ELISA
- Enzyme-Linked Immunosorbent assay, commonly known as ELISA (or EIA), is similar in principle to RIA but depends on an enzyme rather than a radioactive label.
- An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product.
- Such a substrate is called a chromogenic substrate.
- A number of enzymes have been employed for ELISA, including alkaline phosphatase, horseradish peroxidase, and b-galactosidase.
- These assays match the sensitivity of RIAs and have the advantage of beings safer and less costly.
- There are numerous variants of ELISA
- A number of variations of ELISA have been developed, allowing qualitative detection or quantitative measurement of either antigen or antibody.
- Each type of ELISA can be used qualitatively to detect the presence of antibody or antigen.
- Alternatively, a standard curve based on known concentrations of antibody or antigen is prepared, from which the unknown concentration of a sample can be determined.
Indirect ELISA
- Antibody can be detected or quantitatively determined with an indirect ELISA.
- Serum or some other sample containing primary antibody (Ab1) is added to an antigen coated microtiter well and allowed to react with the antigen attached to the well.
- After any free Ab1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody (Ab2) that binds to the primary antibody.
- Any free Ab2 is then washed away, and a substrate for the enzyme is added.
- The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measured the absorbance of all of the wells of a 96-well plate in seconds.
- Indirect ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.
- In this assay, recombinant envelope and core proteins of HIV are adsorbed as solid-phase antigens to microtiter wells. Individuals infected with HIV will produce serum antibodies to epitopes on these viral proteins. Generally, serum antibodies to HIV can be detected by indirect ELISA.
Sandwich ELISA
- Antigen can be detected or measured by a sandwich ELISA.
- In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well.
- A sample containing antigen is added and allowed to react with the immobilized antibody.
- After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen.
- After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.
Competitive ELISA
- Another variation for measuring amounts of antigen is competitive ELISA. In this technique, antibody is first incubated in solution with a sample containing antigen.
- The antigen antibody mixture is then added to an antigen coated microtiter well.
- The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well.
- Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well, as in an indirect ELISA. In the competitive assay, however, then higher the concentration of antigen in the original sample, the lower the absorbance.
- What does ELISA stand for?
a) Enzyme-Linked Immunospecific Assay
b) Electro-Labeled Immunoassay
c) Enzyme-Linked Immunosorbent Assay
d) Enzyme-Linked Isotope Assay
✔️ Answer: c) Enzyme-Linked Immunosorbent Assay - Which label is used in ELISA instead of a radioactive isotope?
a) Dye
b) Enzyme
c) Fluorescent tag
d) Antibody
✔️ Answer: b) Enzyme - The substrate used in ELISA is known as:
a) Reactive substrate
b) Photogenic substrate
c) Chromogenic substrate
d) Fluorogenic substrate
✔️ Answer: c) Chromogenic substrate - Which of the following is NOT a commonly used enzyme in ELISA?
a) Horseradish peroxidase
b) β-galactosidase
c) Alkaline phosphatase
d) Trypsin
✔️ Answer: d) Trypsin - What type of reaction product is generated in ELISA?
a) Fluorescent
b) Radiolabeled
c) Colored
d) Odorous
✔️ Answer: c) Colored
- Which type of ELISA is commonly used to detect HIV antibodies?
a) Competitive ELISA
b) Direct ELISA
c) Sandwich ELISA
d) Indirect ELISA
✔️ Answer: d) Indirect ELISA - In indirect ELISA, the secondary antibody is:
a) Conjugated with antigen
b) Conjugated with enzyme
c) Conjugated with protein
d) Unconjugated
✔️ Answer: b) Conjugated with enzyme - The spectrophotometric plate reader is used to:
a) Detect antigens
b) Read absorbance
c) Wash wells
d) Separate antibodies
✔️ Answer: b) Read absorbance - Which of the following is a key step in sandwich ELISA?
a) Immobilization of antigen
b) Immobilization of antibody
c) Use of radioactive isotope
d) Use of free enzyme
✔️ Answer: b) Immobilization of antibody - Sandwich ELISA detects the presence of:
a) Antibodies
b) Proteins
c) DNA
d) Antigens
✔️ Answer: d) Antigens
- In competitive ELISA, the higher the antigen concentration, the:
a) Higher the absorbance
b) Lower the absorbance
c) More enzyme needed
d) Faster the reaction
✔️ Answer: b) Lower the absorbance - The enzyme-linked antibody used in indirect ELISA binds to:
a) Antigen directly
b) Substrate
c) Primary antibody
d) Secondary antigen
✔️ Answer: c) Primary antibody - Which ELISA variant allows quantitative detection of antigens?
a) Western blot
b) Sandwich ELISA
c) Immunoprecipitation
d) Immunofluorescence
✔️ Answer: b) Sandwich ELISA - What is used to coat microtiter wells in indirect ELISA?
a) Secondary antibody
b) Chromogen
c) Antigen
d) Substrate
✔️ Answer: c) Antigen - Which one of the following is NOT an advantage of ELISA over RIA?
a) Safer
b) Less expensive
c) Uses radioactive material
d) Equally sensitive
✔️ Answer: c) Uses radioactive material
- Which of the following can be detected by ELISA?
a) DNA mutations
b) Protein expression
c) Hormones and antibodies
d) mRNA levels
✔️ Answer: c) Hormones and antibodies - In indirect ELISA, the color development is proportional to the amount of:
a) Substrate added
b) Antigen bound
c) Secondary antibody used
d) Primary antibody present
✔️ Answer: d) Primary antibody present - In ELISA, enzyme-substrate reaction results in:
a) DNA cleavage
b) mRNA production
c) Color change
d) Heat generation
✔️ Answer: c) Color change - In competitive ELISA, free antibodies in the well indicate:
a) Low antigen in sample
b) High antigen in sample
c) High substrate binding
d) Antibody loss
✔️ Answer: a) Low antigen in sample - The standard curve in ELISA is used to:
a) Determine substrate purity
b) Calculate antigen/antibody concentration
c) Plot enzyme activity
d) Compare antibody isotypes
✔️ Answer: b) Calculate antigen/antibody concentration
- In ELISA, washing steps are essential to:
a) Denature proteins
b) Inhibit enzymes
c) Remove unbound reagents
d) Kill microorganisms
✔️ Answer: c) Remove unbound reagents - ELISA is mostly performed in which type of plate?
a) Glass slide
b) Test tube
c) Microtiter plate
d) Petri dish
✔️ Answer: c) Microtiter plate - The chromogenic substrate for horseradish peroxidase is often:
a) X-gal
b) ONPG
c) TMB (tetramethylbenzidine)
d) IPTG
✔️ Answer: c) TMB (tetramethylbenzidine) - Which ELISA method is most useful for detecting small antigen molecules?
a) Direct
b) Sandwich
c) Indirect
d) Competitive
✔️ Answer: d) Competitive - ELISA test is preferred for disease diagnosis because it is:
a) Invasive and time-consuming
b) Complicated and expensive
c) Sensitive, specific, and easy to automate
d) Only suitable for research labs
✔️ Answer: c) Sensitive, specific, and easy to automate