ELISA Techniques PDF

ELISA by: dr. sura zeki Dr. sausan ELISA *ELISA is an antigen antibody reaction *It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens in blood. *ELISA is a plate based assay technique which is used for detecting and quantifying substance...

ELISA by: dr. sura zeki Dr. sausan ELISA *ELISA is an antigen antibody reaction *It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens in blood. *ELISA is a plate based assay technique which is used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. Principle Enzyme immunoassays (EIAs) use the catalytic properties of enzymes to detect and quantify immunologic reactions. Enzyme-linked immunosorbent assay (ELISA) is a heterogeneous EIA technique used in clinical analyses. In this type of assay, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates the separation of bound and free-labeled reactants Specific antibodies in a sample can also be quantified using an ELISA procedure in which antigen instead of antibody is bound to a solid phase. The second reagent is an enzyme-labeled antibody specific to the analyte antibody. In addition, ELISA assays have been used extensively to detect antibodies to viruses and autoantigens in serum or whole blood. In addition, enzyme conjugates coupled with substrates that produce visible products have been used to develop ELISA-type assays with results that can be interpreted visually. Such assays are very useful in screening, point-of-care, and home testing applications Types of ELISA There are four major types of ELISA: 1. Direct ELISA (antigen-coated plate; screening antibody) 2. Indirect ELISA (antigen-coated plate; screening antigen/antibody) 3. Sandwich ELISA (antibody-coated plate; screening antigen) 4. Competitive ELISA (screening antibody) Direct ELISA In a direct ELISA, the primary detection antibody binds directly to the protein of interest. the plate is rewashed to remove any unbound antibodies. An enzyme, such as alkaline phosphatase (AP) or horseradish peroxidase (HRP), is added to the plate, which results in a color change. The color change of the sample occurs by either the hydrolysis of phosphate groups from the substrate by AP or by the oxidation of substrates by HRP. The advantages of using direct ELISA include eliminating secondary antibody cross-reactivity, and due to fewer steps, it is rapid compared to indirect ELISA. Its disadvantages include its low sensitivity compared to the other types of ELISA and its high cost of reaction. 2. Indirect ELISA Indirect ELISA requires two antibodies: a primary detection antibody that sticks to the protein of interest and a secondary enzyme-linked antibody complementary to the primary antibody. The primary antibody is added first, followed by a wash step, and then the enzyme-conjugated secondary antibody is added and incubated. After this, the steps are the same as the direct ELISA, which includes a wash step, the addition of substrate, and the detection of a color change. The indirect ELISA has a higher sensitivity when compared to the direct ELISA. It is also less expensive and more flexible due to the many possible primary antibodies that can be used. The only major disadvantage of this type of ELISA is the risk of cross-reactivity between the secondary detection antibodies.[ 2. Sandwich ELISA Antigen can be detected by sandwich ELISA. In this technique, antibody is coated on the microtiter well. A sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming antigen-antibody complex. 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. The major disadvantages of this type of ELISA are the time and expense and the necessary use of “matched pair” (divalent/multivalent antigen) and secondary antibodies.  3. Competitive ELISA The competitive ELISA tests for the presence of an antibody specific for antigens in the test serum. This type of ELISA utilizes two specific antibodies: an enzyme-conjugated antibody and another antibody present in the test serum (if the serum is positive). Combining the two antibodies into the wells will allow for competition for binding to antigens. The presence of a color change means that the test is negative because the enzyme-conjugated antibody bound the antigens (not the antibodies of the test serum). The absence of color indicates a positive test and the presence of antibodies in the test serum. The competitive ELISA has a low specificity and cannot be used in dilute samples. However, the benefits are that there is less sample purification needed, it can measure a large range of antigens in a given sample, it can be used for small antigens, and it has low variability.. Diagnostic Tests 1. Detect and Measure the Presence of Antibodies in the Blood Hepatitis A, B, C, HIV, etc. 2. Detect and Estimate the Levels of Tumor Markers Prostate-specific antigen (PSA) 3. Detect and Estimate Hormone Levels Luteinizing hormone ,Follicular stimulating hormone 4. Tracking Disease Outbreaks, Cholera, HIV 5. Detecting Past Exposures, HIV, Lyme disease, Hepatitis 6. Screening Donated Blood for Possible Viral Contaminants Anti-HIV-1/2, Anti-HCV, HBsAg 7. Detecting Drug Abuse Amphetamine, Cocaine Interfering Factors 1. Plate Assay: the shape and quality of the wells, the material of the plate, potential pre- activation, and even or uneven coating. 2. Buffer: pH, contamination. 3. Capture and detection antibody: incubation time, temperature, specificity, titer, affinity. 4. Blocking buffer: cross-reactivity, concentration, contamination. 5. Target antigen: conformation, stability, epitopes. 6. Enzyme conjugate: type, concentration, function, cross-reactivity. 7. Washes: contamination, frequency, volume, duration, composition. 8. Substrate: quality/manufacturer. 9. Detection: instrument-dependent factors 10. Reader/human error. Thank you