Immunoassays and ELISA Overview

Immunoassay Auto Analyzer

• Immunoassay is a biochemical test that determines the presence or concentration of a large molecule or small molecule in a solution using an antibody or antigen.
• An immunoassay auto analyzer is a lab instrument that automates many manual immunoassay techniques.

ELISA Technique

• ELISA stands for enzyme-linked immunosorbent assay.
• It's a plate-based technique used to detect and measure soluble substances like peptides, proteins, antibodies, and hormones.
• The technique relies on the highly specific interaction between antigens and antibodies.

Types of ELISA Assays

• ELISA tests are categorized based on the antigen-antibody binding methods:
  • Direct ELISA: Antibody is coated onto the microtiter well.
  • Indirect ELISA: Antigen is coated onto the well, followed by antibody binding.
  • Sandwich ELISA: Primary antibody is coated, then antigen binds, followed by a secondary antibody binding to the antigen.
  • Competitive ELISA: Antigen competes with labeled antigen to bind to the antibody coated on the well.

Direct ELISA

• This Sandwich ELISA variant detects antigens in a sample.
• The microtiter well is coated with antibody.
• Sample containing antigen is added and unbound antigen is removed.
• Enzyme-specific substrate is added, yielding a measurable colored product.

Indirect ELISA

• Indirect ELISA identifies antibodies in a sample.
• Antigen is coated onto the microtiter well.
• Sample containing antibodies is added to bind to the antigen.
• Enzyme-conjugated secondary antibody is used to detect the antigen-antibody complex.
• Specific substrate creates a measurable colored product.

Sandwich ELISA

• This ELISA type detects antigens through a dual antibody capture.
• A coated antibody on the well binds the antigen.
• An enzyme-linked secondary antibody binds to another epitope on the antigen.
• The enzyme substrate produces a measurable colored product

Competitive ELISA

• This ELISA detects antigen concentration by competition.
• The microtitre well is coated with antigen.
• Sample containing antibodies and antigen incubate together.
• The amount of free antibody is inversely correlated to the concentration of antigen in the sample.

ELISA Test Requirements

• Purified antigen or antibody
• Standard solutions (positive and negative controls)
• Sample to be tested
• Microwell plates
• Wash buffer
• Enzyme-labeled antibody and substrate

Enzymes used in ELISA

• Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are commonly used.

ELISA Reader Components

• ELISA reader is a sophisticated instrument to detect proteins in a liquid sample.
• The system components include:
  • ELISA plate
  • Light source
  • Optical system
  • Optic fiber

ELISA Results and Interpretation

• ELISA result is expressed with OD450 values and a correlation is used to quantify the amount of antigen or antibody. The concentration of antigen in the sample is shown in a graph.
• The value is plotted against the known concentrations of positive and negative solutions.

Explore the basics of immunoassays and the ELISA technique through this quiz. Learn about the different types of ELISA assays and how immunoassay auto analyzers enhance laboratory efficiency. Test your knowledge on these essential biochemical testing methods.