Components in ELISA assay, ELISA Kit
ELISA assays play important roles in clinical detection. Three necessary ELISA reagents include: immunosorbent, conjugate and substrate. All the components of ELISA reagents for a complete ELISA assay, or an ELISA kit are as follows:
Immunosorbent (Solid support which has been coated antibodies or antigens)
Solid supporter which has been coated with antigens and antibodies can be stored in low-temperature (2~8℃) and drying condition for six months. There are some kits that provide coating antigens or antibodies, so inspector do it by themselves.
In ELISA test, solid carrier is adsorbent and container, so do not react. There are lots of materials, usually polystyrene, that can be used in ELISA. Polystyrene is strong in adsorbing protein. Antibody or protein antigen remains activity after adsorbed on it. In addition, it is widely used because of cheap price. Polystyrene is plastic material, so it can be made all kinds of shapes.
There are three shapes of ELISA carrier: microtiter plate, small ball and small tube. Usually, microplate which is 96 wells plate is generally used. The character of ELISA plate is that it can be used to detect large amount of simples at the same time and the results are quickly calculated on a colorimeter. Polystyrene is strong in adsorbing protein, usually immunoglobulin, after radiation. It can increase antibodies in solid supporters.
The good ELISA plate is strong adsorbing, low-value blank and high-transparency in the bottom of well. The performance among plates and wells in a plate is similar.
Conjugate (Antigen or antibodies conjugated enzyme)
Conjugate is antibody (antigen) linked with enzyme, it is the key substance in ELISA. The good conjugate possess not only catalytic activity of enzyme but also immunological competence of antibody. Antibodies (antigens) are in proportion to enzymes in order to reduce uncombined enzymes or unlinked antibodies (antigens). In addition, the conjugate must be favorable stability. If the kit requires you to prepare a "working" conjugate solution, be sure to follow the instructions closely. Prepare only what you immediately need, and do not save leftover solution for future use. If conjugates are contaminated or improperly stored, they may lose enzymatic activity or may have an apparent increase in background. Most kits supply a ready-to-use conjugate.
Enzymes used in ELISA should meet requirements such as high purity, high conversion rate, favorable specificity, stable properties, rich resources, cheap price and remaining active component and catalytic capacity after becoming conjugate. It is good that there is no same enzyme in simple. In addition, corresponding substrate is easy to be made and stored. Non-ferrous products is easy to be detected. In ELISA, horseradish peroxidase (HRP) and alkaline phosohatas (AP) are usually used.
2) Antigen and antibody
High purified IgG is usually used in conjugate in order to avoid the interference of other proteins when it is linked with enzyme. It is the best to utilize antibodies that is purified through affinity chromatography. These enzyme conjugates are all specific in immunocompetence and can react in low concentration.
Substrate (The substrate of enzyme)
The chemical activity of the substrate will be compromised if it is exposed to light or comes into contact with metal. Protect this solution by storing it in a dark container until ready for use.
Most kits are formulated with prediluted controls. However, some require that you dilute them in the same manner as your sample. Controls should be added to the plate in the same method and at the same time as the samples.
Sample Dilluent and Wash Solution
Make sure the sample diluent and wash solution have come to room temperature (18–25°C) before use. These are usually the largest bottles in a kit and require the most time to equilibrate. If the wash solution still shows crystal formation after reaching room temperature, mix it by inverting it several times. In plate form of ELISA, 0.05 % of tween 20 of PBST is widely used as diluent.
Be sure to use the stop solution included in the kit. Follow any safety precautions in the package insert. The stop solution should be at room temperature before use. If the stop solution shows crystal formation after reaching room temperature, mix it by inverting several times. The stop solution may crystalize at lower temperatures. Before use, make sure that it is completely dissolved and appears clear. H2SO4 is widely used as stop solution for HRP reaction. Its concentration is based on the final volume of solution, usually 2 mol/L in ELISA plate.