ELISA (Enzyme-Linked Immunosorbent Assay) Methodology
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Questions and Answers

What are the two regions of an antibody fragment resulting from papain cleavage?

Fab region and Fc region

What is the key feature of antibodies that allows them to precisely recognize toxins and pathogens?

Specificity

What process during B-cell maturation generates an enormous repertoire of antigen-binding sites?

Gene rearrangement

What feature of the immune system protects the body from repeated infection by pathogens?

<p>Immunological memory</p> Signup and view all the answers

What mechanism prevents B cells from attacking self-tissues?

<p>Immune tolerance</p> Signup and view all the answers

What is the main purpose of ELISA?

<p>To capture and quantify target antigen or antibody in samples using specific antibodies and enzyme reactions.</p> Signup and view all the answers

What is the function of the variable region (V region) of an antibody?

<p>It provides high degree of three-dimensional structural diversity, allowing antibodies to bind to a wide range of antigens.</p> Signup and view all the answers

What is the term for an antibody's ability to recognize and bind to a specific antigen?

<p>Antibody specificity</p> Signup and view all the answers

What is the Y-shaped part of an antibody molecule that binds to an antigen?

<p>The antigen-binding site at the tip of the 'Y'</p> Signup and view all the answers

What is the name of the protein molecule produced and secreted by B cells that binds to foreign substances?

<p>Immunoglobulin (Ig), also referred to as an antibody</p> Signup and view all the answers

What is the primary function of IgG antibodies in the human body?

<p>IgG detoxifies harmful substances and is important in the recognition of antigen-antibody complexes by leukocytes and macrophages. It is also transferred to the fetus through the placenta and protects the infant until its own immune system is functional.</p> Signup and view all the answers

What is the unique feature of IgM antibodies that gives them higher avidity for antigens?

<p>IgM has a pentameric structure in which five basic Y-shaped molecules are linked together.</p> Signup and view all the answers

What is the main function of IgA antibodies in the human body?

<p>IgA protects the gastrointestinal tract of neonates from pathogens, and is also found in serum, nasal mucus, saliva, and intestinal fluid.</p> Signup and view all the answers

What is the process by which B cells switch from producing IgM and IgD to producing other immunoglobulin isotypes?

<p>Immunoglobulin class switching, which is directed by factors in the B cell environment, such as cytokines secreted by T cells.</p> Signup and view all the answers

What is the term for antibodies generated through a process of immunizing animals with an antigen, and then collecting and purifying the resulting antibodies from their blood?

<p>Polyclonal antibodies.</p> Signup and view all the answers

What is the main difference between IgM and IgG in terms of their affinity for the antigen after repeated immunization?

<p>The affinity of IgM for the antigen does not increase after repeated immunization, whereas the affinity of IgG increases with repeated immunization.</p> Signup and view all the answers

What is the purpose of artificially fusing antibody-producing B cells with immortalized cancer cells (myeloma) to generate hybridomas?

<p>To produce monoclonal antibodies that can be stably produced indefinitely.</p> Signup and view all the answers

How do monoclonal antibodies produced by different clones differ?

<p>They react to different epitopes on the same antigen and have different suitable applications.</p> Signup and view all the answers

What is the main advantage of using phage display method for producing antibodies?

<p>It allows for the selection of antibodies against a specific target molecule from a large mixed population of antibody phages.</p> Signup and view all the answers

What is the main difference between polyclonal and monoclonal antibodies in terms of their specificity?

<p>Polyclonal antibodies have lower specificity due to the presence of multiple types of antibodies, whereas monoclonal antibodies have high specificity if good quality antibodies are selected.</p> Signup and view all the answers

What is the advantage of using polyclonal antibodies as a secondary antibody in detection?

<p>Higher sensitivity of detection (amplification of the signal) due to multiple antibodies binding to a primary antibody molecule.</p> Signup and view all the answers

Why are polyclonal antibodies more amenable to modification compared to monoclonal antibodies?

<p>Because multiple different antibody molecules are present, making them less sensitive to protease digestion and less likely to lose antigen-binding ability.</p> Signup and view all the answers

What is the purpose of the three steps involved in purifying antibodies?

<p>To remove contaminants, isolate antibodies, and further purify and exchange the buffer.</p> Signup and view all the answers

What is the specific property of Protein A that allows it to capture IgG?

<p>Its ability to specifically bind to the Fc region of mammalian IgG.</p> Signup and view all the answers

What is the advantage of antigen-affinity purification compared to Protein A/G affinity chromatography?

<p>It provides a higher yield of antigen-specific antibodies.</p> Signup and view all the answers

Study Notes

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.

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Description

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.

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