Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. Being one of the most sensitive immunoassays, ELISA offers commercial value in laboratory research, diagnostic of disease biomarkers, and quality control in various industries.
There are different types of ELISA according to the analyte detection method. Each of them have its own protocol but the basic principle is similar. An ELISA is a generally a five-step procedure:
1) Antigen coating;
2) Blocking all unbound sites to prevent non-specific absorption;
3) Add analyte and incubation;
4) Add labeled antibody and incubation;
5) Add reagents for colouration/ luminescence, thus give a positive result.
We have already discussed the ELISA format and general protocol in ELISA Guide and ELISA Development Guide. Here we mainly discuss about some basic knowledge and practical tips during the operation process of ELISA. If you need more information about commercial ELISA kit, please visit our ELISA Kit Product.
2. ELISA Plate (Solid Phase)
There are varies types of solid phase that can be used for ELISA, such as membrane, well plate and beads. Their characterizations are described in the following Table 1.
Table 1. Types of Immunoassay Solid Phases
|Materiala||Binding Capacity||Type of Interaction|
Plates and tubes
Covalent, Hydrophobic, Hydrophilic
Covalent and Hydrophobic
Most commonly, ELISAs are performed in 96-well (or 384-well) plates. Most plates are either polystyrene or derivatives of polystyrene obtained by chemical modification or irradiation of the surface. The capture protein can be either passively absorbed on the surface of polystyrene plate or covalent coupled through modifications that leave amine or reactive groups such as maleimide, hydrazine, or N-oxysuccinimide groups on the surface (Figure 1). It is this binding and immobilization of reagents that makes ELISA so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from unbound 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.
Figure 1. The surface chemistry of polystyrene plate and immobilized protein.
Polystyrene will bind a wide variety of proteins in an increasing amount depending on their concentration in the coating solution. The specific and optimal amount needs to be determined for each protein. Carbohydrates and heavily glycosylated proteins do not adsorb well to polystyrene by the forces described above because they have very little ability to participate in hydrophobic interactions. In order to adhere these molecules, one must resort to the covalent linkages.
A number of modifications have been made to the polystyrene surface that allow for covalent linking of molecules to the plastic surface (Figure 1c). Maleimide groups react with a sulfhydryl forming a covalent link between the plastic surface and a protein or peptide. Hydrazine reacts with aldehydes generated by periodate oxidation of carbohydrates. N-Hydroxysuccinimide (NHS) reacts with amines on peptides or proteins. Peptides either through the COOH end by using a cross linker such as carbodiimide or through the amine by using a homobifunctional cross linker such as disuccinimidyl suberate (DSS). Table 2 shows recommended method to immobilize different antigens on polystyrene plate.
Table 2. Different antigens and their recommended immobilizing method.
|Direct Adsorption||Covalent Linkage|
Peptides longer than 15-20 amino acids
Small molecule epitopes attached to a protein
Bacteria and virus
Heavily glycosylated proteins
Proteins in the presence of detergents
3. Antigen Coating
A key feature of the plate based ELISA is that capture protein (antibody or antigen) can be attached to surfaces easily by passive adsorption or covalent linkage. This process is commonly called coating. Multiple factors affect the antigen coating process. The rate and extent of the coating mostly depends on the following factors:
• Diffusion coefficient of the attaching molecule
• Ratio of the surface area being coated to the volume of the coating solution
• Concentration of the antigen being adsorbed
• Time of adsorption
These factors are linked. It is most important to determine the optimal antigen coating concentration for each systems. Time and temperature are also important factors controlling the amount of protein adsorbed. A concentration range of 1–10 µg/mL of protein, in a volume of 50-100 µL, is a good guide to the level of protein needed to saturate available sites on a polystyrene plate. Commonly used coating solutions are: 50 mM carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; and 10 mM PBS, pH 7.2. The most thorough adsorption and lowest well-to-well variation occurs overnight (16–18 hours) at 4°C with the wells sealed to prevent evaporation. Adsorption time can be speeded up by incubation at room temperature for 4–8 hours or 37°C for 1-4h. There are many more variations, and ultimately, each scientist must titrate a particular antigen to obtain a standardized regime. After coating process, don’t forget to remove the coating solution and wash the plate by filling the wells with 200μl PBS for 3 times. Then remove the wash solutions by flicking the plate gently over a sink. Remove any remaining drops by patting the plate on a paper towel.
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4. Blocking Process
Coating of wells with the specific binding partner, either antigen or antibody, leaves unoccupied hydrophobic sites on the plastic. These sites must be blocked in order to prevent nonspecific binding of subsequent reactants. If this is not effectively accomplished, the assay will suffer from high background signal and lowered specificity and sensitivity (Figure 2). These blockers work by reducing non-specific binding to increase the signal-to-noise ratio. To prevent non-specific binding, blocking buffers are used after the solid-phase coating step to block any remaining open binding sites.
Blocking reagents are typically chosen in an empirical manner. The optimum blocker for one assay may not perform well in other assays. The two major classes of blocking agents that have been tested are proteins and detergents.
Figure 2. ELISA plate with and without blocking process.
Detergents come in three classes: nonionic, ionic, and zwitterionic. Both ionic and zwitterionic are not recommend to use as blocker because they disrupt the hydrophobic interactions that bind proteins coated to the surface of the plastic. Typically, detergents used as blocking reagents are non-ionic such as Tween 20 and Triton X-100. Detergents are considered temporary blockers; they do not provide a permanent barrier to biomolecule attachment to the surface because their blocking ability can be removed by washing with water or aqueous buffer. Unlike non-ionic detergents, proteins are permanent blockers and only need to be added once after the surface is coated with the capture molecule. Some of the most commonly used protein blockers are listed in Table 3 as well as their advantages and disadvantages.
Table 3. Advantages and disadvantages of commonly used protein blockers.
|Bovine serum albumin (BSA)||1%~5% in PBS at pH 7||
Stored dry or as a sterile solution at 4°C.
Compatibility with Protein A
Cross-reactions with anti-BSA-hapten conjugates.
lack of diversity required to block some covalent surfaces
|Non-fat dry milk (NFDM)||0.1%~0.5%||
compatible with Protein A
Tendency to deteriorate rapidly
|Casein or caseinate||1%~5%.||Main component of NFDM lacks of the impurities||Same as NFDM|
Effectively blocks non-specific reaction
|cross-reactivity with Protein A and anti-IgG antibodies|
|Fish Gelatin||1% ~ 5%||Lack of cross reactivity with mammalian antibodies and Protein A||
Least effective biomolecule surface blocker
Ideal blocking agents have the following characteristics:
• Effectively block nonspecific binding of assay reactants to the surface of the well
• Do not disrupt the binding of assay components that have been adsorbed to the well
• Act as a stabilizer (prevent denaturation) of assay reactants on the solid phase
• Do not cross-react with other assay reactants
• Do not possess enzymatic activity that might contribute to signal generation of the substrate or degradation of the reactants
• Perform consistently across various lots
5. Antibody Preparation
As the antibodies are cornerstone of ELISA test, the choice of antibodies is obviously of prime importance. The most frequently faced problem is how to choose an antibody, monoclone (MAb) or polyclone (PAb)? In general, a MAb is often chosen as the primary antibody to establish the highest level of specificity in an assay, and a PAb is chosen as the secondary antibody, to amplify the signal via multiple binding events. However, any combination can be used. All candidate antibodies must be tested together with the intended sample type in order to select the best performers.
Primary/secondary antibody should be diluted in blocking solution to help prevent non-specific binding. A concentration of 0.1-1.0 μg/ml is usually sufficient.
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Table 4. Key performance differences between MAb and PAb
|Monoclonal antibodies (MAb)||Polyclonal antibodies (PAb)|
a. Generally produced in mice or recombinantly, these antibodies recognize a single epitope.
b. Since only one antibody molecule can bind to the antigen, the interaction is highly specific but can lack sensitivity.
a. Produced in goats, sheep, chicken, rabbits and other animals.
b. Polyclonal sera is a heterogepneous composite of antibodies with unique specificities and the concentration of specific antibody (PAb) is typically 50-200mg/mL.
c. PAbs are able to recognize multiple epitopes on any one antigen which makes them less sensitive to antigen mutational changes.
d. PAbs are useful when the nature of the antigen is not well known. However, their quantity is limited by the lifespan of the animal.
6. Sample Preparation
The samples contains the main analytes which we need to detect by ELISA. An excellent preparation of sample solution may improve the ELISA test quality. The sample type could be variety such as serum, plasma, urine, cell or tissue lysates, saliva, milk, cell culture supernatant etc. Each sample may need a specific preparation process. A general protocol for sample preparation as following:
a. Protein extract concentration is at least 1-2 mg/mL.
b. Cell and tissue extracts are diluted by 50% with binding buffer.
c. Samples are centrifuged at 10,000 rpm for 5 min at 4°C to remove any precipitate before use.
And for each sample details, see Table 5.
Table 5 Common used ELISA sample types and their treatments.
|Serum||Collect whole blood into a tube without additives; Keep at room temperature for 20 minutes. Centrifuge 10 minutes at 3,000 rpm. Aliquot into small tubes and store at -80°C until use. Minimize freeze/thaw cycles.|
|Plasma||Collect whole blood into an EDTA, Citrate or Sodium heparin tube; Centrifuge 10 minutes at 3,000 rpm at 4°C; Aliquot into small tubes and store at -80°C until use. Minimize freeze/thaw cycles.|
|Urine||Collect urine without adding stabilizers. Centrifuge the samples hard (eg. 10,000 x g for 1 min or 5,000 x g for 2 min). Aliquot, quick freeze in dry ice/methanol bath, and store at -80°C until use.|
|Saliva||Collect samples and centrifuge at 10,000 x g for 2 min at 4°C. Aliquot supernatant and store samples at -80°C. Minimize freeze/thaw cycles.|
|Cell lysates||Place tissue culture plates on ice. Remove the media and gently wash cells once with ice-cold PBS. Remove the PBS and add 0.5 ml extraction buffer per 100 mm plate. Tilt the plate and scrape the cells into a pre-chilled tube. Vortex briefly and incubate on ice for 15-30 min. Centrifuge at 13,000 rpm for 10 min at 4°C (this creates a pellet from the insoluble content). Aliquot the supernatant into clean, chilled tubes (on ice) and store samples at -80°C, avoiding freeze/thaw cycles.|
|Tissue lysates||Dissect the tissue of interest with clean tools, on ice preferably and as quickly as possible to prevent degradation by proteases. Place the tissue in round bottom microfuge tubes and immerse in liquid nitrogen to "snap freeze". Store samples at -80°C for later use or keep on ice for immediate homogenization. For a ~5 mg piece of tissue, add ~300 µL complete extraction buffer to the tube and homogenize with an electric homogenizer. Rinse the blade twice using 300 µL complete extraction buffer for each rinse, then maintain constant agitation for 2 h at 4°C. Centrifuge for 20 min at 13,000 rpm at 4°C. Place on ice, aliquot supernatant (this is the soluble protein extract) to a fresh, chilled tube and store samples at -80°C. Minimize freeze/thaw cycles.|
|Cell culture supernatants||Centrifuge cell culture media at 1,500 rpm for 10 min at 4°C. Aliquot supernatant immediately and hold at -80°C, avoiding freeze/thaw cycles.|
7. Buffer Solution
There are 5 mainly buffer solutions used in ELISA test: coating buffer, blocking buffer, washing buffer, substrate buffer and stop buffer.
Coating buffer usually 0.05 M carbonate buffer with pH=9.6.
|Coating buffer||Blocking buffer||Washing buffer||Substrate buffer||Stop buffer|
|0.05M carbonate buffer, pH=9.6||See Table3||0.01M PBS-Tween 20, pH=7.4||Phosphoric-citric acid buffer, pH=5.0||2M H2SO4|
|NaHCO3||2.93g||KH2PO4||0.5g||0.1M Citric acid||24.3mL|
|adjust pH to 9.6||Tween 20||0.5mL||OPD(Substrate)||4mg|
|storage in 4°C||ddH2O add to 1000mL||30% H2O2||0.015mL|
8. Washing Steps
The incubations that are performed in an ELISA allow high-affinity specific interactions to form among reactants. By washing several times between each incubation, the excess reactants are diluted to an undetectable background level. In order to effectively dilute the excess reactants, it is necessary to wash 3–5 times after each incubation. It is also a good idea to allow a 5 to 10 minute soak with wash buffer at each wash step. If the wash steps are being performed by hand, tap out the excess wash buffer at each step by banging the plate upside down on dry paper towels. Do not allow the plate to dry for extended periods between wash steps as this can lead to a reduction of activity.
9. Data Analysis
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 vs log concentration to produce a sigmoidal curve. 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 (Figure 3).
Figure 3. The standard curve of ELISA.
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|3.||ELISA [M]. Springer New York, 2015.|
|4.||Gan S D, Patel K R. Enzyme immunoassay and enzyme-linked immunosorbent assay [J]. Journal of Investigative Dermatology, 2013, 133(9): 1-3.|
|5.||Gibbs J, Kennebunk M E. Effective blocking procedures [J]. ELISA Technical Bulletin. Kennebunk, ME: Corning Incorporated Life Sciences, 2001.|