1. ELISA and RIA are plate-based assay techniques used to detect and quantify peptides, proteins, antibodies, and hormones using the principle of antigen-antibody binding.
2. ELISA is most commonly performed in 96-well polystyrene plates where antibodies or antigens are immobilized, and detection involves an enzyme-linked secondary antibody binding which produces a measurable color change.
3. RIA uses radioactive labeling of antigens or antibodies, and detection involves measuring unbound radioactive material after competition between labeled and unlabeled antigens or antibodies for antibody binding sites.
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. There are several key steps: extraction of proteins from a sample, separation by size using gel electrophoresis, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubation with primary and secondary antibodies to detect the target protein, and use of a substrate to visualize the antibody-protein complex. Western blotting has applications in disease diagnosis, detecting defective proteins, and confirming the presence of viruses or bacteria.
Polyclonal antibodies are produced by injecting an antigen into a host animal which causes its immune system to produce various antibodies that recognize different epitopes of the antigen. The antibodies are then purified from the animal's blood plasma. Polyclonal antibodies are a heterogeneous mixture that can bind to multiple epitopes of the same antigen, whereas monoclonal antibodies are all clones that recognize the same epitope.
Immunoblotting assays such as Western blotting allow detection of specific proteins in complex mixtures by separating proteins by gel electrophoresis, transferring them to a membrane, and using antibodies to identify target proteins on the membrane. The presentation provides details on the key steps of tissue preparation, gel electrophoresis, protein transfer, blocking, detection using labeled antibodies, analysis, and applications of immunoblotting assays like Western blotting.
ELISA- Principle, procedure , types and applicationsJaskiranKaur72
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.
The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result.
The document discusses enzyme-linked immunosorbent assay (ELISA), including its introduction, principle, equipment, procedure, types, advantages, and disadvantages. ELISA is a qualitative or quantitative immunological procedure that detects antigens or antibodies using enzyme-labeled antibodies and chromogenic substrates. It relies on antibody-antigen interactions and uses an enzyme-labeled antibody to generate a colored reaction, allowing detection of a particular antigen. The document outlines the basic equipment, general procedure involving coating wells with antibodies and adding samples and enzyme-labeled antibodies, and the three main types of ELISA - indirect, sandwich, and competitive.
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies and antigens in a liquid sample. It relies on an enzyme-linked antibody or antigen to detect the target protein. There are different types of ELISA including direct, indirect, sandwich, and competitive. The ELISA process involves coating a plate with an antigen or antibody, adding a sample and enzyme-linked antibody, washing unbound material, and detecting the enzyme's product to quantify the target. ELISAs are widely used in medical testing, food safety, and disease detection.
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. There are several key steps: extraction of proteins from a sample, separation by size using gel electrophoresis, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubation with primary and secondary antibodies to detect the target protein, and use of a substrate to visualize the antibody-protein complex. Western blotting has applications in disease diagnosis, detecting defective proteins, and confirming the presence of viruses or bacteria.
Polyclonal antibodies are produced by injecting an antigen into a host animal which causes its immune system to produce various antibodies that recognize different epitopes of the antigen. The antibodies are then purified from the animal's blood plasma. Polyclonal antibodies are a heterogeneous mixture that can bind to multiple epitopes of the same antigen, whereas monoclonal antibodies are all clones that recognize the same epitope.
Immunoblotting assays such as Western blotting allow detection of specific proteins in complex mixtures by separating proteins by gel electrophoresis, transferring them to a membrane, and using antibodies to identify target proteins on the membrane. The presentation provides details on the key steps of tissue preparation, gel electrophoresis, protein transfer, blocking, detection using labeled antibodies, analysis, and applications of immunoblotting assays like Western blotting.
ELISA- Principle, procedure , types and applicationsJaskiranKaur72
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.
The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result.
The document discusses enzyme-linked immunosorbent assay (ELISA), including its introduction, principle, equipment, procedure, types, advantages, and disadvantages. ELISA is a qualitative or quantitative immunological procedure that detects antigens or antibodies using enzyme-labeled antibodies and chromogenic substrates. It relies on antibody-antigen interactions and uses an enzyme-labeled antibody to generate a colored reaction, allowing detection of a particular antigen. The document outlines the basic equipment, general procedure involving coating wells with antibodies and adding samples and enzyme-labeled antibodies, and the three main types of ELISA - indirect, sandwich, and competitive.
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies and antigens in a liquid sample. It relies on an enzyme-linked antibody or antigen to detect the target protein. There are different types of ELISA including direct, indirect, sandwich, and competitive. The ELISA process involves coating a plate with an antigen or antibody, adding a sample and enzyme-linked antibody, washing unbound material, and detecting the enzyme's product to quantify the target. ELISAs are widely used in medical testing, food safety, and disease detection.
Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
This document discusses isoelectric focusing (IEF), a technique that separates molecules like proteins based on differences in their isoelectric point (pI). IEF works by placing proteins in an immobilized pH gradient gel and applying an electric field, which causes the proteins to migrate until they reach the pH that matches their pI and become neutrally charged. Common applications of IEF include separating, characterizing, and purifying proteins and peptides, as well as its use in forensic analysis, taxonomy research, and genetic marker typing.
The document provides an overview of the ELISA (enzyme-linked immunosorbent assay) technique. It discusses the principle, basic procedure, materials needed, types (indirect, sandwich, competitive), advantages, disadvantages, and applications of ELISA. ELISA is a sensitive and specific quantitative immunological technique used to detect antigens, antibodies, or other proteins in biological samples. It has various applications in detecting hormones, proteins, infectious agents, and for vaccine quality control among others.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to identify a substance. It involves using an enzyme to detect antigen-antibody binding, which converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA can provide quantitative or qualitative results and has applications like screening donated blood and measuring hormone levels.
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
This document describes detailed information about Radio immuno assay (RIA) including its principle, procedure, advantages, disadvantages, application etc
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by gel electrophoresis, transferring them to a membrane, and using antibodies to identify target proteins. The key steps are tissue preparation, gel electrophoresis, protein transfer to a membrane, blocking of the membrane to prevent nonspecific antibody binding, incubation with primary and secondary antibodies, and detection of bound antibodies through methods like colorimetric reactions or chemiluminescence. Western blotting has applications in detecting conditions like HIV and hepatitis B. While sensitive, it takes longer than other tests and may be more costly.
This document discusses Western blotting and ELISA techniques. It provides details on:
- The basic process of Western blotting including sample preparation, gel electrophoresis, protein transfer, antibody probing, detection and analysis.
- The basic principle and process of ELISA including indirect, direct, sandwich and competitive ELISA techniques.
- The use of antibodies, enzymes and other probes in Western blotting and ELISA.
- The applications of these techniques in medicine, research and industry.
This document discusses and compares polyclonal and monoclonal antibodies. Polyclonal antibodies are derived from different B cell lineages and can have batch-to-batch variation, making them less suitable for clinical diagnostic tests. Monoclonal antibodies are derived from a single B cell clone and have greater homogeneity, specificity, and ability to produce unlimited quantities of antibody, enabling their use in diagnostic tests. The document also outlines methods for generating recombinant antibodies and the various therapeutic and diagnostic applications of monoclonal antibodies.
This document provides an overview of electrophoresis. It discusses how electrophoresis works, involving the movement of charged particles through an electrolyte when subjected to an electric field. It then covers the history, factors affecting electrophoresis, types of electrophoresis including paper, SDS-PAGE, native gel, gradient gel, IEF gels, 2D gel electrophoresis, protein blotting, pulsed field gel electrophoresis, and capillary electrophoresis. It also discusses instrumentation, reagents, and applications of various electrophoresis techniques.
The western blot is a technique used to detect specific proteins in a sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are sample preparation, gel electrophoresis, blotting, blocking, antibody probing, and detection. Western blotting allows researchers to identify proteins from complex mixtures and is widely used in molecular biology and medical diagnosis, such as detecting HIV, HBV, and HSV infections.
Monoclonal antibodies & hybridoma technologyAjay Dominic
Monoclonal antibodies are produced from a single clone of cells and bind to the same epitope. They are produced using hybridoma technology which involves fusing antibody-secreting B cells with myeloma cells to form immortal hybridoma cells that secrete monoclonal antibodies. This technique was developed by Kohler and Milstein in 1975 and they received the Nobel Prize.
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.
Production and applications of monoclonal antibodiesKaayathri Devi
production and applications of monoclonal antibodies, monoclonal antibodies ,applications of monoclonal antibodies, production of monoclonal antibodies,
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.
Gel electrophoresis is a method to separate biomolecules like proteins, nucleic acids, and lipids based on their charge and size. During gel electrophoresis, an electric current is applied across a gel, causing negatively charged molecules to migrate toward the positive electrode and positively charged molecules to migrate toward the negative electrode. Smaller molecules migrate faster through the gel than larger molecules. Factors like the charge, size, and shape of molecules, as well as the electric current, gel composition, and buffer used, determine how far each type of molecule will migrate through the gel. Gel electrophoresis has applications in separating DNA, RNA, proteins, and other biomolecules.
The document describes a seminar on the ELISA (Enzyme Linked ImmunoSorbent Assay) technique. It discusses the principle, types (direct, indirect, sandwich, competitive), applications in detecting antigens and antibodies, and advantages and disadvantages of the ELISA method. ELISA is a widely used technique that detects antigens or antibodies by using an enzyme-linked secondary antibody to generate a colored reaction product, which is then read quantitatively or qualitatively.
The document provides an introduction to ELISA (enzyme-linked immunosorbent assay), which is a biochemical technique used mainly in immunology to detect the presence of an antibody or antigen in a sample. It describes the basic principles and steps of the ELISA process, which involves detecting antibodies or antigens using an enzyme-labeled secondary antibody and color changing reaction. Key aspects covered include antigen-antibody binding, use of enzyme labels, substrate conversion, and quantitative/qualitative applications of ELISA for detecting various molecules.
A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.
Hybridoma technology involves fusing antibody-producing B cells (splenocytes) with myeloma cells to produce immortal hybridoma cell lines that secrete monoclonal antibodies of a single specificity. Georges Köhler and César Milstein developed the technique in 1975 and were awarded the 1984 Nobel Prize for this discovery. The process involves immunizing an animal to generate B cells that produce antibodies against the target antigen. The B cells are isolated from the spleen and fused with myeloma cells using polyethylene glycol or electrofusion. The fused cells are selected in HAT medium, which allows only hybridomas expressing the hypoxanthine-guanine phosphoribosyl transferase enzyme to survive and multiply indefinitely. The resulting stable monoclonal antibody-
The document discusses enzyme-linked immunosorbent assay (ELISA), including its uses, variations, and applications. ELISA uses an enzyme-conjugated antibody that reacts with a substrate to generate a detectable color change. There are several types of ELISA including indirect, sandwich, and competitive ELISA that can be used either qualitatively or quantitatively to detect antigens or antibodies. ELISA has numerous medical and industrial applications such as detecting HIV, food allergens, drugs, and various pathogens.
Radioimmunoassay (RIA) is an immunoassay technique that uses radiolabeled molecules to detect and quantify antigens or antibodies. RIA involves attaching a radioactive isotope to an antigen or antibody, then measuring the unbound antigen after the labeled and unlabeled antigens compete for binding sites on antibodies. RIAs are highly sensitive and can detect very low concentrations of substances like hormones, drugs, and vitamins in samples. They have various applications in disease diagnosis, drug monitoring, and pharmaceutical research.
Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
This document discusses isoelectric focusing (IEF), a technique that separates molecules like proteins based on differences in their isoelectric point (pI). IEF works by placing proteins in an immobilized pH gradient gel and applying an electric field, which causes the proteins to migrate until they reach the pH that matches their pI and become neutrally charged. Common applications of IEF include separating, characterizing, and purifying proteins and peptides, as well as its use in forensic analysis, taxonomy research, and genetic marker typing.
The document provides an overview of the ELISA (enzyme-linked immunosorbent assay) technique. It discusses the principle, basic procedure, materials needed, types (indirect, sandwich, competitive), advantages, disadvantages, and applications of ELISA. ELISA is a sensitive and specific quantitative immunological technique used to detect antigens, antibodies, or other proteins in biological samples. It has various applications in detecting hormones, proteins, infectious agents, and for vaccine quality control among others.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to identify a substance. It involves using an enzyme to detect antigen-antibody binding, which converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA can provide quantitative or qualitative results and has applications like screening donated blood and measuring hormone levels.
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
This document describes detailed information about Radio immuno assay (RIA) including its principle, procedure, advantages, disadvantages, application etc
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by gel electrophoresis, transferring them to a membrane, and using antibodies to identify target proteins. The key steps are tissue preparation, gel electrophoresis, protein transfer to a membrane, blocking of the membrane to prevent nonspecific antibody binding, incubation with primary and secondary antibodies, and detection of bound antibodies through methods like colorimetric reactions or chemiluminescence. Western blotting has applications in detecting conditions like HIV and hepatitis B. While sensitive, it takes longer than other tests and may be more costly.
This document discusses Western blotting and ELISA techniques. It provides details on:
- The basic process of Western blotting including sample preparation, gel electrophoresis, protein transfer, antibody probing, detection and analysis.
- The basic principle and process of ELISA including indirect, direct, sandwich and competitive ELISA techniques.
- The use of antibodies, enzymes and other probes in Western blotting and ELISA.
- The applications of these techniques in medicine, research and industry.
This document discusses and compares polyclonal and monoclonal antibodies. Polyclonal antibodies are derived from different B cell lineages and can have batch-to-batch variation, making them less suitable for clinical diagnostic tests. Monoclonal antibodies are derived from a single B cell clone and have greater homogeneity, specificity, and ability to produce unlimited quantities of antibody, enabling their use in diagnostic tests. The document also outlines methods for generating recombinant antibodies and the various therapeutic and diagnostic applications of monoclonal antibodies.
This document provides an overview of electrophoresis. It discusses how electrophoresis works, involving the movement of charged particles through an electrolyte when subjected to an electric field. It then covers the history, factors affecting electrophoresis, types of electrophoresis including paper, SDS-PAGE, native gel, gradient gel, IEF gels, 2D gel electrophoresis, protein blotting, pulsed field gel electrophoresis, and capillary electrophoresis. It also discusses instrumentation, reagents, and applications of various electrophoresis techniques.
The western blot is a technique used to detect specific proteins in a sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are sample preparation, gel electrophoresis, blotting, blocking, antibody probing, and detection. Western blotting allows researchers to identify proteins from complex mixtures and is widely used in molecular biology and medical diagnosis, such as detecting HIV, HBV, and HSV infections.
Monoclonal antibodies & hybridoma technologyAjay Dominic
Monoclonal antibodies are produced from a single clone of cells and bind to the same epitope. They are produced using hybridoma technology which involves fusing antibody-secreting B cells with myeloma cells to form immortal hybridoma cells that secrete monoclonal antibodies. This technique was developed by Kohler and Milstein in 1975 and they received the Nobel Prize.
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.
Production and applications of monoclonal antibodiesKaayathri Devi
production and applications of monoclonal antibodies, monoclonal antibodies ,applications of monoclonal antibodies, production of monoclonal antibodies,
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.
Gel electrophoresis is a method to separate biomolecules like proteins, nucleic acids, and lipids based on their charge and size. During gel electrophoresis, an electric current is applied across a gel, causing negatively charged molecules to migrate toward the positive electrode and positively charged molecules to migrate toward the negative electrode. Smaller molecules migrate faster through the gel than larger molecules. Factors like the charge, size, and shape of molecules, as well as the electric current, gel composition, and buffer used, determine how far each type of molecule will migrate through the gel. Gel electrophoresis has applications in separating DNA, RNA, proteins, and other biomolecules.
The document describes a seminar on the ELISA (Enzyme Linked ImmunoSorbent Assay) technique. It discusses the principle, types (direct, indirect, sandwich, competitive), applications in detecting antigens and antibodies, and advantages and disadvantages of the ELISA method. ELISA is a widely used technique that detects antigens or antibodies by using an enzyme-linked secondary antibody to generate a colored reaction product, which is then read quantitatively or qualitatively.
The document provides an introduction to ELISA (enzyme-linked immunosorbent assay), which is a biochemical technique used mainly in immunology to detect the presence of an antibody or antigen in a sample. It describes the basic principles and steps of the ELISA process, which involves detecting antibodies or antigens using an enzyme-labeled secondary antibody and color changing reaction. Key aspects covered include antigen-antibody binding, use of enzyme labels, substrate conversion, and quantitative/qualitative applications of ELISA for detecting various molecules.
A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.
Hybridoma technology involves fusing antibody-producing B cells (splenocytes) with myeloma cells to produce immortal hybridoma cell lines that secrete monoclonal antibodies of a single specificity. Georges Köhler and César Milstein developed the technique in 1975 and were awarded the 1984 Nobel Prize for this discovery. The process involves immunizing an animal to generate B cells that produce antibodies against the target antigen. The B cells are isolated from the spleen and fused with myeloma cells using polyethylene glycol or electrofusion. The fused cells are selected in HAT medium, which allows only hybridomas expressing the hypoxanthine-guanine phosphoribosyl transferase enzyme to survive and multiply indefinitely. The resulting stable monoclonal antibody-
The document discusses enzyme-linked immunosorbent assay (ELISA), including its uses, variations, and applications. ELISA uses an enzyme-conjugated antibody that reacts with a substrate to generate a detectable color change. There are several types of ELISA including indirect, sandwich, and competitive ELISA that can be used either qualitatively or quantitatively to detect antigens or antibodies. ELISA has numerous medical and industrial applications such as detecting HIV, food allergens, drugs, and various pathogens.
Radioimmunoassay (RIA) is an immunoassay technique that uses radiolabeled molecules to detect and quantify antigens or antibodies. RIA involves attaching a radioactive isotope to an antigen or antibody, then measuring the unbound antigen after the labeled and unlabeled antigens compete for binding sites on antibodies. RIAs are highly sensitive and can detect very low concentrations of substances like hormones, drugs, and vitamins in samples. They have various applications in disease diagnosis, drug monitoring, and pharmaceutical research.
Immunological assays use antibodies or antigens to detect the presence or concentration of a molecule in a solution. There are several types of immunoassays including radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), and bioluminescence assays. RIA uses radioactive labels on antigens or antibodies for highly sensitive detection, but requires special safety precautions. ELISA is a common plate-based assay that uses enzyme labels for detection and has advantages like sensitivity, reproducibility, and flexibility. Bioluminescence assays convert chemical energy from reactions involving luciferins, luciferases, and oxygen into detectable light for applications like cell proliferation analysis.
Theoretical basis and optimization of immunoassayAkankshDas
This document discusses the theoretical basis and optimization of immunoassays. It begins by introducing immunoassays and their importance in pharmaceutical analysis due to their specificity and sensitivity. It then describes the basic principles of immunoassays, including the competitive binding reaction between labeled and unlabeled analyte for antibody binding sites. The document categorizes different types of immunoassays and provides examples of their use. It also discusses optimization of immunoassays through techniques like minimizing background signals, improving precision and reproducibility, stabilizing conjugates, and increasing shelf-life. A key immunoassay method discussed is ELISA.
The document describes various types of ELISA (enzyme-linked immunosorbent assay) tests, including direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA. It explains the basic principles and procedures for each type. ELISA tests use enzyme and antibody or antigen reactions to detect substances like proteins, hormones, antibodies, or drugs in samples. The tests are used for medical diagnostic purposes like detecting infections and allergies.
This document discusses homogeneous and heterogeneous immunoassays. It defines immunoassays as biochemical tests that measure macromolecules or small molecules using antibodies or antigens. Heterogeneous immunoassays involve separating bound and unbound components, while homogeneous immunoassays do not require separation. Examples of immunoassays discussed include ELISA (enzyme-linked immunosorbent assay) and EMIT (enzyme multiplied immunoassay technique).
ELISA is a widely used technique to detect antigens or antibodies. It works by using an enzyme-conjugated antibody that reacts with a chromogenic substrate to generate a colored product, allowing detection of antigen-antibody complexes. There are several variants of ELISA including indirect, sandwich, competitive, and ELISPOT assays that can be used qualitatively or quantitatively. ELISA has various applications such as detecting antibodies and allergens and is advantageous for being sensitive, having widely available equipment, and not using radiation.
1. ELISA (Enzyme-linked Immunosorbent Assay) is an immunoassay technique used to detect the presence of antibodies and antigens in a solution. It relies on antibodies and antigens binding specifically together.
2. There are several types of ELISA including direct, indirect, sandwich, competitive, and reverse ELISA. Each uses a different approach but all rely on an enzyme-labeled antibody or antigen binding to produce a detectable color change.
3. ELISA is a very sensitive technique that can detect ng/mL to pg/mL concentrations and is widely used for applications like detecting hormones, infectious agents, drugs and more.
Radioimmunoassay (RIA) is a sensitive technique introduced in 1960 that uses radioactive isotopes and antibodies to measure antigens or hormones. It involves competitive binding between radiolabeled and unlabeled antigens for antibodies. Enzyme-linked immunosorbent assay (ELISA) is a common technique introduced in 1971 that uses enzymes to detect and quantify substances like proteins. It can be indirect, sandwich, or competitive based on antigen-antibody binding structure. Both RIA and ELISA are sensitive techniques used to detect substances like hormones, vitamins, drugs, and antigens in clinical medicine and research.
This document summarizes various serological tests used to detect antigens and antibodies, including:
- Primary tests like ELISA, IFAT, RIA that detect markers
- Secondary tests like agglutination, complement fixation, precipitation that detect interactions
- Tertiary tests that assess protective value of antiserum in animals
It then provides details on specific tests like agglutination, Coombs test, hemagglutination inhibition, precipitation, complement fixation, ELISA and their applications in medicine, food/plant pathology, and quality control.
This document summarizes various serological tests used to detect antigens and antibodies. It describes primary, secondary and tertiary serological tests including ELISA, immunofluorescence, radioimmunoassay and more. It also details different types of agglutination tests like qualitative and quantitative tests. Additional tests covered are precipitation tests, complement fixation tests, passive hemagglutination and sandwich ELISA. The document provides information on applications and procedures for many antibody and antigen detection techniques.
This document describes various serological tests used to detect antigens and antibodies. It discusses primary tests like ELISA, IFAT, and RIA. Secondary tests include agglutination, complement fixation, precipitation, and neutralization tests. Tertiary tests determine antibody protective value. Agglutination tests can qualitatively and quantitatively detect particulate antigens. Coombs tests detect non-agglutinating antibodies. ELISA is then explained in detail, including indirect, sandwich, and competitive formats. ELISA is widely used to detect antigens and antibodies in applications like HIV and food allergen testing.
Immunological techniques are methods used by immunologists to induce, measure, and characterize immune responses. Key techniques described in the document include ELISA, RIA, immunoprecipitation, and western blotting. ELISA uses antibodies to detect antigens and can be used to diagnose conditions like HIV, Lyme disease, and hepatitis. RIA is a sensitive technique that uses radiolabeled antigens or antibodies to detect proteins at very low levels. Immunoprecipitation isolates specific proteins from cell lysates using antibodies, while western blotting separates and identifies proteins by molecular weight.
ELISA involves an antigen-antibody reaction between a solid surface like a microwell plate coated with the target antigen and an enzyme-labeled antibody specific to that antigen. There are four main types of ELISA - direct, indirect, sandwich, and competitive - which differ in the order and number of antibodies used. ELISA is commonly used to detect antigens and antibodies and has applications in fields like medical diagnostics, food testing, and environmental monitoring.
elisaLecture_based on new way to medical lab.pptmainakg09
The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses antibodies and antigen-enzyme conjugates to detect the presence and quantify antigens or antibodies in a sample. It is a sensitive, specific immunoassay that is usually performed in microwell plates, where antibodies or antigens bind to the wells and are detected using enzyme-linked antibodies and color-changing substrates. There are different types of ELISA including sandwich, competitive, and indirect formats.
This document discusses various types of labeled immunoassays, including fluorescent, radioactive, chemiluminescent, and enzyme labels. It describes the key characteristics and procedures for competitive and noncompetitive assays. Specific enzyme immunoassay techniques are covered in detail, such as competitive and noncompetitive ELISA, capture (sandwich) assays, rapid immunoassays, and homogeneous assays. The advantages of enzyme immunoassays include safety compared to radioimmunoassays and the ability to perform assays without expensive instrumentation. Challenges include potential interference from inhibitors or nonspecific binding.
Immunoassay( theoretical basis and optimization of immunoassay)Rashmi116
An immunoassay is a biochemical test that uses antibodies and antigens to detect the presence or concentration of a molecule. It works by utilizing the binding between antibodies and antigens. There are various types including competitive and non-competitive immunoassays. Immunoassays have many applications such as disease diagnosis, drug testing, and environmental testing because they are highly sensitive, specific, and cost-effective tests.
The document provides an overview of the enzyme-linked immunosorbent assay (ELISA) technique. It describes three main types of ELISA - the sandwich ELISA, indirect ELISA, and competitive ELISA. For each type, it provides a brief example of clinical applications, such as detecting neural proteins in urine to diagnose Alzheimer's disease (sandwich ELISA) or detecting HIV antibodies in serum (indirect ELISA). It emphasizes that ELISA is a sensitive, cost-effective, and widely used assay for diagnosing infections and diseases.
This presentation explains about the principle and procedure involved in elisa method of immunoassay, development o f elisa , application advantages and disadvantages of elisa
ELISA (Enzyme-Linked Immunosorbent Assay) is a biochemical technique used to detect antibodies or antigens in a sample. There are three main types of ELISA: competitive ELISA, sandwich ELISA, and indirect ELISA. Sandwich ELISA coats a plate with capture antibodies and detects antigens bound between the capture and detection antibodies. Indirect ELISA coats antigens on a plate and detects antibodies in samples using enzyme-linked secondary antibodies. ELISA is used to test samples like blood, urine, and tissue extracts for proteins, hormones, antibodies, and other molecules.
This document discusses general considerations for pilot plant scale up techniques. It outlines 12 key areas that should be considered when scaling up a formulation from the laboratory to a pilot plant scale, including reporting responsibilities, personnel requirements, space requirements, reviewing the formula, raw materials, processing equipment, production rates, process evaluation, manufacturing procedures, product stability and uniformity, GMP compliance, and transferring analytical methods to quality assurance. The goal is to produce a formulation on an intermediate batch scale that represents the procedures used for commercial manufacturing.
Important for D. Pharmacy, B. Pharmacy and M. Pharmacy.
A brief introduction to the basic of pilot plant scale up and its objectives, significance, applications and importance
A biosensor is an analytical device containing a biological element and transducer. The biological element interacts specifically with an analyte to produce a measurable signal. There are several types of biosensors including electrochemical, optical, thermal, and piezoelectric biosensors. Biosensors find various applications in pharmaceutical industries such as detection of pathogens in food and drugs, monitoring of bioprocesses, and environmental monitoring. They provide fast, accurate, and portable detection compared to conventional analytical methods.
1) Combinatorial chemistry allows for the simultaneous synthesis of multiple compounds using a set of building blocks. This approach can generate thousands of compounds quickly and efficiently.
2) Traditional synthesis is slow, producing one compound at a time. Combinatorial chemistry addresses this through parallel and split-and-mix techniques, generating library of compounds on solid resin beads or in solution.
3) The library is then screened to identify compounds with desired biological activity, accelerating drug discovery. Encoding methods like positional or chemical tagging are used to identify the active compounds within complex mixtures.
Temple of Asclepius in Thrace. Excavation resultsKrassimira Luka
The temple and the sanctuary around were dedicated to Asklepios Zmidrenus. This name has been known since 1875 when an inscription dedicated to him was discovered in Rome. The inscription is dated in 227 AD and was left by soldiers originating from the city of Philippopolis (modern Plovdiv).
A Visual Guide to 1 Samuel | A Tale of Two HeartsSteve Thomason
These slides walk through the story of 1 Samuel. Samuel is the last judge of Israel. The people reject God and want a king. Saul is anointed as the first king, but he is not a good king. David, the shepherd boy is anointed and Saul is envious of him. David shows honor while Saul continues to self destruct.
Leveraging Generative AI to Drive Nonprofit InnovationTechSoup
In this webinar, participants learned how to utilize Generative AI to streamline operations and elevate member engagement. Amazon Web Service experts provided a customer specific use cases and dived into low/no-code tools that are quick and easy to deploy through Amazon Web Service (AWS.)
This presentation was provided by Rebecca Benner, Ph.D., of the American Society of Anesthesiologists, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
How Barcodes Can Be Leveraged Within Odoo 17Celine George
In this presentation, we will explore how barcodes can be leveraged within Odoo 17 to streamline our manufacturing processes. We will cover the configuration steps, how to utilize barcodes in different manufacturing scenarios, and the overall benefits of implementing this technology.
This presentation was provided by Racquel Jemison, Ph.D., Christina MacLaughlin, Ph.D., and Paulomi Majumder. Ph.D., all of the American Chemical Society, for the second session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session Two: 'Expanding Pathways to Publishing Careers,' was held June 13, 2024.
Level 3 NCEA - NZ: A Nation In the Making 1872 - 1900 SML.pptHenry Hollis
The History of NZ 1870-1900.
Making of a Nation.
From the NZ Wars to Liberals,
Richard Seddon, George Grey,
Social Laboratory, New Zealand,
Confiscations, Kotahitanga, Kingitanga, Parliament, Suffrage, Repudiation, Economic Change, Agriculture, Gold Mining, Timber, Flax, Sheep, Dairying,
1. Guided by: Presented By :
Dr . Mahesh Kumar Kataria Nitish Chugh
Professor and Head, Department of Pharmaceutics M.Pharmacy (Pharmaceutics)
SETH G. L. BIHANI S. D. COLLEGE OF TECHNICAL EDUCATION,
SRI GANGANAGAR (RAJ.)
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 1
2. ELISA (enzyme-linked immunosorbent assay) is a plate-based assay
technique designed for detecting and quantifying peptides, proteins, antibodies
and hormones. In an ELISA, an antigen must be immobilized to a solid surface
and then complexed with an antibody that is linked to an enzyme. Detection is
accomplished by assessing the conjugated enzyme activity via incubation with a
substrate to produce a measureable product. The most crucial element of the
detection strategy is a highly specific antibody-antigen interaction.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which
will passively bind antibodies and proteins. It is this binding and immobilization
of reagents that makes ELISAs so easy to design and perform. Having the
reactants of the ELISA immobilized to the microplate surface makes it easy to
separate bound from non-bound material during the assay. This ability to wash
away nonspecifically bound materials makes the ELISA a powerful tool for
measuring specific analytes within a crude preparation.
ELISA is an antigen antibody reaction. In 1971, ELISA was introduced by Peter
Perlmann and Eva Engvall at Stockholm University in Sweden. It is a common
laboratory technique which is usually used to measure the concentration of
antibodies or antigens in blood. An enzyme conjugated with an antibody reacts
with colorless substrate to generate a colored product. Such substrate is called
chromogenic substrate. A number of enzymes have been used for ELISA such as
alkaline phosphatase, horse radish peroxidase and beta galactosidase. Specific
substrate such as ortho-phenyldiamine dihydrochloride (for peroxidase),
paranitrophenyl phosphate (for alkaline phosphatase) are used which are
hydrolysed by above enzymes to give colored end product.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 2
4. ELISAs are typically performed in 96-well polystyrene plates. The
serum is incubated in a well, and each well contains a different
serum. A positive control serum and a negative control serum
would be included among the 96 samples being tested.
Antibodies or antigens present in serum are captured by
corresponding antigen or antibody coated on to the solid
surface. After some time, the plate is washed to remove serum
and unbound antibodies or antigens with a series of wash
buffer. To detect the bound antibodies or antigens, a secondary
antibodies that are attached to an enzyme such as peroxidase
or alkaline phosphatase are added to each well.
After an incubation period, the unbound secondary antibodies are
washed off. When a suitable substrate is added, the enzyme
reacts with it to produce a color. This color produced is
measurable as a function or quantity of antigens or antibodies
present in the given sample. The intensity of color/ optical
density is measured at 450nm. The intensity of the color gives
an indication of the amount of antigen or antibody.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 4
5. Indirect ELISA
Sandwich ELISA
Competitive ELISA
Direct ELISA
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 5
6. Antibody can be detected or quantitatively determined by indirect
ELISA. In this technique, antigen is coated on the microtiter well.
Serum or some other sample containing primary antibody is added
to the microtiter well and allowed to react with the coated antigen.
Any free primary antibody is washed away and the bound antibody
to the antigen is detected by adding an enzyme conjugated
secondary antibody that binds to the primary antibody.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 6
7. Antigen can be detected by sandwich ELISA. In this technique,
antibody is coated on the microtiter well. A sample containing
antigen is added to the well and allowed to react with the
antibody attached to the well, forming antigen-antibody
complex. After the well is washed, a second enzyme-linked
antibody specific for a different epitope on the antigen is
added and allowed to react with the bound antigen. Then
after unbound secondary antibody is removed by washing.
Finally substrate is added to the plate which is hydrolyzed by
enzyme to form colored products.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 7
8. In this test, antibody is first incubated in solution with a
sample containing antigen. The antigen-antibody mixture is
then added to the microtitre well which is coated with
antigen. The more the antigen present in the sample, the less
free antibody will be available to bind to the antigen-coated
well. After the well is washed, enzyme conjugated secondary
antibody specific for isotype of the primary antibody is added
to determine the amount of primary antibody bound to the
well. The higher the concentration of antigen in the sample,
the lower the absorbance.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 8
9. For direct detection, an antigen coated to a multi-
well plate is detected by an antibody that has been
directly conjugated to an enzyme. This detection
method is a good option if there is no
commercially available ELISA kits for your target
protein
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 9
10. Presence of antigen or the presence of
antibody in a sample can be evaluated.
Determination of serum antibody
concentrations in a virus test.
Used in food industry when detecting
potential food allergens.
Applied in disease outbreaks- tracking the
spread of disease e.g. HIV, bird flu, common,
colds, cholera, STD etc.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 10
11. RIA or Radioimmunoassay is an in vitro assay that measures
the presence of an antigen with very high sensitivity. Basically
any biological substance for which a specific antibody exists
can be measured, even in minute concentrations. RIA has
been the first immunoassay technique developed to analyze
nanomolar and picomolar concentrations of hormones in
biological fluids.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 11
12. Radioimmunoassay is based upon the competition between
labeled and unlabeled antigen for specific antibody sites,
forming antigen-antibody complexes. This reaction is
described by the expression see journal for formula. At
equilibirum, the radioactive complex (bound) is separated from
the radioactive antigen (free). The B/F ratio is dependent upon
the amount of nonradioactive antigen. Antigen concentration in
unknown samples is determined by comparing the B/F ratio to
the B/F ratios obtained by incubating varying amounts of
known nonradioactive antigen with the same amount of
antibody as in the unknown sample under similar assay
conditions. Sensitivity of the order of 10-12 moles/liter may be
achieved through the preparation and use of a labeled antigen
of high specific activity and the production and selection of
antisera with appropriately high affinity constants. Specificity is
dependent upon the ability of the antiserum to recognize
subtle structural features of the antigen molecule. The ability to
conveniently assay large numbers of samples with good
precision has led to the application of this technique to
quantitate substances (such as steroids) already measurable
but by more cumbersome methods.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 12
13. The target antigen is labeled radioactively and bound to its specific
antibodies; a limited and known amount of the specific antibody has to be
added. A sample, for example a blood-serum, is then added in order to
initiate a competitive reaction of the labeled antigens from the preparation,
and the unlabeled antigens from the serum-sample, with the specific
antibodies. The competition for the antibodies will release a certain amount
of labeled antigen. This amount is proportional to the ratio of labeled to
unlabeled antigen. A binding curve can then be generated which allows the
amount of antigen in the patient’s serum to be derived.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 13
14. As an example of how this technique works,
let’s apply it to insulin. To measure insulin,
the first step is to mix known amounts of
radioisotope-tagged insulin and antibodies.
These combine chemically. Next, a small
amount of the patient’s blood is added. The
insulin contained in the blood displaces some
of the tagged insulin. The free-tagged insulin
is then measured with isotope detectors and
the patient’s insulin level is calculated
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 14
15. A plot of the distribution of radioactivity as a
function of the amount of unlabeled antigen present
is known as the standard or dose response curve.
Standard curves can be plotted in a variety of ways.
The most commonly used response curves are the
bound-free (B/F), free-bound (F/B), fraction bound
(B) of occasionally the percent bound B/Bo ratios.
The dose can be plotted on either the arithmatic or
logarithmic scale. To obtain response curves that
are essentially linear over a large part of the antigen
concentration, the logit function defined as follows
has been used: Logit(y) = where y = either B/Bo or
(B/F)/(Bo/Fo) When logit (y) is plotted against the
log concentration of the antigen, a linear dose-
response curve results for most of the assay system.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 15
17. Radioimmunoassay (RIA) has many uses, including narcotics (drug)
detection, blood bank screening for the hepatitis a highly contagious
condition virus, early cancer detection, measurement of growth
hormone levels, tracking of the leukemia virus, diagnosis and
treatment of peptic ulcers, and research with brain chemicals called
neurotransmitters.
1) Detection of Narcotic Drugs- Heroin & Morphine can be detected in
hair with the use of Radioimmunoassay (RIA). In a research hair
samples obtained from morphine treated mice and heroin user
contained Nano gram levels of drug per milligram of hair . The result
of the hair analysis for all subject admitting the use of heroin were
positive where as the result of only 30% of thin layer chromatographic
urine analysis of these same subjects were positive.
2) Radioimmunoassay of Hydromorphone & Hydrocodone in Human
Plasma- Hydromorphone & Hydrocodone belongs to morphine group
of drugs and are used in combination with antitussive & analgesic
antipyretic mixture. The RIA method is capable of estimating the above
drug within a range of 2.5 to 20 ng/mL using standard 100 µl plasma
sample. RIA is carried out using morphine-6-antiserum &
dihydromorphine. Free drug is separated from the bound drug using
dextran coated charcoal & an aliquot of the supinate containing the
antiserum bound drug is subsequently counted for radioactivity.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 17
18. 3) Radioimmunoassay of Flunisolide in human plasma- Flunisolide is a
fast-acting corticoid designed for the treatment of allergic rhinitis,
asthma, and other allied respiratory disorders in humans. As the
quantum of drug delivered by inhalation (i.e., the usual route of
administration of the drug), is invariably small, the plasma-levels
attained can also be fairly small. Hence, there is a need for a sensitive
method of plasma concentration evaluation which is satisfied by
radioimmunoassay.
4) Measurement of Ferritin- Serum ferritin levels are indicative of iron
stores present in a patient. Levels are useful in differentiating true iron
deficiency from the body's failure to utilize these stores.
5) Detection of Digoxin- This allows direct measurement of serum
digoxin levels quickly and accurately. It is important to rule out Digi
toxicity quickly and accurately. We are also able to filter out Digi bind
to let the physician know how much the level has dropped after Digi
bind has been administered.
6) Thyroid Testing- This is used to determine the patient's thyroid status
and to follow patients after iodine-131 therapy to see if the dose was
indeed effective.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 18
19. • Prolonged reaction time (in days) as a
consequence highly diluted reagent is used.
• Radioisotopes are costly.
• Possible health hazards due to handling of
radioisotopes.
• Limited assay range.
• Lack of direct linear relationship between
analyte concentration and signal response.
• Difficult of automation.
• Lengthy counting time.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 19
20. 1. Abraham, G.E. snd Grover, P.K. Covalent linkage of hormonal haptens
to protein carriers for use in radioimmunoassay. In Odell, W.D. and
Daughadsy, W.H. editors: Principles of competitive protein binding
assays. Philadelphia, J.B. Lippincott Co., 1971.
2. Rodbsrd, D., Rayford, P.L, Cooper, i.A. and Ross, G.T. Statistical
quality control and routine data processing for radioimmunoassays
(RIA) and immunoradiometric assays (IRMA). din. Chem.,
1974;20:1255-70.
3. Rodbsrd, D., Bridson, W. and Rayford, P.L Rapid calculation of
radioimmunosssay results. 3. Lab. dIm. Med., 1969;74:770-81.
4. Garcia, E.J. and Fiori, A. Radioimmunoasssy and competitive binding
analysis. In text book of nuclear medicine: BasicSciences (Eds. A.
Fernando snd.1.C. Harbert): Lea and Febiger Publishers, 1978; pp.
344360.
5. Hunter, W.M. and Greenwood, P.C. Preparation of 1-131 labeled
growth hormone of high specific activity. Nature (London),
1962;194:495-96.
Dr. Mahesh Kumar Kataria Email: kataria.pharmacy@gmail.com 20