The methods we describe here were adopted by our NIH COBRE sponsored, University of Kentucky Protein Core Facility to help researchers produce single-domain antibodies in alpacas. Single-domain antibodies are an important tool used to answer key questions in biomedical research. The main advantage of the techniques we demonstrate is that it allows production of single-domain antibodies to up to five distinct proteins in a single alpaca.
The implications of this technique extend to human health and disease treatment since these single-domain antibodies can be used as therapeutics, as vaccines, and as diagnostics. The most significant hurdle for most established biomedical investigators is handling large animals, such as the alpaca. Visual demonstration of handling an alpaca, the injection of antigens, and the collection of blood, are critical for the successful production of single-domain antibodies in these camelids.
Demonstrating the procedure will be Doctor Jeff Smiley, University Veterinarian, Ms.Kathy Boaz, Veterinary Technician, and Ms.Erin Jones, Lab Animal Technician. Handling of the blood will be demonstrated by Doctor Martin Chao, Research Associate. The real stories of this production are Blue Eyes, Big Boy, or affectionately known as the Gorilla, and Freddie, our Kentucky alpacas.
To begin, restrain the animal with a halter and lead. Then trim the alpaca's hair with electric clippers in a crescent moon pattern along the base of it's neck, from shoulder to shoulder. Carefully hold the alpaca by the head and neck, and slowly inject the antigens subcutaneously with a 22 gauge needle along the base of the neck, near the bow lymph nodes.
Perform five injections of approximately 200 microliters each, about five centimeters apart. After this, monitor the animal for about 20 minutes for signs of anaphylaxis. Inspect the injection site for localized inflammation at the injection sites weekly.
To draw blood, clip the alpaca's hair and disinfect the collection site with alcohol swabs. Restrain the alpaca gently, ensuring that the neck is held upright and straight. Using the ventral projections of the transverse process of the vertebrae as a landmark, occlude the vein by applying pressure just medial to the ventral process.
Next, insert the needle at a 45 degree angle, slightly medial to the projection, toward the center of the neck. Manipulate the needle until the blood flows. First, dilute 15 milliliters of the collected blood with five milliliters of PBS.
Then, pipette blood into a lymphocyte separation tube to isolate peripheral blood lymphocytes, or PBLs, via density gradient centrifugation. Gently remove the top plasma layer, saving it in a separate tube. Then, gently remove the PBL layer, and transfer it to a sterile 50 milliliter tube.
Add 45 milliliters of pre-chilled PBS to the PBLs and spin the tube for 20 minutes at 800 times G, and four degrees Celsius. Remove the supernatant with a pipette, leaving the pellet containing the lymphocytes. Wash the pellet two more times with five milliliters of pre-chilled PBS to ready the PBLs for RNA isolation.
Using this protocol, single-domain antibodies were generated against a range of protein antigens. Immune monitoring indicated that the majority of antigens showed robust response beginning at three weeks, and that the optimal time to obtain blood is at six weeks. Sequencing the positive colonies identified diverse single-domain antibody sequences for different antigens.
For example, three unique colonies were isolated to the reference antigen maltose-binding protein. Generally, the single-domain antibodies contained significant sequence diversity and highly variable length of antigen-binding loops. These features are particularly important in single-domain antibody antigen interactions.
This procedure allows production of single-domain antibodies for up to five antigens per animal. A single blood collection, and library construction, is utilized for each animal. With phage display-based selection utilized to identify antigen-specific, single-chain antibodies.
While doing the antigen injections and blood sampling, it's important to keep the alpacas in a group, which keeps them calmer. This can be done using halters and leads, and tying them in close proximity during the procedures.
