ELISA and Western Blotting Notes

ELISA (Enzyme-Linked Immunosorbent Assay)

  • A widely used biochemical assay to detect the presence and quantity of proteins (e.g., hormones, antibodies, bacteria, viruses) in a sample.

  • Relies on the coupling of antigens and antibodies, utilizing the specificity and affinity of antibodies for antigens.

    • Specificity: Ability to discriminate among diverse proteins.

    • Affinity: Ability to tightly bind to molecules.

  • Can determine the amount of antibody by starting with an antigen, or determine the amount of antigen/hormone by starting with an antibody.

Antibodies

  • Large glycoprotein molecules produced by B-lymphocytes during the humoral immune response to antigens introduced into the body (adaptive immunity).

  • Lymphocytes include B-lymphocytes (B-cells) and T-lymphocytes (T-cells), which are white blood cells formed from hematopoietic (blood) stem cells in the bone marrow.

Antibody-Antigen Interaction

  • Antibodies bind to antigens following the lock-and-key model; antigens bind to the receptor sites.

  • Secondary antibodies can also bind to antibodies following the same principle, either at the receptor site or the tail part.

The Name ELISA Explained

  • Antigen/antibody of interest is adsorbed on a plastic surface (sorbent).

  • Antigen is recognized and binds to a specific antibody (immuno).

  • The antibody is recognized by the second antibody, which has an enzyme attached (enzyme-linked).

  • Substrate reacts with the enzyme to produce a product, usually colored.

  • ELISA has been used to detect hepatitis B, rabies, and HIV through antibodies in blood serum, and to measure amounts of hormones, toxins, and allergens.

Indirect ELISA (Detect antibodies in the sample)

  • a) Binding Known Antigen: Known antigen bound to wells of a microtiter plate.

  • b) Blocking: Unoccupied sites in each well are bound by a concentrated solution of non-interacting protein (e.g., casein or bovine serum albumin) to prevent other proteins in the test sample from adhering.

  • c) Washing: Rinse to remove unbound antigen and non-interacting protein.

  • d) Adding Test Sample Primary Antibody: Test sample of serum containing primary antibodies (e.g., HIV, rabies, or hepatitis B antibodies) is added to each well.

  • e) Washing: Rinse to remove antibodies that did not bind to the known antigen.

  • f) Adding Enzyme-linked Secondary Antibody: An enzyme-linked secondary antibody is added to bind to the test sample antibodies; enzymes on the secondary antibodies are proteins such as horseradish peroxidase or alkaline phosphatase.

  • g) Washing: Rinse to remove any secondary antibodies that did not bind to the primary antibody.

  • h) Adding Substrate: A substrate is applied, which is converted by the enzyme to give a color, fluorescence, or electrochemical signal. TMB turns blue in the presence of horseradish peroxidase.

  • i) Reading Results: Using a spectrophotometer, spectrofluorometer, or electrochemical device, results are read and recorded. The amount of color produced is proportional to the amount of primary antibody bound to the antigen proteins on the bottom of the wells.

  • In ELISA, TMB (3,3',5,5'-Tetramethylbenzidine) acts as a chromogenic substrate and is highly sensitive for HRP. When added to HRP, HRP reduces hydrogen peroxide and oxidizes TMB, turning it from colorless to blue-green.

Reading Results of ELISA

  • Spectrophotometers (microplate readers) are commonly used to read ELISA results.

Direct ELISA vs. Indirect ELISA

  • Indirect ELISA has the advantage of signal amplification.

Sandwich ELISA (Detect antigens in the sample)

  • Most commonly used format.

  • Requires two antibodies specific for different epitopes of the antigen (matched antibody pairs).

  • Highly specific, minimizes false positives.

ELISA Advantages and Disadvantages

  • Advantages:

    • High sensitivity and specificity: Can detect antigens at the picogram level.

    • High throughput: Commercial ELISA kits are available in 96-well plate format but can be adapted to 384-well plates.

    • Easy to perform: Protocols are easy to follow and involve little hands-on time.

    • Quantitative: Can determine the concentration of antigen in a sample.

    • Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine, and saliva, among others.

  • Disadvantages:

    • Temporary readouts: Detection is based on enzyme/substrate reactions, requiring readout in a short time span.

    • Limited antigen information: Information is limited to the amount or presence of the antigen in the sample.

ELISA as Test Kits

  • ELISA tests are widely available as test kits such as pregnancy tests, which detect Human Chorionic Gonadotropin (hCG).

Blots

  • Techniques for transferring DNA, RNA, and proteins onto a carrier for separation, often following gel electrophoresis.

    • Southern blot – DNA

    • Northern blot – RNA

    • Western blot – proteins (immunoblot)

    • Eastern blot – post-translational proteins

  • Protein blotting is an analytical method that involves immobilizing proteins on membranes before detection using antibodies.

  • SDS-PAGE technique is a prerequisite for Western blotting.

SDS-PAGE

  • SDS-PAGE (Polyacrylamide Gel Electrophoresis) is an electrophoresis method that allows protein separation by mass.

  • Sodium dodecyl sulfate (SDS) is a negatively charged detergent used to denature and linearize proteins.

SDS-PAGE Details

  • Application of an electric field causes the migration and separation of proteins.

  • Polyacrylamide is used to form a gel that provides a matrix of pores through which molecules migrate at different rates.

Visualization of Protein Bands

  • Visualize the band under UV light.

  • Visualize by staining:

    • Coomassie Blue (most common)

    • Silver stain (most sensitive)

Western Blot - Transferring

  • To make proteins accessible to antibody detection, they are transferred onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF).

  • Transfer methods:

    • Diffusion transfer

    • Electro transfer