1: ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of _______ in a sample.
a.) amino acid
b.) DNA
c.) antigen
d.) Protein
Answer: c
Explanation: ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of antigen in a sample.
2: Please arrange the following steps about ELIZA (Enzyme-linked immunosorbent assay) in chronological order
i. incubate with antibody-enzyme complex that binds primary antibody
ii. coat surface with antigen, block unoccupied sites with nonspecific protein
iii. add substrate, formation of colored product indicates presence of specific antigen
iv. incubate with primary antibody against specific antigen
a.) i, iv, ii, iii
b.) i, iv, iii, ii
c.) ii, iv, iii, i
d.) ii, iv, i, iii
Answer: d
Explanation: The steps for ELIZA are
1. Coat surface with antigen, block unoccupied sites with nonspecific protein.
2. Incubate with primary antibody against specific antigen.
3. Incubate with antibody-enzyme complex that binds primary antibody.
4. Add substrate, formation of colored product indicates presence of specific antigen.
3: What is the role of goat anti-rabbit IgG horseradish peroxidase conjugate in the experiment?
a.) Antigen
b.) Primary antibody
c.) Second antibody
d.) Substrate
Answer: c
Explanation: Goat anti-rabbit IgG horseradish peroxidase conjugate is the second antibody in the experiment.
4: In order to stop the reaction between enzyme and substrate, what process is needed?
a.) Incubate the sample at 100oC
b.) Add strong acid to sample
c.) Wash sample with PBS-Tween
d.) Add blocking solution to sample
Answer: b
Explanation: In the experiment, strong acid H2SO4 is added to sample to stop the enzyme reaction.
5: In the experiment, product formation (monitored as color intensity) is _________ the concentration of the antigen (BSA) solution in the sample.
a.) proportional to
b.) inversely proportional to
c.) independent of
d.) none of the above
Answer: a
Explanation: Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample.
6. In Rocket Immunodiffusion the length of the rocket is
a.) proportional to the amount of antibody placed in each well
b.) inversely proportional to the amount of antibody placed in each well
c.) inversely proportional to the amount of antigen placed in each well
d.) proportional to the amount of antigen placed in each well
Answer: d
Explanation: In Rocket Immunodiffusion the length of the rocket is proportional to the amount of antigen placed in each well.
7. What would happen if serum is omitted from the ELISA, but all other steps remain same?
a.) Anti-human Ig-conjugate would not bind and be washed away
b.) The OD values would be nearly the same as the assay control
c.) Both (a) and (b)
d.) Anti-human Ig-conjugate would bind non-specifically to the ELISA plate
Answer: c
Explanation:
Omitting serum from the ELISA can lead to decreased sensitivity of the assay due to the lack of a blocking factor [1], as well as improper orientation, denaturation, and poor immobilization efficiency of the target molecule [2]. Additionally, the lack of serum may lead to binding of contaminants along with the target molecule [2], and can cause greater inhibition of the readout of many cytokines than plasma [3]. Therefore, it is important to include serum in the ELISA in order to ensure accurate results.
Sources:
- https://www.researchgate.net/post/How-does-serum-in-culture-media-affect-ELISA
- https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html
- https://pubmed.ncbi.nlm.nih.gov/24522699/
8. Immunoelectrophoresis techniques are used to separate the components of a mixture using electrophoresis.
a.) prior to reaction with antibody
b.) prior to reaction with antigen
c.) after reaction with antibody
d.) after reaction with antigen
Answer: a
Explanation:
Immunoelectrophoresis is a technique used to separate the components of a mixture using electrophoresis. It combines the principles of zone electrophoresis and immunodiffusion, and requires immunoglobulins, also known as antibodies, to react with the proteins to be separated or characterized [1]. Immunoelectrophoresis is used to detect normal and abnormal proteins in human serum, such as myeloma proteins [2]. It is also used to analyze complex protein mixtures containing different antigens [3]. The method combines the principles of zone electrophoresis and immunodiffusion, and is still used by some clinical laboratories [4]. It is also used to identify and characterize proteins within complex mixtures [5].
Sources:
- https://en.wikipedia.org/wiki/Immunoelectrophoresis
- https://microbenotes.com/immunoelectrophoresis-principle-procedure-results-and-applications-advantages-and-limitations/
- https://microbenotes.com/immunoelectrophoresis-principle-procedure-results-and-applications-advantages-and-limitations/
- https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/immunoelectrophoresis
- https://pubmed.ncbi.nlm.nih.gov/30426422/
9. The antibody class whose serum levels are highest is:
a) IgE
b) IgM
c) IgD
d) IgG
Answer: d
Explanation: Immunoglobulin G (IgG), which makes up approximately 80% of all antibodies, has the highest serum levels.
10. All of the following—apart from which one—are advantages of EIA (Enzyme immunoassay) over RIA (Radioimmunoassay).
a.) Decrease in hazardous waste
b.) Shorter shelf life of kit
c.) No need for expensive equipment
d.) Ease of adaptation to automated techniques
Answer: b
Explanation:
The main advantages of Enzyme Immunoassay (EIA) or EnLinked Immunosorbent Assay (ELISA) are that it is a simple test that does not cause side effects, has high sensitivity and specificity, offers more accuracy compared to other techniques such as radioimmunoassay (RIA) tests, and is usually in a 96 well microplate format.
