Skip to main content

Radioimmunoassay (RIA)

 INTRODUCTION

Radioimmunoassay (RIA) stands as a remarkably sensitive laboratory method employed to quantify specific molecules, such as cytokines, within biological specimens.


Principle of RIA:
RIA hinges on the competition between a radioactively labeled antigen (of known quantity) and an unlabeled antigen (found in the sample) for binding to a limited number of specific antibodies.

Radioactive Labeling:
A measured amount of the cytokine of interest gets tagged with a radioactive isotope, like I-125. This labeled cytokine serves as a tracer in the assay.

Antibody Preparation:
Polyclonal or monoclonal antibodies that specifically target the cytokine are generated. These antibodies exhibit a high affinity and specificity for the target cytokine.

Sample Preparation:
The sample (e.g., serum or plasma), containing an unknown concentration of the cytokine, is introduced into the assay tube.

Addition of Labeled Antigen:
A known quantity of the radioactively labeled cytokine is added to the assay tube containing the sample. This labeled antigen competes with the endogenous (sample) cytokine for antibody binding.

Incubation:
The assay tube is then incubated to allow for competition between the labeled antigen and endogenous cytokine for antibody binding. This step ensures that equilibrium is attained.

Separation of Bound and Unbound Antigen:
Following incubation, the contents of the tube undergo a separation step, often involving a solid-phase support (e.g., precipitating agent or beads coated with anti-immunoglobulin antibodies). This separation step distinguishes between the bound (antibody-bound) and unbound (free) antigen.

Washing:
Unbound labeled antigen is removed from the tube through a series of washes. This step eliminates any excess tracer that has not bound to antibodies.

Radioactivity Measurement:
The radioactivity of either the bound or unbound fraction is gauged using a gamma counter. This provides a quantitative measure of the amount of labeled antigen in the sample.

Calculating Cytokine Concentration: The cytokine concentration in the sample is inversely proportional to the measured radioactivity. Higher radioactivity implies a lower cytokine concentration in the sample.

Calibration Curve:
To determine cytokine concentration, a calibration curve is established using known cytokine concentrations (standards). These standards undergo the same assay procedure as the samples.

Sensitivity and Specificity
RIA boasts high sensitivity, thanks to the use of radioactive tracers, which enables the detection of minute cytokine concentrations. The assay's specificity is contingent on the quality of the antibodies employed.

Safety Considerations:
Given the use of radioactive isotopes, RIA mandates specialized handling, storage, and disposal procedures to ensure safety.

Applications:
RIA finds widespread utility in both research and clinical laboratories for quantifying cytokines, hormones, and other small molecules in various biological samples.

Advantages and Limitations:
Advantages encompass its high sensitivity and capability to measure low cytokine concentrations. Limitations encompass the necessity for specialized equipment, safety precautions, and regulatory considerations due to the use of radioisotopes.
Labels:

Comments

Post a Comment

Popular posts from this blog

Active Transport

  Active Transport Active transport is a vital biological process that enables cells to move ions and molecules against their concentration gradients, from regions of lower concentration to regions of higher concentration. This lecture will explore the principles, mechanisms, and importance of active transport in various physiological processes. Key Concepts of Active Transport Energy Requirement : Active transport requires energy input, usually in the form of adenosine triphosphate (ATP) or a proton gradient generated by primary active transport. This energy is used to move substances against their concentration gradients. Ion Pumps and Transporters : Active transport is carried out by specialized proteins known as ion pumps or transporters. These proteins actively move ions and molecules across cell membranes or within cellular compartments. Concentration Gradients : Active transport serves to maintain or establish concentration gradients of specific ions or molecules. These grad...
Post a Comment
Read more

Metabolism of Carbohydrates QnA

Short Questions and answers of Metabolism of Biomolecules   Topic - Carbohydrate metabolism : Glycolysis  and its regulation 1. What is glycolysis?    Answer: Glycolysis is a fundamental metabolic pathway in which glucose is broken down into two molecules of pyruvate, generating ATP and NADH in the process. 2. Where does glycolysis take place in the cell.    Answer: Glycolysis occurs in the cytoplasm of the cell. 3. What are the main substrates and products of glycolysis? Answer: The substrates of glycolysis are glucose, and the products are two molecules of pyruvate, two molecules of NADH, and a net gain of two ATP molecules. 4. What is the role of ATP in glycolysis?    Answer: ATP is both consumed and generated in glycolysis. Two ATP molecules are used in the early steps of glycolysis, and four ATP molecules are produced, resulting in a net gain of two ATP molecules. 5. What is the significance of NADH in glycolysis?   ...
Post a Comment
Read more

Concepts of Biomolecules

Introduction Biomolecules are the molecules that make up the essential components of living organisms. They play crucial roles in various biological processes and are fundamental to life itself. Classification of Biomolecules Biomolecules can be broadly categorized into four major groups: Carbohydrates : Carbohydrates are organic compounds composed of carbon, hydrogen, and oxygen in a 1:2:1 ratio (CH2O). They serve as a primary source of energy and provide structural support in cells. Examples include glucose, cellulose, and glycogen. Lipids : Lipids are hydrophobic molecules that include fats, oils, and phospholipids. They function as energy storage molecules, cell membrane components, and signalling molecules. Examples include triglycerides, phospholipids, and cholesterol. Proteins : Proteins are complex macromolecules composed of amino acids. They have diverse functions, including enzyme catalysis, structural support, transport, and signaling. Examples include enzymes, antibodies, a...
Post a Comment
Read more