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 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 document provides an overview of 2D NMR spectroscopy techniques, specifically HETCOR. It discusses the principles behind 2D NMR, describing how it plots data in two frequency axes rather than one, providing more information about a molecule's structure. It then explains the four periods that occur in a 2D NMR experiment: preparation, evolution, mixing, and detection. The document focuses on HETCOR, describing it as a heteronuclear experiment that provides correlations between different nuclei like protons and carbons. Examples of HETCOR spectra are provided to show how they indicate couplings between protons and the carbons they are attached to. Related techniques like HSQC and HMBC are also briefly described.
This document provides an overview of LC-FTIR, a hyphenated technique that combines liquid chromatography (LC) and Fourier transform infrared spectroscopy (FTIR). LC is used to separate mixtures into individual components, which are then analyzed by FTIR to obtain structural information. There are two main interfaces used: flow cell interfaces analyze components in real-time without solvent removal, while solvent elimination interfaces remove interfering solvent before analysis. LC-FTIR has applications in trace analysis, isomer detection/distinction, and analysis of pharmaceuticals, polymers, and environmental pollutants.
This document presents an overview of mass spectrometry. It discusses fragmentation rules, rearrangements like McLafferty rearrangement, the ring rule for calculating unsaturation, and isotopic peaks. It also examines the mass spectrometry of specific functional groups like alkanes, alkenes, alcohols, alkyl halides, and provides references for further reading. The presentation aims to cover basic concepts in mass spectrometry including how different functional groups fragment in the mass spectrometer.
CHEMISTRY OF PEPTIDES [M.PHARM, M.SC, BSC, B.PHARM]Shikha Popali
THE CHEMISTRY OF PEPTIDES THE DIFFICULT TO COLLECT DATA FOR READERS , THREFORE HERE WE HAVE COLLECTED ALL THE DATA AT A PLACE AND PROVIDED EASIER TO CHEMISTRIANS.
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.
This document discusses common side reactions that can occur during peptide synthesis, including initiation by proton abstraction, protonation, and overactivation. Proton abstraction can lead to carboxylate anion formation or racemization. Protonation can cause racemization through carbocation formation or alkylation. Overactivation occurs when the carboxyl component is too reactive, leading to unwanted acylation of amino or hydroxyl groups. Understanding these side reactions is important for optimizing peptide synthesis techniques.
This document discusses ion exclusion chromatography, which uses an ion exchange stationary phase to separate ionic and nonionic substances. Ionic substances pass quickly through the column while nonionic substances are retained longer. Separation depends on whether substances are ionized and repelled by the resin or able to enter the resin network if nonpolar or partially ionized. Detection methods include conductivity detectors and UV-visible or fluorescence detectors. Applications include separation of carboxylic acids, inorganic anions, amino acids, and determination of water in organic solvents.
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 document provides an overview of 2D NMR spectroscopy techniques, specifically HETCOR. It discusses the principles behind 2D NMR, describing how it plots data in two frequency axes rather than one, providing more information about a molecule's structure. It then explains the four periods that occur in a 2D NMR experiment: preparation, evolution, mixing, and detection. The document focuses on HETCOR, describing it as a heteronuclear experiment that provides correlations between different nuclei like protons and carbons. Examples of HETCOR spectra are provided to show how they indicate couplings between protons and the carbons they are attached to. Related techniques like HSQC and HMBC are also briefly described.
This document provides an overview of LC-FTIR, a hyphenated technique that combines liquid chromatography (LC) and Fourier transform infrared spectroscopy (FTIR). LC is used to separate mixtures into individual components, which are then analyzed by FTIR to obtain structural information. There are two main interfaces used: flow cell interfaces analyze components in real-time without solvent removal, while solvent elimination interfaces remove interfering solvent before analysis. LC-FTIR has applications in trace analysis, isomer detection/distinction, and analysis of pharmaceuticals, polymers, and environmental pollutants.
This document presents an overview of mass spectrometry. It discusses fragmentation rules, rearrangements like McLafferty rearrangement, the ring rule for calculating unsaturation, and isotopic peaks. It also examines the mass spectrometry of specific functional groups like alkanes, alkenes, alcohols, alkyl halides, and provides references for further reading. The presentation aims to cover basic concepts in mass spectrometry including how different functional groups fragment in the mass spectrometer.
CHEMISTRY OF PEPTIDES [M.PHARM, M.SC, BSC, B.PHARM]Shikha Popali
THE CHEMISTRY OF PEPTIDES THE DIFFICULT TO COLLECT DATA FOR READERS , THREFORE HERE WE HAVE COLLECTED ALL THE DATA AT A PLACE AND PROVIDED EASIER TO CHEMISTRIANS.
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.
This document discusses common side reactions that can occur during peptide synthesis, including initiation by proton abstraction, protonation, and overactivation. Proton abstraction can lead to carboxylate anion formation or racemization. Protonation can cause racemization through carbocation formation or alkylation. Overactivation occurs when the carboxyl component is too reactive, leading to unwanted acylation of amino or hydroxyl groups. Understanding these side reactions is important for optimizing peptide synthesis techniques.
This document discusses ion exclusion chromatography, which uses an ion exchange stationary phase to separate ionic and nonionic substances. Ionic substances pass quickly through the column while nonionic substances are retained longer. Separation depends on whether substances are ionized and repelled by the resin or able to enter the resin network if nonpolar or partially ionized. Detection methods include conductivity detectors and UV-visible or fluorescence detectors. Applications include separation of carboxylic acids, inorganic anions, amino acids, and determination of water in organic solvents.
PRINCIPLES of FT-NMR & 13C NMR
Fourier Transform
FOURIER TRANSFORM NMR SPECTROSCOPY
THEORY OF FT-NMR
13C NMR SPECTROSCOPY
Principle
Why C13-NMR is required though we have H1-NMR?
CHARACTERISTIC FEATURES OF 13 C NMR
Chemical Shifts
NUCLEAR OVERHAUSER ENHANCEMENT
Short-Comings of 13C-NMR Spectra
The slides covers brief description of ion exclusion chromatography. i hope the slides will be helpful
for any further details you can contact me through email.
mail id - sobhigaba@gmail.com
Gas chromatography-atomic absorption spectroscopy (GC-AAS) is an analytical technique used to separate and quantify elemental components in a sample. It combines gas chromatography, which separates compounds, with atomic absorption spectroscopy, which measures elemental concentrations. Key components of a GC-AAS system include a gas chromatograph unit with a carrier gas, injection port, column, and detector; an atomic absorption spectrometer with a light source, atomizer, monochromator, and detector; and an interface connecting the two instruments. GC-AAS is applied to quantify metals in various samples like industrial waste, alloys, foods, and more.
In Silico methods for ADMET prediction of new moleculesMadhuraDatar
The document discusses the importance of predicting absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of new molecules in silico during drug design. It describes how ADMET prediction techniques have evolved since 1863 and helped advance drug development. Factors considered in developing ADMET prediction models include the model purpose, required prediction speed and accuracy. Common molecular descriptors used in these models are also discussed. The document outlines methods for predicting various ADMET properties like permeability, solubility, distribution and metabolism in silico. Recent tools for computational ADMET prediction are also mentioned.
NMR stands for Nuclear Magnetic Resonance. It is a scientific technique used to study the structure, composition, and dynamics of molecules. In NMR spectroscopy, a sample is placed in a strong magnetic field and subjected to radiofrequency radiation. The atomic nuclei in the sample, particularly those with a nonzero spin, absorb and re-emit electromagnetic radiation at specific frequencies. By measuring the frequencies at which the nuclei resonate, valuable information about the chemical environment and connectivity of the atoms in the molecule can be obtained. It is a powerful tool for chemists and other scientists working in fields related to molecular analysis and characterization.
Similarities and differences between 1D and 2D NMR techniques are broadly illustrated here:
This document provides an overview of LC-NMR spectroscopy and its applications. It discusses the history, principles, instrumentation, and modes of LC-NMR. The key components of an LC-NMR system include the LC unit, interface, and NMR unit. Modes of analysis include continuous flow, stopped flow, time slicing, peak parking, and peak trapping. Technology such as online SPE, high field magnets, and solvent suppression methods can improve sensitivity. LC-NMR is useful for structural elucidation of organic compounds.
NOESY (Nuclear Overhauser Effect Spectroscopy) is a 2D NMR technique used to identify nuclear spins undergoing cross-relaxation and measure their rates. It provides information about which proton resonances are from protons close in space. NOESY experiments exploit the nuclear Overhauser effect to observe through-space dipolar couplings. One application is in protein NMR to assign structures by sequential walking. It is useful for determining the stereochemistry of biomolecules in solution.
The document discusses structure-based in-silico virtual screening protocols. It describes virtual screening as using computer methods to discover new ligands based on a target protein's biological structure. The main goal is to reduce the enormous chemical space of potential compounds to a manageable number with the highest likelihood of becoming drug candidates. Molecular docking is a key method, involving sampling potential ligand positions in the protein's binding site and scoring the interactions. The document also discusses force field, empirical, and knowledge-based scoring functions used to evaluate docking poses. Applications mentioned include designing Hsp90 inhibitors for cancer treatment and identifying novel BACE1 inhibitors.
Ion exclusion chromatography is a technique,introduced by Wheaton and Bauman, used to separate ionic compounds from non-ionic compounds and to separate mixtures of acids.
This document discusses various aspects of stereochemistry. It begins by explaining Fischer's D and L notation system for assigning configurations based on a compound's relation to glyceraldehyde. It then discusses pseudo asymmetric centers in meso compounds and cis-trans isomerism that can occur due to restricted bond rotation around double bonds. Finally, it introduces the E-Z system for naming geometric isomers with three or more different groups, which is based on Cahn-Ingold-Prelog priority rules to determine whether higher priority groups are on the same or opposite sides of the double bond.
Interpretation of organic compounds by IR, NMR and Mass SpectrometryAshitoshPanchal
This document discusses various spectroscopy techniques for analyzing organic compounds, including infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry. It describes attenuated total reflectance (ATR)-IR spectroscopy and how it works. It also explains 2D NMR techniques like COSY spectroscopy and how they provide more structural information than 1D NMR. Finally, it discusses how mass spectrometry works and common fragmentation patterns seen in mass spectra for different functional groups like alkanes, cycloalkanes, and compounds containing isotopes.
The document discusses 2D-QSAR (Quantitative Structure-Activity Relationship) analysis methods. It defines QSAR as mathematical relationships linking chemical structure and pharmacological activity. It describes several common 2D-QSAR methods including Hansch analysis, Free Wilson analysis, and various statistical methods. Cluster analysis is discussed as a way to group similar molecules and select a diverse subset for analysis. Molecular descriptors that encode structural, electronic, and topological properties are also introduced.
2D NMR provides more information than 1D NMR by plotting data in a space defined by two frequency axes. There are several types of 2D NMR experiments including COSY, NOESY, and HETCOR. COSY identifies spin-coupled protons by showing cross peaks between protons that are directly bonded. NOESY correlates protons that are near each other in space but not necessarily directly bonded. HETCOR plots 1H and 13C spectra on separate axes and connects carbon signals to bonded proton signals. 2D NMR techniques provide additional structural information about molecules compared to traditional 1D NMR.
This document summarizes an ultrasound assisted reaction presentation. It discusses how ultrasound differs from conventional energy sources and how it can be used in organic synthesis and green and pharmaceutical chemistry. It describes how sonochemistry works through cavitation, where bubbles form and violently collapse, generating high pressures and temperatures. This can enhance chemical reactivity in homogeneous liquid, heterogeneous solid/liquid, and heterogeneous liquid/liquid phase reactions. Examples of synthetic applications where ultrasound switching altered reaction pathways are provided. The conclusion discusses how bubble collapse concentrates energy that can be used to heat bubble contents and enhance reactivity.
This document discusses the coupling of liquid chromatography (LC) with Fourier transform infrared spectroscopy (FTIR). It describes how LC and FTIR can be combined to detect and identify separated compounds. Various interfaces for coupling LC to FTIR are presented, including flow cell interfaces, solvent elimination interfaces, and different types of each. Application areas like trace analysis and analysis of pharmaceuticals and environmental pollutants are also mentioned.
PHARMACOHORE MAPPING AND VIRTUAL SCRRENING FOR RESEARCH DEPARTMENTShikha Popali
THE PHARMACOPHORE MAPPING AND VIRTUAL SCRRENING , THESE PRESENTATION INCLUDES THE DEATIL ACCOUNT ON PHARMACOPHORE, MAPPING, ITS IDENTIFIATION FEATURES, ITS CONFORMATIONAL SEARCH, INSILICO DRUG DESIGN, VIRTUAL SCREENING, PHARMACOPHORE BASED SCREENING
The document provides information about various 2D NMR techniques including HETCOR, INADEQUATE, and guidelines for interpreting NMR data. It discusses how HETCOR spectra show carbon-proton coupling and provides an example spectrum of 2-methyl-3-pentanone. INADEQUATE is introduced as a technique for determining carbon-carbon coupling constants using natural abundance. Examples of NMR data interpretation are also provided for small molecules such as methane, benzene, adenine, cytosine, naphthalene, and quinine to demonstrate analyzing chemical shifts and spin multiplicity.
Quantum Mechanics in Molecular modelingAkshay Kank
This slides gives you the information related to computer aided drug design and its application in drug discovery. Also you learn the Quantum mechanics related to the molecular mechanics. Theory related to molecular modeling and how the molecular modeling helps in drug discovery.
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.
Some Clinical Laboratory Measurement of Immune FunctionsAmany Elsayed
This document describes several clinical laboratory techniques for measuring immune functions, including antibody-based assays that detect antigens or quantify them. It discusses methods like agglutination assays, precipitation assays, immunoassays using radioisotopes, enzymes or fluorescence to label antibodies or antigens. It also summarizes techniques like immunofluorescence and flow cytometry to detect epitopes on or within cells, assays to assess immune functions like phagocytosis and proliferation, and methods for evaluating hypersensitivity reactions.
PRINCIPLES of FT-NMR & 13C NMR
Fourier Transform
FOURIER TRANSFORM NMR SPECTROSCOPY
THEORY OF FT-NMR
13C NMR SPECTROSCOPY
Principle
Why C13-NMR is required though we have H1-NMR?
CHARACTERISTIC FEATURES OF 13 C NMR
Chemical Shifts
NUCLEAR OVERHAUSER ENHANCEMENT
Short-Comings of 13C-NMR Spectra
The slides covers brief description of ion exclusion chromatography. i hope the slides will be helpful
for any further details you can contact me through email.
mail id - sobhigaba@gmail.com
Gas chromatography-atomic absorption spectroscopy (GC-AAS) is an analytical technique used to separate and quantify elemental components in a sample. It combines gas chromatography, which separates compounds, with atomic absorption spectroscopy, which measures elemental concentrations. Key components of a GC-AAS system include a gas chromatograph unit with a carrier gas, injection port, column, and detector; an atomic absorption spectrometer with a light source, atomizer, monochromator, and detector; and an interface connecting the two instruments. GC-AAS is applied to quantify metals in various samples like industrial waste, alloys, foods, and more.
In Silico methods for ADMET prediction of new moleculesMadhuraDatar
The document discusses the importance of predicting absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of new molecules in silico during drug design. It describes how ADMET prediction techniques have evolved since 1863 and helped advance drug development. Factors considered in developing ADMET prediction models include the model purpose, required prediction speed and accuracy. Common molecular descriptors used in these models are also discussed. The document outlines methods for predicting various ADMET properties like permeability, solubility, distribution and metabolism in silico. Recent tools for computational ADMET prediction are also mentioned.
NMR stands for Nuclear Magnetic Resonance. It is a scientific technique used to study the structure, composition, and dynamics of molecules. In NMR spectroscopy, a sample is placed in a strong magnetic field and subjected to radiofrequency radiation. The atomic nuclei in the sample, particularly those with a nonzero spin, absorb and re-emit electromagnetic radiation at specific frequencies. By measuring the frequencies at which the nuclei resonate, valuable information about the chemical environment and connectivity of the atoms in the molecule can be obtained. It is a powerful tool for chemists and other scientists working in fields related to molecular analysis and characterization.
Similarities and differences between 1D and 2D NMR techniques are broadly illustrated here:
This document provides an overview of LC-NMR spectroscopy and its applications. It discusses the history, principles, instrumentation, and modes of LC-NMR. The key components of an LC-NMR system include the LC unit, interface, and NMR unit. Modes of analysis include continuous flow, stopped flow, time slicing, peak parking, and peak trapping. Technology such as online SPE, high field magnets, and solvent suppression methods can improve sensitivity. LC-NMR is useful for structural elucidation of organic compounds.
NOESY (Nuclear Overhauser Effect Spectroscopy) is a 2D NMR technique used to identify nuclear spins undergoing cross-relaxation and measure their rates. It provides information about which proton resonances are from protons close in space. NOESY experiments exploit the nuclear Overhauser effect to observe through-space dipolar couplings. One application is in protein NMR to assign structures by sequential walking. It is useful for determining the stereochemistry of biomolecules in solution.
The document discusses structure-based in-silico virtual screening protocols. It describes virtual screening as using computer methods to discover new ligands based on a target protein's biological structure. The main goal is to reduce the enormous chemical space of potential compounds to a manageable number with the highest likelihood of becoming drug candidates. Molecular docking is a key method, involving sampling potential ligand positions in the protein's binding site and scoring the interactions. The document also discusses force field, empirical, and knowledge-based scoring functions used to evaluate docking poses. Applications mentioned include designing Hsp90 inhibitors for cancer treatment and identifying novel BACE1 inhibitors.
Ion exclusion chromatography is a technique,introduced by Wheaton and Bauman, used to separate ionic compounds from non-ionic compounds and to separate mixtures of acids.
This document discusses various aspects of stereochemistry. It begins by explaining Fischer's D and L notation system for assigning configurations based on a compound's relation to glyceraldehyde. It then discusses pseudo asymmetric centers in meso compounds and cis-trans isomerism that can occur due to restricted bond rotation around double bonds. Finally, it introduces the E-Z system for naming geometric isomers with three or more different groups, which is based on Cahn-Ingold-Prelog priority rules to determine whether higher priority groups are on the same or opposite sides of the double bond.
Interpretation of organic compounds by IR, NMR and Mass SpectrometryAshitoshPanchal
This document discusses various spectroscopy techniques for analyzing organic compounds, including infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry. It describes attenuated total reflectance (ATR)-IR spectroscopy and how it works. It also explains 2D NMR techniques like COSY spectroscopy and how they provide more structural information than 1D NMR. Finally, it discusses how mass spectrometry works and common fragmentation patterns seen in mass spectra for different functional groups like alkanes, cycloalkanes, and compounds containing isotopes.
The document discusses 2D-QSAR (Quantitative Structure-Activity Relationship) analysis methods. It defines QSAR as mathematical relationships linking chemical structure and pharmacological activity. It describes several common 2D-QSAR methods including Hansch analysis, Free Wilson analysis, and various statistical methods. Cluster analysis is discussed as a way to group similar molecules and select a diverse subset for analysis. Molecular descriptors that encode structural, electronic, and topological properties are also introduced.
2D NMR provides more information than 1D NMR by plotting data in a space defined by two frequency axes. There are several types of 2D NMR experiments including COSY, NOESY, and HETCOR. COSY identifies spin-coupled protons by showing cross peaks between protons that are directly bonded. NOESY correlates protons that are near each other in space but not necessarily directly bonded. HETCOR plots 1H and 13C spectra on separate axes and connects carbon signals to bonded proton signals. 2D NMR techniques provide additional structural information about molecules compared to traditional 1D NMR.
This document summarizes an ultrasound assisted reaction presentation. It discusses how ultrasound differs from conventional energy sources and how it can be used in organic synthesis and green and pharmaceutical chemistry. It describes how sonochemistry works through cavitation, where bubbles form and violently collapse, generating high pressures and temperatures. This can enhance chemical reactivity in homogeneous liquid, heterogeneous solid/liquid, and heterogeneous liquid/liquid phase reactions. Examples of synthetic applications where ultrasound switching altered reaction pathways are provided. The conclusion discusses how bubble collapse concentrates energy that can be used to heat bubble contents and enhance reactivity.
This document discusses the coupling of liquid chromatography (LC) with Fourier transform infrared spectroscopy (FTIR). It describes how LC and FTIR can be combined to detect and identify separated compounds. Various interfaces for coupling LC to FTIR are presented, including flow cell interfaces, solvent elimination interfaces, and different types of each. Application areas like trace analysis and analysis of pharmaceuticals and environmental pollutants are also mentioned.
PHARMACOHORE MAPPING AND VIRTUAL SCRRENING FOR RESEARCH DEPARTMENTShikha Popali
THE PHARMACOPHORE MAPPING AND VIRTUAL SCRRENING , THESE PRESENTATION INCLUDES THE DEATIL ACCOUNT ON PHARMACOPHORE, MAPPING, ITS IDENTIFIATION FEATURES, ITS CONFORMATIONAL SEARCH, INSILICO DRUG DESIGN, VIRTUAL SCREENING, PHARMACOPHORE BASED SCREENING
The document provides information about various 2D NMR techniques including HETCOR, INADEQUATE, and guidelines for interpreting NMR data. It discusses how HETCOR spectra show carbon-proton coupling and provides an example spectrum of 2-methyl-3-pentanone. INADEQUATE is introduced as a technique for determining carbon-carbon coupling constants using natural abundance. Examples of NMR data interpretation are also provided for small molecules such as methane, benzene, adenine, cytosine, naphthalene, and quinine to demonstrate analyzing chemical shifts and spin multiplicity.
Quantum Mechanics in Molecular modelingAkshay Kank
This slides gives you the information related to computer aided drug design and its application in drug discovery. Also you learn the Quantum mechanics related to the molecular mechanics. Theory related to molecular modeling and how the molecular modeling helps in drug discovery.
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.
Some Clinical Laboratory Measurement of Immune FunctionsAmany Elsayed
This document describes several clinical laboratory techniques for measuring immune functions, including antibody-based assays that detect antigens or quantify them. It discusses methods like agglutination assays, precipitation assays, immunoassays using radioisotopes, enzymes or fluorescence to label antibodies or antigens. It also summarizes techniques like immunofluorescence and flow cytometry to detect epitopes on or within cells, assays to assess immune functions like phagocytosis and proliferation, and methods for evaluating hypersensitivity reactions.
This ppt file represents a simple overview on what is antibody validation & how to validate an antibody before performing any research.
Used references are also included.
This document summarizes monoclonal antibodies and gene therapy. It discusses the discovery of monoclonal antibodies by Kohler and Milstein in 1975. It describes the production process of monoclonal antibodies which involves immunizing mice, fusing spleen cells with myeloma cells to form hybridomas, and cloning cell lines. The document also discusses types of monoclonal antibodies including murine, chimeric, and humanized, as well as applications in cancer therapy, diagnostics, and immunosuppression. Gene therapy techniques like ex vivo and in vivo delivery are summarized along with strategies for cancer like suicide gene therapy using thymidine kinase.
Radioimmunoassay (RIA) and enzyme immunoassay (EIA) techniques allow for the quantitative detection of analytes at trace levels. RIA uses radioisotopes as labels, while EIA uses enzymes. Both techniques can be formatted competitively or non-competitively. Variations include immunoradiometric assays, enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and apoenzyme reactivation immunoassay (ARIS). These assays find wide application in research, clinical medicine, and drug monitoring due to their high sensitivity and specificity.
This document provides an overview of monoclonal antibodies and gene therapy. It discusses the discovery of monoclonal antibodies by Kohler and Milstein in 1975. It also describes the multi-step process of producing monoclonal antibodies through cell fusion and hybridoma technology. Several types of monoclonal antibodies are outlined, along with their purification techniques and therapeutic applications in cancer treatment and other diseases. Gene therapy approaches including ex vivo and in vivo methods are briefly introduced.
Monoclonal antibody as targeting drug delivery systemsagarwani560
Monoclonal antibodies (Mabs) can be used for targeted drug delivery. Mabs are produced through cell fusion between B cells and myeloma cells, generating hybridomas that secrete antibodies against a specific antigen. This allows production of antibodies that are homogeneous and specific. Mabs can be conjugated to drugs to actively target delivery to sites like tumors, reducing side effects. Targeted delivery using Mabs aims to achieve the desired pharmacological response at selected sites without undesirable interactions elsewhere.
This document discusses various types of immunoassays and their applications. It describes enzyme immunoassays including ELISA and EMIT, fluoroimmunoassays using fluorescent dyes, chemiluminescence immunoassays with luminescent labels, optical immunoassays detecting changes in light reflection, and radioimmunoassays using radioactive antigens. Common applications include detecting drugs, hormones, infectious diseases, and cancer biomarkers in serum, plasma, urine or tissue samples. Immunoassays offer high specificity and sensitivity for quantitative analysis of various analytes in biological specimens.
The document discusses immunoassay of digoxin. It provides an overview of immunoassays including the basic principles of competitive and non-competitive immunoassays. It describes how digoxin works to treat conditions like congestive heart failure and its mechanisms of action. The document also outlines the procedure for performing an immunoassay to measure digoxin levels and lists several analytical methods used like enzyme immunoassay, cloned enzyme donor immunoassay, and fluorescence polarization immunoassay.
The document discusses two types of immunological assays: radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). RIA uses radioactively labeled antigens or antibodies to detect and quantify antigens or antibodies. It relies on competitive binding and can detect very low concentrations. ELISA uses enzymes to detect antigen-antibody binding and comes in indirect, sandwich, and competitive formats. Both techniques are sensitive and specific methods to detect proteins, hormones, drugs and other molecules through antibody-antigen reactions.
monoclonal antibodies and engineered antibodiesMunawar Ali
This document provides an overview of monoclonal antibodies and engineered antibodies. It discusses the advantages and disadvantages of monoclonal versus polyclonal antibodies. Production methods like hybridoma technology and fermentation are described. Problems associated with monoclonal antibody therapy like HAMA response are covered. Applications in diagnosis, therapy and analytical uses are mentioned. Finally, the document discusses engineering antibodies by modifying regions to reduce immunogenicity and enhance functions.
Immunochemical techniques utilize the specific binding of antibodies and antigens. They are simple, sensitive methods for detecting and quantifying proteins in tissues or cells. Major techniques include immunoprecipitation, immunoelectrophoresis, immunoassays like ELISA, and methods using particle agglutination. These techniques are important for clinical diagnosis and analyzing protein expression and function.
This document provides an overview of various immunological techniques including radioimmunoassay (RIA), rocket electroimmunodiffusion, chemical immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and allergen testing. It describes the basic principles, methodologies, applications, advantages, and disadvantages of each technique. Radioisotopes commonly used in RIA and the steps of the RIA methodology are detailed. The document also discusses specific chemiluminescent compounds, types of ELISAs, and in vivo and in vitro allergen testing methods.
This document discusses the history and development of radioimmunoassay (RIA) by Rosalyn Yalow, who won the Nobel Prize for her work. RIA involves labeling an antigen with a radioactive isotope, then using the specificity of the antibody-antigen reaction to separate bound labeled antigen from unbound antigen. This allows for quantification of antigens even at very low concentrations. The document outlines the principles, procedures, applications, advantages and disadvantages of RIA.
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.
ria-151214062348 (1) (1).pptx radio immuno.assayAakanksha38925
This document provides an overview of radioimmunoassay (RIA). It discusses how RIA uses radioactive isotopes and the antibody-antigen reaction to detect trace amounts of substances. Key points covered include:
- RIA was developed in 1959 to measure insulin levels and allows detection of substances at the trillionth of a gram.
- It involves mixing a radiolabeled antigen with antibodies and using competition between labeled and unlabeled antigens to determine the concentration of the unlabeled antigen.
- Common radioactive isotopes used are iodine-125, carbon-14, and tritium. Iodine-125 is most commonly used due to its optimal half-life and ability to label antigens.
Radioimmunoassay (RIA) is an immunoassay technique that uses radiolabeled antigens or antibodies to detect and quantify antigens or antibodies in a sample. RIA uses the principle of competitive binding - radiolabeled and unlabeled antigens or antibodies compete for binding sites on an antibody or antigen. The amount of radiolabeled binding is inversely proportional to the concentration of unlabeled antigen or antibody in the sample. RIA is a highly sensitive and specific technique that can detect picogram quantities of antigens or antibodies. It has applications in areas like endocrinology, pharmacology, and clinical diagnostics.
This presentation explains about the principle and procedure involved in elisa method of immunoassay, development o f elisa , application advantages and disadvantages of elisa
This presentation gives a overview about the microvolume UV/VIS spectroscopy. Instrumentation , working ,merits, demerits, cleaning of the sample platform. mainly explains about the measurement of sample using nano or micro volume samples.
ADVANCED ANALYSIS OF CARBOHYDRATES ,ANALYSIS OF CARBOHYDRATES IN FOOD MATRICESsuriyapriya kamaraj
This document discusses advanced analysis methods for carbohydrates in food. It begins with an introduction to carbohydrate classification, including simple sugars, oligosaccharides, and polysaccharides. Sample preparation methods are outlined, such as extraction and fractionation. Advanced analytical techniques for carbohydrate analysis are then described, including gas chromatography, high performance liquid chromatography, capillary electrophoresis, and spectroscopic methods like NMR and mass spectrometry.
This presentation explains about qualifications of HPTLC, types of qualifications, design qualification , installation qualification ,operational qualification, performance qualification ,documentation of qualification .
This slide shows about the Intellectual property rights, Intellectual property laws, Law of protection, Patent, Copyrights, Trade Marks ,Trade secrets, Geographical Indication, Industrial Design, Registration process of Intellectual Property, Period of Validation. Protection of Intellectual Property, WIPO
The document discusses array detectors used in spectroscopy. It describes photodiode array detectors and charged coupled device (CCD) detectors. Photodiode array detectors contain an array of silicon photodiodes on a single chip that can simultaneously measure radiation intensities at all wavelengths. CCD detectors contain an array of linked capacitors that can transfer electric charges between neighboring capacitors, allowing detection of low intensity light signals. Both detector types offer advantages like low noise, wide spectral response, and simultaneous detection of emissions at different wavelengths.
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
Does Over-Masturbation Contribute to Chronic Prostatitis.pptxwalterHu5
In some case, your chronic prostatitis may be related to over-masturbation. Generally, natural medicine Diuretic and Anti-inflammatory Pill can help mee get a cure.
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RADIO IMMUNO ASSAY
1. RADIOIMMUNO ASSAY
PRESENTED BY,
SURIYAPRIYA .K,
1st yr M.PHARM
DEPARTMENT OF PHARMACEUTICAL ANALYSIS,
ADVANCED PHARMACEUTICAL ANALYSIS,
KMCH COLLEGE OF PHARMACY,
COIMBATORE.
KMCH COLLEGE OF PHARMACY
1
7/25/2021
2. OVERVIEW
IMMUNOASSAY
ANTIBODY ,ANTIGEN, ANALYTE
RADIOIMMUNOASSAY INTRODUCTION
HISTORY
PRINCIPLE
REQUIREMENTS FOR ASSAY
STEPS INVOLVED IN RIA
PROCEDURE INVOLVED IN RIA
INSTRUMENTATION
RIA IN DIGITALIS
RIA IN INSULIN
MERITS OF RIA
DEMERITS OF RIA
DRAWBACK
REFERENCE 7/25/2021
KMCH COLLEGE OF PHARMACY 2
3. IMMUNO ASSAY
An immunoassay is a biochemical test ,that measures the presence or
concentration of a macromolecule or a small molecule in a solution through
the use of an antibody or an antigen.
An immunoassay is a test that uses antibody and antigen complexes .
An antibody and antigen complex is also known as an immune- complex .
refers to an immune response that causes the body to generate
antibodies .
refers to a test .
“Immuno”
“Assay”
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KMCH COLLEGE OF PHARMACY
3
5. ANTIBODIES, ANTIGENS AND ANALYTES
An antibody is a protein that is produced by the body immune response to an
“invading” (foreign) substance.
Antibodies are produced as part of the body’s immune response to protect
itself.
An antigen is the substance that the body is trying to “fight off” by mounting an
immune response.
For example, Drug is the antigen that binds to the antibody.
An immunogen is a substance that elicits immune response.
E.g. drug-protein conjugate.
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6. An analyte is anything measured by a laboratory test.
In immunoassay testing, the analyte may be either an antibody, or an antigen.
Immunoassays utilize one or more selected antibodies to detect analytes of
interest.
The analytes being measured may be:-
1. That are naturally present in the body (such as a thyroid hormone)
2. The body produces but are not typically present (such as a cancer antigen)
3. Do not naturally occur in the body (such as an abused drug)
TO BE CONTINUED ,
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6
7. STRUCTURE OF
ANTIBODIES
Antibodies are produced by the B lymphocytes , Y
shaped (Immunoglobulin )
The most important one is immunoglobulin G
(IgG).
IgG is a protein composed of two main structural
and functional regions:
Fab region: Contains the antigen (Ag) binding
site that varies between different antibodies.
Fc region: Region of constant structure within an
antibody class.
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8. RADIOIMMUNO ASSAY
Radioimmunoassay is a technique for determining antibody levels by
introducing an antigen labelled with a radioisotope and measuring the
subsequent radioactivity of the antibody component.
For example, Hormone levels in blood (insulin ) by use of antibodies .
The RIA technique is extremely sensitive and extremely specific .
It is the technique used for the estimation of any ligand / antigen in
biological fluids ,based on antigen antibody reaction .
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9. HISTORY
Developed in 1959 by Rosalyn Yalow and Solomon Berson
for measurement of insulin in plasma.
Rosalyn Yalow was a nuclear physicist.
She developed radioimmunoassay (RIA) together with
doctor Solomon Berson
It represented the first time that hormone levels in the blood
could be detected by an in vitro assay.
In 1977 Yalow received the Nobel Prize for her and Berson’s
development of RIA
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10. REQUIREMENTS
Micro titer plate / Polypropylene Test tubes
Pure antigen
Pipette
Radio labelled antigen
Antibodies
Standard’s / Reference
Vortex mixer
Centrifuge
Radioactive counter
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11. RADIO IMMUNO ASSAY
TWO METHODS EMPLOYED
FOR DRUG DETECTION IN BIOLOGICAL
MATRICES
WITH DOUBLE ANTIBODY RIA WITH COATED TUBE RIA
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12. PREPARATION AND RADIOLABELING OF THE ANTIGEN
Antigens prepared by
synthesis of the molecule
isolation from natural sources
Radiolabeling [Tagging procedure ]
3H,14C,125I are used as radioactive tags
Antigens are tagged to 3H , 14c , 125I
Tagging should NOT affect antigenic specificity and activity 7/25/2021
KMCH COLLEGE OF PHARMACY
12
13. PREAPRATION OF SPECIFIC ANTIBODY
Antibody can be either polyclonal or monoclonal .
Antigen injected intradermally into rabbit or guinea pigs antibody
production .
Antibodies recovered from the serum .
Some ligands are not antigenic.
With the low molecular weight substances such as the thyroxine, steroid
hormones, and drugs conjugation is essential ..
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14. POLYCLONAL ANTIBODY MONOCLONAL ANTIBODY
Cheap to produce Expensive to produce
Mixed population of
antibodies
Single antibody species
May bind to different areas
of the target molecules
Will only bind single
specific site
Tolerant of small changes in
protein structure
May recognise a particular
protein form
Monoclonal antibody
Polyclonal antibody
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15. PRINCIPLE OF RIA :
The basic principle of radioimmunoassay is competitive binding, where a radioactive antigen
("tracer") competes with a non-radioactive antigen for a fixed number of antibody or receptor binding
sites.
The radio labeled antigen should have similar biological activity and immunogenicity like that of
native antigen .
LABELED ANTIGEN + SPECIFIC ANTIBODY LABELED ANTIGEN Ag *ANTIBODY complex
Ag* Ab Ag*Ab
UNLABELED ANTIGEN
Ag
UNLABELED ANTIGEN - ANTIBODY COMPLEX
Ag Ab
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16. TO BE CONTINUED ,
The free antigen and labelled antigen are separated and washed out .
The radioactivity of the labeled antigen antibody complex is measured
using scintillation counter or gamma counter.
The measured radioactivity is inversely proportional to the concentration of
antigen .
More is the concentration of antigen , lesser is the radioactivity of the
complex and vice versa .
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17. CALIBRATION CURVE :
From these data, a standard binding curve, like the one shown in red, can be drawn.
The samples to be assayed (the unknowns) are run in parallel .
After determining the ratio of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve.
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19. PREPARATION AND CHARACTERIZATION OF ANTIGEN
The antigen whose concentration is to be
measured , should be available in pure form.
Also required to create antibodies ,in assay
process to form complex .
RADIO LABELING OF ANTIGEN
TAGGING , where antigen is tagged with
radio active elements like 125I , 14C
PREPARATION OF SPECIFIC ANTIBODY
Specific antibodies are produced by injecting the antigen in
mouse , rabbit, guinea pig & collect & purify the antibodies .
In case of drug or other compounds (Eg . morphine ,
hormones ) which are not antigenic .
They combined with albumin , to become antigenic .
DEVELOPMENT OF ASSAY
VALIDATION OF METHOD Parameter like sensitivity ,specificity,
precision, linearity etc
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21. THE ASSAY IS CARRIED OUT IN WELL PLATES (TYPICALLY 96 WELL PLATE)
Where inner surface is coated with antibodies
RADIO ACTIVE ANTIGEN IS ADDED TO ALL THESE WELLS
Which forms a complex with antibodies
THE UNLABELED ANTIGEN OF KNOWN CONCENTRATION AND ANTIGEN OF
UNKNOWN CONCENTRATION (SAMPLE)
Where known conc. Added to multiple wells and
unknown conc. Added to one of the well
COMPETITION BETWEEN LABELED AND UNLABELED ANTIGEN TAKE PLACE
Due to limited binding sites present in antibodies
RADIO LABELED ANTIGEN BOUNDED TO ANTIBODY
Separated by precipitation/chromatography/gel
filtration
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22. ANTIBODY COMPLEX FORMED
• This leads to displacement of radio labeled antigen by
unlabeled antigen
AFTER INCUBATION TIME THE PLATE ARE REMOVED AND WASHED
• This remove free antigen and unbound
antigen
GAMMA OR SCINTILLATION COUNTER
• Measuring the radio activity
MORE CONC.OF FREE ANTIGEN LESSER RADIO ACTIVITY IN COMPLEX
FROM THE GRAPH OF RADIO ACTIVITY VS CONC.THE UNKNOWN ,CONC.OF ANTIGEN CAN
BE DETERMINED
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24. Figure illustrates how a limited amount of antibody binding sites contained in a test tube or
microplate well can either bind unlabeled ligand (in this example, Hepatitis B Surface Antigen) or
radiolabeled ligand. As the amount of unlabeled ligand increases, there is consequently less
radiolabeled ligand bound. The unlabeled ligand can come from either a “calibration standard”
or the sample that you are trying to measure.
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25. SEPARATION TECHNIQUE
To separate free from bound labelled antigen separation techniques are :
a ) physical methods:-filtration ,chromatography ,electrophoresis, charcoal
dextran adsorption etc.
b) Chemical methods:-organic solvents such as ethanol, dioxane , PEG,
ammonium sulfate for precipitating antibody .
C) Immunological precipitation :- 2nd antibody
d)Solid phase systems :- with antibodies on solid support or matrix
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27. swing- bucket rotor
CENTRIFUGE
Fixed angle head rotor
generating 1200-2500
rpm
generating 3500-4000
rpm
pellet is formed at an
angle
pellet is formed at the
bottom of the test tube
The latter type is
preferred
The former type is
preferred
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30. RIA OF DIGITALIS
Digitalis Commonly known as Foxglove leaves,
Generic name – Digitalis purpurea
Family - Scrophulariaceae.
It is also used for drug preparations that contain cardiac glycosides, like
Digoxin & Digitoxin extracted from various parts of the plant.
USES : Increase cardiac contractility
Antiarrhythmic agent
Atrial flutter
Atrial fibrillation
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31. PRINCIPLE OF RIA FOR DIGITALIS
The assay is based on the use of 125-iodine-labelled digoxin and of a gel equilibration technique
for the separation of antibody- bound and free digoxin.
Digoxin in serum samples competes with radio-labelled (125-1) digoxin derivative for binding sites
on the antibody to digoxin.
The unbound digoxin is then separated from bound form.
It is then quantified by counting radioactivity & concentration of unlabeled digoxin in serum
sample is calculated by comparison to digoxin standard .
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32. Digoxin Standards - From 0.5- 8.0 ng/ml
Anti-serum and Tracer solutions
Digoxin antibodies raised in rabbits by subcutaneous
Injections of digoxin-bovine serum albumin
Phosphate saline buffer
Digoxin Radio Immuno assay kit, with 3-0-succinyl
Digoxigenin tyrosine 125-1.
Materials Used For Assay
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33. The assay is carried out using 3-O-succinvl digoxigenin tyrosine 125-Ⅰ.
1 ml of phosphate buffer solution + 0-50 µl of standard digoxin solution
+ 10 µl of 3-0-succinyl digoxigenin tyrosine 125-Ⅰ
Add 10 µl of digoxin anti-serum, all contents are mixed well.
Standard
Curve
PROCEDURE FOR ASSAY
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34. To 50µl of patient's plasma, + 1 ml of phosphate buffered saline.
To this add 10µ of labelled solution, & 10µl of digoxin anti-serum,
All contents are mixed well
All the tubes are allowed to stand and 0.5ml of charcoal solution is added to all tubes.
The tubes are then centrifuged, gamma radioactivity.
Procedure For
Unknowns
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KMCH COLLEGE OF PHARMACY 34
35. RIA measurements of insulin
RIA measurements of insulin combined with glucose estimations.
Can diagnose hyperinsulinism due to insulin secreting tumors of the pancreas.
Insulin (in micro units per ml) to glucose (in mg/100 ml) ratios of more than 0.30 is indicative
of this condition.
Measurement of insulin and C-peptide concentrations following arginine stimulation Test .
In this test, 300 ml of a 10% solution of arginine is infused into a fasting patient who has not
had insulin, over a period of 30 minutes and blood taken at - 0, 15, 30, 45, 60 and 90
minutes.
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36. In Type I, insulin dependent diabetes, basal levels of C-peptide would be less than 0.3 ng/ml
and there would be no response to arginine.
If basal C-peptide is low, say 0.3-1.0 ng/ml, and there is some response to arginine (a 100%
rise), the patient is still insulin deficient.
In typical Type II diabetes, basal levels would be 2.5 ng/ml or over, and a 100% rise on
stimulation would be seen.
RIA for microalbuminuria is becoming popular as a predictor of the development of clinical
nephropathy in diabetics.
TO BE CONTINUED ,
7/25/2021
KMCH COLLEGE OF PHARMACY 36
37. MERITS OF RADIOIMMUNOASSAY
High specificity and hence there is no interference from other substances.
High sensitivity – Immuno reaction are very sensitive , so when antibody with high
affinity are used , it is possible to detect antigen up to a few picogram (10-12 g).
High precision and accuracy
Applicable to wide variety of compounds in various fields
pharmacology
Endocrinology
Oncology
Epidemiology
Clinical immunology
7/25/2021
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38. DEMERITS OF RADIOIMMUNOASSAY
Expensive compared to other methods ,as radioactive materials are used .
Radiation hazards because of Usage of radio labelled reagents .
Requires specially trained persons .
Labs require special license to handle radioactive material.
Requires special arrangements for Requisition, storage of radioactive material ,radioactive
waste disposal.
Development of specific antibodies to the antigen ..
Compliant technique than other methods compared to Tlc , Hptlc , etc.. 7/25/2021
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38
39. APPLICATION OF RADIO IMMUNOASSAY
1.The test can be used to determine very small quantities (e.g. nanogram) of antigens and antibodies
in the serum.
2.The test is used for quantitation of hormones, drugs(Digitoxin or digoxin ), HBsAg in blood ,HIV 1& 2
Virus and other viral antigens.
3.Analyze nanomolar and picomolar concentrations of hormones in biological fluids.
4.Certain abused drugs , Eg . MORPHINE
5.Ria has been used to assay plasma levels of :
1.Most of hormones
2.Insulin in human plasma
3.Beta - HCG in females
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40. DRAWBACKS :
The cost of equipment and reagents are expensive .
Short shelf life of radiolabeled compounds .
The problems associated with the disposal of radioactive waste.
Hazards if preparing and handling the radioactive antigen.
Both 125I or 131I emit gamma radiation that requires special
counting equipment . 7/25/2021
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41. THE SPECIFICITY OF AN RIA IS IMPROVED BY:
(a) The use of pure immunogens or conjugates.
(b) Pure labelled compounds.
(c) Pure analyte for assay, pre-extracted and purified if needed.
(d) Immunization with specific subunits, e.g. b-hCG
(e) Saturation with cross reactants, e.g. TSH antibody with hCG
(f) Use of monoclonal antibodies of high specificity 7/25/2021
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