoprecipitation technique for analysis of protein–p...Qing Chen
This document describes a visual chip-based coimmunoprecipitation (vChip-coIP) technique for analyzing protein-protein interactions. Key points:
1. The technique combines advantages of antibody microarrays, traditional coIP, and silver enhancement detection. Antibodies are spotted onto slides to capture interacting protein pairs from cell lysates.
2. Interactions are detected using a biotinylated antibody, colloidal gold-labeled streptavidin, and silver enhancement. This makes interaction signals visible without further processing.
3. The technique is shown to be simple, cost-effective, and efficient for comprehensive study of protein-protein interactions using small amounts of crude cell lysate.
The document discusses various drug delivery methods including conventional methods like oral, injection, and transdermal delivery as well as novel methods like liposomes, peptides, nanoparticles, and nanocomposites. Liposomes are described as tiny bubbles enclosed by a phospholipid bilayer that can encapsulate drug molecules and target delivery to specific tissues. Peptide drug delivery uses short chains of amino acids linked by peptide bonds to deliver drugs. Nanocomposites embed nano-sized particles in a polymer matrix, exhibiting enhanced properties while reducing cytotoxicity. Novel drug delivery provides benefits over conventional methods like reduced side effects, targeted delivery, and controlled release of drugs.
Western Blot Assessment of Polyclonal Anti-Host Cell Protein Antibody ProductionCovance
BEBPA HCP 2019 -- The most critical part of developing an ELISA for measuring Host Cell Protein (HCP) impurities in Biotherapeutics is generation of specific antibodies with appropriate recognition of the total population of HCPs potentially present in the product. The generation of the antiserum is dependent upon the sum of the individual biological responses (i.e., antibodies) of multiple animals to the antigen. Response is monitored throughout the program qualitatively by 1D (one dimension) Western Blot and adjustments are made to the antigen (immunogen) as needed to yield the broadest antigen recognition. Ultimately, the highest quality antisera from multiple animals are pooled. The quality of the final product is demonstrated through evaluation of coverage which is typically by 2D (two dimension) Western Blot or more recently by mass spectrometry. The BioCMC Group at Covance was tasked with development of a process capable of supporting this type of assessment. This capability will be illustrated through discussion of a typical antibody reagent production program.
Antibody Coupling - Single Coupling Approach To Bind Antibodies Diverse Speci...Anteo Technologies
Traditional covalent chemistries use harsh chemicals and require expertise in the techniques of diverse covalent coupling methodologies. The Antibody Coupling Kit was made to address issues such as: difficulties in binding certain antibodies with traditional covalent chemistries, antibody wastage, and incorrect antibody orientation. Anteo’s technology offers scientists the flexibility to bind any antibody onto a solid support surface through the use of polymeric metal complexes. The polymeric metal nature of the technology allows multi-valent binding of the target antibody through chelation to the electron donating groups located in the Fc region of the antibody. Anteo’s kit promotes gentle monolayer binding, meaning proteins assemble in the correct orientation while reducing the amount of damaged proteins, leading to increased functionality of antibodies and less antibodies used for the experiment.
This application note demonstrates the ability of the Antibody Coupling Kit to bind Mouse IgG, Mouse IgM, Rabbit IgG, Human IgG and Human IgM antibodies onto 200 nm magnetic particles using a particle-based fluorescent antibody loading assay.
This document describes the development of a novel fluorescent protein-based sensor for detecting 2-oxoglutarate (2OG) levels in living cells. The sensor, termed mOGsor, was created by inserting the 2OG-binding domain GAF from the NifA protein into yellow fluorescent protein (YFP). mOGsor exhibits increased fluorescence intensity upon binding to 2OG in a concentration-dependent manner. Testing showed mOGsor has high specificity for 2OG and fast kinetics. Using mOGsor, the authors were able to monitor real-time changes in 2OG levels in E. coli cells under different nutrient conditions. mOGsor represents an improvement over previous FRET-based 2OG sensors by providing a
Combined effects of PEGylation and particle size on uptake of PLGA particles ...Nanomedicine Journal (NMJ)
Abstract
Objective:
At the present study, relationship between phagocytosis of PLGA particles and combined effects of particle size and surface PEGylation was investigated.
Materials and Methods:
Microspheres and nanospheres (3500 nm and 700 nm) were prepared from three types of PLGA polymers (non-PEGylated and PEGylation percents of 9% and 15%). These particles were prepared by solvent evaporation method. All particles were labeled with FITC-Albumin. Interaction of particles with J744.A.1 mouse macrophage cells, was evaluated in the absence or presence of 7% of the serum by flowcytometry method.
Results:
The study revealed more phagocytosis of nanospheres. In the presence of the serum, PEGylated particles were phagocytosed less than non-PEGylated particles. For nanospheres, this difference was significant (P<0/05) and their uptake was affected by PEGylation degree. In the case of microsphere formulation, PEGylation did not affect the cell uptake. In the serum-free medium, the bigger particles had more cell uptake rate than smaller ones but the cell uptake rate was not influenced by PEGylation.
Conclusion:
The results indicated that in nanosized particles both size and PEgylation degree could affect the phagocytosis, but in micron sized particles just size, and not the PEGylation degree, could affect this.
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1. Brief/Technical Note
Theme: Emerging Technologies in the BioAnalytical Space Applied to Large Molecule Determinations
Guest Editors: Johanna Mora and Binodh DeSilva
Development of a Generic Anti-PEG Antibody Assay Using BioScale’s Acoustic
Membrane MicroParticle Technology
Huijin Dong,1,3
Johanna R. Mora,1
Catherine Brockus,1
Shannon D. Chilewski,1
Robert Dodge,1
Colin Merrifield,2
W. Matthew Dickerson,2
and Binodh DeSilva1
Received 7 January 2015; accepted 8 June 2015
Abstract. Immunogenicity testing for PEGylated biotherapeutics should include methods to detect both
anti-protein and anti-PEG antibodies (anti-PEG). Although some methods have been published for the
detection of anti-PEG antibodies, the information is incomplete and, in some cases, reagents used (such
as Tween-20) are known to interfere with detection. This rapid communication describes the use of
BioScale’s Acoustic Membrane MicroParticle (AMMP®) technology using the ViBE® Workstation to
measure anti-PEG antibodies in human serum samples. Briefly, a sample spiked with monoclonal human
IgG anti-PEG antibody is diluted in buffer and incubated with paramagnetic beads coated with linear
chain mPEG to capture anti-PEG antibodies. The complex is then captured on an acoustic membrane
coated with Protein A. The change in mass on the membrane caused by the binding of the complex to the
membrane results in a signal proportional to the mass of anti-PEG antibodies. The data indicate that an
assay with a sensitivity of less than 1000 ng/mL for IgG is achievable. This level of sensitivity is better
than current published reports on IgG anti-PEG antibody detection.
KEY WORDS: acoustic membrane microparticle technology; anti-peg antibodies; emerging technology;
immunogenicity assays; pegylated biotherapeutics.
INTRODUCTION
PEGylation is a well-documented modification used to
increase therapeutic protein half-life. However, immune
responses to the PEG itself have caused, in some cases, loss
of product efficacy and adverse safety consequences, which
highlights the importance of developing a strategy to monitor
anti-PEG antibodies based on risk assessment (1). Also, the
recently published FDA Guidance for Industry on Immuno-
genicity Assessment for Therapeutic Protein Products has
recommended that for PEGylated therapeutic protein prod-
ucts, anti-drug antibody (ADA) assays should be able to
detect both anti-protein antibodies and antibodies against the
PEG moiety (2). This recommendation has proven to be a tall
order, as developing and validating assays to detect antibod-
ies against a PEG moiety is a major challenge. In a review
paper by Schellekens et al. (3), the authors concluded that
most, if not all, assays used for detecting anti-PEG antibodies
are flawed due to the lack of specificity as well as poor
characterization of positive controls (3, 4). Until recently,
traditional bridge immunoassay format assays have been able
to detect anti-PEG IgM antibodies but have struggled to
detect IgG isotype antibodies with sufficient sensitivity in
human matrix (5, 6), suggesting that the type of PEG and/or
protein therapeutic may play a role. In addition to previously
published comments (7), our own observations during assay
development have noted that high levels of IgG in a sample
make detection of low affinity anti-PEG IgG antibodies
difficult in a plate-based or non-plate-based assay format
using anti-human IgG detection reagents. A well-
characterized antibody positive control and robust assay to
detect anti-PEG IgG isotype will help to understand the
mechanism of induced anti-PEG response following
PEGylated therapeutic protein injection in human (8, 9). In
this rapid communication, we report preliminary results for
detecting IgG anti-PEG antibodies using an Acoustic Mem-
brane MicroParticle (AMMP) platform.
The Acoustic Membrane MicroParticle platform is an
emerging technology that utilizes a non-optical detection
system to determine analyte concentration by measuring the
change in the oscillating frequency of a piezoelectric mem-
brane (10). This rapid communication describes a method in
which human serum spiked with monoclonal chimeric IgG is
diluted in buffer and incubated with paramagnetic beads
coated with either PEGylated therapeutic protein or biotinyl-
ated PEG to capture anti-PEG antibodies. The complex is
then detected by magnetically pulling all paramagnetic beads
1
Bioanalytical Sciences Biologics, Bristol-Myers Squibb, Princeton,
New Jersey 08543, USA.
2
BioScale, Billerica, Massachusetts 01821, USA.
3
To whom correspondence should be addressed. (e-mail:
Huijin.dong@bms.com)
The AAPS Journal (# 2015)
DOI: 10.1208/s12248-015-9799-4
1550-7416/15/0000-0001/0 # 2015 American Association of Pharmaceutical Scientists
2. onto an acoustic membrane sensor coated with Protein A.
Beads that are complexed with anti-PEG antibodies remain
bound to the membrane sensor through the Protein A,
following removal of the magnet. The change in mass on
the membrane results in a signal proportional to the mass of
anti-PEG antibodies.
Biotinylated PEG of various molecular weights can be
coupled to streptavidin-coated paramagnetic beads, making
this technology able to detect anti-PEG antibodies against a
variety of PEG molecules. The work presented here focuses
on the implementation of AMMP for the detection of anti-
PEG antibodies for immunogenicity assessment.
MATERIALS AND METHODS
Materials
Commercial Reagents
Biotin-PEG 20 kDa and biotin-PEG 40 kDa were
purchased from Nanocs (www.nanocs.net, New York, NY).
All biotinylated PEG molecules used in this study as well as
in positive control characterization were linear chain mole-
cules with a single biotin attached at one end and a methyl
cap at the other end except for the PEG (40 kDa branched)
attached to BMS drug A. The following buffers were
purchased from Thermo Fisher Scientific, Waltham, MA:
Blocker Casein in phosphate-buffered saline (PBS) with 1%
(w/v) casein (Hammarsten grade), pH 7.4; Super Block buffer
in PBS with proprietary protein, pH 7.4; and Super Block
buffer in Tris-buffered saline (TBS) with proprietary protein,
pH 7.4. Normal human sera were purchased from
Bioreclamation LLC, Westbury, NY. AMMP Type I Labeling
Kit for Assay Discovery, AMMP Type II Labeling Kit for
Assay Discovery, and Protein A Cartridges with ViBE
Cartridge Regeneration Buffer were purchased from
BioScale, Billerica, MA. Polypropylene plates (96-well) for
inline incubations were purchased from BioScale.
Proprietary Reagents
Drug A is a BMS biotherapeutic with a 40 kDa PEG
attached to a 12 kDa protein.
Custom-made monoclonal anti-PEG antibody (PEG.2):
Briefly, mice were immunized at BMS with a panel of
PEGylated BMS therapeutics and hybridomas were selected
that showed binding to PEG coupled to unrelated protein to
ensure anti-PEG reactivity. The variable region genes from
antibody production cell line were sequenced, subcloned into
human IgG1 Fc bearing constructs, stably expressed in CHO
cell lines and purified. The binding of PEG.2 was analyzed on
a Biacore T200 SPR instrument (GE Healthcare, Piscataway,
NJ, USA) by immobilization of different MW biotin-mPEG
onto a Neutravidin-coated sensor. PEG.2 showed the highest
normalized binding response units (RU) to the 20 kDa PEG
compared to 5, 10, 30, and 40 kDa PEG. Also, amine-PEG-
amine (aPEGa from Creative PEGWorks with a linear
formula NH2(CH2)2O(CH2CH2O)n(CH2)2NH2 and MW
20,000) was a stronger inhibitor than triethylene glycol
monomethyl ether (mTEG from Sigma-Aldrich Corp. with
linear formula CH3(OCH2CH2)3 and MW 164.20) in the
binding of PEG.2 to biotin-PEG on a plate suggesting that
PEG.2 is a backbone unit-specific antibody (11).
METHODS
Capture Reagent
Epoxy Bead Labeling
PEGylated BMS drug A was directly conjugated to
epoxy beads (BioScale’s AMMP Type II Labeling Kit) per
manufacturer’s procedure.
Streptavidin Bead Labeling
An AMMP Type I Labeling Kit was used for binding of
biotinylated PEG of 20 and 40 kDa to paramagnetic
streptavidin beads. The method for binding biotinylated
PEG to the beads was modified slightly. Briefly, biotinylated
PEG was diluted in PBS buffer and reacted with streptavidin
beads. The manufacturer’s recommendations were followed
except that biotinylated bovine serum albumin (BSA) was
not used to saturate open binding sites that were not
specifically blocked with a biotinylated reagent.
Protein A Detection With or Without Off-Line Bead Wash
The ViBE platform, for this assay, uses Protein A on a
piezoelectric membrane to capture the antibody complex. In
the detection step, bound antibody complexes are separated
from other matrix components present in the sample by
magnetically attracting the beads to the membrane surface,
but allowing beads without bound anti-PEG to fall away from
the protein A-coated membrane as the magnet is then
removed.
Experiments were designed to compare the assay with or
without off-line wash, that is, homogenous vs. a non-
homogeneous assay. PEG.2 dilutions ranging from 0.250 to
4.0 μg/mL were prepared in PBS with 1% BSA (w/v) and
incubated with epoxy beads conjugated with drug A for 1 h.
The plate was then placed either on a BioTek washer
equipped with a magnet block to allow aspiration of non-
binding components, or analyzed on the ViBE without the
wash step. In both cases, a Protein A surface was used for
detection.
Buffer Selections
Multiple buffers were tested for suitability, and three
buffers (Blocker Casein in PBS, Super Block in PBS, and
Super Block in TBS) were further tested for bead blocking
and sample dilution. Conjugated type I beads were diluted in
each buffer and blocked for 2 h at ambient temperature prior
to use. Control samples were prepared by adding PEG.2
positive control to normal human serum pool at 0.625 to
40 μg/mL and diluted to a final concentration of 5% (v/v) in
each of the buffers. Detection of the controls was evaluated
using both sets of biotin-20 kDa PEG and biotin-40 kDa PEG
type I beads (20 and 30 μg PEG/mg beads) without off-line
wash. Blocking and serum dilutions were paired for these
comparisons.
Dong et al.
3. Assay Sensitivity and Reproducibility Using Optimized Assay
Format
The optimized assay format using 10% serum (v/v) is
shown in Fig. 1. Control samples were prepared by
adding PEG.2 positive control to normal human serum
pool at 0.625 to 40 μg/mL and stored at −70°C for 24 h
prior to use. The spiked controls were thawed at room
temperature and diluted tenfold in Blocker Casein in
PBS. To perform the assay, Biotin-PEG 20 kDa labeled
beads were first diluted in Blocker Casein in PBS and
incubated for 1.5 h at room temperature. Then, 80 μL of
each positive control diluted to 10% serum were com-
bined with 40 μL of blocked bead solution in a 96-well
polypropylene plate and incubated for 1 h on the ViBE
instrument integrated shaker. Once the incubation was
complete, the online assay steps were initiated and data
were collected by the ViBE software version 0.7.4.14126.
The controls were run a total of 12 times over 2 days,
one plate per day. Each plate contained six replicates.
The mean response and intra- and inter-run %CV were
calculated.
RESULTS
Assay Format Using Protein A Detection With or Without
off-Line Bead Wash
The assay using drug A on epoxy (type II) beads
with Protein A cartridges had better sensitivity without
using the off-line wash than when the off-line wash was
performed. Accumulation values recorded by the ViBE
are the measurement of the number of beads in the
reaction. The data indicated that magnet-separated sam-
ples (pre-treatment step) had lower and less consistent
bead accumulation counts than the samples without a
magnetic separation pre-treatment step. Mean bead values
for samples in 96 wells without a magnetic separation pre-
treatment step were more than twofold greater than for
magnetic separated reactions and bead values in 96 wells
had a good precision (%CV≤6.7). These data are
summarized in Fig. 2.
Buffer Selections for Blocking and Sample Dilution
Using streptavidin (SA) coated type I beads and two
loading concentrations of biotin-PEG 20 kDa, Blocker
Casein in PBS had the lowest background signal and
resulted in the best assay sensitivity as shown in Fig. 3.
Differences between the 20 and 30 μg/mg loads indicated
that the lower coating concentration was more effective at
providing higher sensitivity (<1000 ng/mL) as well as a
greater range of detection to up to 40 μg/mL, the
maximum level tested. Similar results were obtained using
40 kDa biotin-PEG beads (not shown).
Assay Sensitivity Using Optimized Format
The sensitivity of the optimized assay was assessed in
human serum using PEG.2 anti-PEG antibody as a positive
control. Data were normalized to the negative control value
for each dilution buffer on each plate. A normalized curve is
shown in Fig. 4. Approximate sensitivity of the ViBE method
using 10% human serum in Blocker Casein in PBS and
20 kDa PEG/20 μg/mg beads with the PEG.2 positive control
is 800 ng/mL based on lowest control concentration above a
cut point. The cut point was calculated according to the
formula of average + 1.645 x SDMean, where the average of
0.14 and standard deviation of 12 replicates of unspiked
human serum pool were used. Assay reproducibility was
evaluated using the same runs as for sensitivity, one plate per
day for each of 2 days. Each plate consisted of six replicates
of the titration curve prepared as tenfold dilutions in Blocker
Casein in PBS against 20 kDa PEG /20 μg/mg beads. Inter-
plate and intra-plate variability for the replicates were
indicated by ≤6.7% CV and 1.8% CV, respectively, as shown
in Table I.
DISCUSSION
Reports (12, 13) of both pre-existing and therapeu-
tically induced anti-PEG antibodies highlight the impor-
tance of developing a reliable assay for their detection.
The development of an assay for the detection of anti-
PEG antibodies has proven to be challenging. Some
assays have been developed to measure IgM response,
but the measurement of IgGs is more complicated
because of the high levels of other human IgGs
interfering when using an anti-human Fc in the assay.
Also, we hypothesize that anti-PEG IgGs may be less
successful at bridging two distinctly labeled PEG mole-
cule instead of both antibody Barms^ binding two units
within the same molecule.
Using PEG.2 as a positive control antibody, a
generic Acoustic Membrane Micro Particle assay using
the ViBE instrument platform for the detection of anti-
PEG antibodies in human serum has been successfully
developed. It is possible that this assay on the ViBE can
overcome issues observed in other assays regarding high
Anti-PEG Sample
Biotin-PEG
Protein A
Fig 1. AMMP assay format: anti-PEG detection with biotin-PEG
20 kDa immobilized on the magnetic bead and detection of complex
on the cartridge coated with Protein A. Biotin-PEG is coupled onto
streptavidin-coated paramagnetic beads. Anti-PEG in the human
serum forms complexes on the coated beads under homogeneous
conditions. When samples flow across the sensor cartridge, the bead
complex is captured by Protein A bound to the sensor surface, a
piezoelectric membrane. This change in mass on the sensor generates
a signal proportional to the mass of bound anti-PEG
Development of a Generic Anti-PEG Antibody Assay
4. background signal due to non-specific binding thanks to
the following factors:
a) In the ViBE platform, bound antibody complexes are
separated from other matrix components present in the
sample by attracting beads to the membrane surface
magnetically, but allowing unbound beads to fall away
from the membrane as the magnet is then removed.
Other proteins in sample may bind to the Protein A
surface, but if they are not complexed to magnetic
Fig 2. Comparative 3-D graphs of bead accumulation values across assay plates using a an
off-line bead wash and b without off-line bead wash prior to loading onto the ViBE
analyzer. The bead accumulation value is the number of beads captured at sensor surface
when the magnet is engaged. Magnetic separation was achieved by using a BioTek
microwell plate washer fitted with a magnetic plate to draw down beads suspended in
solution, followed by aspiration of supernatant. Bead accumulation without off-line bead
wash was superior to results obtained with an off-line bead wash because some of the
beads were lost during the wash. Assay format without off-line bead wash was selected for
the final method development
Fig 3. Buffer effect on assay background and sensitivity. Positive control curves were
prepared in buffer containing 5% (v/v) human serum and assay was performed with biotin-
PEG 20 kDa using type I streptavidin beads coated with 20 or 30 μg biotin-PEG. Reduced
slopes, sensitivity, and higher background values were found using 30 μg coated beads,
regardless of buffer used. The combination including Blocker Casein in PBS with 20 μg
coated beads was selected for further testing
Dong et al.
5. beads, they would not contribute significantly to the
change in mass on the membrane and would not be
detected.
b) The assay format does not require an off-line wash
step. This may help improve the detection of low
affinity antibodies.
c) The format utilizes a protein A detection surface.
Protein A has five repeated IgG binding domains
which increases assay sensitivity compare to utilization
of an anti-IgG as the detector antibody.
In our internal assay development, evaluation of other
assay formats and technologies (MSD and AlphaLISA) with
PEG.2 as the positive control only achieved sensitivities in the
50–500 μg/mL range (data not shown). Furthermore, exper-
iments on the ViBE (data not shown) using anti-fluorescein
on the membrane and FITC-labeled anti-human Fc antibod-
ies as the detection system resulted in high background and
apparent non-specific binding that could not be overcome
with washes or separation steps which suggested the need for
an alternative format.
Some of the key factors to be considered in the
development of a sensitive anti-PEG assay are:
1. Positive Control
We have found that not all positive controls are
detectable across all platforms and assay formats.
Hence, it is very important to use well-characterized
monoclonal or polyclonal antibodies to try to under-
stand advantages and disadvantages across assay
formats and platforms.
2. Assay Buffer
In the development of this assay, multiple commercial
and Bhome-made^ blocking buffers were tested. For
this particular format, Casein in PBS from Thermo
Fisher Scientific provided the lowest signal for the
0
2
4
6
8
0010111.0
Concentration of PEG.2 ( g/mL)
SignaltoNoiseRatio
Fig 4. Dose–response curve (4PL regression using GraphPad) of the immunogenicity
ViBE assay for the detection of anti-PEG antibodies. The observed sensitivity based on the
95th percentile confidence interval was 800 ng/mL. Calibrators were prepared by spiking
PEG.2 positive control into the normal human serum at 0.625 to 40 μg/mL and normal
human serum was used as negative control. Calibrators and negative control were diluted
tenfold in Blocker Casein in PBS. Biotin-PEG 20 kDa labeled beads at 20 μg/mg were
reacted in a homogeneous mixture with 80 μL of each spiked control in 10% serum in a 96-
well polypropylene plate and incubated for 1 h on the ViBE instrument integrated shaker.
Signal-to-noise ratio=mean instrument response of positive control (n=12)/mean instru-
ment response of negative control (n=12)
Table I. Intra- and Inter-Assay Precision Values for Anti-PEG AMMP Assay Reproducibility
Nominal concentration (μg/mL)
Statistic 40.0 20.0 5.0 1.3 0.8 0.6 0.0
N 12 12 12 12 12 12 12
Mean AMMP Signal 0.822 0.725 0.461 0.218 0.182* 0.159 0.14
SD 0.017 0.026 0.065 0.048 0.023 0.042 0.019
Normalized Signal 5.87 5.18 3.29 1.56 1.30 1.14 1.0
Intrabatch (%CV) 0.0 0.1 0.7 1.2 1.2 1.8 1.7
Interbatch (%CV) 0.0 0.1 1.3 3.9 2.9 6.7 6.4
Assay sensitivity level using calculated cut point as described in text (0.17)
Development of a Generic Anti-PEG Antibody Assay
6. NQC and in turn the best assay sensitivity. However,
we have observed lot-to-lot variability in assay
performance.
3. Labeling Ratio of PEG/mg Bead
During assay development, we found that beads
labeled with 30 or 40 μg of a PEGylated protein per
mg of beads gave lower background signal compared
to the beads labeled with 10 or 20 μg of protein per
mg of bead in buffers containing 5% human serum.
This finding suggested that higher protein coverage of
the bead surface helped decrease non-specific binding
of other serum proteins to the beads. However, when
beads were labeled with PEG alone (no protein),
20 μg of PEG per mg beads had lower background
and better assay sensitivity than beads labeled with
30 μg. In the future, studies around the orientation of
the PEG on the beads may help better understand the
results.
4. Removal of Tween-20 or Any Other Surfactants that
Resemble PEG from the Assay Buffer
Sherman et al. (14, 15) previously reported on the
detrimental effects of such surfactants on the sensitiv-
ity of detection for anti-PEG antibodies. Since our
positive control antibody is PEG backbone unit
specific, the interference was expected. In our internal
assay development, we also observed that Tween-20 in
the wash buffer reduced assay sensitivity. In a direct
MSD buffer assay with a polyclonal antibody as
positive control, nice dose–response curves were
observed with three different PEGs tested (0.35,
0.75, and 40 kDa) when only PBS was used as wash
buffer. The dose responding curves disappeared when
0.05% Tween 20 was added to PBS wash buffer. We
are currently exploring other detergents which do not
have structural resemblance to PEG.
This manuscript is the first report in which the ViBE
platform is used to detect anti-PEG antibodies and the
first report on adequate sensitivity for detection of an IgG
anti-PEG control. As a rapid communication, it only
touches on some of the key aspects to the development
of an anti-PEG assay. We have recently undertaken the
task of comparing a several anti-PEG positive controls
from our inventory across at least two platforms to
deepen our understanding of advantages and potential
pitfalls of each.
CONCLUSION
An Acoustic Membrane MicroParticle method for de-
tection of anti-PEG antibodies using the ViBE instrument
from BioScale has been found to be reproducible (% CV<7)
and with a sensitivity of near 1000 ng/mL in human serum
samples. This preliminary work is intended to assist re-
searchers developing assays for detection of anti-PEG
antibodies and will be expanded upon.
ACKNOWLEDGMENTS
The authors would like to acknowledge the work done
by Murli Krishna and Holly Palmé in the oversight of reagent
generation for this program; Steven Piccoli for review of this
manuscript.
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