ELISA Assay Solutions & Protocol

ELISA Assay Solutions & Protocol

Enzyme-linked immunosorbent assays (ELISA’s) are plate-based enzyme assays used for identifying and quantifying sample proteins, peptides, antigens, and antibodies that are targets in research of a specific compound. The identification of a specifically targeted protein in an ELISA array is completed by using antibodies to immobilize the protein and detect the presence of the protein targeted. The general ELISA applications are often referred to as a sandwich assay because the measured analyte is between two antibodies. One of the antibodies serves as the capture antibody, while the other is the detection antibody.

How to Perform ELISA Assay

A specific protocol is recommended for standard sandwich ELISA’s. A standard protocol is completed using a 96-well plate for colorimetric detection:

  1. Coat Capture Antibody:
    • Using your starting concentration, add 100 ul capture antibody to appropriate wells
      • Starting Concentration Example: 240 μg/mL --> 1:240 --> 1.0 μg/mL in COAT (1X)
    • Incubate 1 hour at Room Temperature
    • Wash 3X with wash buffer
    • Recommended reagents for this step:
      • BioFX™ Tris Buffered Saline Wash Solution - 10X Concentrate (Product Code:WSHW)
      • BioFX ELISA Plate Coating Solution - 10X Concentrate (Product Code:COAT)
  2. Blocking and Plate Stabilization:
  3. Continue and Finish Assay in Same Day:
    • Aspirate plate or wash with wash buffer
    • Proceed to step 5
    • Recommended reagents for this step:
      • BioFX Tris Buffered Saline Wash Solution - 10X Concentrate (Product Code:WSHW)
  4. Long Term Plate Stability and Storage:
    • Aspirate blocker/stabilizer from wells and blot plate dry to remove residual liquid.
    • Dry plates using methods optimized by end user.
    • Recommended drying methods:
      • Dry in a humidity-controlled chamber (less than 15% humidity) until dry (4-24 hours)
      • Dry 2-4 hours in a sealed desiccated container
      • Dry plates at 30-40C in a vacuum over for 4 hours
      • Package plates in temperature sealed moisture proof pouch with desiccant
  5. Standards, Samples and Controls: (concentrations pre-determined by end user titration):
    • Using your starting concentration, add 100 μL of standards, samples and controls to appropriate wells across plate
    • Incubate at Room Temperature for 2 hours
    • Wash 3X with wash buffer
    • Recommended reagents for this step:
  6. Primary Antibody (concentration pre-determined by end user titration):
    • Using your starting concentration, add 100 μL to all wells on plate
      • Starting concentration example: 2.0 mg/mL --> 1:20,000 --> 100 ng/mL in StabilZyme™ Stabilizer
    • Incubate 1 hour at Room Temperature
    • Wash 3X with wash buffer
    • Recommended reagents for this step:
  7. Secondary HRP/AP Conjugated Antibody (concentration pre-determined by end user titration)
    • Using your starting concentration, add 100 μL to all wells on plate
      • Example Starting Concentration: 2.0 mg/mL --> 1:20,000 --> 100 ng/mL in StabilZyme Conjugate Stabilizer
    • Incubate 1 hour at Room Temperature
    • Wash 3X with wash buffer
    • Recommended reagents for this step:
  8. Detection:

Please contact us here to connect with one of our R&D scientists directly to learn more about our ELISA protocol recommendations and request free samples for evaluation!

ELISA: Enzyme Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay (ELISA) is a method used to identify and quantify substances such as proteins, peptides, antibodies, hormones, and other analytes. ELISA kits have become one of the most used assay techniques for research laboratories, clinical diagnostics companies and more. ELISA is used in other formats such as testing individual samples and automated screening. ELISA is used as a common diagnostic tool for medicine, biotechnology, plant pathology, and quality assurance in many industries.

An assay is completed using a specific antibody which is used to coat a microtiter plate. After the sample is added, a primary antibody will bind the target protein of interest. A secondary conjugated detection antibody will then bind to the primary antibody [TJ3] Reagents that are unbound are removed through washing. Once a substrate is added, a color reaction develops proportional to the protein concentration that is bound. The concentration of the target protein in the sample is determined by comparing to a known concentration of the protein in the standard curve.

The high sensitivity ELISA technology depends on the affinity of the antibodies as well as the system one is using. A standard range for the assay shows the upper and lower limits of analyte concentration. The accuracy of the ELISA is calibrated with an external reference. Standards can be found available from the National Institute of Biological Standards and Control.

Common Questions:

How does an ELISA assay work?

Enzyme-linked immunosorbent assays (ELISA) are similar to other immunoassay technologies. ELISA’s use specific antibodies that bind a specific antigen, and a system indicates the identification and quantification of antigens that are bound. To maximize precision, the plate is carefully coated with highly specific antibodies. The test sample is then added, and if the analyte is present, it will bind to the coated antibody. A matching detection antibody will then detect the presence of the analyte. The antibodies that are not bound are then removed. These unbound antibodies are then washed away. The binding subsequent reaction products are detectable signals.

What is an ELISA test used for?

An ELISA test is used to measure proteins, glycoproteins, antigens, and antibodies in specific biological samples.[TJ6]  Examples of biological samples that use available ELISA tests include HIV infection diagnosis, pregnancy tests, and the measurement of soluble receptors in a serum. 

What is the basic principle of ELISA?

The ELISA principle uses procedures to detect analytes recognized by an antibody in biological specimens. The procedures follow a basic principle including:

  1. Coating: coating solution is used to adsorb protein to plate surface
  2. Blocking: Sites are blocked using a buffer designed to reduce non-specific binding and matrix interferences
  3. Detection Antibody: conjugated protein binds to analyte if present
  4. Substrate Addition: Colorimetric substrate added to wells catalysed by enzyme
  5. Analysis: ELISA reader analyzes data

What is ELISA and its types?

ELISA is an acronym for enzyme-linked immunosorbent assay. This is a biochemical assay used commonly to detect proteins in a liquid sample. The detection antibody is directed against the proteins that are being measured. There are three main types of ELISA, broken down into tests based on how the antibodies and analytes are bonded. 

Sandwich ELISA

Sandwich assays are the most commonly used type of ELISA. This process uses two specific antibodies, which then sandwich the antigen or antibody of interest. These are commonly referred to as matched antibody pairs. The capture antibody is then coated on a microplate, and if present target protein binds to the captured antibody. A conjugated antibody is added and binds to detect the target protein. An ELISA substrate is added, producing a signal proportional to the analyte in the sample. These ELISA are highly specific assays, but often need additional research. This is due to risk for false positive results.

Direct ELISA

The Direct ELISA process is when an antigen or other sample of interest is immobilized directly onto the plate. It is then detected by a conjugated antibody that binds to the target protein. A substrate is then added, which produces a signal proportional to the amount of analyte concentration within the sample. These tests are less specific than the common sandwich ELISA. This is because only one antibody is being used in a direct ELISA.

Competitive ELISA

Competitive assays are typically used for small molecules. This ELISA is used when the target protein is too small to sandwich with two antibodies. A capture antibody is coated on a microplate. Then a conjugated antigen is used to compete with the sample antigen to bind with the capture antibody on the plate. The more antigen that is present in a sample, the less conjugated antigen will bind to the captured antibody. A substrate is then added, so the signal produced is inversely proportional to the amount of protein in the sample.