ELISA Protocol
TThe Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used technique for detecting and quantifying antigens or antibodies in a sample. This highly sensitive assay relies on the specific interaction between an antibody and its corresponding antigen. ELISA can be applied to various sample types, including serum, plasma, and cell culture supernatants, making it a versatile tool in scientific research and diagnostics.
In this protocol, we describe a general procedure for performing a sandwich ELISA, which is commonly used for the detection of proteins or biomarkers. The protocol can be easily adapted for different sample types or detection methods, depending on the specific requirements of the experiment. The assay consists of several key steps, including the immobilization of a capture antibody, sample binding, detection by a secondary antibody, and signal generation using a substrate. This guide provides a standardized approach to ELISA for consistent and reliable results across different applications.
Standard ELISA Protocol
Materials
Materials:
• Coating buffer: Carbonate-bicarbonate buffer (pH 9.6)
• Blocking buffer: 1% BSA in PBS or a suitable blocking buffer
• Wash buffer: PBS with 0.05% Tween-20 (PBST)
• Capture antibody (for sandwich ELISA)
• Detection antibody (for sandwich ELISA)
• Sample: Serum, plasma, or cell culture supernatant
• Substrate solution: TMB (3,3',5,5'-Tetramethylbenzidine)
• Stop solution: 1M H2SO4 or 2N HCl
• Microplate reader (450 nm)
• ELISA plate (96-well)
Procedure
1. Plate Coating:
• Dilute the capture antibody in the coating buffer to the desired concentration (typically 1-10 µg/mL).
• Add 100 µL of the diluted capture antibody to each well of the 96-well plate.
• Incubate the plate overnight at 4°C or 2 hours at room temperature (RT).
2. Washing:
• Wash the plate 3 times with 300 µL/well of PBST (phosphate-buffered saline with 0.05% Tween-20). Tap the plate on absorbent paper to remove excess liquid between washes.
3. Blocking
• Add 200 µL of blocking buffer to each well to block non-specific binding sites.
• Incubate for 1-2 hours at RT.
4. Sample Addition
• Dilute the samples and standards in the appropriate buffer (dilution may vary depending on the sample).
• Add 100 µL of diluted sample or standard to each well.
• Incubate for 1-2 hours at RT.
5. Washing
• Wash the plate 3 times with PBST as described above.
6. Detection Antibody Addition
• Dilute the detection antibody in the blocking buffer.
• Add 100 µL of the detection antibody solution to each well.
• Incubate for 1 hour at RT.
7. Washing
• Wash the plate 3 times with PBST.
8. Substrate Addition
• Add 100 µL of TMB substrate solution to each well.
• Incubate the plate in the dark at RT for 15-30 minutes or until the desired color intensity develops.
9. Stop Reaction
• Add 50 µL of stop solution (1M H2SO4 or 2N HCl) to each well to stop the reaction.
• The color will change from blue to yellow.
10. Reading
• Measure the absorbance of each well at 450 nm using a microplate reader.
Algent ELISA Kits
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