ELISA principle

ELISA protocols and Troubleshooting guide for high quality and reliable data

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 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 measurable 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. The binding and immobilization of reagents makes ELISAs simple to design and perform. Having the reactants of the ELISA immobilized to the microplate surface enables easy separation of bound from non-bound material during the assay. This ability to wash away non-specifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

How ELISA Works?

Enzyme-linked immunosorbent assays (ELISA) principles are very similar to other immunoassay technologies. ELISAs 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.

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Bosterbio Your ELISA Power House

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Our 2300+ ELISA kits are validated in multiple sample matrices from serum and saliva to urine and feces, ensuring wide application ranges for you to select from. Boster's 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. Here are the list of our most popular ELISA kit.

4 TYPES OF ELISA

Comparisons on Direct, Indirect, Sandwich, Competitive ELISA

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 cpture 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.

direct-ELISA

DIRECT 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.

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DIRECT ELISA

Advantages:

Quick because only one antibody and fewer steps are used.

Cross-reactivity of secondary antibody is eliminated.

Disadvantages:

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.

indirect-ELISA

INDIRECT ELISA

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

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INDIRECT ELISA

Advantages:

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.

Disadvantages:

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-ELISA

SANDWICH ELISA

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.

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SANDWICH ELISA

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

competitive-ELISA

COMPETITIVE ELISA

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.

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COMPETITIVE ELISA

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.

THE ELISA PROCEDURE

General ELISA Workflow, a step-by-step ELISA protocol

ELISA FORMAT COMPARISON

STEPS INDIRECT DIRECT SANDWICH COMPETITIVE
Capture Ab Coating
Antigen Coating
Blocking
Sample (Antigen) Incubation
Primary Ab Incubation
Secondary Ab Incubation
Substrate Prep
Signal Detection
Data Analysis

ELISA data interpretation -3 steps

  1. 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.
  2. 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.
  3. 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 interpretation

ELISA data is typically graphed with optical density (or fluorescence) vs concentration to produce a sigmoidal curve as shown above.

picokine™ elisa kit benefits

Picokine ELISA kits are Boster Bio manufactured ELISA kits that have Picokine level sensitivity. Our ELISA kits come with over 20 years of manufacturing expertise and proprietary methods which provide the precision you need.

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™ 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™ ELISA kits.

Sensitivity

Boster's Picokine™ ELISA kits are made with high affinity antibodies that can detect native form proteins with picogram and subpicogram level sensitivity.

Specificity

Boster's QC department validates our ELISA kits against proteins in relevant superfamilies and proteins with similar immunogenicities to ensure specificity to the analytes of interests.

Selection

Our 2300+ ELISA kits are validated in multiple sample matrices from serum and saliva to urine and feces, ensuring wide application ranges for you to select from.

Service

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popular picokine™ ELISA kits

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