ELISA Technique


Enzyme-Linked Immunosorbent Assay (ELISA) is a common immunoassay, in which antibodies, peptides, proteins, and small molecules can be detected and quantified using a multi-well plate. This assay is the preferred method to determine the titer of antisera and purified antibodies. ELISA can also be successfully employed for the quantitative assessment of an antigen in a sample, often available in convenient, easy-to-use kit formats.


Key characteristics that make ELISA an ideal method of testing:



Can be run in a variety of settings, with little equipment investment and many formats using different combinations of reagents.



Multiple samples can be tested in a single assay, providing high capacity, rapid, and inexpensive assays.



Readouts from enzyme catalysts or fluorescent tags efficiently amplify the signal and provide a broad range of sample detection.



The signal detected by multi-channel spectrophotometers allows data to be stored and analyzed statistically.

Although different immunoassay systems operate within common parameters, each configuration differs in the nature and number of stages needed. The common parameters include immobilization to a solid phase, separation of bound and free reagents by washing steps, and readout from colorimetric, fluorescent, or luminescent signals.

Rockland offers custom assay development services including qualification, validation, and lot release testing. Our ability to generate antibodies and supporting reagents within our own US-based facilities allows for efficiency and first-hand expertise throughout the assay development process.


ELISA Comparisons

  Direct Indirect Sandwich Direct Sandwich Indirect
Coating (adsorption to solid phase) Antigen Antigen Capture antibody Capture antibody
Blocking Addition of blocking agent to prevent non-specific binding
Wash Separate bound/unbound analytes
Analyte (addition of testing sample) Enzyme- or fluorescence-conjugated antibody Unconjugated antibody Antigen sample Antigen sample
Wash Separate bound/unbound analytes
Secondary reagent N/A Enzyme- or fluorescence-conjugated antibody Enzyme- or fluorescence-conjugated detection antibody Biotin-conjugated or
unconjugated detection antibody
Wash Separate bound/unbound analytes
Additional reagent N/A N/A N/A Enzyme- or fluorescence-conjugated streptavidin or secondary antibody
Wash Separate bound/unbound analytes
Signal development Addition of substrate for enzyme-conjugated antibodies
Stop signal development For end-point reading of enzyme-based detection systems
Signal Detection Colorimetric, fluorescent, or chemiluminescent detection


Types of ELISA


Direct ELISA

It is the simplest configuration in which the antigen is bound by passive adsorption to the solid phase, washed to remove any unbound molecules and then directly incubated with a conjugated antibody. Following the incubation period and additional washing, the substrate is added to produce a signal that is allowed to develop. After a certain time, the substrate reaction is stopped and the resulting signal quantified. It is commonly used for tittering conjugated secondary antibodies and is very useful to estimate antigen cross-reactivity.

Direct ELISA


Indirect ELISA

In this system, initial antigen binding and washing steps are the same as the direct method. The main difference, in this case, is the use of an unconjugated antibody to bind the immobilized antigen upon incubation. Following a washing step to remove unbound antibodies, the remaining antigen-bound antibodies are targeted by a conjugated secondary antibody that will generate the readout signal as described for direct ELISA. This system allows multiple sample evaluations using different primary antibodies to be screened with a single conjugated secondary antibody.

Indirect ELISA


Sandwich ELISA

This assay requires a compatible antibody pair that recognize different antigenic targets (epitopes) on the same antigen. The first antibody, called the capture antibody, is coated on the plate and used to immobilize the antigen upon binding during incubation with the sample. Free antigen is removed by a washing step and then a detection antibody is added to bind the captured antigen and enable subsequent detection. Sandwich ELISA is divided into two main systems:


Direct Sandwich

Direct Sandwich uses a detection antibody conjugated to an enzyme or fluorescence tag. Following incubation with the antibody-antigen complex immobilized on the plate well, signal detection is performed upon successive addition of substrate and stopping solution or appropriate excitation/emission of the fluorescent tag.

Direct Sandwich ELISA


Indirect Sandwich

Indirect Sandwich uses an unconjugated antibody bound to the antibody-antigen complex on the well. Following a washing step to remove unbound antibodies, the remaining antigen-bound antibodies are targeted by a conjugated secondary detection antibody. Signal detection is performed upon successive addition of substrate and stopping solution or appropriate excitation/emission of the fluorescent tag.

Indirect Sandwich ELISA


Competitive and Inhibition ELISA

Each of the core systems described above can be further modified to measure molecules based on their ability to interfere with a well-known pre-titrated assay. Such assays can be used to study either antigens or antibodies and they can be competitive or inhibitory based on the specific conditions of each assay. In both types of assays, a pre-titrated system is challenged with the presence of the testing sample whose binding activity is then determined from the degree of the resulting interference in the established system.

Competitive ELISA


For the most part, inhibition assays give the testing sample the advantage of binding (i.e. incubated in advance) before the other system’s components are added whereas in competitive assays the testing sample is mix incubated and mixed with the other components of the pre-titrated system.

Tittering ELISA