ELISA Fundamental Principle, How ELISA Works
What is ELISA?
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies and hormones. In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.
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ELISA basics/ELISA principle
Enzyme-linked immunosorbent assays operate on principles very similar to other immunoassay technologies. ELISA’s rely on specific antibodies to bind the target antigen, and a detection system to indicate the presence and quantity of antigen binding. In order to maximize the sensitivity and precision of the assay, the plate must be carefully coated with high-affinity antibodies – a process that Boster Bio has mastered.
Benefits of Boster's Picokine™ ELISA Kits
Get Better Results with Boster Picokine™ ELISA Kits
Boster Bio is proud to offer over 1,000 ELISA kits for a wide range of targets. With over 20 years of experience in antibody and ELISA kit manufacturing, Boster Picokine™tm enzyme linked immunosorbent assay (ELISA) kits are guaranteed to be sensitive, specific, and stable. We rigorously validate every lot against a wide range of samples to ensure consistent, reliable results. Join over 14,000 scientists who put their trust in Boster Picokine™tm ELISA kits.
Why choose Picokine™?
- Sensitivity – single-digit picogram detection limits
- Specificity – rigorously validated capture antibodies have low cross-reactivity
- Stability – guaranteed to remain sensitive for up to a year
- Service – Boster offers fast, insightful technical support
Choose the ELISA kit for your application from a selection of over 1,000 ELISA kits
At Boster, our mission is to support research in areas such as immunology, neuroscience, cancer, and more by providing the high-quality ELISA kits needed to get better results. We supply ELISA kits that detect IL-6, IL-8, IL-10, EGF, VEGF, and over 1,000 other targets. We thoroughly validate every kit using biologically relevant matrices, including cell culture supernatant, serum, plasma, cell lysate, and tissue to ensure our kits will provide consistent results with any sample type. Browse our catalog to find your target, or take a look at one of our research area mini-catalogs to see ELISA kits in your field of research.
General ELISA Procedure
*This is conceptual explanation for how ELISA works. Click here for a step-wise ELISA protocol.
Unless you are using a kit with a plate that is pre-coated with antibody, an ELISA begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate. This is followed by a blocking step in which all unbound sites are coated with a blocking agent. Following a series of washes, the plate is incubated with enzyme-conjugated antibody. Another series of washes removes all unbound antibody. A substrate is then added, producing a calorimetric signal. Finally, the plate is read.
Because the assay uses surface binding for separation, several washes are repeated in each ELISA step to remove unbound material. During this process, it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next assay step. To ensure uniformity, specialized plate washers are often used.
ELISAs can be quite complex and include multiple intervening steps, especially when measuring protein concentration in heterogeneous samples such as blood. The most complex and varying step in the overall process is detection, where multiple layers of antibodies can be used to amplify signal.
Four Most Common ELISA Types
ELISAs can be performed with a number of modifications to the basic procedure: direct, indirect, sandwich or competitive. The key step, immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then detected either directly (enzyme-labeled primary antibody) or indirectly (enzyme-labeled secondary antibody). The detection antibodies are usually labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP). A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).
Among the standard assay formats discussed and illustrated below, where differences in both capture and detection were the concern, it is important to differentiate between the particular strategies that exist specifically for the detection step. However an antigen is captured to the plate (by direct adsorption to the surface or through a pre-coated "capture" antibody, as in a sandwich ELISA), it is the detection step (as either direct or indirect detection) that largely determines the sensitivity of an ELISA.
For direct detection, an antigen coated to a multi-well plate is detected by an antibody that has been directly conjugated to an enzyme. This detection method is a good option if there is no commercially available ELISA kits for your target protein.
- Quick because only one antibody and fewer steps are used.
- Cross-reactivity of secondary antibody is eliminated.
- Cell Smear: Adhere non-adherent cells on coverslip with chemical bond
- Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags.
- Labeling primary antibodies for each specific ELISA system is time-consuming and expensive.
- No flexibility in choice of primary antibody label from one experiment to another.
- Minimal signal amplification.
For indirect detection, the antigen coated to a multi-well plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal. The indirect assay, the most popular format for ELISA, has the advantages and disadvantages:
- A wide variety of labeled secondary antibodies are available commercially.
- Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
- Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
- Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.
- Cell Smear: Adhere non-adherent cells on coverslip with chemical bond
- Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
- An extra incubation step is required in the procedure.
Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. A first antibody (known as capture antibody) is coated to the wells. The sample solution is then added to the well. A second antibody (known as detection antibody) follows this step in order to measure the concentration of the sample. This type of ELISA has the following advantages:
- High specificity: the antigen/analyte is specifically captured and detected
- Suitable for complex (or crude/impure) samples: the antigen does not require purification prior to measurement
- Flexibility and sensitivity: both direct or indirect detection methods can be used
This ELISA kit is of competitive format. Competitive ELISA, also known as inhibition ELISA, is a surface/plate based assay, where the plate is coated with capture antibodies reactive to the molecule of interest. The sample (containing native molecule of interest) and enzyme conjugated recombinant protein (the competing molecule) are added to the coated wells. Since the amount of enzyme conjugated molecule in each well is constant, the level of native molecule in the sample will determine the binding ratio of enzyme conjugated molecule vs. native molecule. After an incubation period, any unbound antibody is washed off. Enzyme substrate (for example, TMB for HRP) is added to each well and will be transformed into a blue precipitate, the amount of which is linearly proportional to the amount of enzyme in the well. The precipitate is then turned into yellow by adding the acid stop solution and the concentration of yellow precipitate is read at 450nm for light absorbance (O.D. value). The O.D. is then used to calculate the amount of molecule of interest in each well, by comparing each sample well against the standard curve. The standard curve is generated using the same principle but instead of adding samples, a series of recombinant molecules with known concentrations are added to 6-8 wells.
Summary of Key Steps in Different ELISA Types
|Capture Ab Coating|
|Sample (Antigen) Incubation|
|Primary Ab Incubation|
|Secondary Ab Incubation|
ELISA Data Interpretation
The ELISA assay yields three different types of data output:
- Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.
- Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.
- Semi-Quantitative: ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.
ELISA data is typically graphed with optical density (or fluorescence) vs concentration to produce a sigmoidal curve as shown below. Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve. In fact, it is the relatively long linear region of the curve that makes the ELISA results accurate and reproducible. The unknown concentration can be determined directly on the graph or with curve fitting software which is typically found on ELISA plate readers.
BOSTER FEATURED ELISA KITS
"Good price. Worked as advertised."
--Lu Yang, UCSD, CMMW, GRADUATE STUDENT
|Applications||Protein level screen|
|Sample||Cell culture media from MCF7 cells|
"The TFF1 ELISA kit from Boster was competitively priced and worked with cell culture media taken from MCF7 cells...The kit is very sensitive, detecting TFF1 protein at levels of 10pg/mL. Good ELISA kit for human TFF1 protein."
MEDICINE, DUKE UNIVERSITY
"The standards and ELISA kit worked great."
--Jason Somarelli, DUKE UNIVERSITY, MEDICINE, RESEARCH ASSOCIATE
|Applications||Detection of secreted IL-2 in media of Jurkat T cells|
|Sample||Jurkat T cell media (RPMI, 10% FBS, 1% pen/strep)|
"I was using this kit to detect IL-2 secretion from Jurkat T-cells upon activation with PMA and PHA. I followed the protocol exactly. I had extremely low levels of IL-2 in my experiment, which were just above background. In this case, it was essential for me to use the standards and blanks to ensure that my samples were above background. All components come with the kit, unlike some other kits from other companies in which only some of the necessary components are provided. This is a great kit that is a bit expensive, but worth the cost for an easy-to-use and sensitive assay."
ELISA SPECIALIST, MOLECULAR INNOVATIONS
"Specific and low variability between lots"
--Tabitha Doci, ELISA DEPARTMENT, MOLECULAR INNOVATIONS, ELISA SPECIALIST
SUMMARY: Anti-Prolactin Antibody (RP1021)
|Sample||Purified mouse prolactin and plasma|
"We were looking for an antibody that detects mouse prolactin in ELISA, this antibody performs well and I particularly liked that there's no lot to lot variability. Low background and fast high OD. Will purchase again."
--- Unattributed Review* (Reviews submitted to our site where we are unable to verify the identity of the author are considered “unattributed reviews".)
|Applications||IL-1RA detection in rat serum|
"I used this kit to detect a recombinant IL1RA in rat serum and it worked very well. The procedure was easy to follow and my standard curve was right on target. If needed we will order this kit and use it again. The kit was able to detect our rIL1RA but was less sensitive in detecting an altered formulation of the protein."
BIOMED SCIENCES, E. TENNESSEE STATE UNIV.
"Easy to Use GDNF ELISA Kit"
--Hannah Oakes, EAST TENNESSEE STATE UNIVERSITY, BIOMEDICAL SCIENCES, GRADUATE ASSISTANT
|Sample||Brain Tissue (Hippocampus)|
"We used this kit and the BDNF kit, because we were looking at neurogenesis within the hippocampus. We used the ELISA to complement our immunohistochemistry. It was easy to optimize the kits and the instructions were extremely easy to follow. Additionally, the instructions do not include using samples from tissue homogenates, but we did and the plates came out great. This product was selected after speaking with a representative about it at a conference. I am extremely glad we decided to go with this kit. This is a great kit. The protocol was easy to follow and optimize. We will be using these kits again."
NUTRITIONAL SCIENCES, UCONN
"Great TNF alpha Kit"
--Tho Pham, UCONN, NUTRITIONAL SCIENCES, GA
|Sample||Cell culture supernatant|
"This ELISA kit worked as advertised. Wells can be removed as strips, so not everything has to be used. Great product. Results are good and have low %CV. Quick assay kit and cheaper than other companies in my opinon."
DENTISTRY , UIC
--J L, UIC, DENTISTRY, SCIENTIST
|Sample||Skin tissue homogenates|
"This product was used to investigate the dynamic change of FGF1 during mouse skin wound healing. The reasons to choose this product are: a) Easy to use. b) Less expense, c) Standard covers a wide range of concentrations, d) It works for tissue homogenates. FGF1 significantly increased at the proliferation/remolding stage. Easy to use and less expensive. Highly recommended."
MICROBIO AND IMMUNOLOGY, UNIV OF N. CAROLINA
"Kit worked great"
--Charles Stopford, UNIVERSITY OF NORTH CARLINA, MICROBIOLOGY AND IMMUNOLOGY, RESEARCH SPECIALIST
"This kit was very straightforward and easy to use. Followed the protocol given in the kit...Straight forward experiment, no tips needed. Kit worked great - positive control was clearly positive. Nothing bad about the kit, although our experimental results were negative. We would buy this kit or similar ELISA kits again from BosterBio."
SCIENTIST II, JECURE THERAPEUTICS
"Good quality budget option"
--Milos Lazic, JECURE THERAPEUTICS, JECURE THERAPEUTICS, SCIENTIST II
|Sample||protein concentrations in cell culture media|
"We are a pharmaceutical startup optimizing cell based drug assays where one of our endpoints was observing the differences in mature IL-1b production in the media. I have compared several kits from the most expensive versions of R&D, over eBioscience/Affymetrix to a more budget Boster's kit. The aim was to decide what is the best price for the value and quality we are getting for reliable and replicable ELISA analyses. This assay was used to test the effect of increasing concentrations of a drug on IL-1b levels (dose response). The kit reported similar data obtained from the other two kits (R&D and eBiosciences), with a slightly larger standard deviations. Results replicated the findings of the previous kits and the assay had good sensitivity/ instructions on how to perform assay and analyze data. I would use this assay in an academic lab for a project where no more than 2-3 96-well plates will be used. It should be done carefully by a scientist that has previously performed ELISA assays. There are many steps to it. It is a good quality budget option to the other leading kits on the market. I would not recommend it for a larger drug screen assay where more than 5 ELISAs kits ought to be used."