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Importance of ELISA Experiment

To detect and measure the concentration of an analyte (usually antibodies, antigens, hormones, proteins or peptides) in solutions.

ELISA Experiment Method

Sandwich ELISA.

ELISA Experiment: Theoretical Background

ELISA begins with a coating step, where the first layer either an antigen or an antibody is absorbed to a polystyrene 96 well plate. Coating is followed by blocking and detection steps and finally reading the results on ELISA reader.

There are four basic ELISA formats, allowing for a certain amount of flexibility which can be adjusted based on the antibodies available, the results required, or the complexity of the samples:

• Direct ELISA.
• Indirect ELISA.
• Sandwich ELISA.
• Competition or Inhibition ELISA.

ELISA is one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng.

Sandwich ELISA Uses

Sandwich ELISA uses are very important in many fields like: Food allergens identification Authenticity of the food products corroboration Vaccine Development Measuring and monitoring the changes of the immune response for understanding immune disease.

 

Advantages of Sandwich ELISA

The main advantages of Sandwich ELISA are: The sample does not have to be purified before analysis, so it is suitable for complex samples. Flexibility and sensitivity since both direct and indirect detection methods can be used. High specificity since the antigen is specifically detected and captured.

 

The Sandwich ELISA Principle of Work

The Enzyme Linked Immunosorbent Assay (ELISA) is a common laboratory technique which is used to measure the concentration of the analyte (usually antibodies or antigens) in solutions. The steps of the ELISA result in a colored end product which correlates to the amount of the analytes present in the original sample. In this experiment we will be performing Sandwich ELISA in order to detect the presence of an antigen. Sandwich ELISA typically requires the use of matched antibody pairs, where each antibody is specific for different, non-overlapping parts of the antigen molecule. The first antibody termed the capture antibody is coated to the plate. Next, the analyte or sample solution is added to the well. A second antibody layer, the detection antibody, follows this step in order to measure the concentration of the analyte. If the detection antibody is conjugated to an enzyme, then the assay is called a direct sandwich ELISA. If the detection antibody is unlabeled, then a second detection antibody will be needed resulting in an indirect sandwich ELISA.