Non-Specific Binding – What It Is, How It Occurs and How to Reduce or Eliminate It
Both specific and nonspecific binding occurs in nature when molecules interact with one another. The struggle, however, to overcome this, through the use of artificial systems like biosensor technologies, or biomedical diagnosis and monitoring of disease treatment and progression, as described here is one that many facilities are still facing to date.
The main reason for this is the nonspecific interactions of binding events. This hinders aspects such as the causes, identities and influences which are rampant within the scientific communities. So, what exactly is non-specific binding? We take a look at this concept, as well as how it occurs within various scenarios and solutions to help eliminate or reduce it.
Non-Specific Binding (NSB)
In its simplest definition, it is the binding of assay antibodies, which do not associate with specific analytes and antibodies. It is the occurrence where antibodies bind to assays such as receptors, unintended proteins and transporters.
There are two NSBs, indistinguishable from one another and that commonly occur in laboratories. These are:
- The binding that occurs to Western blotting membranes or ELISA wells: when this occurs, it results in false positives or high background in ELISA. It binds to elements within solutions, that are especially in a high concentration such as immunoglobulin and albumin.
- Non-specific binding of analytes: causing the same effect of false positives or negatives, and here is it either partially bound by the substances inside a solution or any types of sample constituents. This too is difficult to detect especially with assays.
When there is an attraction to either the main receptor or the secondary ones such as the FcRs or Fc Receptors, it leads to unwanted binding, and this is where the non-specificity action occurs.
To avoid these errors within results of such high importance for medical professionals, and patients, there are some solutions that researchers are trying to achieve, some of which have worked to avoid such endogenous enzyme activities
A Solution That Works
Because of the necessity for accuracy, especially within medical and research fields, it is imperative to come up with the best solution to avoid any problems with such a significant area. There are a few things that can be used to avoid unnecessary situations and some of these include using diluents, blockers and ‘dried protein stabilizers.
Structures that are on the surface of certain types of cells are known as FcRs or Fluid Collection and Reinjection System, and various studies have been conducted in regards to this issue such as the one mentioned in this online publication: https://www.nature.com/articles/srep00028
They bind with certain regions and the cross-linking that takes place between them and other substances provides some insight into the relationship between humoral and cellular branches of the immune system. This action produces several responses such as:
- The release of inflammatory mediators
- The enhancement of antigens
- An antibody-dependent cytotoxicity
- Endocytosis
- Phagocytosis
All or any of these responses take place depending on the cell type.
It has been researched, and positive results have shown when preincubation of samples such as serums from secondary Abs are conducted and help to prevent non-specific binding of enzymes to endogenous FcRs.
However, in some cases, goat serum is also used when dealing with human immunohistopathology, which does not bind to the FcRs human cells. Solutions that contain this type of serum, when preincubated, also prevent background activity such as staining and various unfavorable interactions. This type of blocking of non-specific binding is observed by many immune research laboratories and manufacturers, who now offer ready-made blocking formulations and solutions.
By using optimal chemical components in immunoassays, the risk of this activity is reduced significantly and in turn, so is the risk of false negatives and false positives in patient samples. In this case, dried proteins and stabilizers act as the blockers and diluents, and as such, manufacturers specifically design them to maintain and, in some cases, increase the signal of the intended assay.
Comparative immunostaining experiments done with and without these protein blockers conclude much of the results and showed the lack of retention to bind the Fc to the Abs, and such experimentations are usually done using various other components such as alcohol, formaldehyde and acetone for more accurate results.
The costs and time involved in trying to negate such issues are lessened with newer innovations and technologies and although conclusions have been noted, there may still be some further experiments to be done to find an even better solution.
In the end, this is a complex and sometimes tedious task but those who are keen on improving the necessary, yet, time-consuming process, are very close to coming up with the ultimate solutions, for now, this is what works and many have seen positive outcomes from it, as opposed to unnecessary and false assumptions. It can help save patients lives and has become a primary research topic in many medical and immune technology sectors.
