Introduction The Enzyme-Linked Immunosorbent Assay (ELISA) technique is one of the most important and widely used immunological methods in clinical laboratories, disease diagnosis, and biological research. This technique is characterized by high sensitivity, accuracy, and the ability to measure the concentration of various biomolecules, such as hormones, antibodies, proteins, cytokines, and viruses. The principle of ELISA is based on the binding of an antigen to an enzyme-linked antibody, which generates a colorimetric signal measured by an absorbance reader. First: Principle of ELISA The technique relies on the specific interaction between an antigen and an antibody, where one of them is immobilized on the surface of a specialized microplate containing 96 wells. After adding a detection reagent containing an enzyme-linked antibody, the substrate is converted into a color product due to the enzymatic reaction. The intensity of the color is directly proportional to the concentration of the target biomolecule in the sample. Second: Types of ELISA 1. Indirect ELISA Used to measure antibodies in blood. Steps: · Coating the antigen onto the wells. · Adding the sample containing the antibodies. · Adding a secondary enzyme-linked antibody. · Color development. 2. Direct ELISA Uses a single enzyme-linked antibody directly. Advantageous for speed but less sensitive. 3. Sandwich ELISA Most commonly used for measuring hormones, cytokines, and proteins. Employs two antibodies that bind to the antigen from both sides (capture and detection). Known for high accuracy and sensitivity. 4. Competitive ELISA Used for small molecules such as hormones or drugs. Based on the principle of competition between the target substance and a standard for antibody binding. Third: Steps of Performing an ELISA Test · Preparation of the microplate (coating) and immobilization of the antibody or antigen. · Addition of the sample and incubation to allow binding. · Washing the wells to remove unbound materials. · Addition of the enzyme-linked antibody. · Addition of the substrate, such as TMB. · Reading the absorbance using an ELISA reader at a specific wavelength. · Calculating the concentration based on a standard calibration curve. Fourth: Advantages of ELISA · High sensitivity and accuracy. · Rapid results. · Capability to measure multiple samples simultaneously. · Low cost compared to molecular techniques. · Applicability in diagnosing hormones such as LH, FSH, Prolactin, Insulin, and Testosterone, as well as cytokines like OSM and IL-6. Fifth: Applications of ELISA in Medical Diagnosis · Diagnosing viruses such as Hepatitis and HIV. · Measuring hormones in endocrine disorders. · Assessing inflammation via cytokines. · Diagnosing food allergies via IgE. · Measuring tumor markers such as AFP and CA-125. · Monitoring diabetic patients by measuring insulin and C-peptide. Sixth: Factors Affecting the Accuracy of the Test · Efficiency of the washing process. · Purity of the antibodies. · Validity of reagents and enzymes. · Temperature and incubation time. · Quality of the calibration curve. Conclusion The ELISA technique is a cornerstone in medical laboratories due to its ability to quantitatively and qualitatively detect various biomolecules. With the continuous development of highly sensitive reagents and enzymes, this technique has become one of the most important tools in diagnosing immune disorders, hormonal imbalances, infectious diseases, and modern medical research. --- University of Al-Mustaqbal Ranks First Among Iraqi Private Universities