ELISA and Direct ELISA Technique

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.

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.