Frequently Asked Questions

What is a primary antibody?

Primary antibodies are antibodies used in detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease. Primary antibodies bind to target proteins, antibodies or antigens.

What is a secondary antibody?

Antibody that attaches to the primary antibody to detect target antigen is referred as secondary antibody. Secondary antibody have specificity for isotype of primary antibody and often is conjugated. Technically, the secondary antibody’s FAB domain binds to primary antibody’s Fc domain.

Secondary antibodies find application in ELISA, western blot, Immunostaining, Immunohistochemistry and Immunocytochemistry studies.

What is an antibody conjugate?

Conjugated antibody is a monoclonal or polyclonal antibody linked to probe enzyme or agents. It helps in detection in wide range of assay techniques. Also, conjugated antibodies help in detection, purification and microscopy application.

What is the difference between western blot and ELISA?

Both the tests measure immune system’s response to infectious agents. But, ELISA is less sophisticated and cost effective compared with Immunoblotting. The advantage of the Western Blot over the ELISA test is that far more indicators are examined than the single antigen in ELISA.

 What is a western blot?

Western blot is also a detection assay. Unlike ELISA, viral proteins are separated, immobilized and visualized. The proteins are collected into a gel slab and electric current is passed through them. Different proteins based on their size move at different velocities. Once these proteins are segregated by size, the procedure continue like ELISA.

What is ELISA test used for?

ELISA is used quantifying antibody/antigen concentration in viral tests, and as a consumer product ELISA finds application in home pregnancy tests. It is also used in detecting potential food allergens in food products such as milk, peanuts, almonds and eggs. ELISA is used widely in the fields of Immunology, Toxicology and Diagnostics.

Is ELISA test reliable?

As a biochemical assay, ELISA is reliable and effective in detecting antibodies/antigens. In some medical cases, the positive or negative results are always confirmed by Western Blotting. This is mainly done to confirm the test results.

What is the Difference between Direct and Indirect ELISA?

In direct ELISA, the method of antigen mobilization is not specific. When serum is used as the source of target antigen, the proteins in the sample may stick to microtiter plate well and analyte in serum must compete with other proteins to bind with the well surface. This problem is eliminated by using secondary antibody specific for the test antigen in Indirect or sandwich ELISA. This is the key difference between direct and Indirect ELISA.

What is the Difference between Sandwich ELISA and Competitive ELISA?

Competitive ELISA is known for precision and reproducibility whereas Sandwich ELISA is known for sensitivity and specificity. The competitive ELISA is more attractive if no antibody pair can be identified for sandwich ELISA, and when Analyte is too small to bind with a primary and secondary antibody.

What are the variants of Sandwich ELISA?

Sandwich ELISA is known for specificity and sensitivity, but the inherent disadvantage lies in finding antibody pairs to carry out the assay. If the detection antibody is conjugated with an enzyme, the assay is called direct sandwich ELISA and if the secondary antibody is unlabelled, the need for an antibody enzyme conjugate arises, these assays are called Indirect Sandwich ELISA.

What is the difference between Indirect and Sandwich ELISA?

The most popular od ELISAs, Indirect ELISA uses a secondary antibody that binds with primary antibody for detection. The secondary antibody has specificity for primary antibody.

The most powerful in terms of specificity and sensitivity, Sandwich ELISA captures the analyte between two primary antibodies – the capture and detection antibody.

Why is PBS used in ELISA?

Phosphate Buffered Saline is a liquid formulation of buffers and saline. It is used in ELISA procedure to balance PH without disrupting protein binding sites.

What are the commonly used substrates for ELISA?

Horseradish peroxide, Alkaline Phosphatase, β-Galactosidase and Urease. Substrates allow direct visualization and enable kinetic studies.

What is a chromogenic reporter?

Substrates that allow direct visualization and enable kinetic studies are chromogenic reporters. They are far less sensitive compared with fluorescent or chemiluminescent substrates. The detection is assisted with standard absorbance plate readers common to many laboratories. The ease with which one can use chromogenic reporters make them a popular choice as reporting agents.

What are the factors that could affect signal generation in ELISA?

Shape and material of the plate, pH of the buffer, specificity of the capture antibody, incubation time and temperature, conformation and stability of target antigen, specificity, affinity and cross reactivity of detection antibody, concentration and cross reactivity of enzyme conjugate, sensitivity and age of the substrate, and filters and exposure time of the imaging instrument.

Can non-purified antibody be used in ELISA?

Non-purified antibody can be used but may result in higher background. It is highly recommended that purified antibodies be used for optimal signal to noise ratio.

What are some commonly used coating and detection antibodies?

Polyclonal serum, crude ascites, affinity purified monoclonal and affinity purified polyclonal antibody.

What are the recommended secondary antibody concentrations for ELISA?

For Horseradish peroxide, the concentration for colorimetric system should be 20 – 200 ng/ml, the concentration for fluorescent system should be 25 – 50 ng/ml, 10-100 ng/ml for chemiluminescent system.

For Alkaline Phosphatase, 100 – 200 ng/ml for colorimetric system and 40 – 200 ng/ml for chemiluminescent system.

How long does it take to run ELISA?

The assay can be run in less than 3 hours in any formats.