Loading Control Antibody Guide
Knockout-validated primary antibodies
Secondary antibodies resources
Alexa Fluor secondary antibodies
Biotinylated secondary antibodies
Gold-labelled secondary antibodies
How to prepare Phosphate Buffered Saline (PBS)
How to design a flow cytometry panel
Enhancing Detection of Low-Abundance Proteins
9 tips for detecting phosphorylation events using a Western Blot
Western Blotting with Tissue Lysates
Western blot membrane stripping protocol
Immunohistochemistry and Immunocytochemistry
Chromogenic and Fluorescent detection
Preparing paraffin-embedded and frozen samples for Immunohistochemistry
Competitive ELISA assay protocol
Measuring analyte concentration using serial dilutions and standard curve
Types of ELISA plates, buffers and antibodies:
Types of plates:
The most common type of plates are polyvinyl chloride or polystyrene flat-bottomed ones, containing 96 wells. Sometimes using a strip plate can be more appropriate. This type of plate is the same size as a 96 plate, but it consists of the required amount of 8 or 12-well strips for the experiment.
If processing a larger number of samples per plate is needed, 384-well plates and higher can be used. Because they need special automated handling, their uses are limited.
Various buffers are required when performing this assay. One buffer is needed for coating, the second one for blocking, another one for the washing step and sometimes an additional one for diluting the sample or antibody.
Every ELISA assay begins with a coating step. During this step, a diluted antigen or antibody is incubated until fixed to the well. This passive process is carried out by the hydrophobic interactions between the plastic well surface and the amino acid side chains, that can be found in the antibody.
Adsorption relies on the temperature, time, the coating buffer’s pH and the coating agent’s concentration being perfect.
These types of buffers act as stabilizers to the antibody, that is used for coating the assay’s plate. It also enhances the adsorption and improves the interaction with the detection antibody.
This step ensures that non-specific binding of the detection antibodies to the plate is eliminated. The two types of blocking agents are detergents and proteins.
Proteins are a permanent solution, so they can be added only after the capture antibody has completely adsorbed to the well.
On the other hand, detergents offer a temporary blocking, because their function is removed after a washing step.
Upon completely blocking the plate, the sensitivity is increased due to the lack of non-specific binding.
Removing unwanted materials from the wells is a crucial part of this assay. Usually ELISA wells are filled with a PBS buffer containing small amounts of non-ionic detergent.
The process of washing occurs around five times, between each assay step. It is important to note that the buffer has only a brief contact with the well and should be thoroughly removed in the last wash, otherwise the reagents can become diluted and the readings inaccurate.
Both monoclonal and polyclonal antibodies can be used for this assay, alongside a combination of the two types. These types of antibodies have their strengths and weaknesses, so knowing them and using them to their full potential can greatly improve this experiment.
The major factors for antibody-antigen interaction are avidity, affinity, and specificity. When performing an ELISA, these three can conduct the amount of antibody concentration that is needed, combined with other forms of optimisation.
Avidity is a factor, that describes the overall stability of the antigen-antibody interaction. This term depends on the antibody valency, the number of antigen binding sites and the other factor-affinity. Note that avidity can vary with different isotopes and the state of the antibody-intact or fragmented. Another variable is the spatial arrangement of the complex.
Affinity corresponds to the strength of the antibody binding to a single epitope. Because binding is a reversible process, affinity describes how much antibody is bound to the antigen, the time needed for this to happen and how long it lasts. Antibodies with high affinity are always the better choice, because they can provide more sensitive detection.
Specificity defines if the antibody will bind only to a unique epitope from a single species’ antigen, or if it will also bind to similar epitopes. When specificity is low, cross-reactivity can occur.
Matched pair antibodies:
This type of antibodies is commonly used in sandwich ELISAs. These antibodies have the ability to detect different epitopes of the same antigen, making them a good choice for capture and detection of one antigen in a sandwich ELISA assay. The matched pair can be two monoclonal, two polyclonal or one of each antibody type.