The Enzyme-Linked Immunosorbent Assay (ELISA), also called Enzyme ImmunoAssay (EIA), is an effective method for detecting and quantifying the presence of specific biomolecules like antigens, antibodies and hormones in biological fluids such as serum, plasma, saliva and urine.
ELISA technology uses a biomolecule (typically an antigen or antibody) labeled with an enzyme (e.g., peroxidase or alkaline phosphatase), resulting in a conjugate with both immunological and enzymatic activity. During the antigen-antibody reaction, one of the components (antigen or antibody) is immobilized on a plate. When a substrate reacts with the enzyme, it produces a color change that is quantifiable using a spectrophotometer.
ELISA assays are among the most sensitive and reproducible plate-based technologies. The process is rapid, straightforward, and easily automated.
Key Applications of ELISA in Diagnostics and Research
ELISA plate-based test are widely used to diagnose and monitor various diseases and conditions, including:
Infectious Diseases: Detecting antibodies against bacterial, viral, or fungal infections.
Tumor Markers: Evaluating levels of tumor markers to assess the presence of cancer.
Autoimmune Diseases: Measuring autoantibodies that the immune system produces to attack the body’s cells.
Allergic Diseases: Identifying antibodies against allergens that cause rhinitis, asthma, dermatitis, and other allergic conditions.
Endocrinology: Hormone testing, including:
LH, FSH, Prolactin
T3, T4, TSH
Steroid hormones (e.g., Testosterone, Cortisol)
hCG
Blood Transfusion Screening:
Hepatitis A, B, C
HIV
Syphilis
An ELISA test includes different steps and reagents:
The first step is performed on a 96-well polystyrene plate, the solid phase. The function of the solid phase is to immobilize a specific lipoprotein target, which is passively adsorbed. This step is called coating.
In a second step the bound target is complexed with an excess of a specific anti-target linked to an enzyme, called conjugate. This is the case of a direct ELISA test (see below). Other types of ELISA tests require different steps and reagents (see below).
After an incubationstep, the plate is washed to eliminate any excess of unbound conjugate in the reaction medium.
Finally, the enzyme activity of the bound conjugate is measured using a chromogen-substrate that changes color when modified by the enzyme. Light absorption of the product, formed after chromogen-substrate addition, is measured and converted to numeric values.
Depending on the lipoprotein target-antibody combination, the assay is called:
Direct ELISA
Indirect ELISA
Competitive ELISA
Sandwich ELISA
Direct ELISA
The target lipoprotein (antigen in the picture) is bound to the bottom of the microplate well and it is only recognized by the specific antibodies (primary antibody in the picture) against the target antigen. The primary antibody is labelled with the enzyme that allows detection through the Chromogen/Substrate reaction.
It is important to underline that, according to the reaction flow, the antigen bound to the well is the target to be evaluated.
Indirect ELISA
The target lipoprotein (antigen in the picture) is bound to the bottom of the microplate well, then an antigen-specific antibody (primary antibody in the picture) is added. A secondary enzyme-conjugated antibody that binds to the primary antibody is added, allowing its detection through the Chromogen/Substrate reaction.
It is important to underline that, according to the reaction flow, the primary antibody is the target to be evaluated.
Direct ELISA
Indirect ELISA
Competitive ELISA
The target lipoprotein (antigen in the picture) is bound to the bottom of the microplate well.
The sample, containing the same type of antigen bound to the well, plus an anti-antigen -conjugated antibody (primary antibody conjugated in the picture) are added to the wells. If the sample includes the antigen (inhibitor antigen in the picture), it competes with the antigen bound to the well for binding to the specific antibody (Primary antibody conjugated). Unbound material is washed away. The more antigen is in the sample, the less specific antibody ends up being bound to the coated antigen, and the lower is the final signal.
The Chromogen/Substrate reaction allows detecting the specific enzyme-conjugated antibody bound to the antigen.
It is important to underline that, according to the reaction flow, the inhibitor antigen is the target to be evaluated.
Sandwich ELISA
For this type of ELISA, two antibodies specific to two different epitopes on the target lipoprotein (antigen in the picture) are used. The capture antibody (primary antibody in the picture) is bound to the bottom of the microplate well and binds to one antigen epitope. The detection antibody (secondary antibody in the picture) binds to a different antigen epitope and is conjugated to an enzyme, allowing detection through the Chromogen/Substrate reaction.
It is important to underline that, according to the reaction flow, the antigen is the target to be evaluated.
Competitive ELISA
Sandwich ELISA
The figure represents the typical curve of an indirect ELISA test, where the respective optical density increases proportionally as the target concentration to be evaluated increases.
The calibration curve refers to the dosage of anti-Tetanus Toxoid antibodies. The assay is the typical indirect ELISA, where the microplate is coated with the Tetanus Toxoid antigen (the solid phase of the test). The concentration of anti-Tetanus Toxoid antibody being evaluated are shown on the x-axis, while the corresponding optical densities are shown on the y-axis.
It is important to point out that in addition to the ELISA test, there are two other tests, namely the CLIA and the FIA, which have the same reaction principle, but differ in the reagents and the type of microplate to be used.
In the CLIA test, white microplates are used, while in FIA test, black ones.
Discover Biomat’s most requested ELISA 96-Well plates and their technical guide:
Biomat ‘s full range of both Medium & High Binding ELISA 96 well plates include:
Breakable strip plates: on single well-holding frame
Strip plates: assembled on 12 x 8 well-holding frame
Solid plates
allowing the maximum flexibility for the user.
They are made in Transparent, White and Black Polystyrene and can be used for ELISA, Luminescence (White) and Fluorescence (Black) assays.
Biomat’s ELISA microplates are produced in polystyrene, the most widely used material for this application. Polystyrene is hydrophobic, however its properties can be modified by performing surface treatments or coatings. In this way it is possible to get different requirements for diverse applications and its characteristics allow it to be the best suited for microscopic imaging and optical measurements.
Polystyrene is composed of an aliphatic carbon chain with pendant intermittent benzene rings. This provides a very hydrophobic surface and microplates of this type are typically referred to as Medium Binding, in terms of binding capacity towards hydrophilic biomolecules.
The binding capacity can be enhanced through a surface treatment, such as irradiation, which breaks a certain number of benzene rings, yielding carboxyl (COOH) and hydroxyl (OH) groups. The presence of these groups provides an opportunity for hydrophilic interactions. Microplates modified in this way are typically referred to as High Binding.
The resulting surface is primarily hydrophobic with intermittent carboxylic and hydroxylic groups capable of ionic interactions with positively charged groups on biomolecules.
The forces that passively adsorb biomolecules through the surface of ELISA Medium Binding Plates and ELISA High Binding Plates in order of increasing strength are:
hydrophobic interactions
ionic interactions
van der Waals forces
hydrogen bonding
The pictures describe the four physical adsorptions that occur when the bio- molecule comes in contact with the bottom of polystyrene microplate.
QUALITY / RELIABILITY
Biomat has selected the best raw materials medical grade to produce its microplates. We mould our ELISA plates in clean conditions and following strict quality controls in complete absence of contaminants.
Thanks to the continue feedback of the clients we have improved the stability of our wells in our frames that come off easily but are also stable in the washing phase.
All lots are tested to guarantee their quality.
HIGH BINDING vs. MEDIUM BINDING CAPACITY ELISA PLATES
The High Binding Surface provides an opportunity for hydrophilic interactions, whereas Medium Binding Surface provides a hydrophobic surface.
HIGH BINDING CAPACITY
Biomat High binding surface is a hydrophilic surface suitable for passive adsorption of proteins with different grades of hydrophilicity. This surface is ideal for immunoassays with a binding capacity of 400 to 500 ng IgG/cm2.
Furthermore, this surface is highly selective and shows high affinitytowards the adsorption of molecules also when they are present in very small amounts (it is possible to obtain an efficient coating even starting from a concentration of 1 µg/ml of protein to be coated *) allowing to obtain the maximum sensitivity of the test.
* The good practice for carrying out a coating with low concentrations is to couple the protein with an inert protein, such as BSA, which serves as a stabilizer of the primary protein during the coating.
a comparable binding capacity of proteins of both Biomat HB8 and competitor’s strips
the capacity of both types of samples to assure the specific binding between the coated protein and the protein to be revealed: 100% of results of our tests (on 232 sera, 94 positive and 128 negative) were confirmed stating the sensitivity and specificity of both types of samples with all the tested sera
the capacity of both types of strips to assure the specific binding between the coated protein and the protein to be revealed was confirmed carrying out an ELISA test: 100% of results of the ELISA test with our strips (on 232 sera, 94 positive and 128 negative) were confirmed stating the sensitivity and specificity with the competitor’s strips with all the tested sera
the result of regression analysis, whose acceptable value had been fixed at R 0.95 has been fully respected
the ranges of coefficients and values obtained in equation y= a+bx where the a values must not significantly differ from 0 and the b values must fall in the range 0.8 – 1.2 proved the strict correspondence of results
MEDIUM BINDING CAPACITY
Biomat Medium binding surface is a hydrophobic surface suitable for passive adsorption of proteins with different grades of hydrophobicity.
It is used in assays in which the adsorbed molecule present large hydrophobic regions, such as antibodies.
Furthermore, this surface is highly selective and high affinity towards hydrophobic polypeptides that must present molecular weight higher than 10-20kDa.