Question

Summary

An enzyme-linked immunosorbent assay (ELISA) is typically performed to detect the presence and/or amount of a target protein of interest within an experimental sample. Detection of the target protein is made possible by antibodies, which make the ELISA an immunoassay. Through a series of incubation and washing steps, these antibodies, which are frequently linked, or conjugated, to an enzyme, will detect protein coating the bottom of a well on a microtiter plate. When exposed to a substrate, antibody-bound enzyme will cause a color change, thereby indicating the presence of the protein-of-interest in the sample.

In this video, the theory behind how ELISAs work is explained, including a discussion of both primary and secondary antibody binding and the importance of blocking steps. Theory is followed by practice, as the video progresses to an explanation of the step-by-step procedure. Finally, variations of the standard ELISA such as the sandwich and competitive ELISAs are introduced, and real world applications of this method, such as in over-the-counter pregnancy tests are explained.

1. For which of the following applications could an ELISA be used?

- To neutralize trypsin within a sample.

- To determine the size of a plasmid within a sample.

- To determine the presence or absence of contamination within a sample.

- To determine the presence or absence of a specific protein within a sample.

2. The target protein is recognized by...

- ...the substrate.

- ...buffer enzymes.

-...unlabeled viral particles.

-...the primary antibody.

3. The absorbance measured for each well is _____to the amount of target protein present in

each sample. (cell culture media harvested from human anti-body- producing cell lines).

- equal

- not directly related

- inversely proportional

- directly proportional

Answer 1

1. ELISA could be used for -

To determine the presence or absence of a specific protein within a sample.

ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform.

2. The target protein is recognized by--

The primary antibody

The direct detection method uses a labeled primary antibody that reacts directly with the antigen. Direct detection can be performed with an antigen that is directly immobilized on the assay plate or with the capture assay format. Direct detection while not widely used in ELISA is quite common for immunohistochemical staining of tissues and cells.

The indirect detection method uses a labeled secondary antibody for detection and is the most popular format for ELISA. The secondary antibody has specificity for the primary antibody. In a sandwich ELISA, it is critical that the secondary antibody be specific for the detection primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen. Generally, this is achieved by using capture and primary antibodies from different host species .

3. The absorbance measured for each well is directly proportional to the amount of target protein present in each sample.

ELISAs are usually performed in 96-well plates, where many conditions and repeat samples can be tested at once, and then measured using micro plate reader.
An absorbance-based plate reader can measure the absorbance of light at chosen wavelengths relevant to whatever is produced from the enzyme reaction, providing quantitative differences between positive and negative
samples.