Model

Rh (D) Antibody

A Single-chain variable fragment (scFv) is a small, engineered antibody fragment consisting of the variable regions of both the heavy (VH) and light (VL) chains of an antibody, connected by a flexible peptide linker. This structure preserves the antigen-binding specificity of a full-sized antibody while being significantly smaller in size (about 25-30 kDa). The linker allows the two variable regions to fold correctly and form an antigen-binding site, similar to that of the original antibody.

What is special about Single-chain variable fragment (scFv)?

1. Size and Structure: Due to their smaller size, scFvs have better tissue penetration and faster diffusion rates compared to full-sized antibodies. The structure is maintained by a peptide linker, usually about 10-25 amino acids long, which keeps the VH and VL domains together in the correct orientation.
2. Binding Specificity: Although scFvs are smaller, they retain the full antigen-binding specificity of the original antibody. They can be engineered to bind to a wide range of targets, from proteins to small molecules.
3. Versatility and Modifiability: scFvs are highly versatile and can be genetically engineered to enhance their stability, affinity, or specificity. They can be fused to other proteins or molecules, such as enzymes, toxins, or nanoparticles, to create multifunctional therapeutic agents or diagnostic tools.
4. Production: scFvs are produced using recombinant DNA technology, which allows them to be expressed in various systems, including bacteria, yeast, or mammalian cells. This makes them relatively easy and cost-effective to produce at scale.

Introduction to Single-domain Antibody (sdAb)

A Single-domain antibody (sdAb), also known as a nanobody, is a small, engineered antibody fragment consisting of a single monomeric variable domain. Unlike conventional antibodies, which are composed of two heavy and two light chains, an sdAb is derived from the variable domain of a heavy-chain-only antibody found in camelids (like llamas and camels) or sharks. These antibodies are the smallest functional units of an antibody that can specifically bind to antigens.

What is special about Single-domain Antibody?

1. Size and Structure: sdAbs are extremely small, usually around 12-15 kDa, which is about one-tenth the size of a conventional antibody. This small size enhances their ability to penetrate tissues and cross biological barriers, making them particularly useful in targeting dense tissues or crossing the blood-brain barrier.
2. Stability: sdAbs are highly stable, even under extreme conditions, such as high temperatures or low pH. Their stability comes from their unique single-domain structure, which lacks the flexible linker regions found in other antibodies, making them less susceptible to denaturation or aggregation.
3. High Affinity and Specificity: Despite their small size, sdAbs maintain high binding affinity and specificity to their target antigens. Their structure allows them to recognize and bind to unique or hidden epitopes (binding sites) that are often inaccessible to larger antibodies, including those in small crevices or active sites of proteins.
4. Ease of Production: sdAbs can be produced efficiently in a variety of expression systems, such as bacteria, yeast, or mammalian cells, due to their simple structure and small size. This makes them cost-effective and suitable for large-scale production.

Origin of antibodies

The origins of antibodies can be traced back to the late 1800s. Kitasato Shibasaburō and Emil von Behring discovered that human blood contains substances that can neutralize the toxins caused by the infection of bacteria. This incident led to the usage of diphtheria antitoxin serum from infected animals on humans. The biological term “antibody” originated from Paul Ehrlich’s proposal of the ‘side chain model’. He believes that every cell has a variety of side chains that can act as antitoxins or antibodies in the blood. In 1901, Karl Landsteiner discovered the ABO blood group system, and blood cells carry specific antigens that can be used to determine blood types. His work had a great impact on transfusion medicine. However, further research in the 1930s to 1940s on antigens showed that their functions are beyond the recognition of blood types. Antigens could be any molecule that induces an immune response, such as proteins, polysaccharides, or even small chemicals, and antibodies are highly specific in recognizing their corresponding antigens. In 1969, Gerald Edelman and his co-workers realized the antibody molecules are y-shaped. Each antibody perfectly matches the structure of the epitope on the antigen. By the 1970s, monoclonal antibodies revolutionized medicine by providing targeted therapies. Despite the fact that the discovery of antibodies and antigens were more than a century ago, they are still crucial to modern medicine. Scientists are able to distinguish the differences among the immune system, such as self and non-self, which prompted the discovery of autoimmune diseases, where the immune system mistakenly attacks the body's own tissues. The concept of immune memory was also established around this time. When the body is exposed to an antigen that it has previously encountered before, the immune system would react faster and stronger. In addition to dealing with immunity-related diseases, antibodies and antigens are being used for vaccine development and cancer treatment.

Lac Operon and IPTG

The lac operon is a set of genes in E. Coli and other bacteria that regulate lactose metabolism. It includes structural genes (lacZ, lacY, and lacA), which code for enzymes involved in lactose breakdown. For example, lacZ produces β-galactosidase, an enzyme that splits lactose into glucose and galactose, while lacY encodes lactose permease, a protein that transports lactose into the cell. The operon also contains promoter and operator regions where RNA polymerase binds to initiate transcription and where the lac repressor protein binds to block transcription when lactose is absent. When lactose or its analog IPTG is present, it binds to the lac repressor, causing it to release from the operator and allowing the genes to be transcribed. IPTG, often used in laboratories, mimics lactose by inactivating the repressor and inducing gene expression without being broken down by the cell.

Overall structure: three chamber design. To implement our project, we designed a two-step blood group conversion kit to 1) attach the protein to the Rh antigen and 2) purify the converted red blood cells in preparation for transfusion. We plan to pass washed red blood cells through a series of chambers where protein attachment occurs. We then separate the red blood cells into successfully attached proteins by filtration through centrifugation and leukocyte removal filters. Made of transparent or translucent polycarbonate or medical-grade plastic, users can clearly observe the liquid flow and reaction status. Connect pipe uses medical grade silicone or polyethylene tubing, ensuring non-toxic and good chemical resistance. The containers and lines are disposable, which ensures sterility. The freshly extracted blood is first washed repeatedly with red blood cell concentrate using 0.9% normal saline in a red blood cell washing machine. The Hct value of the resulting red blood cell product should be below 75%. During the washing process, almost all plasma should be removed, and white blood cells and platelets are removed at the same time. The capacity of each chamber should be designed according to the volume of blood that needs to be processed. For example, each chamber can hold 100-400 ml. A flow rate adjustment device in the pipeline to control the liquid flow rate.