ELISA Overview

Enzyme-linked immunosorbent assay (ELISA) is a microplate-based immunoassay to detect and quantify the target antigen from a sample of complex mixtures. Essentially ELISA utilizes the specificity and selectivity of antigen-antibody interaction to recognize the captured or immobilized antigen of interest on a solid surface by an antibody linked to an enzyme. Upon subsequent incubation with the enzyme substrate develops a colored end-product, which correlates to the amount of target antigen present in the original sample, thus provides the measurable signal for antigen detection.

To develop an ELISA, the necessary steps are to select the ELISA format, collect the components required for the assay, and determine an effective assay protocol. ELISA optimization is an important process in developing the assay that comprises testing of variable components and systemic adjustments to ensure the accuracy and reproducibility of the developed assay. ELISA validation determines the degree of conformity, confidence, and validity of the assay to measure the target antigen efficiently.

Different formats of ELISA development

The fundamental factor to consider for ELISA development is to select the assay format suitable for the antigen of interest or the desired sensitivity. Depending on the way of capturing and detecting the target antigen, there are four major types of ELISA- direct, indirect, sandwich, and competitive ELISA.

Both direct and indirect ELISA involve target antigen-coated assay plate surface while the antigen is detected by a labeled primary antibody (direct ELISA) or a primary antibody with a labeled secondary antibody recognition (indirect ELISA).

In sandwich ELISA, as the name suggests, use two layers of primary antibodies-capture and detection antibodies (either direct or indirect detection), where the antigen is sandwiched in between, followed by a labeled secondary antibody detection. Sandwich ELISA is widely known as the most robust and sensitive format of the assay.

In competitive ELISA, also known as inhibition ELISA, the presence of target antigen is measured by the assay signal inhibition or interference where the target antigen competes with a reference antigen to bind to the detection primary antibody and generates assay signal inversely proportional to the target antigen concentration. Each ELISA type can be implemented and adjusted to a competitive format.

Each format of ELISA offers different level of preference over the others, depending on the objective of detection, reagent availability, time requirement, efficiency, and simplicity of the assay.

Table 1: Brief description of the suitability and challenges of different types of ELISA: 




Direct ELISA

Applicable for simple and fast detection of immunological response to high molecular weight antigens

Labeled primary antibody is required, making it time-consuming, expensive and a rare assay format

Indirect ELISA

Commonly used to quantify total antibody concentration in samples; highly sensitive and more versatile than direct ELISA

With the need of a labeled secondary antibody, possesses risk of cross-reactivity and non-specific binding

Sandwich ELISA

Ability to detect low-to-high molecular weight antigens from complex samples with high sensitivity and specificity

Difficult to optimize a mutually compatible antibody pair for antigen recognition; chance of cross-reactivity between the capture and detection antibodies.

Competitive ELISA

Suitable for low concentration and low molecular weight antigens with a limited number of antibody-binding sites, e.g., small molecules

Requirement of a reference antigen contributes to the complexity of protocol development and optimization


Once the ELISA format is selected, it is necessary to collect the components for the assay that include the microplate, coating agent (antigen or capturing agent), blocking solution, wash buffer, sample diluent, sample standard, detection primary antibody, enzyme conjugate, enzyme substrate, stop solution, and microplate reader. For detailed information on ELISA formats and different elements of ELISA, see our technical guide “ELISA Handbook”.

The basic design of an ELISA is as follows:

  1. Coating the solid phase provided by the microplate well surface with either the analyte or the capture primary antibody.
  2. The remaining unoccupied binding sites on the solid phase are blocked using blocking solution.
  3. If the analyte is coated on the solid phase, anti-analyte primary antibody is added for detection
  4. On the other hand, if the solid phase is coated with anti-analyte primary antibody, the analyte is added in the well to get captured by the antibody, followed by a washing step. Afterwards, a second anti-analyte primary antibody is added to detect the captured analyte
  5. The excess reagent gets washed out to separate the bound from the free analytes.
  6. Enzyme conjugate is added that can recognise the detection anti-analyte primary antibody followed by a washing step
  7. The substrate to the enzyme is added to develop colored product that is proportional to the level of target analyte.

If it is necessary to develop ELISA for novel target of interest, compatible or matched pair antibodies may not be available and custom antibodies need to be produced. Otherwise, it is recommended to use available commercial ELISA kits for the target analyte. We have the facility and expertise to develop ELISA kits optimized and standardized to detect specific targets, containing antibody pre-coated microplates and reagents equipped to perform the assay. Each of our ELISA kit development proceeds through validation and quality checkpoints that eliminates the need for laborious optimization steps by the end-users. The following reference data were produced from our developed VEGF ELISA kit detecting human VEGF recombinant protein.


Figure 1. Detection of recombinant human VEGF protein by ELISA.

Sandwich ELISA analysis of human VEGF was performed using a Human VEGF Colorimetric ELISA kit by loading 100 µl per well of human VEGF recombinant protein at 50 to 0.78 pM with six-point dilution by factor 1:2, and 0 pM across anti-human VEGF pre-coated plate and incubating for 2 hours at room temperature along with 100 µl per well of anti-human VEGF biotinylated antibody. The plate was washed and incubated with 100 µl per well of Streptavidin-HRP in all test wells for 30 minutes at room temperature. Detection was performed using TMB Substrate for 30 minutes at room temperature in the dark. The assay in the plate was stopped with 50 µl of stop solution, developing yellow colored end-product respective to the amount of analyte present in the sample. Absorbances were read on a spectrophotometer at 450-540 nm.