ELISA (Enzyme-Linked Immunosorbent Assay) Methodology

Enzyme-Linked Immunosorbent Assay (ELISA)

• ELISA is a method of target antigen or antibody capture in samples using a specific antibody or antigen and detection/quantitation using an enzyme reaction with its substrate.
• Various antigen-antibody combinations are used, including an enzyme-labeled antigen or antibody.
• Enzyme activity is measured colorimetrically using a substrate that changes color when modified by the enzyme.

Antibodies

• Antibodies are proteins produced and secreted by B cells that bind to foreign substances, such as pathogens.
• The term "antibody" refers to its function of binding to an antigen.
• Another name for this protein molecule is immunoglobulin (abbreviated Ig).
• Antibodies are Y-shaped molecules consisting of two heavy chains (H chains) and two light chains (L chains).
• Each antibody recognizes a specific antigen, known as "antibody specificity."

Variable and Constant Regions

• The N-terminal domains of the H and L chains are called the variable regions (V regions).
• The rest of the molecule is called the constant region (C region).
• The amino acid sequence of the V region varies from antibody to antibody, accounting for the high degree of three-dimensional structural diversity of immunoglobulin chains.

Fab and Fc Regions

• The protease papain cleaves antibodies above the disulfide bonds that connect the two H chains, generating three fragments.
• The two N-terminal fragments are called the Fab region, and the C-terminal fragment is called the Fc region.
• The Fab region includes the antigen-binding site.

Antibody Diversity and Specificity

• Antibodies against a variety of antigens preexist in the body, known as "antibody diversity."
• Each B cell produces one kind of antibody, but tens to hundreds of millions of different B cells are circulating in the body.
• Antibodies precisely recognize toxins and pathogens, known as "antibody specificity."

Gene Rearrangement

• Antibody-producing B cells are produced in the bone marrow and mature in the periphery.
• During B-cell maturation, the antibody genes (immunoglobulin genes) undergo recombination, generating an enormous repertoire of antigen-binding sites (the variable region).

Immunoglobulin Class Switching

• B cells expressing plasma membrane-bound IgM and IgD (mature B cells) are activated upon encounter with a specific antigen and begin to proliferate and produce secretory IgM and IgD.
• With further activation, these mature B cells differentiate into cells that produce increasing amounts of secreted immunoglobulins and start to produce immunoglobulin isotypes other than IgM and IgD.

Polyclonal and Monoclonal Antibodies

• Polyclonal antibodies are generated by injecting an antigen into an animal, which induces the production of multiple antibodies that react to the antigen.
• Monoclonal antibodies are produced by artificially fusing a single B cell producing an antibody with immortalized cancer cells.
• Monoclonal antibodies react to a single epitope on an antigen, while polyclonal antibodies react to multiple epitopes.

Antibody Purification

• Antibodies are usually purified by centrifugation or filtration, followed by affinity chromatography (purification with Protein A/G or antigen-affinity purification).
• Protein A is a cell wall protein of Staphylococcus aureus that specifically binds to the Fc region of mammalian IgG.
• Antigen-affinity purification involves using a column packed with immobilized antigen to isolate antibodies that bind to the antigen.

Learn about the ELISA method, a laboratory technique used to detect and quantify specific antigens or antibodies in samples. It involves an enzyme-catalyzed colorimetric reaction to measure the binding of antibodies to antigens.