32 Questions
What is the primary purpose of immunoassay techniques?
To identify and quantify specific antigens in a sample
What is the role of B-lymphocytes in the immune system?
To produce antibodies in response to antigens
What is the term for the biochemical compound in a sample that is detected or analyzed in immunoassays?
Analyte
What is the percentage of human glycoprotein antibodies that are in the immunoglobulin class IgG?
80%
What is the shape of IgG antibodies?
Y-shaped
What is the term for the part of the antigen that an antibody binds to?
Epitope
What is the function of T-lymphocytes in the immune system?
To mark antigens-antibody complex to be destroyed
What is the term for the large glycoprotein molecules produced by B-lymphocytes?
Antibodies
What is the molecular weight of IgG molecules?
150,000 Daltons
Where are the genes for the heavy chains of IgG molecules located?
Chromosome 14
What type of bonds connect the heavy and light chains of IgG molecules?
Di-sulphide bonds
What is the antigen binding site of an IgG molecule?
Determinant specific region
What is an example of a naturally occurring antigen in the body?
HCG hormone
What is the method used to produce large quantities of antibodies in the lab?
Injecting antigens into goats, sheep, or horses
What is the characteristic of monoclonal antibodies?
They attach only to a specific antigen binding site
What is the purpose of ELISA technique?
To detect the presence of an antibody or an antigen in a sample
What is the purpose of an enzyme-conjugated anti-immunoglobulin in an ELISA?
To detect the antibodies being tested for
What is the result of the reaction between the substrate and the enzyme in an ELISA?
A color change that is proportional to the amount of antigen bound
What is the main difference between direct and indirect ELISA?
The sequence of antibody addition
What is the purpose of the spectrophotometer in an ELISA?
To measure the intensity of the color produced
What type of ELISA is used to detect very small analyte concentrations in a biological sample?
Sandwich ELISA
What is the shape formed by the antibodies and the antigen in a sandwich ELISA?
Sandwich
What is the advantage of using two antibodies in a sandwich ELISA?
It is highly specific, since two antibodies are required to bind to the protein of interest
What is the purpose of the ELISA reader?
To measure the intensity of the color produced
What is the main characteristic of a competitive ELISA?
Using a conjugated antigen to compete with the antigen present in the sample
What happens when there is more antigen present in the sample in a competitive ELISA?
Less conjugated antigen will bind to the capture antibody
What is the purpose of the first wells in a 96-well plate in a competitive ELISA?
To draw the standard curve
Why is it important to read the kit instructions carefully before starting a competitive ELISA?
To avoid disturbance and loss of expensive kits
What is the purpose of the quality control samples provided with the kit?
To test the performance of the assay
How is the standard curve drawn in a competitive ELISA?
By plotting the concentration on the X-axis and the absorbance on the Y-axis
What is the purpose of the standard curve in a competitive ELISA?
To determine the unknown concentration of each sample
What is the characteristic of a positive result in a competitive ELISA?
The solution changes color
Study Notes
Immunoassay
- Immunoassay is a technique that uses the binding between an antigen and its homologous antibody to identify and quantify specific antigens or antibodies in a sample.
- Samples can be urine, saliva, tears, or any biochemical material.
Antigens and Antibodies
- Antigens are substances that stimulate the production of antibodies when they enter the body.
- Antibodies are large glycoprotein molecules produced by B-lymphocytes in response to antigens.
- Antibodies are designed to bind to specific surface binding sites or epitopes on the antigen.
Structure of Antibodies
- Over 80% of human glycoprotein antibodies are in the immunoglobulin class IgG.
- IgG molecules have a molecular weight of 150,000 Daltons and are made of 2 long (heavy) chains and 2 short (light) chains connected by di-sulphide bonds.
Antibody Production in Lab
- Small quantities of antibody can be produced by injecting an antigen into small mammals, such as mice, rats, or rabbits.
- Large quantities of antibody can be produced by injecting antigens into goats, sheep, or horses to obtain anti-sera with polyclonal antibodies.
- Monoclonal antibodies can be produced by combining antibody-secreting B-lymphocytes with cancer cells (hybridomas).
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 can be used to detect hormones, drugs, toxins, and genetic modified crops.
Non-Competitive ELISA Requirements
- Antigens (Ag) fixed to a solid surface (immobilized) on a 96-well plastic plate.
- Antibodies (Ab) in solution to be tested.
- Enzyme-conjugated Anti-immunoglobulin (Ab) against the antibodies being tested for.
- Substrate binds to enzyme and produces color.
ELISA Types
- Direct ELISA: antigen is immobilized on a plate, and primary detection antibody is added.
- Indirect ELISA: antigen is immobilized on a plate, primary detection antibody is added, and then a secondary antibody conjugate with the enzyme is added.
- Sandwich ELISA: two specific antibodies are used, forming a sandwich shape with the antigen.
- Competitive ELISA: a capture antibody is coated on a microplate, and a conjugated antigen is used to compete for binding with the antigen present in the sample.
ELISA Equipment
- ELISA reader
- ELISA washer
- Washer solutions
- ELISA monitor
- ELISA kits
Analysis of Results
- Classic method: read the results using a spectrophotometer, and draw the standard curve.
- Soft ware method: specify the standard concentrations on the x-axis and the reading of each standard on the y-axis, and draw the standard curve.
Results
- The quality control sample concentration is determined from the standard curve.
- Positive results are determined by the color change in the solution, and negative results are determined by the absence of color change.
Learn about the competitive ELISA technique, a type of enzyme-linked immunosorbent assay used to detect and measure small proteins. Understand how it works and its application.
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