This document provides an overview of immunohistochemistry (IHC). IHC is a technique that uses antibodies to identify antigens in cells and tissues. It involves using a primary antibody that binds to the antigen of interest, followed by detection with a labeled secondary antibody or enzyme. Key steps include antigen retrieval to unmask antigens, blocking to reduce background staining, and using enzyme labels or other detection methods to visualize antibody binding sites. Common enzymes used are horseradish peroxidase and alkaline phosphatase. The document discusses different IHC methods including direct, indirect, PAP, ABC, and streptavidin-biotin techniques. It also covers antigen retrieval methods, fixatives, controls, and interpretation of IHC results.
Immunohistochemistry is a technique used to identify antigens in tissue samples using antigen-antibody interactions. It has made a large impact in disease diagnosis since its development in the 1940s-1970s. The technique involves using labeled antibodies that specifically bind to antigens in tissue sections. This binding is then visualized using markers like enzymes or fluorescent dyes. Several methods have been developed to increase the signal and reduce background noise, including indirect labeling techniques and polymer-based methods. Proper tissue processing and antibody selection are important for obtaining high quality results with immunohistochemistry.
Immunohistochemistry (IHC) is a highly sensitive method that allows the localization of antigen within a cell or a tissue with high resolution. The method is based on the use of a primary antibody that specifically binds to its complementary antigen. The bound antibody may then be visualized by a variety of methods such as colorimetric end points.
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 provides an overview of several techniques used to study proteins, including:
- Protein quantitation methods like BCA, Bradford, and Lowry assays.
- Peptide sequencing techniques like Edman degradation and mass spectrometry.
- Antibody applications such as monoclonal antibody production, ELISA, Western blotting, and immunofluorescence.
- Structural determination methods including NMR spectroscopy, X-ray crystallography, and cryo-electron microscopy.
This document discusses various techniques used in immunoblotting and blotting. It begins by defining blotting as techniques used to visualize specific DNA, RNA, and proteins among contaminants. It then describes three main types of blotting - western blotting for proteins, northern blotting for RNA, and southern blotting for DNA. The document focuses on western blotting and immunoblotting. It provides details on tissue preparation, gel electrophoresis, protein transfer, blocking, detection, analysis, and applications of western blotting and immunoblotting techniques.
Immunohistochemistry, the basics and applications.pptxAmirRaziq1
This document provides information about immunohistochemistry techniques. It discusses antigen retrieval methods like heat-induced epitope retrieval and protease digestion to expose buried epitopes. It describes primary and secondary antibody incubation steps and the differences between monoclonal and polyclonal antibodies. Detection methods like direct immunofluorescence, indirect immunofluorescence, peroxidase anti-peroxidase, and avidin-biotin complex are outlined. Chromogen substrates and counterstaining are also summarized. Automated immunohistochemistry systems and the differences between IHC and hematoxylin and eosin staining are briefly covered.
principle of Immunohistochemistry and its use in diagnosticsEkta Jajodia
Immunohistochemistry (IHC) localizes antigens in tissues based on antigen-antibody recognition. The principle is visualizing target compounds in tissues with high signal-to-noise ratio. IHC was developed in the 1960s using enzyme labels instead of fluorescent labels to visualize targets under a light microscope. Key steps in IHC include antigen retrieval to unmask antigens, blocking endogenous enzymes, primary antibody incubation, secondary antibody or polymer incubation, and signal development with chromogens. IHC is commonly performed on formalin-fixed paraffin-embedded sections and can identify cell types and localization of proteins to characterize tissues.
Immunohistochemistry is a technique used to identify antigens in tissue samples using antigen-antibody interactions. It has made a large impact in disease diagnosis since its development in the 1940s-1970s. The technique involves using labeled antibodies that specifically bind to antigens in tissue sections. This binding is then visualized using markers like enzymes or fluorescent dyes. Several methods have been developed to increase the signal and reduce background noise, including indirect labeling techniques and polymer-based methods. Proper tissue processing and antibody selection are important for obtaining high quality results with immunohistochemistry.
Immunohistochemistry (IHC) is a highly sensitive method that allows the localization of antigen within a cell or a tissue with high resolution. The method is based on the use of a primary antibody that specifically binds to its complementary antigen. The bound antibody may then be visualized by a variety of methods such as colorimetric end points.
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 provides an overview of several techniques used to study proteins, including:
- Protein quantitation methods like BCA, Bradford, and Lowry assays.
- Peptide sequencing techniques like Edman degradation and mass spectrometry.
- Antibody applications such as monoclonal antibody production, ELISA, Western blotting, and immunofluorescence.
- Structural determination methods including NMR spectroscopy, X-ray crystallography, and cryo-electron microscopy.
This document discusses various techniques used in immunoblotting and blotting. It begins by defining blotting as techniques used to visualize specific DNA, RNA, and proteins among contaminants. It then describes three main types of blotting - western blotting for proteins, northern blotting for RNA, and southern blotting for DNA. The document focuses on western blotting and immunoblotting. It provides details on tissue preparation, gel electrophoresis, protein transfer, blocking, detection, analysis, and applications of western blotting and immunoblotting techniques.
Immunohistochemistry, the basics and applications.pptxAmirRaziq1
This document provides information about immunohistochemistry techniques. It discusses antigen retrieval methods like heat-induced epitope retrieval and protease digestion to expose buried epitopes. It describes primary and secondary antibody incubation steps and the differences between monoclonal and polyclonal antibodies. Detection methods like direct immunofluorescence, indirect immunofluorescence, peroxidase anti-peroxidase, and avidin-biotin complex are outlined. Chromogen substrates and counterstaining are also summarized. Automated immunohistochemistry systems and the differences between IHC and hematoxylin and eosin staining are briefly covered.
principle of Immunohistochemistry and its use in diagnosticsEkta Jajodia
Immunohistochemistry (IHC) localizes antigens in tissues based on antigen-antibody recognition. The principle is visualizing target compounds in tissues with high signal-to-noise ratio. IHC was developed in the 1960s using enzyme labels instead of fluorescent labels to visualize targets under a light microscope. Key steps in IHC include antigen retrieval to unmask antigens, blocking endogenous enzymes, primary antibody incubation, secondary antibody or polymer incubation, and signal development with chromogens. IHC is commonly performed on formalin-fixed paraffin-embedded sections and can identify cell types and localization of proteins to characterize tissues.
Immunohistochemistry utilizes labeled antibodies to localize specific cell and tissue antigens through antigen-antibody interactions. It is a highly sensitive technique that can visualize the distribution of cellular components in tissues. Tissues are prepared through processes like fixation, sectioning, and antigen retrieval before antibodies are applied. Labeled antibodies then bind to target antigens, allowing their visualization through markers like fluorescent dyes or enzymes. Controls are used to validate the protocol and antibody specificity.
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.
Immunohistochemistry utilizes labeled antibodies to localize specific antigens within cells and tissues. It combines histological, immunological, and biochemical techniques to visualize the distribution of cellular components through antigen-antibody reactions tagged with visible labels. Tissues are prepared through fixation and sectioning, then exposed to labeled antibodies targeting specific antigens. This allows visualization of targeted proteins and structures under a microscope. IHC is a sensitive technique useful for cancer diagnosis, differential diagnosis, and research applications.
Techniques for the study of molecular pharmacologypradnya Jagtap
This document provides an overview of several molecular pharmacology techniques:
1. Western blotting is used to detect specific proteins and involves separating proteins by size, transferring them to a membrane, and using antibodies to visualize target proteins.
2. Immunostaining uses antibodies to detect specific proteins in tissue samples and can be used for diagnostic purposes.
3. RT-PCR is used to analyze gene expression by converting mRNA to cDNA and then amplifying specific sequences using PCR for detection.
4. Cloning techniques such as DNA cloning are used to generate identical copies of DNA fragments or genes using vectors like bacterial plasmids.
Immunohistochemistry (IHC) utilizes labeled antibodies to localize specific antigens in tissue sections through antigen-antibody interactions visualized by markers like fluorescent dyes or enzymes. IHC allows visualization of the distribution and localization of cellular components within tissues. The process involves raising antibodies to target antigens, labeling the antibodies, and applying them to tissue sections using techniques like direct, indirect, or peroxidase anti-peroxidase (PAP) methods. IHC is a sensitive and specific technique that is useful for cancer diagnosis and research applications.
Immunoprecipitation: Procedure, Analysis and Applicationsajithnandanam
Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
This document discusses antigen retrieval in immunohistochemistry. It begins with a brief history and defines key terms like antigen, antibody, affinity, and sensitivity. It describes how antigens can become masked during tissue fixation and various techniques to retrieve masked antigens, including heat-induced epitope retrieval (HIER), protease-induced epitope retrieval (PIER), and combinations of the two. Specific methods of HIER using microwave ovens, pressure cookers, steamers and other devices are outlined. The document also discusses newer antigen retrieval techniques like those for frozen sections. In summary, antigen retrieval techniques aim to expose masked antigens by reversing the effects of tissue fixation through the use of heat and enzymes.
Immunohistochemistry description of the fluorescence mehodes and enzymetic m...HadeelAlboaklah
This document defines immunohistochemistry and describes techniques for identifying cellular or tissue constituents using antigen-antibody interactions. It discusses antigens, antibodies, antibody-antigen binding, and two main methods - immunofluorescence and enzymatic. The immunofluorescence method uses fluorescent dyes to label antibodies and allow detection under a fluorescence microscope. The enzymatic method uses enzyme-labeled antibodies and reaction with a substrate to yield a colored product detectable by light microscope.
priciples and applications Immunohistochemistry Markos Tadele
This document provides an overview of immunohistochemistry (IHC), including the principle, general protocol, and key steps. IHC combines histology and immunology to identify specific tissue components using antigen-antibody reactions tagged with visible labels. The protocol involves fixing, embedding, sectioning tissues, performing antigen retrieval, incubating with primary/secondary antibodies, and visualizing the antigen-antibody complex through detection systems like peroxidase or fluorescence. Proper controls and interpretation by a pathologist are needed for accurate results.
This document provides an overview of immunohistochemistry methods. It defines key terms like antigens, antibodies, affinity, and sensitivity. It discusses the history of immunohistochemistry from the 1930s to current techniques. The principles of immunohistochemistry are described, including production of primary reagents, tissue fixation, antigen retrieval, staining, and limitations. Various immunohistochemistry methods are summarized such as direct, indirect, polymer, peroxidase-antiperoxidase, and alkaline phosphatase techniques.
This document provides information about the virology and parasitology course code 320 at semester 6. It discusses various immunological and molecular techniques used for diagnosis of viruses and parasites including radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), immunoblotting, DNA probes, polymerase chain reaction (PCR) amplification, and real-time PCR.
Endotoxin Testing is performed to ensure that injectable preparations and medical devices are free from pyrogens and safe for human use.
Pyrogens constitute a heterogeneous group of fever causing substances which comprise both microbial and non-microbial substances. The most potent and most widely known are the endotoxins or lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. Gram-positive bacteria are also sources of pyrogens, in particular lipoteichoic acid (LTA), as are particles from yeasts and viruses. Non-microbial pyrogens often emanate from production environments. Small particles of packaging materials are a typical example.
ELISA is an antigen-antibody reaction technique used to detect substances like proteins, peptides, antibodies, and hormones. There are three main types of ELISA - indirect ELISA detects antibodies, sandwich ELISA detects antigens using two antibodies that bind to different sites, and competitive ELISA measures antigen concentration based on competition between sample antigen and antigen coated on the plate for a limited number of antibody binding sites. ELISA involves capturing the antigen or antibody of interest on a plate using its binding partner, then detecting it using an enzyme-linked secondary binding partner and colorimetric substrate.
The document provides information about two medical diagnostic techniques: the quantitative buffy coat (QBC) test and enzyme-linked immunosorbent assay (ELISA). It describes the QBC test procedure which uses a stained capillary tube to detect malaria parasites in centrifuged blood samples based on their fluorescence. It also gives an overview of the principles and types of ELISA, including indirect, sandwich, and competitive ELISA, which are immunochemical techniques used to detect and quantify antigens or antibodies.
- ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay technique used to detect substances like proteins, peptides, antibodies, and hormones. There are four main types of ELISA: direct, indirect, sandwich, and competitive.
- Direct ELISA involves coating an antigen onto a plate and adding an enzyme-labeled primary antibody that binds directly to the antigen. Indirect ELISA uses an unlabeled primary antibody and enzyme-labeled secondary antibody. Sandwich ELISA uses two antibodies that bind to different sites on an antigen. Competitive ELISA measures antigen concentration based on competition between antigen in a sample and antigen coated on a plate for binding to an antibody.
- ELISA has various applications like screening donated blood,
Food borne pathogens causes various diseases. So it is very important to detect them. Rapid methods help to detect pathogens in a very short period of time.
Immunohistochemistry uses antibodies to detect antigens in tissue samples in order to localize proteins and study biological processes. It has various applications including cancer prognosis, tumor identification, and research. The process involves deparaffinizing tissue, antigen retrieval, blocking, primary/secondary antibody incubation, signal development using enzymes, counterstaining, and mounting slides. Different methods like direct, indirect, PAP, ABC, and LSAB are used depending on the desired sensitivity and specificity. Careful antibody selection and protocol optimization are important for obtaining high quality results.
Immunohistochemistry uses antibodies to detect antigens in tissue samples in order to localize proteins and study biological processes. It has various applications including cancer prognosis, tumor identification, and research. The process involves deparaffinizing tissue, antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, signal development using enzymes, counterstaining, and mounting slides. Different methods like direct, indirect, PAP, ABC, and LSAB are used depending on the desired sensitivity and signal amplification needed. Careful antibody selection and protocol optimization are important for obtaining high quality results.
Radioimmunoassay (RIA) is a technique that uses the specificity of antibody-antigen binding and radioactivity to separate and quantify proteins. RIA revolutionized research and clinical practice by allowing the detection of hormone levels in blood. It works by measuring the displacement of labeled antigen from an antibody when unlabeled antigen is added. While useful, RIA requires radioactive materials and specialized equipment. Enzyme-linked immunosorbent assay (ELISA) was developed as a safer alternative, using enzyme-labeled antibodies or antigens. ELISA and other techniques like agglutination reactions, complement fixation tests, immunodiffusion, and precipitation reactions exploit the binding properties of antibodies to detect or quantify antigens.
Immunohistochemistry (IHC) is a technique that combines immunology and histology to detect antigens in tissues using antibodies, with the main steps including preparing tissue sections, using primary and secondary antibodies to target antigens, developing signals using reporters and counterstains, and observing under a microscope. IHC is widely used for disease diagnosis by identifying molecular markers characteristic of cellular events involved in diseases like cancer and neurological disorders. Troubleshooting for IHC focuses on reducing background staining and improving antigen detection through optimizing antibody concentrations and incubation times.
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
Immunohistochemistry utilizes labeled antibodies to localize specific cell and tissue antigens through antigen-antibody interactions. It is a highly sensitive technique that can visualize the distribution of cellular components in tissues. Tissues are prepared through processes like fixation, sectioning, and antigen retrieval before antibodies are applied. Labeled antibodies then bind to target antigens, allowing their visualization through markers like fluorescent dyes or enzymes. Controls are used to validate the protocol and antibody specificity.
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.
Immunohistochemistry utilizes labeled antibodies to localize specific antigens within cells and tissues. It combines histological, immunological, and biochemical techniques to visualize the distribution of cellular components through antigen-antibody reactions tagged with visible labels. Tissues are prepared through fixation and sectioning, then exposed to labeled antibodies targeting specific antigens. This allows visualization of targeted proteins and structures under a microscope. IHC is a sensitive technique useful for cancer diagnosis, differential diagnosis, and research applications.
Techniques for the study of molecular pharmacologypradnya Jagtap
This document provides an overview of several molecular pharmacology techniques:
1. Western blotting is used to detect specific proteins and involves separating proteins by size, transferring them to a membrane, and using antibodies to visualize target proteins.
2. Immunostaining uses antibodies to detect specific proteins in tissue samples and can be used for diagnostic purposes.
3. RT-PCR is used to analyze gene expression by converting mRNA to cDNA and then amplifying specific sequences using PCR for detection.
4. Cloning techniques such as DNA cloning are used to generate identical copies of DNA fragments or genes using vectors like bacterial plasmids.
Immunohistochemistry (IHC) utilizes labeled antibodies to localize specific antigens in tissue sections through antigen-antibody interactions visualized by markers like fluorescent dyes or enzymes. IHC allows visualization of the distribution and localization of cellular components within tissues. The process involves raising antibodies to target antigens, labeling the antibodies, and applying them to tissue sections using techniques like direct, indirect, or peroxidase anti-peroxidase (PAP) methods. IHC is a sensitive and specific technique that is useful for cancer diagnosis and research applications.
Immunoprecipitation: Procedure, Analysis and Applicationsajithnandanam
Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
This document discusses antigen retrieval in immunohistochemistry. It begins with a brief history and defines key terms like antigen, antibody, affinity, and sensitivity. It describes how antigens can become masked during tissue fixation and various techniques to retrieve masked antigens, including heat-induced epitope retrieval (HIER), protease-induced epitope retrieval (PIER), and combinations of the two. Specific methods of HIER using microwave ovens, pressure cookers, steamers and other devices are outlined. The document also discusses newer antigen retrieval techniques like those for frozen sections. In summary, antigen retrieval techniques aim to expose masked antigens by reversing the effects of tissue fixation through the use of heat and enzymes.
Immunohistochemistry description of the fluorescence mehodes and enzymetic m...HadeelAlboaklah
This document defines immunohistochemistry and describes techniques for identifying cellular or tissue constituents using antigen-antibody interactions. It discusses antigens, antibodies, antibody-antigen binding, and two main methods - immunofluorescence and enzymatic. The immunofluorescence method uses fluorescent dyes to label antibodies and allow detection under a fluorescence microscope. The enzymatic method uses enzyme-labeled antibodies and reaction with a substrate to yield a colored product detectable by light microscope.
priciples and applications Immunohistochemistry Markos Tadele
This document provides an overview of immunohistochemistry (IHC), including the principle, general protocol, and key steps. IHC combines histology and immunology to identify specific tissue components using antigen-antibody reactions tagged with visible labels. The protocol involves fixing, embedding, sectioning tissues, performing antigen retrieval, incubating with primary/secondary antibodies, and visualizing the antigen-antibody complex through detection systems like peroxidase or fluorescence. Proper controls and interpretation by a pathologist are needed for accurate results.
This document provides an overview of immunohistochemistry methods. It defines key terms like antigens, antibodies, affinity, and sensitivity. It discusses the history of immunohistochemistry from the 1930s to current techniques. The principles of immunohistochemistry are described, including production of primary reagents, tissue fixation, antigen retrieval, staining, and limitations. Various immunohistochemistry methods are summarized such as direct, indirect, polymer, peroxidase-antiperoxidase, and alkaline phosphatase techniques.
This document provides information about the virology and parasitology course code 320 at semester 6. It discusses various immunological and molecular techniques used for diagnosis of viruses and parasites including radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), immunoblotting, DNA probes, polymerase chain reaction (PCR) amplification, and real-time PCR.
Endotoxin Testing is performed to ensure that injectable preparations and medical devices are free from pyrogens and safe for human use.
Pyrogens constitute a heterogeneous group of fever causing substances which comprise both microbial and non-microbial substances. The most potent and most widely known are the endotoxins or lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. Gram-positive bacteria are also sources of pyrogens, in particular lipoteichoic acid (LTA), as are particles from yeasts and viruses. Non-microbial pyrogens often emanate from production environments. Small particles of packaging materials are a typical example.
ELISA is an antigen-antibody reaction technique used to detect substances like proteins, peptides, antibodies, and hormones. There are three main types of ELISA - indirect ELISA detects antibodies, sandwich ELISA detects antigens using two antibodies that bind to different sites, and competitive ELISA measures antigen concentration based on competition between sample antigen and antigen coated on the plate for a limited number of antibody binding sites. ELISA involves capturing the antigen or antibody of interest on a plate using its binding partner, then detecting it using an enzyme-linked secondary binding partner and colorimetric substrate.
The document provides information about two medical diagnostic techniques: the quantitative buffy coat (QBC) test and enzyme-linked immunosorbent assay (ELISA). It describes the QBC test procedure which uses a stained capillary tube to detect malaria parasites in centrifuged blood samples based on their fluorescence. It also gives an overview of the principles and types of ELISA, including indirect, sandwich, and competitive ELISA, which are immunochemical techniques used to detect and quantify antigens or antibodies.
- ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay technique used to detect substances like proteins, peptides, antibodies, and hormones. There are four main types of ELISA: direct, indirect, sandwich, and competitive.
- Direct ELISA involves coating an antigen onto a plate and adding an enzyme-labeled primary antibody that binds directly to the antigen. Indirect ELISA uses an unlabeled primary antibody and enzyme-labeled secondary antibody. Sandwich ELISA uses two antibodies that bind to different sites on an antigen. Competitive ELISA measures antigen concentration based on competition between antigen in a sample and antigen coated on a plate for binding to an antibody.
- ELISA has various applications like screening donated blood,
Food borne pathogens causes various diseases. So it is very important to detect them. Rapid methods help to detect pathogens in a very short period of time.
Immunohistochemistry uses antibodies to detect antigens in tissue samples in order to localize proteins and study biological processes. It has various applications including cancer prognosis, tumor identification, and research. The process involves deparaffinizing tissue, antigen retrieval, blocking, primary/secondary antibody incubation, signal development using enzymes, counterstaining, and mounting slides. Different methods like direct, indirect, PAP, ABC, and LSAB are used depending on the desired sensitivity and specificity. Careful antibody selection and protocol optimization are important for obtaining high quality results.
Immunohistochemistry uses antibodies to detect antigens in tissue samples in order to localize proteins and study biological processes. It has various applications including cancer prognosis, tumor identification, and research. The process involves deparaffinizing tissue, antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, signal development using enzymes, counterstaining, and mounting slides. Different methods like direct, indirect, PAP, ABC, and LSAB are used depending on the desired sensitivity and signal amplification needed. Careful antibody selection and protocol optimization are important for obtaining high quality results.
Radioimmunoassay (RIA) is a technique that uses the specificity of antibody-antigen binding and radioactivity to separate and quantify proteins. RIA revolutionized research and clinical practice by allowing the detection of hormone levels in blood. It works by measuring the displacement of labeled antigen from an antibody when unlabeled antigen is added. While useful, RIA requires radioactive materials and specialized equipment. Enzyme-linked immunosorbent assay (ELISA) was developed as a safer alternative, using enzyme-labeled antibodies or antigens. ELISA and other techniques like agglutination reactions, complement fixation tests, immunodiffusion, and precipitation reactions exploit the binding properties of antibodies to detect or quantify antigens.
Immunohistochemistry (IHC) is a technique that combines immunology and histology to detect antigens in tissues using antibodies, with the main steps including preparing tissue sections, using primary and secondary antibodies to target antigens, developing signals using reporters and counterstains, and observing under a microscope. IHC is widely used for disease diagnosis by identifying molecular markers characteristic of cellular events involved in diseases like cancer and neurological disorders. Troubleshooting for IHC focuses on reducing background staining and improving antigen detection through optimizing antibody concentrations and incubation times.
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
Clinic ^%[+27633867063*Abortion Pills For Sale In Tembisa Central19various
Clinic ^%[+27633867063*Abortion Pills For Sale In Tembisa Central Clinic ^%[+27633867063*Abortion Pills For Sale In Tembisa CentralClinic ^%[+27633867063*Abortion Pills For Sale In Tembisa CentralClinic ^%[+27633867063*Abortion Pills For Sale In Tembisa CentralClinic ^%[+27633867063*Abortion Pills For Sale In Tembisa Central
Our backs are like superheroes, holding us up and helping us move around. But sometimes, even superheroes can get hurt. That’s where slip discs come in.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
Adhd Medication Shortage Uk - trinexpharmacy.comreignlana06
The UK is currently facing a Adhd Medication Shortage Uk, which has left many patients and their families grappling with uncertainty and frustration. ADHD, or Attention Deficit Hyperactivity Disorder, is a chronic condition that requires consistent medication to manage effectively. This shortage has highlighted the critical role these medications play in the daily lives of those affected by ADHD. Contact : +1 (747) 209 – 3649 E-mail : sales@trinexpharmacy.com
TEST BANK For Community and Public Health Nursing: Evidence for Practice, 3rd...Donc Test
TEST BANK For Community and Public Health Nursing: Evidence for Practice, 3rd Edition by DeMarco, Walsh, Verified Chapters 1 - 25, Complete Newest Version TEST BANK For Community and Public Health Nursing: Evidence for Practice, 3rd Edition by DeMarco, Walsh, Verified Chapters 1 - 25, Complete Newest Version TEST BANK For Community and Public Health Nursing: Evidence for Practice, 3rd Edition by DeMarco, Walsh, Verified Chapters 1 - 25, Complete Newest Version Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Pdf Chapters Download Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Pdf Download Stuvia Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Study Guide Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Ebook Download Stuvia Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Questions and Answers Quizlet Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Studocu Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Quizlet Test Bank For Community and Public Health Nursing: Evidence for Practice 3rd Edition Stuvia Community and Public Health Nursing: Evidence for Practice 3rd Edition Pdf Chapters Download Community and Public Health Nursing: Evidence for Practice 3rd Edition Pdf Download Course Hero Community and Public Health Nursing: Evidence for Practice 3rd Edition Answers Quizlet Community and Public Health Nursing: Evidence for Practice 3rd Edition Ebook Download Course hero Community and Public Health Nursing: Evidence for Practice 3rd Edition Questions and Answers Community and Public Health Nursing: Evidence for Practice 3rd Edition Studocu Community and Public Health Nursing: Evidence for Practice 3rd Edition Quizlet Community and Public Health Nursing: Evidence for Practice 3rd Edition Stuvia Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Pdf Chapters Download Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Pdf Download Stuvia Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Study Guide Questions and Answers Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Ebook Download Stuvia Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Questions Quizlet Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Studocu Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Quizlet Community and Public Health Nursing: Evidence for Practice 3rd Edition Test Bank Stuvia
2. INTRODUCTION
Definition:
• Immunohistochemistry (IHC) is a technique
for identifying cellular or tissue constituents
(antigens) by means of antigen-antibody
interactions, the site of antibody binding being
identified either by direct labelling of antibody or
by use of a secondary labelling method.
3. Antigens:
• it is a foreign substance (protein, carbohydrate
or lipid molecule) that bears one or more
antibody binding sites.
- these sites are composed of a small number of
amino acids or monosaccharide units & are known as
antigenic determinant groups or epitopes.
4. Antibodies:
• Also known as immunoglobulins
• Five types: (IgA, IgD, IgE, IgG & IgM)
• Formed by plasma cells
• Comprises of two light chains and two heavy chains
linked by disulphide bonds to form a Y shaped
structure.
5.
6.
7. History
• The principle has existed since the 1930s.
• Started in 1941 when Coons identified
pneumococci using a direct fluorescent method.
• Indirect method
• Addition of horseradish peroxidase
• Peroxidase anti-peroxidase technique in 1979
• Use of Avidin & Biotin complex in early 1980’s
9. Fixation
• To ensure the preservation of tissue architecture
and cell morphology
• There is no one universal fixative that is ideal for
the demonstration of all antigens
10. Coagulant fixative
• Organic & nonorganic compound that coagulate
protein – render them insoluble
• Two types –
• Dehydrant (alcohol , acetone)
• Strong acid (picric acid, trichloracetic acid)
• Histomorphology well maintain but loss of
mitochondria and secretary granules
11. Cross-Linking Fixatives
• Intramolecular and intermolecular cross-links
in proteins , formaldehyde (methylol),
• Intermolecular bonds in some lipids
• Aldehydes – Formaldehyde, Gluteraldehyde,
12.
13.
14.
15. Antigen Retrieval
• Fixation modifies the tertiary structure of proteins(Ags) - many
times making them undetected by specific Abs
• Particularly Necessary when tissues are fixed in cross-linking
fixatives.
• ‘opening the curtains’ making the section like ‘fresh tissue’ again
• Approximately 85% of Ags fixed in formalin require some type of AR
to optimize the immunoreaction
• Reverse the effect of formalin fixation and re-establish
immunoreactivity
16. Methods of Antigen Retrieval
Heat Induced Epitope Retrieval (HIER)-
• Microwave Oven, Pressure Cooker, cooplin jar,
water bath, autoclave and Steamer are the most
commonly used heating devices. Other devices
also include the use of autoclave and water bath
• The heating length of 20 minutes appears to be
the most satisfactory and the cooling usually
takes about 20 minutes
17. Microwave antigen retrieval
• demonstrated the Ki67 antigen in formalin
fixation and paraffin processing.
• The method improved the demonstration of well-
established antibodies such as CD45 and CD20
• Uneven heating and the production of hot spots
have been reported
18. Pressure cooker antigen retrieval
• more uniform than other heating methods.
• unmasking certain nuclear tissue antigens
such as bcl-6, p53, p21, estrogen receptor, and
progesterone receptor.
19. Steamer
• Times in excess of 40 minutes are sometimes
required
• advantage : less damaging to tissues than the
other heating methods.
• Commercially available rice steamers are
adequate
20. Water bath
• temperature to 95–98°C,
• advantage of being gentler on the tissue sections
because the temperature is set below boiling
point.
• By using a lower temperature than other heating
methods the antigen retrieval buffer does not
evaporate
• expensive commercial antigen retrieval solutions
can be safely reused.
• disadvantage in that the antigen retrieval times
are increased compared to other methods.
21. Autoclave
• method offers an alternative form of heat
mediated antigen retrieval
• good results for nuclear antigens such as
MIB1, p21, and p53.
22. AR solution
• Citrate buffer of pH6.0 is the most popularly
used retrieval solution
• The TRIS (hydroxymethyl)aminomethane EDTA
of pH 10 and EDTA of pH8.0 are second most
used retrieval solutions
23. Proteolytic Induced Epitope Retrieval (PIER)-
• proteinase k, trypsin, chymotrypsin, pepsin, pronase and
various other proteases
• breaks down formalin cross-linking and the antigenic sites
for a number of antibodies are uncovered
Combination of Heat Mediated and Proteolytic Enzyme
Method-
• Alternative approach to unmask antigens if other methods
did not work
• Especially useful when performing double or triple labeling
of two or more antigens simultaneously
24. Washing
• After almost every step, except serum
blocking, a wash should be performed. This
will remove unwanted proteins or chemicals
• Wash in Tris(Tris, or trisaminomethane )/ PBS
(Phosphate buffered saline)
25. Serum blocking
• Serum blocking is an important step. The
presence of serum proteins keeps the
antibodies from binding non-specifically to the
slide.
26. Hydrophobic and ionic interaction
Collagen, epithelium and adipocytes
give rise to background staining due to hydrophobic interactions
minimised by addition of a blocking protein or detergent or high
salt concentration to buffer
• non-immunologically to highly charged groups
present on connective tissue elements
– innocuous protein solution
– Traditionally, non immune serum
Major causes of background staining
28. Biotin Blocking
• If endogenous biotin is suspected, and
especially if using a biotin detection system,
use the following method:
– Block the slides for 15 minutes in Avidin buffer.
• Avidin Buffer: 0.001% Avidin in PBS, pH 7
Use AFTER Primary Antibody Incubation
29. Peroxidase Blocking
• If endogenous levels of peroxidase are
suspected, and especially if using a peroxidase
detection system:
– After primary antibody incubation, block the
slides for 10 minutes in peroxidase buffer:
• Peroxidase Buffer: 3%H2O2 in PBS
30. Detection system
• Antibody molecules must be flagged by some
method that permits their visualization.
Enzyme labels
Colloidal metal labels
Fluorescent labels
Radiolabels
31. • The Direct Method uses a conjugated primary
antibody, and avoids further secondary
antibody applications.
Direct Method
32. IHC – Direct method
• The primary antibody solution should contain
antibody, at a concentration recommended by the
manufacturer, and diluted in the same buffer as the
antibody is already in
• Advantages
– Rapid and easy to perform
• Disadvantages
– Helpful only with monoclonal antibody
– For each different antigen it is necessary to conjugate each
primary antibody separately
– High concentration of antibody required
– signal strength will always be lower
33. The Indirect Method uses a primary antibody, and then a
secondary antibody which is conjugated and recognizes the
primary antibody. (The signal may be stronger, because multiple
secondaries can recognize one primary.)
34. IHC Indirect Method
• The secondary antibody should be ‘against’ the animal in which primary
antibody was raised in. Should be diluted in the same buffer as primary
was diluted in.
• After secondary antibody incubation, follow with another wash sequence.
• Counter stain, if desired, and wash again.
• Apply your detection system, which will react with the conjugation of
secondary antibody
• Wash again, then dehydrate in 95% EtOH for 1 minute, then 100% EtOH
2X3 minutes.
• Clear in Xylene for 2X5 minutes.
• Visualize slides as appropriate
35. Advantages over direct method
• Increased versatility -Conjugation process
applied only to secondary antibody
• High specificity
• Increased signal strength
36. Unlabelled Ab methods
• Enzyme bridge technique
• Peroxidase-antiperoxidase method
• Biotin-avidin procedure
• Avidin-biotin conjugate procedure
• Biotin streptavidin systems
• Polyvalent systems
• Alkaline phosphatase anti-alkaline phosphatase method
• Protein A methods
• Enzyme labelled ag methods
• Polymeric labelling two step method
• Tyramine signal amplification
37. Enzyme bridge technique
A second Ab is used to link (bridge) the primary Ab to an
antiperoxidase Ab, which in turns binds to free peroxidase
Avoid the problems inherent in chemical conjugation
38. • The PAP (Peroxidase,
anti-Peroxidase)
Method, uses a third
antibody
immunologically bound
to peroxidase, which
recognizes the
unconjugated secondary
antibody, improving
specificity, and signal.
• alkaline phosphatase-anti-
alkaline phosphatase(APAAP)
IHC PAP Method
39. • The ABC (Avidin-Biotin Complex) Method
exploits the high-affinity binding between
avidin and biotin
IHC ABC Method
41. IHC ABC Method
• Different batches of avidin and biotin have
differing affinities to each other – drastically
affects the sensitivity and reproducibility
• Endogenous biotin – nonspecific / false
positive staining
42. Biotin – Streptavidin System
• Substitution of streptavidin in the place of
avidin
• Streptavidin
– Tetrameric 60kD avidin analog
– Isolated from Streptomyces avidinii
– Bind to biotin with a very high affinity
44. Advantages over Ab system
• Streptavidin contains no carbohydrate which
can bind nonspecifically to lectin like substrate
• Isoelectric point is closed to neutrality
• Highly stable reagent
• High sensitivity with modification of the
reagents
45. Enzyme labels – Horse Radish
peroxidase
• Why HRP?
– Small size – does not hinder binding of antibody to
adjacent sites
– Easily obtainable in purified form
– Stable
– Endogenous activity is easily quenched
• Chromogen
– MC – 3,3’-diaminobenzidene tetrahydrochloride –
dark brown reaction product
– Although initially reported to be a potential
carcinogen, the risk is now thought to be low
47. Enzyme Labels – Alkaline phosphatase
• Most widely used alternative to HRP
– APAAP method using alkaline phosphatase (intestinal)
• Endogenous activity?
– Blocked by
• Levamisole: Inhibits certain types of alkaline phosphatase
(not intestinal and placental)
• 20% glacial acetic acid: Blocks all types but acid can damage
antigens
• Chromogen:
– Fast red TR (napthol AS-MX phosphate sodium salt) –
red
48. Enzyme labels - Others
• Glucose oxidase
– Navy blue reaction
– No need for endogenous blocking as animals lack
glucose oxidase enzyme
• Bacterial B-D-galactosidase
– Permanent blue reaction
– No need for endogenous blocking as mammalian
enzyme requires a different optimal pH for
function
49. Polymeric labeling two step method
EnVision Systems-
• Based on dextran polymer technology
• This unique chemistry permits binding of a large
number of enzyme molecules (horseradish
peroxidase or alkaline phosphatase) to a secondary
antibody via the dextran backbone
• The benefits are
– increased sensitivity,
– minimized non-specific background staining
– reduction in the total number of assay steps as compared
to conventional techniques
51. CHROMOGENS
HORSERADISH
PEROXIDASE
COLOR SOLUBILITY IN ALCOHOL
DIAMINOBENZIDINE(DAB) BROWN INSOLUBLE
DAB WITH ENHANCEMENT BLACK INSOLUBLE
3-AMINO-9-ETHYL
CARBAZOLE(AEC)
RED TO GREEN BROWN SOLUBLE
4-CHLORO-1-NAPHTHOL(4-
CN)
BLUE-BLACK SOLUBLE
HANKER YATES REAGENT BLUE INSOLUBLE
ALPHA NAPHTHOL
PYRONIN
RED SOLUBLE
3,3’, 5,5’-
TETRAMETHYLBENZIDINE(T
NBT)
BLUE INSOLUBLE
52. Counter Staining
• Crystal violet (Methyl violet) stains cell walls
purple.
• Coomassie Blue stains all protein blue.
• Ethidium Bromide binds DNA, and fluoresces
orange.
• Haematoxylin stains nuclei dark purple.
– These are just a few of the stains available!
53. ARTIFACTS IN IHC
• Many different types of artifacts can be observed in IHC. Some of the more common ones
• that I see are listed below.
• 1. "Desquamartifact"
• 2. "Bubble" artifacts.
• 3. Drying artifacts.
• 4. Trapping artifacts
• 5. Edge artifacts.
• 6. Artifacts of inappropriate antibody titers.
• 7. Artifacts of poor fixation.
• 8. Bacterial contamination artifacts.
• 9. Graphite pencil artifacts.
• 10. Endogenous biotin artifacts.
• 11. Precipitated DAB artifact.
• 12. Mold artifact
54. Interpretation of Immunohistology
Signal pattern- Subcellular localization
Four broad categories
1. Membranous pattern
2. Nuclear pattern
3. Cytoplasmic pattern
4. Nuclear-cytoplasmic
Hapten
From Wikipedia, the free encyclopedia
Jump to navigationJump to searchHaptens are minute molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself. (In general, only large molecules, infectious agents, or insoluble foreign matter can elicit an immune response in the body.) Once the body has generated antibodiesto a hapten-carrier adduct, the small-molecule hapten may also be able to bind to the antibody, but it will usually not initiate an immune response; usually only the hapten-carrier adduct can do this. Sometimes the small-molecule hapten can even block immune response to the hapten-carrier adduct by preventing the adduct from binding to the antibody, a process called hapten inhibition.
The mechanisms of absence of immune response may vary and involve complex immunological mechanisms, but can include absent or insufficient co-stimulatory signals from antigen-presenting cells.
Haptens have been used to study allergic contact dermatitis (ACD) and the mechanisms of inflammatory bowel disease (IBD) to induce autoimmune-like responses.[1]
The concept of haptens emerged from the work of Karl Landsteiner[2][3] who also pioneered the use of synthetic haptens to study immunochemical phenomena.[4]
Examples of haptens[edit]
The first researched haptens were aniline and its carboxyl derivatives (o-, m-, and p-aminobenzoic acid).[5]
A well-known example of a hapten is urushiol, which is the toxin found in poison ivy. When absorbed through the skin from a poison ivy plant, urushiol undergoes oxidation in the skin cells to generate the actual hapten, a reactive quinone-type molecule, which then reacts with skin proteins to form hapten adducts. Usually, the first exposure causes only sensitization, in which there is a proliferation of effector T-cells. After a subsequent, second exposure, the proliferated T-cells can become activated, generating an immune reaction that produces typical blisters of a poison ivy exposure.
Some haptens can induce autoimmune disease. An example is hydralazine, a blood pressure-lowering drug that occasionally can produce drug-induced lupus erythematosus in certain individuals. This also appears to be the mechanism by which the anaesthetic gas halothane can cause a life-threatening hepatitis, as well as the mechanism by which penicillin-class drugs cause autoimmune hemolytic anemia.
Other haptens that are commonly used in molecular biology applications include fluorescein, biotin, digoxigenin, and dinitrophenol.
Lastly, nickel allergy is caused by nickel metal ions penetrating the skin and binding to skin proteins.
Hapten Conjugation
Epitope
From Wikipedia, the free encyclopedia
Jump to navigationJump to searchAn epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. For example, the epitope is the specific piece of the antigen to which an antibody binds. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes.
The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope.[1] A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. The proportion of epitopes that are conformational is unknown.[citation needed]
By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen.
T cell epitopes[edit]
T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, whereas most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length,[2] and non-classical MHC molecules also present non-peptidic epitopes such as glycolipids.
Cross-activity[edit]
Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies (originally proposed by Nobel laureate Niels Kaj Jerne). If an antibody binds to an antigen's epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.[citation needed]
B cell Epitopes
cross-linking mechanism involving
the amino groups of lysine and other amino acids
through the formation of pyridine intermediaries.
The use of osmium tetroxide fixation to preserve lipids
is fundamental to TEM
volume of buffer between 400 and 600 ml in a suitably sized
microwave-resistant plastic container,
A pressure cooker at 15 psi (10.3 kPa) reaches a temperature of around 120°C at full pressure.
ethylenediaminetetraacetic acid (EDTA)
3,3α-diaminobenzidine tetrahydrochloride
(DAB),
Pyruvate carboxylase, an enzyme in the Krebs cycle, contains four molecules of biotin,
and is localized in mitochondria within the cytoplasm of cells. Considering this fact, it is not
surprising that endogenous biotin artifact is most prominent in metabolically active cells that
contain numerous mitochondria, such as liver and kidney cells. As
alp is sued in hematological cases n skin as melanocyte also contains browm color
fluorescein gree and rhodamine red
strepta vidin ha good affinity for biotin but poory express its all 4 sites
biotin has isoelectric property so that it can bind to negative charge protein like lectin
Pyruvate carboxylase, an enzyme in the Krebs cycle, contains four molecules of biotin,
and is localized in mitochondria within the cytoplasm of cells. Considering this fact, it is not
surprising that endogenous biotin artifact is most prominent in metabolically active cells that
contain numerous mitochondria, such as liver and kidney cells. As
Other chromogens are available, including:
3-amino-9-ethylcarbazole (Graham et al., 1965;
Kaplow, 1975), which gives a red final reaction product;
4-chloro-1-naphthol (Nakane, 1968), a blue final
reaction product; Hanker-Yates reagent (Hanker et al.,
1977), a dark blue product and α-naphthol pyronin
(Taylor & Burns, 1974), a red-purple final reaction
product. Many of these contain hazardous reagents
and have now largely been superseded by commercial
chromogens available in kit form.
Vector Laboratories, for example produce a wide
range of different colored chromogens suitable as
alternatives to DAB which can be used for multilabeling
techniques. These include Vector Red, Vector
Blue, Vector VIP (purple) and BCIP/NBT(blue/
violet).
Other chromogens are available, including:
3-amino-9-ethylcarbazole (Graham et al., 1965;
Kaplow, 1975), which gives a red final reaction product;
4-chloro-1-naphthol (Nakane, 1968), a blue final
reaction product; Hanker-Yates reagent (Hanker et al.,
1977), a dark blue product and α-naphthol pyronin
(Taylor & Burns, 1974), a red-purple final reaction
product. Many of these contain hazardous reagents
and have now largely been superseded by commercial
chromogens available in kit form.
Vector Laboratories, for example produce a wide
range of different colored chromogens suitable as
alternatives to DAB which can be used for multilabeling
techniques. These include Vector Red, Vector
Blue, Vector VIP (purple) and BCIP/NBT(blue/
violet).
soluble in alcohol – requires aqueous mount (eg. neutral phosphate buffered glycerin jelly)