Radioimmunoassay (RIA)
- One of the most sensitive techniques for detecting antigen or antibody is radioimmunoassay (RIA).
- The technique was first developed in 1960 by two endocrinologists, S.A. Berson and Rosalyn Yalow, to determine levels of insulin-anti-insulin complexes in diabetics.
- Although their technique encountered some skepticism, it soon proved its value for measuring hormones, serum proteins, drugs, and vitamins at concentrations of 0.001 micrograms per milliliter or less.
- In 1977, some years after Berson’s death, the significance of the technique was acknowledged by the award of a Nobel Prize to Yalow.
- The Principle of RIA involves competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody.
- The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody.
- Then test samples of unlabeled antigen of unknown concentration are added in progressively larger amounts.
- The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody.
- As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites.
- The decrease in the amount of radio labeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample.
- The antigen is generally labeled with a gamma-emitting isotope such as 125I, but beta-emitting isotopes such as tritium (3H) are also routinely used as labels.
- The radio labeled antigen is part of the assay mixture; the test sample may be a complex mixture, such as serum or other body fluids, that contains the unlabeled antigen.
- The first step in setting up an RIA is to determine the amount of antibody needed to bind 50% to 70% of a fixed quantity of radioactive antigen in the assay mixture.
- This ratio of antibody to Ag* is chosen to ensure that the number of epitopes presented by the labeled antigen always exceeds the total number of antibody binding sites.
- Consequently, unlabeled antigen added to the sample mixture will compete with radio labeled antigen for the limited supply of antibody.
- Even a small amount of unlabeled antigen added to the assay mixture of labeled antigen and antibody will cause a decrease in the amount of radioactive antigen bound, and this decrease will be proportional to the amount of unlabeled antigen added.
- To determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to separate it from free antigen (antigen not bound to antibody), and the radioactivity in the precipitate is measured.
- A standard curve can be generated using unlabeled antigen samples of known concentration (in place of the test sample), and from this plot the amount of antigen in the test mixture may be precisely determined.
- Several methods have been developed for separating bound antigen from free antigen in RIA. One method involves precipitating Ag-Ab complexes with a secondary anti-isotope antiserum.
- For example, if the Ag-Ab complex contains rabbit IgG antibody, then goat anti-rabbit IgG will bind to the rabbit IgG and precipitate the complex.
- Another method makes use of the fact that protein A of Staphylococcus aureus has high affinity for IgG.
- If the Ag-Ab complex contains an IgG antibody, the complex can be precipitated by mixing with formalin-killed S. aureus.
- After removal of the complex by either of these methods, the amount of free labeled antigen remaining in the supernatant can be measured in a radiation counter; subtracting this value from the total amount of labeled antigen added yields the amount of labeled antigen bound.
- Various solid-phase RIAs have been developed that make it easier to separate the Ag-Ab complex from the unbound antigen.
- In some cases, the antibody is covalently cross-linked to Sepharose beads.
- The amount of radiolabeled antigen bound to the beads can be measured after the beads have been centrifuged and washed.
- Alternatively, the antibody can be immobilized on polystyrene or poly-vinyl chloride wells and the amount of free labeled antigen in the supernatant can be determined in a radiation counter.
- In another approach, the antibody is immobilized on the walls of microtiter wells and the amount of bound antigen determined.
- Because the procedure requires only small amounts of sample and can be conducted in small 96-well microtiter plates (slightly larger than a 3 × 5 card), this procedure is well suited for determining the concentration of a particular antigen in large numbers of samples.
- For example, a microtiter RIA has been widely used to screen for donor blood and has sharply reduced the incidence of hepatitis B infections in recipients of blood transfusions.
- Radioimmunoassay (RIA) was first developed to measure which of the following?
a) Blood pressure
b) Blood glucose
c) Insulin-anti-insulin complexes
d) Red blood cells
✔️ Answer: c) Insulin-anti-insulin complexes - RIA was developed in the year:
a) 1945
b) 1950
c) 1960
d) 1977
✔️ Answer: c) 1960 - Which scientist won the Nobel Prize for developing RIA?
a) Claude
b) Palade
c) Rosalyn Yalow
d) Watson
✔️ Answer: c) Rosalyn Yalow - RIA works on the principle of:
a) Precipitation
b) Diffusion
c) Competitive binding
d) Electrophoresis
✔️ Answer: c) Competitive binding - In RIA, which type of antigen competes with the unlabeled antigen?
a) Soluble antigen
b) Radiolabeled antigen
c) Enzyme-labeled antigen
d) Denatured antigen
✔️ Answer: b) Radiolabeled antigen - Which isotope is commonly used in RIA?
a) C-14
b) 3H
c) 125I
d) 35S
✔️ Answer: c) 125I - Beta-emitting isotope used in RIA is:
a) 125I
b) 3H
c) 14C
d) 32P
✔️ Answer: b) 3H - In RIA, test samples are added in:
a) Decreasing amounts
b) Constant amounts
c) Random volumes
d) Progressively increasing amounts
✔️ Answer: d) Progressively increasing amounts - The binding of labeled antigen to antibody decreases as:
a) Temperature increases
b) Labeled antigen increases
c) Unlabeled antigen increases
d) pH increases
✔️ Answer: c) Unlabeled antigen increases - In RIA, the first step involves:
a) Addition of radiolabeled antigen to serum
b) Fixing antibody to polystyrene
c) Determining antibody amount to bind 50-70% of radiolabeled antigen
d) Measuring free antigen
✔️ Answer: c) Determining antibody amount to bind 50-70% of radiolabeled antigen
- Which method can be used to precipitate Ag-Ab complexes in RIA?
a) Protein A precipitation
b) SDS-PAGE
c) Spectrophotometry
d) Dialysis
✔️ Answer: a) Protein A precipitation - Protein A is derived from:
a) Escherichia coli
b) Bacillus subtilis
c) Staphylococcus aureus
d) Streptococcus pneumoniae
✔️ Answer: c) Staphylococcus aureus - To measure radioactivity in RIA, we use:
a) Colorimeter
b) Flame photometer
c) Radiation counter
d) Spectrophotometer
✔️ Answer: c) Radiation counter - What separates free from bound antigen in solid-phase RIA?
a) Dialysis membrane
b) Radiation counter
c) Centrifugation and washing
d) Chromatography
✔️ Answer: c) Centrifugation and washing - In microtiter-based RIA, the antibody is immobilized on:
a) Nitrocellulose
b) Paper discs
c) Polyvinyl chloride wells
d) Glass slides
✔️ Answer: c) Polyvinyl chloride wells - What is the advantage of using 96-well microtiter plates in RIA?
a) Radioactivity handling
b) Faster centrifugation
c) Large number of samples can be tested simultaneously
d) Greater antigen specificity
✔️ Answer: c) Large number of samples can be tested simultaneously - RIA can detect antigen concentrations as low as:
a) 1 mg/mL
b) 0.1 µg/mL
c) 0.01 µg/mL
d) 0.001 µg/mL
✔️ Answer: d) 0.001 µg/mL - Which of the following is NOT required for RIA?
a) Radiolabeled antigen
b) Antibody
c) Substrate for enzyme
d) Radiation counter
✔️ Answer: c) Substrate for enzyme - Which of the following can be used as a standard in RIA?
a) Antibody
b) Serum
c) Known concentration of unlabeled antigen
d) Plasma membrane
✔️ Answer: c) Known concentration of unlabeled antigen - The amount of antigen in a test sample is determined by comparing with:
a) Radiolabeled antibody
b) Calibration fluid
c) Standard curve
d) Agar plate results
✔️ Answer: c) Standard curve
- Which of the following can be detected using RIA?
a) Vitamins
b) Drugs
c) Hormones
d) All of the above
✔️ Answer: d) All of the above - Rosalyn Yalow received the Nobel Prize in:
a) Chemistry
b) Physics
c) Physiology or Medicine
d) Peace
✔️ Answer: c) Physiology or Medicine - Which of the following is a disadvantage of RIA?
a) Highly specific
b) Requires radioisotopes
c) Sensitive
d) Quantitative
✔️ Answer: b) Requires radioisotopes - What is the main risk in using RIA?
a) Electrical shock
b) Chemical burns
c) Radioactive exposure
d) Fire hazard
✔️ Answer: c) Radioactive exposure - One of the most important benefits of RIA is its ability to:
a) Work without antibodies
b) Avoid centrifugation
c) Detect very small concentrations of substances
d) Use visible dyes
✔️ Answer: c) Detect very small concentrations of substances