This document provides an overview of Enzyme Linked Immunosorbent Assay (ELISA). ELISA is a biochemical technique used to detect the presence of antibodies or antigens in a liquid sample. It relies on the principle of using an enzyme to detect the binding of an antigen-antibody complex. The document discusses the basic components and steps of ELISA including antigen, antibody, equipment, reagents, and different types like direct, indirect, sandwich and competitive ELISA. It also covers troubleshooting, advantages, and applications of this important laboratory technique.
The document provides guidelines for performing ELISA (Enzyme-Linked Immunosorbent Assay), including:
1. ELISA is a popular immunological technique that uses antibodies and enzymes to detect antigens or antibodies. It is very sensitive and can be used both qualitatively and quantitatively.
2. The basic steps of ELISA involve coating a plate with an antigen, adding a primary antibody, adding a secondary antibody linked to an enzyme, adding a substrate that reacts with the enzyme to produce a colored product.
3. Factors that can cause troubleshooting issues include contamination, improper washing, expired reagents, incorrect procedures, temperature issues, poor pipetting technique, and more. Proper controls and validation
Become an ELISA (enzyme-linked immunosorbent assay) expert! This guide includes critical review of principles, from sample preparation to data analysis, step-by-step protocols, troubleshooting tips, and more. Learn how to generate reproducible, high quality data in your ELISA tests. Slide contents include:
1. ELISA principles review
2. History of ELISA
3. General ELISA Procedure
4. Explanation of ELISA Types:
A. Direct ELISA
B. Indirect ELISA
C. Sandwich ELISA
D. Competitive ELISA
5. ELISA Data Interpretation
6. Sample Preparation for:
A. Cell Culture Supernatants
B. Cell Extracts
C. Conditioned Media
D. Tissue Extract
7. Recommended Protocols for:
A. Reagent Preparation:
1. Standard Solutions
2. Biotinylated Antibody
3. Avidin-Biotin-Peroxidase (ABC)
B. Sandwich ELISA:
1. Capture Antibody Coating
2. Blocking
3. Reagent Preparation
4. Sample (Antigen) Incubation
5. Biotinylated Antibody Incubation
6. ABC Incubation
7. Substrate Preparation
8. Signal Detection
9. Data Analysis
C. Indirect ELISA:
1. Antigen Coating
2. Blocking
3. Reagent Preparation
4. Primary Antibody Incubation
5. Secondary Antibody Incubation
6. Substrate Preparation
7. Signal Detection
8. Data Analysis
D. Direct ELISA:
1. Antigen Coating
2. Blocking
3. Reagent Preparation
4. Primary Antibody Incubation
5. Substrate Preparation
6. Signal Detection
7. Data Analysis
E. Competitive ELISA:
1. Antigen Coating
2. Blocking
3. Reagent Preparation
4. Sample (Antigen) Incubation
5. Primary Antibody Incubation
6. Secondary Antibody Incubation
7. Substrate Preparation
8. Signal Detection
9. Data Analysis
8. High Sensitivity Boster ELISA Kits
9. Cytokine Related ELISA Kits
10. Customer Testimonials
11. Additional Technical Resources
Feel free to contact support@bosterbio.com with any questions. Get better results with Boster!
This document provides an overview of ELISA (enzyme-linked immunosorbent assay) technology and procedures. It describes the basic ELISA formats including indirect, blocking, and antigen-capture. It explains the steps involved in blocking ELISA and lists common components of ELISA kits. The document serves as a technical guide for laboratories to help maintain proficiency in ELISA techniques.
Blog praxilabs com_2021_09_20_elisa_principleAyaFarid2
The Enzyme Linked Immunosorbent Assay (ELISA) is one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng. It is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones.
The document describes the ELISA (enzyme-linked immunosorbent assay) technique for detecting and quantifying substances. ELISA uses an antigen immobilized on a plate that complexes with an antibody linked to an enzyme. Detection involves measuring the activity of the conjugated enzyme after incubation with a substrate. There are four main types of ELISA - direct, indirect, sandwich, and competitive - which differ in their antibody and antigen binding steps. ELISA has advantages like high sensitivity, specificity, and throughput, making it widely used in research and clinical applications.
This document provides an overview of Enzyme Linked Immunosorbent Assay (ELISA). ELISA is a biochemical technique used to detect the presence of antibodies or antigens in a liquid sample using enzymes to produce a detectable color change. The basic principle involves an antigen or antibody being immobilized on a plate and detected by an antibody or antigen linked to an enzyme. The document describes the basic components and steps of ELISA including antigen, antibody, equipment, reagents, and different types like direct, indirect, sandwich and competitive ELISA. Advantages include high specificity and sensitivity, while limitations include potential cross-reactivity.
Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.
This document provides an overview of Enzyme Linked Immunosorbent Assay (ELISA). ELISA is a biochemical technique used to detect the presence of antibodies or antigens in a liquid sample. It relies on the principle of using an enzyme to detect the binding of an antigen-antibody complex. The document discusses the basic components and steps of ELISA including antigen, antibody, equipment, reagents, and different types like direct, indirect, sandwich and competitive ELISA. It also covers troubleshooting, advantages, and applications of this important laboratory technique.
The document provides guidelines for performing ELISA (Enzyme-Linked Immunosorbent Assay), including:
1. ELISA is a popular immunological technique that uses antibodies and enzymes to detect antigens or antibodies. It is very sensitive and can be used both qualitatively and quantitatively.
2. The basic steps of ELISA involve coating a plate with an antigen, adding a primary antibody, adding a secondary antibody linked to an enzyme, adding a substrate that reacts with the enzyme to produce a colored product.
3. Factors that can cause troubleshooting issues include contamination, improper washing, expired reagents, incorrect procedures, temperature issues, poor pipetting technique, and more. Proper controls and validation
Become an ELISA (enzyme-linked immunosorbent assay) expert! This guide includes critical review of principles, from sample preparation to data analysis, step-by-step protocols, troubleshooting tips, and more. Learn how to generate reproducible, high quality data in your ELISA tests. Slide contents include:
1. ELISA principles review
2. History of ELISA
3. General ELISA Procedure
4. Explanation of ELISA Types:
A. Direct ELISA
B. Indirect ELISA
C. Sandwich ELISA
D. Competitive ELISA
5. ELISA Data Interpretation
6. Sample Preparation for:
A. Cell Culture Supernatants
B. Cell Extracts
C. Conditioned Media
D. Tissue Extract
7. Recommended Protocols for:
A. Reagent Preparation:
1. Standard Solutions
2. Biotinylated Antibody
3. Avidin-Biotin-Peroxidase (ABC)
B. Sandwich ELISA:
1. Capture Antibody Coating
2. Blocking
3. Reagent Preparation
4. Sample (Antigen) Incubation
5. Biotinylated Antibody Incubation
6. ABC Incubation
7. Substrate Preparation
8. Signal Detection
9. Data Analysis
C. Indirect ELISA:
1. Antigen Coating
2. Blocking
3. Reagent Preparation
4. Primary Antibody Incubation
5. Secondary Antibody Incubation
6. Substrate Preparation
7. Signal Detection
8. Data Analysis
D. Direct ELISA:
1. Antigen Coating
2. Blocking
3. Reagent Preparation
4. Primary Antibody Incubation
5. Substrate Preparation
6. Signal Detection
7. Data Analysis
E. Competitive ELISA:
1. Antigen Coating
2. Blocking
3. Reagent Preparation
4. Sample (Antigen) Incubation
5. Primary Antibody Incubation
6. Secondary Antibody Incubation
7. Substrate Preparation
8. Signal Detection
9. Data Analysis
8. High Sensitivity Boster ELISA Kits
9. Cytokine Related ELISA Kits
10. Customer Testimonials
11. Additional Technical Resources
Feel free to contact support@bosterbio.com with any questions. Get better results with Boster!
This document provides an overview of ELISA (enzyme-linked immunosorbent assay) technology and procedures. It describes the basic ELISA formats including indirect, blocking, and antigen-capture. It explains the steps involved in blocking ELISA and lists common components of ELISA kits. The document serves as a technical guide for laboratories to help maintain proficiency in ELISA techniques.
Blog praxilabs com_2021_09_20_elisa_principleAyaFarid2
The Enzyme Linked Immunosorbent Assay (ELISA) is one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng. It is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones.
The document describes the ELISA (enzyme-linked immunosorbent assay) technique for detecting and quantifying substances. ELISA uses an antigen immobilized on a plate that complexes with an antibody linked to an enzyme. Detection involves measuring the activity of the conjugated enzyme after incubation with a substrate. There are four main types of ELISA - direct, indirect, sandwich, and competitive - which differ in their antibody and antigen binding steps. ELISA has advantages like high sensitivity, specificity, and throughput, making it widely used in research and clinical applications.
This document provides an overview of Enzyme Linked Immunosorbent Assay (ELISA). ELISA is a biochemical technique used to detect the presence of antibodies or antigens in a liquid sample using enzymes to produce a detectable color change. The basic principle involves an antigen or antibody being immobilized on a plate and detected by an antibody or antigen linked to an enzyme. The document describes the basic components and steps of ELISA including antigen, antibody, equipment, reagents, and different types like direct, indirect, sandwich and competitive ELISA. Advantages include high specificity and sensitivity, while limitations include potential cross-reactivity.
Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.
ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique used to detect the presence of antibodies or antigens in a biological sample. It involves an enzymatic reaction that produces a detectable color change if the target antibody or antigen is present. There are several types of ELISA including direct, indirect, sandwich, and competitive ELISA. ELISA tests are commonly used to screen blood donations, detect infections, measure hormone and toxin levels, and detect allergens and drugs.
This document provides guidelines for performing enzyme-linked immunosorbent assays (ELISAs). It discusses ELISA components, equipment, techniques, timing, washing, reading plates, troubleshooting, and quality control. Regular maintenance and calibration of equipment is emphasized to obtain accurate and reproducible results. Following the procedures outlined in test package inserts and this technical guide helps ensure proper ELISA technique.
Want to learn about immunofluoresence? This presentation will go over some basic and popular immunofluoresence concepts in a concise fashion. Featuring:
Introduction
History
Similarities & Difference between IF and IHC
Types of Immunofluorescence
Popular Terms
Commonly used Fluorophores
Disease Diagnosed by Immunofluorescence
Antibodies, Proteins and Genes associated with Immunofluorescence
A path breaking technology which has made it possible for us to detect HIV. ELISA is an immunological assay nowadays even used to detect food proteins & is the science behind pregnancy color test. This presentation unlocks the working of this assay.
ELISA and its application in clinical researchesKcmscicmt
The document describes the enzyme-linked immunosorbent assay (ELISA), a commonly used laboratory technique for detecting and quantifying antigens and antibodies. It involves immobilizing an antigen or antibody to a plate and detecting it using an antibody or antigen linked to an enzyme. There are four basic ELISA formats - direct, indirect, sandwich, and competitive - that allow flexibility based on the antibodies and results required. The general ELISA procedure involves coating a plate with a capture antibody or antigen, adding the sample, washing unbound material, adding a detection antibody linked to an enzyme, washing again, and detecting the enzyme's activity using a substrate to produce a measurable signal correlated to analyte concentration.
The document describes the ELISA (enzyme-linked immunosorbent assay) technique for detecting and quantifying proteins, antibodies, and hormones. In an ELISA, an antigen is immobilized to a plate and detected using an antibody linked to an enzyme. Detection involves incubating the plate with a substrate to produce a measurable product, allowing quantification of the antigen. There are different types of ELISA including direct, indirect, sandwich, and competitive formats that vary the immobilization and detection steps. Proper sample preparation and reagent dilution are important for accurate ELISA results.
ELISA is a biochemical technique used in immunology to detect the presence of an antibody or antigen in a sample. It involves coating microtiter plate wells with an antigen or antibody and using conjugated enzymes and substrates to produce a colored product to indicate a positive result. There are different types of ELISA including direct, indirect, sandwich, and competitive ELISA which are used to test for various antigens or antibodies. ELISA has many applications such as measuring serum antibody concentrations, detecting food allergens or diseases, and identifying past exposure to diseases.
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
Content of this presentation is only for education purpose.
No conflict of interest.
Thanks to google.com it helps me for pictures & searching references.
The document provides an overview of enzyme-linked immunosorbent assay (ELISA) and how it is used as a diagnostic tool. It describes the basic immune response process, including how antigens are presented and recognized by B and T cells, leading to antibody production. It then explains the principles of ELISA, noting it detects antibodies or antigens based on antibody-antigen binding. The main types of ELISA - indirect, direct, sandwich and competitive - are defined. Applications like detecting disease infections and allergens are highlighted.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to detect the presence of a substance like an antigen. There are four main types of ELISA - direct, indirect, sandwich, and competitive. The sandwich ELISA involves capturing the antigen between two antibodies, one coated on a surface and one linked to an enzyme. This amplified signal allows the ELISA to be a sensitive diagnostic tool for detecting substances related to diseases, food allergens, and other analytes.
The document discusses the topic of ELISA (Enzyme-Linked Immunosorbent Assay), including its principle, types, applications, advantages, and disadvantages. ELISA involves binding antibodies or antigens to a polystyrene plate and detecting antigen-antibody complexes using conjugated secondary antibodies and chromogenic substrates, which produce a colored reaction. There are four main types of ELISA: direct, indirect, sandwich, and competitive. ELISA has various applications in medicine, food testing, and more. It is a sensitive and specific technique with advantages like low cost and long shelf life, but also has some disadvantages.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to identify a substance. It involves using an enzyme to detect antigen-antibody binding, which converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA can provide quantitative or qualitative results and has applications like screening donated blood and measuring hormone levels.
ELISA (Enzyme-Linked Immunosorbent Assay) is a sensitive technique used to detect small quantities of antigens, antibodies, or other proteins in biological fluids like blood or urine. There are several types of ELISA including direct, indirect, sandwich, and competitive ELISA. ELISA works by using an enzyme-linked antibody or antigen to detect the presence of a target protein. This allows very small amounts of the target to be detected through the enzyme's catalytic activity.
Enzyme Linked Immunosorbent Assay ELISA and its types with emphasis on Compet...sakeena gilani
The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA allows for qualitative and quantitative analysis of antigens and antibodies. It is highly specific and sensitive. There are four main types of ELISA - direct, indirect, sandwich, and competitive. ELISA involves an antigen or antibody being immobilized on a plate and subsequent washes and incubations with conjugated enzymes and substrates to produce a detectable color change. ELISA has various applications in fields like quality control, disease diagnosis, and more.
The ELISA (enzyme-linked immunosorbent assay) is a popular biochemistry assay that uses antibodies and color change to identify a substance. It involves using an enzyme-linked antibody to detect antigen-antibody binding, where the enzyme converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA has various applications such as screening blood donations, measuring hormone levels, and detecting infections and allergens.
This document provides an introduction to enzyme-linked immunosorbent assay (ELISA), covering its basic principles, procedures, advantages, and applications. ELISA is an antibody-antigen based assay developed in 1971 to detect the presence and quantify concentrations of antigens, antibodies, and other proteins. It works by utilizing antibodies and enzyme-linked substrates to produce a detectable color change in direct proportion to the presence of an antigen or antibody in a sample. Key advantages of ELISA include its speed, sensitivity, specificity, ability to process many samples at once using small sample sizes, and production of easy-to-read colorimetric results. ELISA kits now exist for detecting a wide range of targets like viruses, hormones, toxins, allerg
ELISA, principle and method by kk sahuKAUSHAL SAHU
What is ELISA.
Principle.
History.
Types of ELISA method.
1.Direct ELISA.
2.Indirect ELISA.
3.Sandwhich ELISA.
Conclusion.
References.
Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
The document describes the enzyme-linked immunosorbent assay (ELISA), including:
- ELISA is an immunological assay used to measure antibodies, antigens, proteins and glycoproteins in biological samples. It is commonly used for diagnosis of diseases like HIV and measurement of cytokines.
- ELISA assays are generally carried out in 96 well plates, allowing multiple samples to be measured at once. Each ELISA measures a specific antigen using capture and detection antibodies.
- There are different types of ELISA including sandwich ELISA, which is described as using two antibodies to detect antigens like cytokines between them in a multi-step process involving antibody coating, sample addition, detection antibody addition, and substrate conversion to a detectable signal. Determination of antigen
ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies or antigens in a sample. It involves affixing an unknown antigen to a surface, applying a specific antibody linked to an enzyme, and adding a substrate that the enzyme converts to a detectable signal, most commonly a color change. ELISA has been used as a diagnostic tool in medicine, plant pathology, and the food industry for quality control.
ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique used to detect the presence of antibodies or antigens in a biological sample. It involves an enzymatic reaction that produces a detectable color change if the target antibody or antigen is present. There are several types of ELISA including direct, indirect, sandwich, and competitive ELISA. ELISA tests are commonly used to screen blood donations, detect infections, measure hormone and toxin levels, and detect allergens and drugs.
This document provides guidelines for performing enzyme-linked immunosorbent assays (ELISAs). It discusses ELISA components, equipment, techniques, timing, washing, reading plates, troubleshooting, and quality control. Regular maintenance and calibration of equipment is emphasized to obtain accurate and reproducible results. Following the procedures outlined in test package inserts and this technical guide helps ensure proper ELISA technique.
Want to learn about immunofluoresence? This presentation will go over some basic and popular immunofluoresence concepts in a concise fashion. Featuring:
Introduction
History
Similarities & Difference between IF and IHC
Types of Immunofluorescence
Popular Terms
Commonly used Fluorophores
Disease Diagnosed by Immunofluorescence
Antibodies, Proteins and Genes associated with Immunofluorescence
A path breaking technology which has made it possible for us to detect HIV. ELISA is an immunological assay nowadays even used to detect food proteins & is the science behind pregnancy color test. This presentation unlocks the working of this assay.
ELISA and its application in clinical researchesKcmscicmt
The document describes the enzyme-linked immunosorbent assay (ELISA), a commonly used laboratory technique for detecting and quantifying antigens and antibodies. It involves immobilizing an antigen or antibody to a plate and detecting it using an antibody or antigen linked to an enzyme. There are four basic ELISA formats - direct, indirect, sandwich, and competitive - that allow flexibility based on the antibodies and results required. The general ELISA procedure involves coating a plate with a capture antibody or antigen, adding the sample, washing unbound material, adding a detection antibody linked to an enzyme, washing again, and detecting the enzyme's activity using a substrate to produce a measurable signal correlated to analyte concentration.
The document describes the ELISA (enzyme-linked immunosorbent assay) technique for detecting and quantifying proteins, antibodies, and hormones. In an ELISA, an antigen is immobilized to a plate and detected using an antibody linked to an enzyme. Detection involves incubating the plate with a substrate to produce a measurable product, allowing quantification of the antigen. There are different types of ELISA including direct, indirect, sandwich, and competitive formats that vary the immobilization and detection steps. Proper sample preparation and reagent dilution are important for accurate ELISA results.
ELISA is a biochemical technique used in immunology to detect the presence of an antibody or antigen in a sample. It involves coating microtiter plate wells with an antigen or antibody and using conjugated enzymes and substrates to produce a colored product to indicate a positive result. There are different types of ELISA including direct, indirect, sandwich, and competitive ELISA which are used to test for various antigens or antibodies. ELISA has many applications such as measuring serum antibody concentrations, detecting food allergens or diseases, and identifying past exposure to diseases.
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
Content of this presentation is only for education purpose.
No conflict of interest.
Thanks to google.com it helps me for pictures & searching references.
The document provides an overview of enzyme-linked immunosorbent assay (ELISA) and how it is used as a diagnostic tool. It describes the basic immune response process, including how antigens are presented and recognized by B and T cells, leading to antibody production. It then explains the principles of ELISA, noting it detects antibodies or antigens based on antibody-antigen binding. The main types of ELISA - indirect, direct, sandwich and competitive - are defined. Applications like detecting disease infections and allergens are highlighted.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to detect the presence of a substance like an antigen. There are four main types of ELISA - direct, indirect, sandwich, and competitive. The sandwich ELISA involves capturing the antigen between two antibodies, one coated on a surface and one linked to an enzyme. This amplified signal allows the ELISA to be a sensitive diagnostic tool for detecting substances related to diseases, food allergens, and other analytes.
The document discusses the topic of ELISA (Enzyme-Linked Immunosorbent Assay), including its principle, types, applications, advantages, and disadvantages. ELISA involves binding antibodies or antigens to a polystyrene plate and detecting antigen-antibody complexes using conjugated secondary antibodies and chromogenic substrates, which produce a colored reaction. There are four main types of ELISA: direct, indirect, sandwich, and competitive. ELISA has various applications in medicine, food testing, and more. It is a sensitive and specific technique with advantages like low cost and long shelf life, but also has some disadvantages.
The ELISA (enzyme-linked immunosorbent assay) is a test that uses antibodies and color change to identify a substance. It involves using an enzyme to detect antigen-antibody binding, which converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA can provide quantitative or qualitative results and has applications like screening donated blood and measuring hormone levels.
ELISA (Enzyme-Linked Immunosorbent Assay) is a sensitive technique used to detect small quantities of antigens, antibodies, or other proteins in biological fluids like blood or urine. There are several types of ELISA including direct, indirect, sandwich, and competitive ELISA. ELISA works by using an enzyme-linked antibody or antigen to detect the presence of a target protein. This allows very small amounts of the target to be detected through the enzyme's catalytic activity.
Enzyme Linked Immunosorbent Assay ELISA and its types with emphasis on Compet...sakeena gilani
The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA allows for qualitative and quantitative analysis of antigens and antibodies. It is highly specific and sensitive. There are four main types of ELISA - direct, indirect, sandwich, and competitive. ELISA involves an antigen or antibody being immobilized on a plate and subsequent washes and incubations with conjugated enzymes and substrates to produce a detectable color change. ELISA has various applications in fields like quality control, disease diagnosis, and more.
The ELISA (enzyme-linked immunosorbent assay) is a popular biochemistry assay that uses antibodies and color change to identify a substance. It involves using an enzyme-linked antibody to detect antigen-antibody binding, where the enzyme converts a colorless substrate into a colored product. There are several types of ELISA including indirect, direct, sandwich, and competitive ELISA. ELISA has various applications such as screening blood donations, measuring hormone levels, and detecting infections and allergens.
This document provides an introduction to enzyme-linked immunosorbent assay (ELISA), covering its basic principles, procedures, advantages, and applications. ELISA is an antibody-antigen based assay developed in 1971 to detect the presence and quantify concentrations of antigens, antibodies, and other proteins. It works by utilizing antibodies and enzyme-linked substrates to produce a detectable color change in direct proportion to the presence of an antigen or antibody in a sample. Key advantages of ELISA include its speed, sensitivity, specificity, ability to process many samples at once using small sample sizes, and production of easy-to-read colorimetric results. ELISA kits now exist for detecting a wide range of targets like viruses, hormones, toxins, allerg
ELISA, principle and method by kk sahuKAUSHAL SAHU
What is ELISA.
Principle.
History.
Types of ELISA method.
1.Direct ELISA.
2.Indirect ELISA.
3.Sandwhich ELISA.
Conclusion.
References.
Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
The document describes the enzyme-linked immunosorbent assay (ELISA), including:
- ELISA is an immunological assay used to measure antibodies, antigens, proteins and glycoproteins in biological samples. It is commonly used for diagnosis of diseases like HIV and measurement of cytokines.
- ELISA assays are generally carried out in 96 well plates, allowing multiple samples to be measured at once. Each ELISA measures a specific antigen using capture and detection antibodies.
- There are different types of ELISA including sandwich ELISA, which is described as using two antibodies to detect antigens like cytokines between them in a multi-step process involving antibody coating, sample addition, detection antibody addition, and substrate conversion to a detectable signal. Determination of antigen
ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies or antigens in a sample. It involves affixing an unknown antigen to a surface, applying a specific antibody linked to an enzyme, and adding a substrate that the enzyme converts to a detectable signal, most commonly a color change. ELISA has been used as a diagnostic tool in medicine, plant pathology, and the food industry for quality control.
ELISA (enzyme-linked immunosorbent assay) is a popular immunological assay technique used to detect the presence of antibodies, antigens, or other substances in a liquid sample. It relies on antibodies and antigen-enzyme conjugates to detect the substance of interest. The basic steps involve immobilizing an antigen onto a plate, adding a sample and antibodies, washing away unbound material, and detecting the bound antigen using an enzyme reaction. There are several types of ELISA including direct, indirect, sandwich, and competitive assays that are used for various applications such as disease diagnosis, food testing, and drug development.
The document provides a history of ELISA (Enzyme-Linked ImmunoSorbent Assay) and describes its components, procedures, types, applications and advantages. It discusses key events in immunology research from the 18th century leading to the development of ELISA in the 1970s. ELISA allows detection and quantification of antigens or antibodies in a simple, sensitive and cost-effective manner using enzyme-labeled antibodies or antigens.
ELISA, Principles of ELISA, Types of ELISA- Direct ELISA
Indirect ELISA, Sandwich ELISA, Competitive ELISA, and other Types i.e. ELISPOT (enzyme-linked immunospot assay) and In-cell ELISA, Advantages and disadvantages of ELISA detection methods, Different types of microplates for ELISA, Detection strategies for ELISA
An ELISA uses antibodies to detect the presence of antigens in biological samples immobilized on a microplate. There are four main types of ELISA - direct, indirect, sandwich, and competitive - which differ in how the antigen is immobilized and detected. ELISA relies on the specific binding of antibodies to antigens, and can detect the target analyte using enzymatic or fluorescent tags to provide a quantifiable signal. It is a versatile technique that allows multiple samples to be tested simultaneously.
ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies and antigens in a liquid sample. It relies on an enzyme-linked antibody or antigen to detect the target protein. There are different types of ELISA including direct, indirect, sandwich, and competitive. The ELISA process involves coating a plate with an antigen or antibody, adding a sample and enzyme-linked antibody, washing unbound material, and detecting the enzyme's product to quantify the target. ELISAs are widely used in medical testing, food safety, and disease detection.
TTI screening testing methods have evolved through four generations. Serological tests include rapid tests and ELISA. There are four main types of ELISA - direct, indirect, sandwich, and competitive/inhibition. Sandwich ELISA has the highest sensitivity but requires optimization of antibody pairs. Competitive ELISA can measure antigen or antibody concentration using crude samples. Chemiluminescence immunoassay (CLIA) is similar to ELISA but uses a chemiluminescent substrate, making it ultrasensitive. CLIA requires a luminometer for detection while ELISA uses color change.
The document discusses ELISA (enzyme-linked immunosorbent assay), a commonly used analytical technique for detecting antigens, antibodies, and proteins. It describes the basic principles of ELISA, including immobilizing an antigen and detecting it using an enzyme-linked antibody. There are four main types of ELISA discussed: direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA. Sandwich ELISA is most common and involves capturing the antigen between two antibodies, followed by detection with enzyme-linked secondary antibodies.
The document describes the ELISA (enzyme-linked immunosorbent assay) technique. ELISA uses antibodies and enzymes to detect the presence of antigens or antibodies in a liquid sample. It involves attaching an antigen or antibody to a solid surface and using a labeled antibody or antigen to detect binding. The enzyme label converts a colorless substrate to a colored product, indicating the presence of the antigen or antibody being tested for. There are several types of ELISA including direct, indirect, sandwich, and competitive ELISA. ELISA has various applications including screening blood donations, measuring hormone levels, and detecting infections.
The document describes the ELISA (enzyme-linked immunosorbent assay) technique. ELISA uses antibodies and enzymes to detect the presence of antigens or antibodies in a liquid sample. It involves coating a plate with capture antibodies, adding the sample and detection antibodies linked to an enzyme, and measuring the enzymatic reaction with a substrate. There are several types of ELISA including direct, indirect, sandwich, and competitive formats. ELISA is a sensitive and widely used technique with applications in diagnosing infections, measuring hormone levels, and screening blood donations.
The document describes the ELISA (enzyme-linked immunosorbent assay) technique, which is used to detect antibodies, antigens, proteins, and glycoproteins. ELISA works by detecting and amplifying antigen-antibody reactions using enzyme-antibody conjugates. There are four main types of ELISA: direct, indirect, sandwich, and competitive. The document explains the basic procedures for each type. ELISA has various applications including detecting serum antibody concentrations, potential food allergens, disease outbreaks, antigens, and past exposure to diseases.
The document provides an overview of ELISA (Enzyme-Linked Immunosorbent Assay), describing it as an antibody-based method used to quantitatively or qualitatively detect specific antigens or antibodies. It discusses different types of ELISA including indirect, sandwich, and ELISPOT. The basic principles of two-step and sandwich ELISA are described, involving binding of primary and secondary antibodies and use of an enzyme to produce a color change indicating the presence of an analyte. Considerations for whether ELISA is suitable for a given experiment are also outlined.
This document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA is used to detect antibodies or antigens in samples. It works by affixing an unknown amount of antigen to a surface, applying a specific antibody linked to an enzyme, and detecting the enzyme's activity through a color change. There are three main types of ELISA - direct, indirect, and sandwich - which differ in their use of primary and secondary antibodies. ELISA has advantages over other techniques like radioimmunoassay in not requiring radioactive materials and being highly sensitive and specific. It has a variety of applications like diagnosing infections and measuring protein levels.
The document discusses Enzyme-Linked Immunosorbent Assay (ELISA), a common immunoassay technique used to detect antigens in biological samples. It describes the basic ELISA principles including immobilizing the antigen and using a detection antibody conjugated to an enzyme. The document outlines the advantages of ELISA including high sensitivity and specificity, high throughput, and ability to test various sample types. It also discusses the different types of ELISA - direct, indirect, sandwich, and competitive/inhibition - and compares their features in a table. The key information provided is an overview of the ELISA technique and a comparison of the different types of ELISA assays.
This document provides information about ELISA (Enzyme-Linked Immunosorbent Assay) techniques. It discusses how ELISAs are typically performed in multi-well plates that passively bind antibodies and proteins. Detection enzymes or tags can then be used to detect bound analytes. The document compares direct ELISA detection using a single labeled antibody to indirect detection using unlabeled primary and labeled secondary antibodies. It notes advantages of indirect detection include increased sensitivity through signal amplification. Finally, it provides step-by-step instructions for performing a sandwich ELISA to detect antigens using capture and detection antibodies between wells.
This document provides information about ELISA (Enzyme-Linked Immunosorbent Assay) techniques. It discusses how ELISAs are typically performed in multi-well plates that passively bind antibodies and proteins. Detection enzymes or tags can then be linked to primary or secondary antibodies. The document compares direct ELISA detection, which uses a single labeled primary antibody, to indirect detection which uses an unlabeled primary antibody detected by a labeled secondary antibody, providing advantages and disadvantages of each. It also describes sandwich assay formats and requirements for performing a sandwich ELISA test.
Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
Immunoassays are biochemical methods that use the specificity of antigen-antibody reactions to detect and quantify target molecules in biological samples. The document defines immunoassay and provides details on various types including enzyme immunoassays (ELISA, EMIT), radioimmunoassay (RIA), counting immunoassay (CIA), fluoroimmunoassay (FIA), and chemiluminescence immunoassay (CLIA). It describes the principles, components, procedures, and applications of each type of immunoassay for detecting molecules like antibodies, hormones, and proteins.
ELISA (Enzyme-Linked Immunosorbent Assay) is an immunological technique used to detect the presence of antigens or antibodies in samples. It involves an antigen-antibody reaction, an enzymatic reaction catalyzed by an enzyme linked to an antibody or antigen, and detection of a colored product to quantify the target protein. There are several types of ELISA including direct, indirect, sandwich, and competitive ELISA which differ in their antigen or antibody detection methods. ELISA is a sensitive and specific technique widely used for medical diagnostics, food quality control, and other applications.
Conjugation is the method of adding an antigen to a larger molecule that ensures that the antigen stimulates the immune response that generates antibodies.
This guide is key to successful IHC experiments. Since no universal tissue preparation method will be ideal for all sample and tissue types, all protocols given here are intended as a starting point from which the experimenter must optimize as needed.
Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.
Fa cs sample preparation & protocolJames Waita
Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different subpopulations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell.
Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different sub- populations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell.
Western blotting protocol & troubleshootingJames Waita
This document provides a recommended protocol for performing a Western Blot, including procedures for protein extraction from cells and tissues, protein quantification, gel electrophoresis, protein transfer to a membrane, membrane blocking, and antibody incubation. The key steps are extracting proteins from samples using lysis buffers, quantifying protein concentration using a BCA assay, separating proteins by SDS-PAGE gel electrophoresis, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, and detecting target proteins by incubating the membrane with primary and secondary antibodies. Troubleshooting tips are also provided throughout the protocol.
Western blotting WB (immunoblotting) is a widely practiced analytical technique to detect target proteins within samples using antigen-specific antibodies.
ELISA troubleshooting guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the ELISA assays.
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
The binding of cosmological structures by massless topological defectsSérgio Sacani
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
2. Introduction
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting
and quantifying peptides, proteins, antibodies and hormones. In an ELISA, an antigen must be
immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.
Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate
to produce a measurable product. The most crucial element of the detection strategy is a highly
specific antibody-antigen interaction.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind
antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to
design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it
easy to separate bound from non-bound material during the assay. This ability to wash away
nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes
within a crude preparation
3. General ELISA Procedure
Unless you are using a kit with a plate that is pre-coated with antibody, an ELISA begins with a coating
step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well
polystyrene plate. This is followed by a blocking step in which all unbound sites are coated with a
blocking agent. Following a series of washes, the plate is incubated with enzyme-conjugated antibody.
Another series of washes removes all unbound antibody. A substrate is then added, producing a
calorimetric signal. Finally, the plate is read.
Because the assay uses surface binding for separation, several washes are repeated in each ELISA step to
remove unbound material. During this process, it is essential that excess liquid is removed in order to
prevent the dilution of the solutions added in the next assay step. To ensure uniformity, specialized plate
washers are often used.
ELISAs can be quite complex and include multiple intervening steps, especially when measuring protein
concentration in heterogeneous samples such as blood. The most complex and varying step in the overall
process is detection, where multiple layers of antibodies can be used to amplify signal.
4.
5. Types of ELISA
ELISAs can be performed with a number of modifications to the basic procedure: direct, indirect, sandwich
or competitive. The key step, immobilization of the antigen of interest, can be accomplished by direct
adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The
antigen is then detected either directly (enzyme-labeled primary antibody) or indirectly (enzyme-labeled
secondary antibody). The detection antibodies are usually labeled with alkaline phosphatase (AP) or
horseradish peroxidase (HRP). A large selection of substrates is available for performing the ELISA with an
HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the
instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).
Among the standard assay formats discussed and illustrated below, where differences in both capture and
detection were the concern, it is important to differentiate between the particular strategies that exist
specifically for the detection step. However an antigen is captured to the plate (by direct adsorption to the
surface or through a pre-coated "capture" antibody, as in a sandwich ELISA), it is the detection step (as
either direct or indirect detection) that largely determines the sensitivity of an ELISA
6.
7. Direct ELISA
For direct detection, an antigen coated to a multi-well plate is detected by an antibody that has been
directly conjugated to an enzyme. This detection method is a good option if there is no commercially
available ELISA kits for your target protein.
Advantages
1. Quick because only one antibody and fewer steps are used.
2. Cross-reactivity of secondary antibody is eliminated.
Disadvantages
1. Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or
tags.
2. Labeling primary antibodies for each specific ELISA system is time-consuming and expensive.
3. No flexibility in choice of primary antibody label from one experiment to another.
4. Minimal signal amplification.
8. Indirect Elisa
For indirect detection, the antigen coated to a multi-well plate is detected in two stages or layers.
First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled
secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species
antibody and is often polyclonal. The indirect assay, the most popular format for ELISA, has the
advantages and disadvantages:
Advantages
1. A wide variety of labeled secondary antibodies are available commercially.
2. Versatile because many primary antibodies can be made in one species and the same labeled
secondary antibody can be used for detection.
3. Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
4.Sensitivity is increased because each primary antibody contains several epitopes that can be bound by
the labeled secondary antibody, allowing for signal amplification.
Disadvantages
1. Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
2. An extra incubation step is required in the procedure.
9. Sandwich ELISA
Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for
a different, non-overlapping part (epitope) of the antigen molecule. A first antibody (known as capture
antibody) is coated to the wells. The sample solution is then added to the well. A second antibody (known
as detection antibody) follows this step in order to measure the concentration of the sample. The
diagram above shows the schematics for Boster’s ELISA assay which is based on the sandwich format.
This type of ELISA has the following advantages:
1. High specificity: the antigen/analyte is specifically captured and detected.
2. Suitable for complex (or crude/impure) samples: the antigen does not require purification prior to
measurement.
3. Flexibility and sensitivity: both direct or indirect detection methods can be used.
10.
11. Competitive ELISA
The key event of competitive ELISA (also known as inhibition ELISA) is the process of competitive
reaction between the sample antigen and antigen bound to the wells of a microtiter plate with the
primary antibody. First, the primary antibody is incubated with the sample antigen and the resulting
antibody–antigen complexes are added to wells that have been coated with the same antigen. After an
incubation period, any unbound antibody is washed off. The more antigen in the sample, the more
primary antibody will be bound to the sample antigen. Therefore, there will be a smaller amount of
primary antibody available to bind to the antigen coated on the well, resulting in a signal reduction. The
main advantage of this type of ELISA arises from its high sensitivity to compositional differences in
complex antigen mixtures, even when the specific detecting antibody is present in relatively small
amounts.
12.
13.
14. ELISA Data Interpretation
The ELISA assay yields three different types of data output:
1. Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a
known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.
2. Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular
antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control
antigen.
3. Semi-Quantitative: ELISAs can be used to compare the relative levels of antigen in assay samples,
since the intensity of signal will vary directly with antigen concentration.
15. Assay
ELISA data is typically graphed with optical
density vs log concentration to produce a
sigmoidal curve as shown below. Known
concentrations of antigen are used to produce a
standard curve and then this data is used to
measure the concentration of unknown samples
by comparison to the linear portion of the
standard curve. In fact, it is the relatively long
linear region of the curve that makes the ELISA
results accurate and reproducible. The unknown
concentration can be determined directly on the
graph or with curve fitting software which is
typically found on ELISA plate readers.
16. Sample Preparation
The procedure below provides a general guidance for the preparation of commonly tested samples for
use in ELISA assays. At Boster, we are working on our detailed sample preparation protocols that cover
more than 20 sample types and expecting to update this presentation in the near future. Please check
with the literature for experiments similar to yours for your new assay development. Generally:
● Protein extract concentration is at least 1-2 mg/mL.
● Cell and tissue extracts are diluted by 50% with binding buffer.
● Samples are centrifuged at 10,000 rpm for 5 min at 4°C to remove any precipitate before use.
17. Sample Preparations
1. Cell Culture Supernatants
Centrifuge cell culture media at 1,500 rpm for 10 min at 4°C. Assay immediately. Aliquot supernatant
immediately and hold at -80°C, avoiding freeze/thaw cycles.
2. Cell Extracts
Place tissue culture plates on ice. Remove the media and gently wash cells once with ice-cold PBS.
Remove the PBS and add 0.5 ml extraction buffer per 100 mm plate. Tilt the plate and scrape the cells
into a pre- chilled tube. Vortex briefly and incubate on ice for 15-30 min. Centrifuge at 13,000 rpm for 10
min at 4°C (this creates a pellet from the insoluble content). Aliquot the supernatant into clean, chilled
tubes (on ice) and store samples at -80°C, avoiding freeze/thaw cycles.
3. Conditioned Media
Plate the cells in complete growth media (with serum) until the desired level of confluence is achieved.
Remove the growth media and gently wash cells using 2- 3 mL of warm PBS. Repeat the wash step.
Remove the PBS and gently add serum-free growth media. Incubate for 1-2 days. Remove the media into
a centrifuge tube. Centrifuge at 1,500 rpm for 10 min at 4°C. Aliquot the supernatant and keep samples
at -80°C, avoiding freeze/thaw cycles.
18. Sample Preparation
4. Conditioned Media
Plate the cells in complete growth media (with serum) until the desired level of confluence is achieved.
Remove the growth media and gently wash cells using 2- 3 mL of warm PBS. Repeat the wash step. Remove
the PBS and gently add serum-free growth media. Incubate for 1-2 days. Remove the media into a centrifuge
tube. Centrifuge at 1,500 rpm for 10 min at 4°C. Aliquot the supernatant and keep samples at -80°C, avoiding
freeze/thaw cycles.
For every 5 mg of tissue, add 300 μL of extraction buffer to the tube and homogenize:
● 100 mM Tris, pH 7.4
● 150 mM NaCl
● 1 mM EGTA
● 1 mM EDTA
● 1% Triton X-100 0.5%
● 0.5% sodium deoxycholate
19. Sample Preparation
(This portion of the buffer can be prepared ahead of time and stored at 4°C. Immediately before use, the
buffer must be supplemented with phosphatase inhibitor cocktail [as directed by manufacturer],
protease inhibitor cocktail [as directed by manufacturer] and PMSF to 1 mM to generate a complete
extraction buffer solution.)
Rinse the blade of the homogenizer twice with 300 μL extraction buffer. Place the sample on a shaker at
4°C for 2 hours.
Centrifuge the sample for 20 min at 13,000 rpm at 4°C. Aliquot the supernatant into pre-chilled tubes
sitting in ice. Keep the samples at -80°C, avoiding freeze/thaw cycles.
Note: Lysis buffer volume must be determined according to the amount of tissue present. Typical
concentration of final protein extract is at least 1 mg/mL.
20.
21. Reagent Preparation
1. Standard Solutions (Best used within 2 hours)
10,000 pg/mL: Add 1 mL of sample diluent buffer into one tube of standard (10 ng per tube) and mix
thoroughly. Note: Store this solution at 4°C for up to 12 hours (or -20°C for 48 hours) and avoid freeze-
thaw cycles.
5,000 pg/mL: Mix 0.3 mL of 10,000 pg/mL with 0.3 mL of sample diluent buffer and mix thoroughly.
2,500 pg/mL: Mix 0.3 mL of 5,000 pg/mL with 0.3 mL of sample diluent buffer and mix thoroughly.
Perform similar dilutions until the standard solutions with these concentrations (pg/mL) are made: 1,250,
625, 312, 156 and 78.
Add 100 μL of each of the diluted standard solutions to the appropriate empty wells. Repeat in duplicate
or triplicate for accuracy.
22. Reagent Preparation
2. Biotinylated Antibody
Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells
required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors.
Generate the required volume of diluted antibody by performing a 1:100 dilution (For each 1 μL
concentrated antibody, add 99 μL antibody dilution buffer) and mixing thoroughly.
3. Avidin-Biotin-Peroxidase (ABC) [Diluted ABC solution shouldn’t be prepared > 1 hr before
experiment]
Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells
required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors.
Generate the required volume of diluted ABC solution by performing a 1:100 dilution (For each 1 μL
concentrated ABC solution, add 99 μL ABC dilution buffer) and mixing thoroughly.