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Questions and Answers
What is the molecular weight of IgG molecules?
Which chromosome codes for the heavy chains of IgG molecules?
What is the purpose of di-sulphide bonds in IgG molecules?
What is the primary function of an antibody in the immune system?
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Which of the following is an example of a naturally occurring antigen in the body?
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Which of the following is NOT a type of immunity?
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What is the purpose of injecting antigens into small mammals to produce antibodies?
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What are hybridomas in the context of antibody production?
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What is the term for the biochemical compound in a sample that is detected or analyzed in immunoassays?
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What is the main purpose of ELISA?
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What is the primary site of antibody production in the immune system?
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What is the term for the binding between an antigen and its homologous antibody?
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What is the main difference between polyclonal and monoclonal antibodies?
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What is the term for the large glycoprotein molecules produced by B-lymphocytes in response to antigens?
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What is the shape of the majority of human glycoprotein antibodies?
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What is the purpose of T-lymphocytes in the immune system?
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What is the primary purpose of an enzyme-conjugated anti-immunoglobulin in an ELISA assay?
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In an indirect ELISA, what is the function of the secondary antibody conjugate?
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What is the main difference between a direct ELISA and an indirect ELISA?
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What is the purpose of the Elisa reader in an ELISA assay?
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What is the advantage of using a sandwich ELISA over a direct ELISA?
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What is the purpose of the capture antibody in a sandwich ELISA?
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What is the typical application of a sandwich ELISA?
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What is the role of the substrate in an ELISA assay?
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What is the primary purpose of using a conjugated antigen in competitive ELISA?
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What is the significance of the signal produced in competitive ELISA?
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What is the purpose of including quality control samples in the ELISA kit?
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How are the results of the competitive ELISA typically analyzed?
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What is the purpose of drawing a standard curve in competitive ELISA?
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What is the significance of the initial positive results in competitive ELISA?
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Why is it important to read the kit instructions carefully before starting the competitive ELISA?
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What is the purpose of using duplicate or triplicate samples in competitive ELISA?
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Study Notes
Immunoassay
- Immunoassay is 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.
- Samples can be urine, saliva, tears, or any biochemical materials.
Antigens
- Antigens are substances that stimulate the production of an antibody when they enter the body.
- Examples of antigens include strange biological particles, bacteria, and viruses.
Analyte
- The analyte is the biochemical compound in a sample that is detected or analyzed in immunoassays.
- The analyte can be either an antibody or an antigen.
Antibodies
- Antibodies are large glycoprotein molecules produced by B-lymphocytes in response to antigens.
- Each antibody is designed to bind to a specific surface binding site or epitope on the antigen.
- There are millions of different types of antibodies circulating in an individual's bloodstream.
Structure of An Antibody
- Antibodies are shaped like a Y and are found in the blood, lymph, and intestine.
- The antibody is composed of 2 long (heavy) chains and 2 short (light) chains connected by di-sulphide bonds.
- The antibody recognizes and binds to the specific antigen and determinant specific region on the antigen surface.
Antibody Production
- Antibodies can be produced in the laboratory through various methods:
- Injecting an antigen into small mammals to produce small quantities of antibody.
- Injecting antigens into larger animals to produce large quantities of antibody.
- Producing monoclonal antibodies by combining antibody-secreting B-lymphocytes with cancer cells.
ELISA (Enzyme Linked Immunosorbent Assay)
- ELISA is a biochemical immunology technique used to detect the presence of an antibody or an antigen in a sample.
- ELISA is used to detect hormones, drugs, some toxins, and genetic modified crops.
Non-Competitive ELISA Requirements
- Antigens (Ag) fixed to a solid surface (immobilized).
- Antibodies (Ab) in solution to be tested.
- Enzyme-conjugated Anti-immunoglobulin (Antibody against the antibodies being tested for).
- Substrate binds to enzyme and produces color.
- Color intensity proportional to bound enzyme-Ab.
Indirect ELISA
- Antigen is immobilized on a plate.
- Primary detection antibody is added and binds to the specific antigen forming an antigen-antibody complex.
- Secondary antibody conjugate with the enzyme is added to the complex.
- Substrate is added and produces a signal proportional to the amount of antigen bound in the well.
Sandwich ELISA
- Capture antibody is coated on a microplate.
- Two specific antibodies are used, forming a sandwich shape with the antigen.
- Conjugated enzyme-detection antibody is added and binds to an additional epitope on the target protein (antigen).
- Substrate is added and produces a signal that is proportional to the amount of analyte present in the sample.
Competitive ELISA
- Capture antibody is coated on a microplate.
- Conjugated antigen is used to compete for binding with the antigen present in the sample.
- Note: more antigen present in the sample means less conjugated antigen will bind to the capture antibody.
- Substrate is added and the signal produced is inversely proportional to the amount of protein present in the sample.
Analysis of Results
- The standard curve is drawn using the absorbance readings from the spectrophotometer.
- The concentration of the unknown samples is determined by finding the corresponding concentration on the standard curve.
- Results can also be analyzed using software, which plots the standard curve and determines the concentration of the samples.
Facts about the Results
- The quality control sample concentration is determined from the standard curve and must be within the range given by the kit manufacturer.
- The initial Positive results are determined by the color change of the solution in the ELISA wells, while negative results are determined by the lack of color change.
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Description
ELISA is a technique used to identify and quantify specific antigens or antibodies in biological samples. It involves the binding of an antigen and its homologous antibody.
