Editing Alpaca Antibody (section)

== Preparation of alpaca lymphocytes ==

<br/>Alpacas were purchased locally and maintained in pasture. All animal experiments were approved by the Wallaceville Animal Ethics Committee. Blood was obtained from the jugular vein and collected into heparinised or serum collection tubes. White blood cells were isolated from about 10 mls of heparinised blood by centrifugation and peripheral blood lymphocytes (PBL) were partially purified by separation over HISTOPAQUE®-1077 (Sigma) using standard procedures and stored in RNAlater (Ambion). Serum was separated by centrifugation and stored at −20 °C until testing.

The local lymph node from each animal was removed surgically under general anaesthesia, induced with sodium thiopentone (20 mg/kg intravenously) and maintained with halothane (1 – 3&nbsp;% in oxygen). The pre-scapular lymph node, which drains the sites of immunizations used in these studies, was removed through a small skin incision and blunt dissection of the fat tissue and muscle overlaying the lymph node. Bleeding was controlled by ligation of the nodal artery and vein. After removal of the node, the edges of the dissected tissue and skin were re-apposed with sutures. Post surgical care included a single subcutaneous application of antibiotics (400mg procaine penicillin + 400mg dihydrostreptomycin sulphate) and an analgesic (flunixin 2 mg/kg). The excised lymph node was cut into 1 – 2 mm thick slices and either stored in RNAlater, or immediately subjected to a phenol/chloroform based RNA extraction protocol.

Alpaca immunization and serum analysis<br/>Two adult male alpacas were given four immunizations at two week intervals, each including six 0.2 ml intra-dermal and sub-cutaneous injections of the immunogen in the pre-scapular region. The immunogen contained a total of '''~400 μg''' of ovTNFα for each immunization prepared with 13 mg/ml aluminium hydroxide gel (Sigma) as an adjuvant. Serum samples were obtained in weekly intervals and tested for TNFα-specific antibody response by ELISA. Alpaca antibody bound to ovTNFα was detected in the ELISA using antisera from a rabbit immunized with alpaca immunoglobulins (Green et al., 1996).

Some serum samples were assayed for TNFα neutralizing activity in a standard bioassay (Flick and Gifford, 1984) which measures a reduction in TNFα cytotoxicity. Briefly, 100 μl/well of murine fibroblast L929 cells were seeded in 96-well plates at 1 × 105 cells/ml and incubated overnight. Dilutions of the serum or purified VHH were prepared in RPMI and incubated with serial twofold dilutions of ovine TNF for 30min. After removing of the supernatants of the cultured L929 cells, 100 μl of the prepared dilutions containing actinomycin D at a final concentration of 1.0 μg/ml were added to the wells. Then the plates were incubated at 37 °C for 24 h, the supernatants were removed and 0.5% crystal violet in methanol was added and incubated at room temperature for 10 min. Plates were rinsed gently with water, and the optical density (OD) of bound dye was determined at 620 nm.