Enzyme vs. fluorescent conjugates

Choosing the best detection method for ELISA/IHC

In order for an ELISA to produce useful data, the proteins that become bound in each well must be detected in such a way that the signal strength correlates linearly to the protein concentration. Toward this end, labelled secondary antibodies are used. The choice of which detection system to use is influenced by the equipment available and the needs of the specific application being considered. Use this guide to help decide whether to use enzyme-linked secondary antibodies, or fluorescently labelled secondary antibodies as your detection system.

Enzyme ConjugatesFluorescent Conjugates
  • Signal amplification gives greater sensitivity
  • Greater shelf-life
  • Able to produce chromogenic, chemiluminescent, or fluorescent signals
  • Can be used with simpler equipment
  • No substrate requirement
  • Signal not affected by endogenous enzymes
  • Less likely to interfere with the binding affinity of antibodies
  • May interfere with binding affinity
  • Require substrate that is sensitive to light and prone to degradation
  • Endogenous enzymes may produce competing signal
  • Less versatile
  • Require more specialized equipment
  • Less sensitive
  • Shorter shelf-life
  • Sample autofluorescence may produce competing signal

Keywords:- ELISA, optimization, enzyme, fluorescent, enzyme, conjugate, secondary antibodies

Antibody/Antigen Concentration Optimization

Checkerboard Titration

In order to get the best results from your ELISA assay, the dilution factors of the sample and the detection antibodies must be optimized. If your sample or antibodies are too concentrated, you risk saturating the assay; if they are not concentrated enough, your signal will be weak and difficult to detect. For strong, quantifiable signal, use a checkerboard titration to test for the optimal concentration of sample and detection antibodies.

A checkerboard titration can be used to assess two variables at once: in this case, antibody concentration and sample concentration. By running each well with a different ratio of sample to antibody, you can find not only the optimal concentration of each, but the optimal ratio of concentrations as well. Using the information gleaned from the checkerboard assay, you can perform your ELISA experiment with the optimal concentrations for your application and get better results.

An example of a checkerboard titration. Each of the columns 1-12 contain the antibody dilution factors, and rows A-H contain sample dilution factors.

Keywords:- ELISA, optimization, antibody, antigen, sample, concentration, checkerboard, titration

ELISA Plate Washing Optimization

One of the defining features of an ELISA assay is the ability to quickly and effectively wash away unbound molecules. This allows ELISA assays to capture specific antigens or antibodies from crude samples, enabling researchers to identify, isolate, and quantify nearly any molecule. Optimizing the parameters of the wash steps is critical to obtaining the best results from your ELISA assay. Use this guide to help decide how you will optimize the parameters of your washes:

Wash Volume

One of the most significant parameters to optimize is the wash volume. The amount of wash solution that is used to wash the ELISA wells influences the stringency of the wash: too little and unbound molecules may remain, greatly increasing background signal; too much, and you risk stripping specifically bound molecules.

As a rule of thumb, the wash volume should be at least as high as the coating volume; manufacturers will list the coating volume on the datasheet, providing a starting point for your optimization. A commonly used industry standard is 200 µl, and manufactures will often recommend 300 µl wash volumes.

Number of Wash Cycles

Another major parameter you might wish to consider is the number of wash cycles used. As with wash volume, too few wash cycles cause high background, while too many washes reduce signal strength. The typical number of wash cycles is three; however, you must consider the manufacturers suggestions and the type of plate you’re using: plates coated by the manufacturer typically require fewer washes than in-house coated plates.

Aspiration Parameters

Aspiration of the wash buffer is important to optimize. There are several variables to consider, including aspiration height, and aspiration method, and well shape.

Aspiration height, the distance from the bottom of the well to the aspiration head, contributes significantly to the residual volume of the wells. Residual volume of wash buffer includes unbound molecules, and greater volumes cause higher background. The optimal height of a rigid aspiration head has a range of just 0.1mm; even slightly too high or too low will drastically increase the residual volume. Precise calibration of the washing step is crucial for rigid aspiration heads. Floating heads can move up and down the well according to the volume, so adjusting the height is much less important.

The shape of the well itself can also affect residual volume. Wells with sharp corners tend to retain the most residual volume, while v-wells retain the least. If you experience high background that’s hard to troubleshoot, investigate this parameter.

Given the significance of the wash step, you may wish to perform tests to calibrate your washes before performing sensitive experiments.

Keywords:- ELISA, optimization, antibody, antigen, sample, concentration, checkerboard, titration

ELISA Blocking Optimization

Selecting the Right Blocking Buffer
The process of coating an ELISA plate relies on the passive binding activity of the solid phase, which immobilizes biomolecules on the well surface. Without appropriate blocking, the plate would bind the detection antibody alongside the antigen or detection antibody, resulting in high background signal and low sensitivity. Exposing the plate to a blocking buffer after coating causes the free binding sites on the well bottoms become saturated, removing the possibility of nonspecific binding and greatly improving the signal-to-noise ratio.

There are a variety of blocking buffers, not one of which is ideal for every combination of plate type, assay format, and detection system. Every blocking buffer represents a compromise between reducing background and maintaining specificity. Use this guide to help decide which type of blocking buffer is best suited for your specific application.

Detergent-based blocking buffers

Non-ionic detergents, such as tween-20, provide a convenient an inexpensive blocking solution. However, detergents are not recommended as the sole blocking method: detergents are temporary blockers, since they can be stripped by washing with water or aqueous buffer. Detergents are primarily useful as a secondary blocking agent; when included in the wash buffer, detergents can actively block sites on the plate surface that become exposed as weakly associated proteins are washed away.

Advantages of non-ionic detergentsDisadvantages of non-ionic detergents
  • Inexpensive, despite higher concentration requirements
  • highly stable, able to be stored as working solutions at room temperature
  • Increase the effectiveness of washes by encouraging the dissociation of weakly bound molecules and blocking the resulting exposed binding sites
  • ineffective as sole blocking method
  • may cause the dissociation of molecules bound by noncovalent interactions
  • may interfere with HRP detection systems
  • incompatible with lipopolysaccharides due to their ability to outcompete these molecules

Protien Blockers

Protein blockers are a permanent blocking solution, and plates only need to be treated once for effective blocking. Protein blockers can also be added to the diluents used in subsequent steps to further reduce background signal. The most common blocking proteins include: bovine serum albumin (BSA), nonfat dry milk, and whole normal serum. Each has its own set of advantages and disadvantages:

BSANonfat dry milkWhole normal serum
  • Inexpensive
  • Effective at concentrations as low as 1-3%
  • Well documented efficacy
  • Compatible with protein A
  • Inexpensive
  • Effective at concentrations as low as 0.1-0.5%
  • Highly stable in dry form
  • More effective at blocking covalent interactions
  • Effective at blocking all nonspecific interactions, including protein-protein interactions
  • Acts as protein stabilizer
  • High lot-to-lot variability due to variable fatty acid content
  • May cross-react with some classes of antibodies
  • Less effective at blocking covalent interactions
  • May cross-react with phospho-specific antibodies
  • Incompatible with alkaline phosphatase
  • May cause overall higher background
  • Cross-reacts with protein A and anti-IgG antibodies
  • Expensive
  • Requires up to 10% concentration

Keywords:- ELISA, optimization, blocking, buffer, protein blocker, detergent blocker