Radioimmunoassay (RIA)- Definition, Principle, Procedure, Results, Uses

Radioimmunoassay (RIA) is a laboratory technique that uses radioisotopes to measure the concentration of a substance in a biological sample. RIA is based on the principle of specific and competitive binding of antigens and antibodies. Antigens are molecules that can elicit an immune response and antibodies are proteins that can recognize and bind to antigens.

In RIA, a known amount of a radiolabeled antigen (an antigen that has been attached to a radioactive atom) is mixed with a known amount of a specific antibody. The antibody binds to the radiolabeled antigen, forming an antigen-antibody complex. Then, a sample containing an unknown amount of the same antigen (unlabeled antigen) is added to the mixture. The unlabeled antigen competes with the radiolabeled antigen for binding to the antibody. The more unlabeled antigen there is in the sample, the less radiolabeled antigen will be bound to the antibody.

The mixture is then separated into two fractions: one containing the bound antigen-antibody complex and one containing the free antigens (both labeled and unlabeled). The radioactivity of each fraction is measured using a device called a gamma counter. The radioactivity of the bound fraction reflects the amount of radiolabeled antigen that is bound to the antibody, while the radioactivity of the free fraction reflects the amount of radiolabeled antigen that is displaced by the unlabeled antigen. By comparing the radioactivity of the two fractions, the concentration of the unlabeled antigen in the sample can be calculated.

RIA is a very sensitive technique that can detect very low levels of substances in biological samples. RIA can be used to measure hormones, drugs, antibodies, viruses, toxins and other molecules of interest. RIA has many applications in biomedical research, clinical diagnosis and environmental monitoring. However, RIA also has some limitations, such as the use of radioactive materials, the high cost of equipment and reagents, and the short shelf-life of radiolabeled substances.

Solomon Berson and Rosalyn Yalow were the pioneers of radioimmunoassay (RIA), a sensitive technique that uses radioactive materials to measure the concentration of substances in biological fluids. They worked together at the Bronx Veterans Administration (VA) Hospital in New York City, where they made many discoveries in clinical biochemistry and endocrinology.

Berson was born in New York City in 1918. He graduated from the City College of New York in 1938 and earned a master`s degree in anatomy from New York University (NYU) in 1939. He then enrolled in NYU medical school, where he met Yalow, who was a physics instructor. He graduated with honors in 1945 and served in the army for two years. He returned to New York and completed his residency in internal medicine at the Bronx VA Hospital.

Yalow was born in New York City in 1921. She grew up in a poor Jewish family and developed an interest in science and math at an early age. She graduated from Hunter College in 1941 with a degree in physics and received a scholarship to pursue a doctorate at the University of Illinois. She completed her PhD in 1945 and married Aaron Yalow, a fellow physics student. She then joined the faculty of Hunter College as a physics lecturer.

Berson and Yalow began their collaboration in 1950, when Berson joined the Radioisotope Service of the Bronx VA Hospital and invited Yalow to work with him as a part-time consultant. They initially studied the metabolism of iodine and albumin, but later shifted their focus to insulin, a hormone that regulates blood sugar levels. They developed RIA to measure insulin levels in blood samples, using radioactive isotopes of iodine to label insulin molecules. They published their first paper on RIA in 1960.

They discovered that some diabetic patients had antibodies against insulin, which interfered with its action and caused insulin resistance. They also found that insulin levels varied according to body weight, diet, and stress. They applied RIA to other hormones, such as corticotropin, gastrin, parathyroid hormone, and growth hormone, and revealed new aspects of their physiology and pathology.

Berson and Yalow received many awards and honors for their work, including the Albert Lasker Award for Basic Medical Research in 1976. Berson was also appointed as the chair of medicine at Mount Sinai School of Medicine in 1968 and elected to the National Academy of Sciences in 1972. Unfortunately, he died of a heart attack later that year at the age of 53.

Yalow continued her research at the Bronx VA Hospital until her retirement in 1991. She received the Nobel Prize in Physiology or Medicine in 1977 for her contributions to RIA. She was the second woman to win the Nobel Prize in this field, after Gerty Cori in 1947. She died in 2011 at the age of 89.

Berson and Yalow`s legacy lives on through their invention of RIA, which has revolutionized the fields of clinical biochemistry, endocrinology, immunology, pharmacology, and oncology. RIA has enabled the detection and quantification of various substances in biological fluids, such as hormones, drugs, antibodies, antigens, vitamins, enzymes, and toxins. RIA has also facilitated the diagnosis and treatment of many diseases, such as diabetes, thyroid disorders, pituitary disorders, gastrointestinal disorders, and cancer.

Radioimmunoassay (RIA) is a sensitive immunoassay technique that requires the following components:

• Radiolabeled antigens: These are antigens that are tagged with radioactive isotopes, such as iodine-125 or tritium. They are also called hot antigens. They emit radiation that can be measured by a gamma counter or a scintillation counter. The amount of radiation is proportional to the amount of radiolabeled antigens in the solution.
• Specific antibodies: These are proteins that can bind to a specific antigen with high affinity and specificity. They are required in smaller amounts than antigens. They can be obtained from animals that have been immunized with the antigen of interest, or from hybridoma cells that produce monoclonal antibodies.
• Unlabeled antigens (sample antigens): These are antigens that are not labeled with radioactive isotopes. They are also called cold antigens. They are derived from the sample that needs to be tested for the presence or concentration of the antigen of interest. They compete with the radiolabeled antigens for binding to the specific antibodies.
• Microtitre plates: These are plastic plates that have 96 wells, each well can hold a small volume of liquid. They are used to perform multiple reactions in parallel, and to separate the bound and free antigens by washing or centrifugation.
• Washing buffer solutions: These are solutions that are used to wash away any unbound antigens or antibodies from the microtitre wells. They usually contain salts, detergents, and stabilizers that prevent nonspecific binding and preserve the activity of the antibodies and antigens. An example of a washing buffer solution is 1% trifluoroacetic acid.

Radioimmunoassay (RIA) is based on the principle of competitive binding or displacement of radiolabeled antigens by unlabeled antigens from a sample to a limited amount of specific antibodies. The amount of radioactivity emitted by the bound or free antigens is measured and used to determine the concentration of the antigen of interest in the sample. The principle of RIA can be explained by the following steps:

• Immune Reaction: The first step is to prepare a specific antibody that can recognize and bind to the antigen of interest. The antibody can be produced by immunizing animals with the antigen and collecting their serum or by using monoclonal antibodies that are derived from a single clone of antibody-producing cells. The antibody is then immobilized on a solid support such as a microtitre plate or a bead.
• Competitive binding or displacement reaction: The second step is to add a known amount of radiolabeled antigen (also called hot antigen) to the antibody-coated support. The radiolabeled antigen can be conjugated with a radioactive isotope such as iodine-125 or tritium that emits gamma or beta rays, respectively. The radiolabeled antigen will bind to the available binding sites on the antibody, forming an antigen-antibody complex. Next, a sample containing an unknown amount of unlabeled antigen (also called cold antigen) is added to the same support. The unlabeled antigen will compete with the radiolabeled antigen for the same binding sites on the antibody. Depending on the relative concentrations of the labeled and unlabeled antigens, some of the labeled antigens will be displaced by the unlabeled antigens, resulting in a decrease in the amount of bound radiolabeled antigens and an increase in the amount of free radiolabeled antigens in the solution.
• Measurement of radio emission: The third step is to separate the bound and free antigens and measure their radioactivity using a device such as a gamma counter or a scintillation counter. The amount of radioactivity emitted by the bound antigens is inversely proportional to the amount of unlabeled antigens in the sample, while the amount of radioactivity emitted by the free antigens is directly proportional to the amount of unlabeled antigens in the sample. A standard curve can be constructed by plotting the percentage of bound radiolabeled antigens against known concentrations of unlabeled antigens, and then using this curve to interpolate the concentration of unlabeled antigens in the sample based on its percentage of bound radiolabeled antigens.

The principle of RIA can be summarized by the following equation:

$$\% B = \frac{B}{B_0} \times 100$$

where B is the amount of bound radiolabeled antigen, B0 is the amount of bound radiolabeled antigen in the absence of unlabeled antigen (maximum binding), and %B is the percentage of bound radiolabeled antigen.

One of the main advantages of radioimmunoassay (RIA) is its extremely high sensitivity. Sensitivity refers to the ability of a test to detect very low concentrations of a substance in a sample. RIA can measure substances in the range of nanograms (10^-9 grams) or even picograms (10^-12 grams) per milliliter of sample. This is because the radioisotopes used to label the antigens emit radiation that can be easily detected and quantified by a gamma counter or a scintillation counter. The radiation signal is proportional to the amount of labeled antigen bound to the antibody, which in turn reflects the amount of unlabeled antigen in the sample.

The high sensitivity of RIA makes it suitable for measuring substances that are present in very low amounts in biological fluids, such as hormones, drugs, vitamins, antibodies, and antigens. For example, RIA can detect insulin levels as low as 0.01 micrograms per milliliter of blood plasma, or human chorionic gonadotropin (hCG) levels as low as 1 milli-international unit per milliliter of urine. These substances are important for diagnosing and monitoring various diseases and physiological conditions, such as diabetes, thyroid disorders, pregnancy, and fertility.

The high sensitivity of RIA also allows for using smaller volumes of sample and reagents, which reduces the cost and time of the assay. Moreover, RIA can be performed in a multiplex format, which means that multiple substances can be measured simultaneously in the same sample using different radioisotopes. This increases the efficiency and accuracy of the assay.

In summary, RIA is a highly sensitive immunoassay technique that can measure very low concentrations of substances in biological samples. This advantage makes RIA useful for various clinical and research applications that require precise and reliable measurements of hormones, drugs, antibodies, antigens, and other substances.

The procedure of RIA involves the following steps:

1. Preparation of radiolabeled antigen: The antigen of interest is labeled with a suitable radioisotope, such as I-125 or H-3, by chemical or enzymatic methods. The radiolabeled antigen should retain its immunological properties and have a high specific activity.
2. Preparation of specific antibody: The antibody that can bind to the antigen of interest is obtained from immunized animals or hybridoma cells. The antibody should have a high affinity and specificity for the antigen and should be purified and standardized.
3. Preparation of standard antigen: The unlabeled antigen of known concentration is used as a standard to generate a calibration curve. The standard antigen should be identical to the antigen in the sample and should have the same immunoreactivity as the radiolabeled antigen.
4. Preparation of sample: The sample that contains the unknown amount of antigen is collected and processed to remove interfering substances and to adjust the pH, volume, and ionic strength. The sample should be stored at low temperature or frozen to prevent degradation of the antigen.
5. Incubation: A fixed amount of radiolabeled antigen and a fixed amount of antibody are mixed and incubated in a suitable buffer solution. Then, varying amounts of standard antigen or sample are added to different tubes or wells and incubated for a sufficient time to allow the formation of antigen-antibody complexes. The incubation conditions should be optimized to ensure equilibrium and maximum binding.
6. Separation: After incubation, the bound and free fractions of radiolabeled antigen are separated by physical or chemical methods, such as precipitation, centrifugation, filtration, chromatography, or solid-phase adsorption. The separation method should be efficient and reproducible and should not affect the stability of the complexes or the radioactivity of the label.
7. Measurement: The radioactivity of either the bound or the free fraction of radiolabeled antigen is measured by a suitable detector, such as a gamma counter or a scintillation counter. The measurement should be accurate and precise and should correct for background and quenching effects.
8. Calculation: The radioactivity data are plotted on a graph as a function of the concentration of standard antigen or sample. A standard curve is obtained by fitting a suitable mathematical model to the data points. The concentration of antigen in the sample is determined by interpolating from the standard curve. The calculation should account for dilution factors, recovery rates, and quality control parameters.

The procedure of RIA can be summarized by the following equation:

$$\text{Antibody} + \text{Radiolabeled Antigen} \rightleftharpoons \text{Antibody-Radiolabeled Antigen Complex}$$ $$\text{Antibody-Radiolabeled Antigen Complex} + \text{Unlabeled Antigen} \rightleftharpoons \text{Antibody-Unlabeled Antigen Complex} + \text{Free Radiolabeled Antigen}$$

The amount of free radiolabeled antigen is inversely proportional to the amount of unlabeled antigen in the sample.

The result of RIA depends on the measurement of radioactivity of the bound or free radiolabeled antigens. The radioactivity can be measured by a device called a gamma counter or a scintillation counter . The radioactivity is usually expressed in counts per minute (cpm) or disintegrations per minute (dpm).

The result of RIA is based on the principle of competitive binding, which means that the amount of bound radiolabeled antigen is inversely proportional to the amount of unlabeled antigen in the sample . Therefore, a high concentration of unlabeled antigen will result in a low radioactivity of the bound fraction, and vice versa .

To interpret the result of RIA, a standard curve is constructed by plotting the percentage of bound radiolabeled antigen against known concentrations of a standardized unlabeled antigen . The percentage of bound radiolabeled antigen can be calculated by using the following formula:

$$\% \text{Bound} = \frac{\text{cpm or dpm of bound fraction}}{\text{cpm or dpm of total fraction}} \times 100$$

The standard curve can then be used to determine the concentration of antigen in the unknown samples by interpolating their percentage of bound radiolabeled antigen on the curve . Alternatively, some kits provide tubes coated with secondary antibodies that capture the antigen-antibody complexes, and the radioactivity of the free fraction is measured instead. In this case, the percentage of free radiolabeled antigen can be calculated by using the following formula:

$$\% \text{Free} = \frac{\text{cpm or dpm of free fraction}}{\text{cpm or dpm of total fraction}} \times 100$$

And the standard curve can be plotted by using the percentage of free radiolabeled antigen against known concentrations of a standardized unlabeled antigen.

The result interpretation of RIA can also be affected by some factors such as non-specific binding (NSB), total binding (TB), and total counts (TC). NSB refers to the binding of radiolabeled antigens to other components in the assay system besides the specific antibodies. TB refers to the maximum amount of radiolabeled antigens that can bind to the specific antibodies. TC refers to the total amount of radiolabeled antigens added to each tube. These factors can be measured by using control tubes that contain either no antibody, no antigen, or both. The NSB, TB, and TC values can be used to correct the cpm or dpm values of the samples and standards before calculating their percentage of bound or free radiolabeled antigens.

Radioimmunoassay (RIA) is a highly sensitive and specific technique that can measure the concentration of antigens or antibodies in biological samples. RIA has a wide range of applications in various fields of science and medicine, such as:

• Detection of peptide hormones: RIA was first used for the detection of insulin in plasma by Rosalyn Yalow and Solomon Berson in 1959. Since then, RIA has been used to measure the levels of various peptide hormones, such as growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, parathyroid hormone, gastrin, glucagon, and many others.
• Detection of viral antigens: RIA can be used to detect the presence of viral antigens in serum or other body fluids. For example, RIA can detect hepatitis B surface antigen (HBsAg), which is a marker of hepatitis B infection. RIA can also detect other viral antigens, such as human immunodeficiency virus (HIV), rubella virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV).
• Detection of hormones and drugs: RIA can be used to measure the levels of hormones and drugs in blood or urine. For example, RIA can detect cortisol, testosterone, estrogen, progesterone, aldosterone, catecholamines, digoxin, morphine, methadone, and many others. RIA can also be used to monitor the therapeutic effects or toxicity of drugs.
• Detection of mycotoxins: RIA can be used to detect the presence of mycotoxins, which are toxic substances produced by fungi. For example, RIA can detect aflatoxin B1, which is a carcinogenic mycotoxin that can contaminate food products. RIA can also detect other mycotoxins, such as ochratoxin A, zearalenone, deoxynivalenol, and fumonisin B1.
• Detection of the early stage of cancer: RIA can be used to detect the presence of tumor markers, which are substances produced by cancer cells or by the body in response to cancer. For example, RIA can detect carcinoembryonic antigen (CEA), which is a marker of colorectal cancer. RIA can also detect other tumor markers, such as alpha-fetoprotein (AFP), prostate-specific antigen (PSA), human chorionic gonadotropin (hCG), and CA 125.

These are some of the applications of RIA that demonstrate its usefulness and versatility in various fields of science and medicine. However, RIA also has some limitations and disadvantages that need to be considered before using it. These include the use of radioactive substances that pose health and environmental risks, the high cost and short shelf-life of reagents and equipment, and the possibility of interference from cross-reactivity or non-specific binding. Therefore, alternative techniques such as enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay (CLIA) have been developed to overcome some of these drawbacks. However, RIA still remains a valuable technique that has contributed greatly to the advancement of knowledge and diagnosis in various fields.

Radioimmunoassay (RIA) is a powerful technique that has many advantages over other immunoassays. Some of the advantages are:

• Extremely high sensitivity: RIA can detect very low concentrations of antigens or antibodies in the sample, ranging from nanograms to picograms. This makes it suitable for measuring hormones, drugs, and other substances that are present in trace amounts in biological fluids.
• High specificity: RIA relies on the specific binding of antigens and antibodies, which minimizes the interference from other substances in the sample. The use of radiolabeled antigens also enhances the specificity, as only the bound antigens emit radiation that can be measured.
• Wide range of applications: RIA can be used to measure a variety of antigens and antibodies, such as hormones, enzymes, drugs, toxins, viruses, and tumor markers. RIA can also be used to study the kinetics and dynamics of antigen-antibody interactions, as well as the structure and function of receptors and ligands.
• Simple and rapid: RIA is relatively simple to perform and does not require complex instrumentation or equipment. The procedure can be completed within a few hours, depending on the incubation time and the washing steps. The results can be obtained quickly by using a gamma counter or a scintillation counter to measure the radioactivity of the samples.
• Quantitative and reproducible: RIA provides quantitative results that can be compared with standard curves or reference values. The results are also reproducible and reliable, as long as the quality and quantity of the reagents are controlled and the protocol is followed consistently.

Radioimmunoassay (RIA) is a powerful technique for detecting and quantifying antigens and antibodies in biological samples. However, it also has some limitations that need to be considered before using it. Some of the major limitations of RIA are:

• Working with radioactive substances makes it a bit risky. Radioactive isotopes such as I-125 and Tritium can pose health hazards to the personnel handling them and the environment. They require special precautions, safety measures, and training to avoid exposure and contamination. They also have a limited shelf-life and need to be stored properly.
• Disposal of radioactive substances can be problematic. The waste generated from RIA experiments contains radioactive materials that need to be disposed of carefully and according to the regulations. Improper disposal can cause environmental pollution and health risks. The disposal process can also be costly and time-consuming.
• Equipment and reagents are expensive. RIA requires specialized equipment such as gamma counters, scintillation counters, and microtitre plates that are not easily available or affordable. The reagents such as radiolabeled antigens, specific antibodies, and washing buffers are also costly and need to be purchased from reliable sources. The cost of RIA can limit its accessibility and feasibility for some laboratories or researchers.
• Radiolabeled substances used have a short shelf-life. The radioactivity of the isotopes used in RIA decreases over time due to decay. This means that the radiolabeled antigens or antibodies have a limited period of usability and need to be replaced frequently. This can affect the accuracy and reproducibility of the results and increase the cost of the experiments.

These limitations of RIA can be overcome by using alternative techniques such as enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), or chemiluminescence immunoassay (CLIA) that do not involve radioactive substances but use enzymes, fluorescent dyes, or chemiluminescent substrates instead. These techniques have their own advantages and disadvantages, but they can offer similar sensitivity and specificity as RIA without the risks and challenges associated with radioactivity.

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