An antigen molecule could have multiple antigenic determinants, or known as epitopes. An antibody produced by the immune system can only bind to a specific epitope, while an antigen molecule could bind to different antibodies targeting different epitopes on the antigen. This unique feature has been widely utilized in different immunodetection methods. Methods that only use a single antibody for capturing the target often show less satisfactory specificity in detecting and qualifying different analytes. On the contrary, if two antibodies can simultaneously bind to the same antigen molecule, the so-called paired antibodies will result in higher specificity and sensitivity.
Paired antibodies now have been taken as a convenient and price-wise stand-in for ready-to-use enzyme-linked immunoassays (ELISAs) due to their greater specificity.
What are Antibody Pairs?
Antibody pairs are two different antibodies that can simultaneously bind to two different and non-overlapping regions or epitopes on the same target. An antibody pair product contains a capture antibody and detection antibody that have differential capabilities of epitope recognition. Their unique ability to attach to the same target makes it possible to form a sandwich ELISA format, presenting a great potential to quantify target analyte in biological research.
Antibody Pairs Screening
In preparing antibody pairs, the point is making sure that two antibodies against different epitopes can bind to the same antigen at the same time. The second antibody may fail to bind to the antigen if the binding of the first antibody changes the configurations of other binding sites or steric hindrance is present. Therefore, it's necessary to screen the prepared antibodies to ensure a simultaneous binding of the antibody pair. The most commonly used method of screening antibody pairs is the double-antibody sandwich ELISA method.
How Do Antibody Pairs Work?
The process of antibody paring starts with coating the capture antibody, a target-specific antibody, to the bottom of the wells of a microplate. After an overnight stay, samples, standards, or controls are then added into these wells so that they can bind to the capture antibody immobilized in the wells. Then the enzyme-linked detection antibody is also added to bind to the same target, forming a sandwich ELISA to measure the amount of the target bound between the matched antibody pair. A substrate solution that reacts with the enzyme-antibody-target complex is necessary to be added to produce measurable signals that indicate the concentration of the target in the original sample.
Both monoclonal antibodies and polyclonal antibodies could be used to develop antibody pairs, resulting in different formats of antibody pairs such as monoclonal antibody pairs (mAb/mAb), polyclonal antibody pairs (pAb/pAb), and combination antibody pairs (mAb/pAb). Each antibody type has different advantages, and the point is to recognize the difference and take advantage of them in different conditions.