Practical ELISA AI generated Flashcards
Describe the role of antibodies in humoral immune response.
Antibodies are produced by activated plasma B-cells and can detect antigens encountered previously.
What are the functions of Fab regions and Fc-domain in antibodies?
Fab regions make antigen binding sites, while the Fc-domain is constant and can bind to APCs.
How do antibodies neutralize pathogens/toxins?
Antibodies cover the pathogen/toxin, preventing it from attaching to a cell.
Define opsonization in the context of antibody functions.
Opsonization is when antibodies cover a pathogen/toxin, making it recognizable by phagocytosing cells for ingestion.
Explain how complement activation works with antibodies to destroy pathogens.
Complement can form pores in the pathogen’s cell membrane, leading to lysis, in coordination with antibodies.
What is the significance of epitopes in the interaction between antibodies and antigens?
Each antibody is specific for one epitope on an antigen, which may have multiple epitopes.
How do antibodies contribute to the immune response against pathogens?
Antibodies can neutralize pathogens, facilitate opsonization, and activate the complement system for pathogen destruction.
Describe the process of a sandwich ELISA.
1) Plate is coated with a capture antibody (not labeled); 2) sample is added, and any antigen present binds to capture antibody; 3) detecting antibody is added, and binds to antigen; 4) enzyme-linked secondary antibody is added, and binds to detecting antibody; 5) substrate is added, and is converted by enzyme to detectable form.
What is the purpose of the second antibody in an ELISA test?
The second antibody is necessary to detect the antigen that is bound to the coated antibody on the plate.
How can an ELISA test be both direct and indirect?
An ELISA test can be both direct and indirect depending on the method used to detect the antigen.
Define ELISA.
ELISA stands for Enzyme-Linked Immunosorbent Assay, a technique used to detect the presence of an antigen or antibody in a sample.
Do antibodies play a crucial role in ELISA tests?
Yes, antibodies are essential components in ELISA tests as they help detect antigens or antibodies in the sample.
Describe what an antibody t is.
antibody titer is a number that expresses the amount of antibodies in a sample, based on dilution steps that halve the number of antibodies at each step.
Define IgA deficiency.
IgA deficiency refers to the inability to produce the antibody IgA, typically synthesized by plasma B-cells at mucosal surfaces.
How does biotin play a role in the detection process of antibodies?
Biotin, when fused to the primary antibody, binds strongly to streptavidin, which can then be labeled with Horse radish peroxidase (HRP) for signal amplification.
Describe the process of adding the substrate TMB in the antibody detection process.
When TMB substrate is added, the HRP bound to the target turns blue, indicating binding. The reaction is stopped with HCl, causing the color to change to yellow.
What is the purpose of using a secondary antibody in antibody detection?
The secondary antibody, such as Streptavidin-Poly-HRP, binds to the biotin on the primary antibody, amplifying the signal for detection.
Do you need a spectrophotometer to measure the color change in the antibody detection process?
Yes, the color change from blue to yellow after adding the substrate TMB needs to be measured using a spectrophotometer.
Describe the process of coating a 96-wells plate in ELISA.
The plate is coated with a capture antibody, such as a polyclonal Goat anti-human IgA unlabeled antibody, to recognize multiple areas of the target molecule.
What is the purpose of blocking in ELISA?
Blocking is necessary to prevent non-specific binding of detection antibodies on unoccupied binding sites on the plate, enhancing assay sensitivity by reducing background noise.
