This article describes a new technique that enhances the production efficiency of antibodies. Antibodies are a special type of protein, produced by plasma cells, and a vital component of the body’s immune system. A molecular target (called an antigen) is detected by antibodies with great specificity, causing the two to bind. The antigen is often a microbe or infected cell targeted for destruction during an immune system response. Antibodies are rather large, containing 2 heavy polypeptide chains and 2 light polypeptide chains, on the order of 150 kilodaltons (kDa, a measure of molecular weight). Nanobodies are engineered and constructed proteins, which are much smaller, but act in the same precise manner as an antibody. At an average size of 15 kDa, ten times smaller than a regular antibody, nanobodies demonstrate potential for use in diagnosing diseases in areas where normal sized antibodies are not effective. Prior techniques of creating nanobodies that match the required molecular target (antigen) have been inefficient, limiting the viability of nanobodies to replace a human’s own antibodies.
Researchers at Rockefeller University recently discovered a method that promises to overcome this barrier. This technique may increase the viability for widespread application of immune system boosting technologies. The Rockefeller researchers used Green Fluorescent Protein (GFP) and mCherry Fluorescent Protein (mCherry) as trial antigens. These two proteins are usually used for visual tagging in cells; having an antibody for them is essential and in high demand for experiments involving western blotting and immunohistochemistry. The researchers decided to use GFP and mCherry in this experiment because, if successful in producing nanobodies, it would help supply the demand that already exists for them. The process includes the following steps: first, llamas’ immune systems were exposed to GFP and mCherry, inducing antibody production. The antibodies that most tightly bound to the GFP and mCherry were isolated and extracted, selected as the most viable for antigens in question. The heavy chains were removed, keeping only binding sites intact and creating the nanobodies. At the same time, samples were taken from the bone marrow of the llama that contained all possible antibodies. RNA sequences, which express genetic codes, were amplified and read to characterize the antibodies. The RNA sequences were then translated into the amino acids and entered into a searchable database of peptides (a unit of up to 50 amino acids). Employing a computer algorithm, the researchers were able to determine the basis for the RNA sequence of the nanobodies by matching them to sections from the original RNA data collected from the bone marrow. Using that RNA sequence, a singularly specific nanobody was created and easily mass-produced using genetic engineering techniques. The result is a large supply of highly specific and organically grown nanobodies.
Further testing concluded that the researchers had correctly and efficiently determined high-affinity nanobodies for a given antigen. Not only did they rapidly determine the highest affinity nanobody, they also used this method to generate 25 types of GFP and 6 types of mCherry high affinity alternatives to create a repertoire of possibilities for selection, depending on application needs. This new method of nanobody production can be altered for specificity for nearly any desired antigen. The application possibilities from here are plentiful in the field of biomedical sciences, specifically in treatment for acute coronary syndrome and brain cancer.
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- Paddock C. Future for nanobodies as alternative research tools to antibodies looks bright. [Internet]. 2014 ;2014. Available from: http://www.medicalnewstoday.com/articles/284806.php
- Citekey <span style="font-size: 11pt; line-height: 115%; font-family: Calibri, sans-serif; background-image: initial; background-attachment: initial; background-size: initial; background-origin: initial; background-clip: initial; background-position: initial; background-repeat: initial;">10.1038/nmeth.3170 not found
While the advantages of nanobodies have already been established, previous methods of producing them have imposed limitations. Finding nanobodies with affinity for a specific molecular target has been a time consuming and costly process, inhibiting widespread application. The new technique, however, allows for production of a large repertoire of high affinity antibodies, providing diversity and specificity. Researchers believe this new source of efficient and precise nanobody production will create opportunities in research, diagnostics, and therapeutics.