Enzyme-Linked Immunosorbent Assay (ELISA)
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Questions and Answers

What are antibodies also known as?

Immunoglobulins, Ig

What is the function of antibodies?

To identify and neutralize foreign objects such as bacteria and viruses.

What is an antigen?

A substance that stimulates the production of an antibody when introduced into the body.

What is an immunoassay?

<p>A technique that uses the binding between an antigen and its homologous antibody to identify and quantify the specific antigen or antibody in a sample.</p> Signup and view all the answers

Specificity is the most important property of an antibody-antigen interaction.

<p>True</p> Signup and view all the answers

How are specific antibodies produced?

<p>By injecting an antigen into a mammal, such as a mouse, rat, or rabbit.</p> Signup and view all the answers

What type of antibodies are produced in the blood of animals after immunization?

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

What are monoclonal antibodies?

<p>Antibodies that are produced by a single clone of B cells and recognize only one specific epitope on an antigen.</p> Signup and view all the answers

Monoclonal antibodies are produced by fusing an antibody-secreting lymphocyte with a cancer cell.

<p>True</p> Signup and view all the answers

What is ELISA?

<p>A biochemical technique used in immunology to detect the presence of an antibody or antigen in a sample.</p> Signup and view all the answers

Which of the following is NOT a type of ELISA technique?

<p>Direct ELISA</p> Signup and view all the answers

What is a competitive ELISA used for?

<p>Detection of &quot;small molecules&quot;</p> Signup and view all the answers

What is a Sandwich ELISA used for?

<p>Detection of &quot;large molecules&quot;</p> Signup and view all the answers

What is an Indirect ELISA used for?

<p>Detecting antibodies in a sample</p> Signup and view all the answers

In a Sandwich ELISA, how is the antigen detected?

<p>By an enzyme-linked secondary antibody that binds specifically to the antigen.</p> Signup and view all the answers

In an Indirect ELISA, a primary antibody is added to the well, followed by a secondary antibody.

<p>True</p> Signup and view all the answers

What is a standard curve used for in an ELISA experiment?

<p>To determine the unknown concentration of each sample by fitting the concentration to the absorbance.</p> Signup and view all the answers

What is a Colloidal gold-based immunoassay test strip?

<p>A type of lateral flow assay that uses colloidal gold nanoparticles conjugated to antibodies or antigens to detect a specific analyte in a sample.</p> Signup and view all the answers

Colloidal gold-based immunoassay test strips can be used in both competitive and sandwich formats.

<p>True</p> Signup and view all the answers

Study Notes

Enzyme-Linked Immunosorbent Assay (ELISA)

  • ELISA is a biochemical technique used in immunology to detect the presence of an antibody or antigen in a sample.
  • The technique has three formats: competitive, sandwich (direct), and indirect.

Definitions

  • Antibodies (immunoglobulins, Ig): Globulin proteins in blood, used by the immune system to identify and neutralize foreign substances (bacteria, viruses).
  • Antigens: Substances that stimulate the production of antibodies when introduced into the body.
  • Immunoassay: A technique using antigen-antibody binding to identify and quantify specific antigens or antibodies in a sample.
  • Analyte: The substance being analyzed in immunoassays; either antibody or antigen.

Antibody Production (Polyclonal Antibodies)

  • Polyclonal antibodies are produced by injecting an antigen into a mammal (e.g., mouse, rabbit, goat, sheep, horse) to elicit an immune response.
  • The resulting blood contains multiple antibodies that bind to the same antigen.
  • Animal selection depends on the desired antibody quantity and size.

Antibody Production (Monoclonal Antibodies)

  • Monoclonal antibodies are specific for a single antigen.
  • Antibody-secreting lymphocytes are isolated from the animal and fused with cancer cells (hybridomas).
  • Hybridomas continuously grow and secrete the same antibody.
  • These antibodies are called monoclonal antibodies.

ELISA Techniques

Competitive ELISA

  • Labeled antigen competes with sample antigen for antibody binding sites.
  • More sample antigen leads to a weaker signal.
  • Primarily used to detect small molecules.
  • Signal intensity correlates with the concentration of the analyte.

Sandwich ELISA (Direct ELISA)

  • ELISA plate is coated with antibody specific to the analyte (target protein).
  • Antigen in the sample is captured and binds to the antibody.
  • Enzyme-linked secondary antibody is added, also specifically binding to the analyte.
  • Substrate is then used, causing a colored change.
  • Intensity of color change reflects analyte concentration, measuring the amount of a particular target protein or other antigen captured by the plate.
  • Primarily used for the detection of larger molecules.

Indirect ELISA

  • Antigen is added to each well, adhering due to charge interactions.
  • A non-reacting protein is used to block any remaining uncoated surfaces.
  • Serum containing antibodies is added, some of which may bind specifically to the coated antigen in the well.
  • A labeled secondary antibody is added, binding to the previously bounded antibodies.
  • A substrate then generates a colored product whose intensity is proportional to the concentration of the primary antibody.
  • This indirectly measures the analyte (the substance of interest in the serum.)

Example: An ELISA Experiment

  • The 96-well plate is appropriately labeled, and the initial wells are used to create a standard curve.
  • Sample is added in duplicates or triplicates for calculations of mean results.
  • Quality control samples, provided with the kit, are treated like the test samples.
  • Standard concentrations are marked on the x-axis (horizontal axis); the readings for each standard are placed on the y-axis (vertical axis) of a graph to create a standard curve.
  • The standard curve is used to find the unknown concentrations of the samples by finding the concentration on the x-axis that corresponds to the absorbance reading (from the experiment) on the y-axis.

Colloidal Gold-based Immunoassay Test Strip

  • This technique demonstrates a lateral flow immunoassay.
  • It involves sample application on a test strip.
  • Results are observed based on colored reactions.

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ELISA Techniques PDF

Description

This quiz explores the Enzyme-Linked Immunosorbent Assay (ELISA), a critical technique used in immunology for antibody and antigen detection. It covers various formats of ELISA, essential definitions, and the production of polyclonal antibodies.

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