Questions and Answers
What is the primary application of direct ELISA?
qualitative analysis of macromolecules
What is the purpose of blocking the surface of microtiter plate with proteins in direct ELISA?
to avoid non-specific adsorption of other proteins
In competitive ELISA, what is the consequence of an increasing amount of target antigen in the sample?
the signal decreases
What is the key event of competitive ELISA?
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Who developed the competitive ELISA in 1973?
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What is the main advantage of using enzyme-labeled secondary antibodies in indirect methods like indirect ELISA and icELISA?
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In sandwich ELISA, what is the role of the capture antibody?
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Why is icELISA suitable for measuring both macromolecules and hapten?
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What is the consequence of an increasing amount of antigen in sandwich ELISA?
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What makes sandwich ELISA a highly specific assay?
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What is the limitation of direct ELISA when detecting low molecular weight compounds?
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What is the key step in indirect ELISA systems?
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What is the advantage of indirect ELISA over direct ELISA?
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What is the difference between competitive ELISA and indirect competitive ELISA?
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What is the common requirement for all ELISA methods?
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What is the typical method for preparing hapten-carrier protein conjugates for glycosides?
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What reaction is used to design hapten-carrier protein conjugates to obtain highly specific antibodies to daidzin?
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How is the number of hapten molecules bound to carrier proteins typically evaluated?
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What is the classification of ginsenosides based on their structure?
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What is the purpose of utilizing antibodies with broad cross-reactivity in icELISA?
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What is the main disadvantage of preparing two antibodies in the sandwich ELISA system?
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In the open sandwich ELISA (OS-ELISA), what is the role of streptavidin-coated microtiter plates?
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What is the advantage of using monoclonal antibodies (MAb) over polyclonal antibodies (PAb) in ELISA?
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Why is competitive ELISA (icELISA) used for the analysis of plant secondary metabolites?
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What is the significance of cross-reactivity (CR) in evaluating the specificity of antibodies against haptens?
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What is the difference between the detection of macromolecules and hapten in competitive ELISA?
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How does the signal change in indirect ELISA as the amount of target antigen increases?
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What is the main advantage of indirect ELISA over direct ELISA?
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What is the limitation of direct ELISA when detecting low molecular weight compounds?
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What is the common requirement for all ELISA methods?
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What is the main difference between direct ELISA and competitive ELISA in terms of signal intensity?
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What is the purpose of using an enzyme-labeled antibody or antigen in direct ELISA?
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In what type of analysis is direct ELISA suitable?
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What is the role of the immobilized antibody in competitive ELISA?
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Why is competitive ELISA limited to measuring macromolecules?
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In indirect competitive ELISA (icELISA), what is the purpose of allowing free target antigen and antibody to incubate?
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What is the advantage of using polyclonal antibody as an enzyme-labeled secondary antibody in indirect methods?
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In sandwich ELISA, what is the purpose of using two antibodies that recognize different epitopes?
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What is the significance of the molecular weight of the target antigen in sandwich ELISA?
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What is the advantage of using a universal secondary antibody in indirect methods?
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What is the advantage of using recombinant antibodies in ELISA?
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What is the role of the VL region in open sandwich ELISA (OS-ELISA)?
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What is the main difference between monoclonal antibodies (MAb) and polyclonal antibodies (PAb) in terms of specificity?
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What is the significance of the sodium periodate (NaIO4) oxidation method in preparing hapten-carrier protein conjugates?
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What is the advantage of using open sandwich ELISA (OS-ELISA) over traditional sandwich ELISA?
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What is the advantage of using antibodies with broad cross-reactivity in icELISA, especially in recognizing bioactive skeletons or groups of bioactive compounds?
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How do the number of hapten molecules bound to carrier proteins affect the specificity of antibodies?
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What is the significance of the Mannich reaction in preparing hapten-carrier protein conjugates for obtaining specific antibodies to haptens?
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What is the classification of ginsenosides based on their structure?
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What is the advantage of using monoclonal antibodies (MAb) over polyclonal antibodies (PAb) in ELISA?
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What is the primary event that occurs in direct ELISA?
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In competitive ELISA, what is the role of the enzyme-labeled target?
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What is the difference between direct ELISA and competitive ELISA in terms of signal intensity?
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What is the purpose of using an enzyme-labeled antibody or antigen in direct ELISA?
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Why is direct ELISA suitable for qualitative analysis of macromolecules?
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What is the limitation of competitive ELISA?
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What is the key feature of direct ELISA that makes it different from other types of ELISA?
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In what type of analysis is competitive ELISA commonly used?
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What is the problem that arises when using low molecular weight compounds as antigens in direct ELISA?
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How does competitive ELISA detect the presence of antibodies in a sample?
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What is the main advantage of indirect ELISA over direct ELISA?
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What is the role of the primary antibody in indirect ELISA?
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What is the purpose of using enzyme-labeled secondary antibodies in indirect ELISA?
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What is the difference between indirect ELISA and indirect competitive ELISA?
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What is the limitation of indirect ELISA when detecting hapten?
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How does the signal change in competitive ELISA as the amount of target antigen increases?
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What is the main advantage of competitive ELISA over direct ELISA?
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What is the role of the enzyme-labeled antibody in competitive ELISA?
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In indirect competitive ELISA (icELISA), what is the role of the enzyme-labeled secondary antibody?
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What is the main advantage of using indirect ELISA methods over direct ELISA methods?
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What is the purpose of the 'capture antibody' in sandwich ELISA?
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What is the consequence of an increasing amount of free antigen in icELISA?
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In sandwich ELISA, what is the purpose of using two antibodies that recognize different epitopes?
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What is the significance of using polyclonal antibodies as enzyme-labeled secondary antibodies in indirect methods?
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What is the main difference between icELISA and sandwich ELISA?
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What is the advantage of using a universal secondary antibody in indirect methods?
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What is the limitation of using indirect ELISA methods?
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What is the significance of using two antibodies that recognize different epitopes in sandwich ELISA?
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What is the major drawback of preparing two antibodies in the sandwich ELISA system?
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What is the purpose of the VH region in open sandwich ELISA (OS-ELISA)?
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How do advances in DNA technology impact the development of immunoassays?
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What is the advantage of using monoclonal antibodies (MAb) over polyclonal antibodies (PAb) in terms of specificity?
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What is the role of streptavidin in open sandwich ELISA (OS-ELISA)?
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Why is competitive ELISA (icELISA) suitable for measuring plant secondary metabolites?
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What is the significance of cross-reactivity (CR) in evaluating the specificity of antibodies against haptens?
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How do the design of hapten-carrier protein conjugates affect the specificity of the resultant antibody?
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What is the advantage of using recombinant antibodies in ELISA?
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What is the role of the phage-displayed VH region in open sandwich ELISA (OS-ELISA)?
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What is the significance of the Mannich reaction in preparing hapten-carrier protein conjugates for obtaining specific antibodies to haptens?
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How do the number of hapten molecules bound to carrier proteins affect the specificity of antibodies?
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What is the advantage of using antibodies with broad cross-reactivity in icELISA?
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What is the significance of cross-reactivity (CR) in evaluating the specificity of antibodies against haptens?
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What is the classification of ginsenosides based on their structure?
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What is the advantage of using MAbs against specific ginsenosides in icELISA?
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What is the significance of the NaIO4 oxidation method in preparing hapten-carrier protein conjugates?
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What is the advantage of using antibodies in icELISA for the analysis of plant secondary metabolites?
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What is the significance of the hapten-carrier protein conjugates in antibody production?
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What is the advantage of using specific antibodies in icELISA for the analysis of plant secondary metabolites?
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Study Notes
ELISA (Enzyme-Linked Immunosorbent Assay)
- Developed in 1971 by Engvall and Perlmann, and Van Weemen and Schuurs
- A type of immunological assay that measures the concentration of a target substance (antigen or antibody) in a sample
Direct ELISA
- The target antigen or antibody is immobilized on a microtiter plate
- An enzyme-labeled antibody or antigen is allowed to react with the immobilized target, followed by color development with appropriate substrates
- Suitable for qualitative analysis of macromolecules
- Signal increases with increasing amount of target antigen
Competitive ELISA
- Involves a competitive reaction between the target antigen or antibody in the sample and an enzyme-labeled antigen or antibody
- The target antigen or antibody is immobilized on a microtiter plate
- Signal decreases with increasing amount of target antigen
- Suitable for measuring macromolecules and hapten (low molecular weight compounds)
Indirect ELISA
- The target antigen is immobilized on a microtiter plate
- The primary antibody (in antisera) binds to the immobilized antigen, followed by reaction with an enzyme-labeled secondary antibody
- Signal increases with increasing amount of target antigen
- Suitable for measuring macromolecules and diagnosing endocrine diseases
Indirect Competitive ELISA (icELISA)
- Combines indirect ELISA and competitive ELISA
- The target antigen is immobilized on a microtiter plate
- Free target antigen and antibody are allowed to incubate, followed by reaction with an enzyme-labeled secondary antibody
- Signal decreases with increasing amount of free antigen
- Suitable for measuring both macromolecules and hapten
Sandwich ELISA
- The target antigen is detected via anchoring between two antibodies that recognize different epitopes
- The capture antibody is immobilized on a microtiter plate, followed by reaction with the target antigen and an enzyme-labeled antibody
- Signal increases with increasing amount of target antigen
- Suitable for measuring macromolecules, but can be labor-intensive and expensive
Open Sandwich ELISA (OS-ELISA)
- A modified version of sandwich ELISA that uses a unique immunoassay based on the interaction of variable regions of heavy and light chains
- Suitable for measuring both macromolecules and hapten
Types of Antibodies
- Polyclonal antibody (PAb): recognizes multiple epitopes of the target antigen
- Monoclonal antibody (MAb): recognizes a single epitope of the target antigen
- Recombinant antibodies: produced through DNA technology, including single-chain variable fragment (scFv), bispecific Bis-scFv, fragment antigen-binding (Fab), and multibody (diabody, triabody, and tetrabody)
ELISA for Plant Secondary Metabolites
- Competitive ELISA or icELISA is used for measuring hapten (low molecular weight compounds)
- MAb tends to exhibit higher specificity than PAb against hapten
- The design of hapten-carrier protein conjugates affects the specificity of the resultant antibody
- The Mannich reaction is an important method for obtaining specific anti-hapten antibodies
ELISA Types
-
Direct ELISA: Developed by Engvall and Perlmann, and Van Weemen and Schuurs in 1971
- Involves immobilizing an antigen or antibody on a microtiter plate surface
- Corresponding enzyme-labeled antibody or antigen is added to react with the immobilized target
- Signal increases with increasing amount of target
- Suitable for qualitative analysis of macromolecules
-
Competitive ELISA: Developed by Belanger in 1973
- Involves competitive reaction between targets in the sample and enzyme-labeled targets
- Signal decreases with increasing amount of target antigen
- Suitable for measuring macromolecules and hapten
-
Indirect ELISA: Developed based on direct ELISA
- Involves two-binding process of primary antibody and enzyme-labeled secondary antibody
- Signal increases with increasing amount of target antigen
- Suitable for measuring macromolecules
- Used to diagnose endocrine diseases
-
Indirect Competitive ELISA: Combination of indirect ELISA and competitive ELISA
- Signal decreases with increasing amount of free antigen
- Suitable for measuring both macromolecules and hapten
-
Sandwich ELISA: Involves anchoring between two antibodies that recognize different epitopes
- Signal increases with increasing amount of target antigen
- Suitable for measuring macromolecules
- Can be modified to indirect system by using primary and enzyme-labeled secondary antibodies
Antibodies
-
Polyclonal antibody (PAb): Recognizes different epitopes of the primary antibody
- Exhibits higher sensitivity and versatility compared to direct methods
- Can be commercially available
-
Monoclonal antibody (MAb): Recognizes only one epitope
- Exhibits higher specificity compared to PAb
- Can be screened for desirable characteristics
-
Recombinant antibodies: Include single-chain variable fragment (scFv), bispecific Bis-scFv, fragment antigen-binding (Fab), bispecific Fab2, trispecific Fab3, bivalent minibody, and multibody (diabody, triabody, and tetrabody)
- Can be applied to ELISA
ELISA Applications
-
Plant secondary metabolites: Competitive ELISA or icELISA is used to analyze low molecular weight compounds (hapten)
- Specificity of antibody against hapten is critical
- MAb tends to exhibit higher specificity than PAb
- Design of hapten-carrier protein conjugates affects the specificity of the resultant antibody
-
ELISA for measuring macromolecules and hapten: Depends on the type of antibody and design of hapten-carrier protein conjugates
- Antibodies exhibiting broad cross-reactivity can be used to recognize a bioactive skeleton or a group of bioactive compounds
- Utilization of antibody in icELISA depends on specificity
Other ELISA Systems
-
Open Sandwich ELISA (OS-ELISA): Developed based on the interaction of variable regions of heavy and light chains
- Involves coating of a solid-phase microtiter plate with streptavidin
- Signal increases with increasing amount of antigen
- Modified to be more easy and effective
Types of ELISA
- Direct ELISA: Developed in 1971 by Engvall and Perlmann, and Van Weemen and Schuurs; involves immobilizing an antigen or antibody on a microtiter plate, blocking with proteins, and then reacting with an enzyme-labeled antibody or antigen; signal increases with increasing amount of target; suitable for qualitative analysis of macromolecules.
- Competitive ELISA: Developed in 1973 by Belanger; involves competition between targets in the sample and enzyme-labeled targets against immobilized antibody or antigen; signal decreases with increasing amount of target; suitable for measuring macromolecules and hapten.
- Indirect ELISA: Developed on the basis of direct ELISA; involves indirect detection of antigen using a secondary antibody; signal increases with increasing amount of target; suitable for measuring macromolecules.
- Indirect Competitive ELISA: Involves combination of indirect ELISA and competitive ELISA; signal decreases with increasing amount of target; suitable for measuring both macromolecules and hapten.
- Sandwich ELISA: Involves detecting antigen via anchoring between two antibodies recognizing different epitopes; signal increases with increasing amount of target; suitable for measuring macromolecules; highly specific but expensive and labor-intensive.
Characteristics of ELISA Methods
- Advantages and Disadvantages: Each type of ELISA has its own advantages and disadvantages, such as simplicity, sensitivity, versatility, and potential for cross-reactivity.
- Labeling and Inactivation: Direct and competitive ELISA require labeling of antibodies, which may lead to inactivation.
- Target and Signal: Different ELISA methods have different targets and signal responses, such as macromolecules, hapten, and signal increase or decrease.
Antibodies in ELISA
- Types of Antibodies: Polyclonal antibodies (PAb), monoclonal antibodies (MAb), and recombinant antibodies (e.g., scFv, Fab, bispecific antibodies) can be used in ELISA.
- Specificity: MAb tends to exhibit higher specificity than PAb due to recognition of only one epitope.
- Design of Hapten-Carrier Proteins: The design of hapten-carrier proteins affects the specificity of the resultant antibody, with the Mannich reaction being a more effective method for obtaining specific anti-hapten antibodies.
Applications of ELISA
- Plant Secondary Metabolites: ELISA is used for the analysis of plant secondary metabolites, such as hapten, with high specificity and sensitivity.
- Ginsenosides: ELISA is used for the determination of ginsenosides, which are classified into two groups according to their structure, and are focused as targets for quantitative/qualitative analysis in ELISA.
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Description
Learn about the enzyme-linked immunosorbent assay (ELISA) and its direct variant, a type of immunological assay that measures the concentration of a target substance in a sample.