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In the laboratory setup for enzyme-linked immunosorbent assay (ELISA), the primary objectives are to determine the presence and quantify the amount of a specific protein in a given sample. ELISA is a highly sensitive immunoassay that utilizes an enzyme linked to an antibody or antigen as a marker for the detection of the target protein, which can be either an antigen or an antibody.
Principle of ELISA:
The fundamental principle of ELISA involves the use of an enzyme-linked reagent to detect the analyte in a liquid sample.
This can be carried out using either a wet lab approach with liquid reagents or a dry lab approach with dry strips. In the dry analysis, the strip can be read through reflectometry, and quantitative readings are typically based on the detection of the intensity of transmitted light by spectrophotometry at a specific wavelength.
The sensitivity of detection in ELISA relies on signal amplification during the analytic reaction. Enzymatic reactions generate signals, and these enzymes are linked to the detection reagent in fixed proportions to ensure accurate quantification.
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This is known as enzyme-linked, a crucial aspect of ELISA that enhances the precision and sensitivity of the assay.
Variations in ELISA:
There are two main variations of the ELISA method, each serving a different purpose:
In this case, the assay involves detecting the antibodies present in the sample that bind to the specific antigen.
Laboratory Setup:
1. Materials and Reagents:
2. Sample Preparation:
3. Coating the Microplates:
4. Blocking:
5. Antibody Incubation:
6. Secondary Antibody Addition:
7. Substrate Reaction:
8. Stop Reaction:
9. Reading and Calculation:
Calculations and Formulas:
1. Standard Curve:
2. Sensitivity Calculation:
3. Precision Analysis:
The ELISA laboratory described above provides a systematic approach to detect and quantify specific proteins in a sample. The use of enzyme-linked antibodies or antigens, along with meticulous sample preparation and standard curve analysis, ensures the accuracy and sensitivity of the assay. By following these steps, researchers can confidently determine the presence and concentration of the target protein in a given sample, making ELISA a powerful tool in molecular biology and diagnostics.
Enzyme-linked immunosorbent assay (ELISA), also referred to as enzyme immunoassay (EIA), is a biochemical method widely employed in immunology to identify the presence of antibodies or antigens in a sample. This technique has found applications in medical diagnostics, plant pathology, and quality control across various industries, including its use in the food industry. In essence, ELISA involves affixing an unknown amount of antigen to a surface, followed by the application of a specific antibody that binds to the antigen. This antibody is coupled with an enzyme, and a substance is introduced in the final step, which the enzyme transforms into a detectable signal, often resulting in a color change in a chemical substrate.
Before the advent of ELISA, the primary method for immunoassays was radioimmunoassay, utilizing radioactively labeled antigens or antibodies. Due to potential health risks associated with radioactivity, a safer alternative was sought. Enzymes, such as peroxidase, reacting with suitable substrates, offered a nonradioactive signal. The linkage of enzymes to antibodies in this process was independently developed by Stratis Avrameas and G. B. Pierce. To prepare the immunosorbent, where unbound antibody or antigen needs removal through washing, a technique published by Wide and Jerker Porath in 1966 was crucial.
In 1971, Peter Perlmann and Eva Engvall in Sweden, along with Anton Schuurs and Bauke van Weemen in the Netherlands, published papers synthesizing this knowledge into methods for performing EIA/ELISA. Traditional ELISA involves chromogenic reporters and substrates that result in observable color changes to indicate the presence of the antigen. More recent variations use fluorogenic, electrochemiluminescent, and quantitative PCR reporters for quantifiable signals, offering advantages like increased sensitivity and multiplexing. While technically different, these newer assays are often grouped with ELISAs due to similar underlying principles.
In 2012, an ultrasensitive enzyme-based ELISA test using nanoparticles as a chromogenic reporter demonstrated the capability to provide a naked-eye color signal even for minuscule amounts of analyte. The process involves immobilizing at least one antibody with specificity for a particular antigen on a solid support. The sample, containing an unknown amount of antigen, is then immobilized on the support, either nonspecifically or specifically in a "sandwich" ELISA. Following antigen immobilization, the detection antibody is added, forming a complex. This detection antibody may be covalently linked to an enzyme or detected by a secondary antibody linked to an enzyme through bioconjugation. Washing steps between each incubation remove non-specifically bound proteins or antibodies. The final step involves developing the plate by adding an enzymatic substrate, generating a visible signal indicating the quantity of antigen in the sample.
In this laboratory setup, we are conducting an Enzyme-Linked Immunosorbent Assay (ELISA) to quantify the concentration of Antigen 1 (BSA) in various samples. The materials and methods provided guide us through the experimental procedures, including the preparation of reagents, sample incubation, and the subsequent analysis of absorbance readings. The calculations involve plotting standard curves, determining the best time point, and calculating the concentration of the antigen in the experimental samples.
Materials:
Sample Preparation: Transfer 100 μl of all samples to a microtiter plate, avoiding the wells closest to the edge. Allow the plate to stand at room temperature overnight without letting the wells dry.
Coating ELISA Plate: Wash the coated ELISA plate three times in washing solution (PBST). Add 200 μl/well of blocking solution (0.5% gelatin in PBS) and incubate at room temperature for 30 minutes.
Antibody Incubation: Wash the coated ELISA plate again and add 100 μl of anti-BSA antibody diluted 1/2000 in incubation buffer (PBST). Incubate at room temperature for 30 minutes.
Conjugate Incubation: Wash the coated ELISA plate once more and add 100 μl of Alkaline-Phosphatase conjugated goat anti-rabbit antibody diluted 1/1000 in incubation buffer (PBST). Incubate at room temperature for 30 minutes.
Substrate Reaction: After washing the plate, add 100 μl of enzyme substrate solution to all treated wells. Read the absorbance at 405 nm using an ELISA plate reader.
Calculations:
Standard Curve: Plot the absorbance against the log dilution of BSA to create a calibration curve for all time points from the ELISA results.
Optimal Time Point: Choose the time point with a smooth negative sigmoidal curve from the standard curve and create a graph for its respective absorbance to log concentration values, resulting in a smooth positive sigmoidal curve.
Slope Calculation: Calculate the slope for the Absorbance vs. Log concentration graph. This slope is crucial for determining the concentration of the antigen in the experimental samples.
Concentration Calculation: Use the calculated slope to determine the concentration of antigen in the experimental samples. Apply the mean concentration calculation for all dilutions at the selected time point.
In the laboratory analysis, we first focus on the materials, ensuring that each component is prepared and handled correctly. The microtiter plate acts as the platform for sample incubation, with precise volumes of antigens, antibodies, and conjugates added at each step. The washing steps with PBST are crucial for removing unbound components and ensuring the specificity of the assay.
Moving to the calculations, the standard curve is a fundamental aspect, providing a reference for the concentration of the antigen in the samples. The sigmoidal curves depict the relationship between absorbance and log concentration, guiding us to select the optimal time point for accurate measurements. The slope calculation is central to translating absorbance values into concentration units, ensuring the reliability of the assay.
In the results, a clear presentation of the standard curve, optimal time point graph, and concentration calculations using the slope should be illustrated. Tables can be employed to tabulate absorbance readings, dilution factors, and final concentrations. An Excel sheet is recommended for plotting the graphs and performing calculations systematically.
In conclusion, this ELISA laboratory serves as a comprehensive guide for researchers and technicians aiming to quantify antigen concentrations in various samples. The detailed steps, materials, and calculations contribute to the accuracy and reliability of the assay, making it a valuable tool in immunology and diagnostic research.
Mastering ELISA: A Comprehensive Guide to Antigen Quantification and Detection. (2024, Feb 25). Retrieved from https://studymoose.com/document/mastering-elisa-a-comprehensive-guide-to-antigen-quantification-and-detection
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