The presence of heavy and light chains is confirmed in western blot and quantified using ImageJ.7 Pause point: The purified mAbs (in DPBS buffer) can be stored at ?80C for a couple of years. Expected outcomes In Circulation cytometry, debris and aggregated cells are eliminated by and SSC-A/W and FSC-A/H, respectively. of bovine heavy and light variable genes into human expression vectors ? Expression of chimeric bovine-human monoclonal antibodies in the human Expi293 cell collection Publishers notice: Starting any experimental protocol requires adherence to local institutional guidelines for laboratory security and ethics. We describe herein a protocol for production of chimeric bovine-human monoclonal antibodies (mAbs) from vaccinated cows. The genes of HIV-1-specific single B cells are amplified by reverse transcription-polymerase (-)-Gallocatechin gallate chain reaction (RT-PCR), cloned into human expression vectors, and expressed in human cell lines. This protocol provides an efficient step-by-step methodology to produce HIV-1 chimeric mAbs and could be widely adapted for other antigens. Before you begin This protocol explains how to produce antigen-specific chimeric bovine-human mAbs with the following actions: Isolation of antigen-specific B cells, sequencing and cloning of bovine antibody variable genes and expression of chimeric bovine-human mAbs. The current protocol is an adaptation of the methods explained previously1,2,3 which resulted in isolation and production of ultra-potent cross-clade neutralizing chimeric bovine-human mAbs. Institutional permissions All bovine experiments will need to comply with protocols approved by a local animal ethics committee (This work was conducted under animal ethics approval 2015C17 from your Victorian State Government DEDJTR Research and Extension Animal Ethics Committee). Important resources table We were successful using blood draws made five days after a booster vaccination when memory B-cells are likely to be in peripheral blood circulation. Simultaneously blood without anticoagulants need to be collected to confirm the reactivity of serum against vaccine immunogen. 2. Dilute blood with 20CC25C DPBS-2?mM EDTA (1:1 v/v). 3. Add 20?mL Ficoll-Paque to an empty 50?mL conical centrifuge tube. 4. Carefully layer 30?mL of diluted blood onto Ficoll-Paque. Do not mix the blood and Ficoll- Paque media. Seven 50?mL conical centrifuge tube containing Ficoll-Paque (GE-Healthcare) is required if collecting 100?mL blood. 5. Centrifuge at 800??for 20?min at 20CC25C (brake: off; acceleration: slow). Set the centrifuge at 4C after this step. 6. Using a sterile pipette, cautiously discard the upper layer made up of plasma and platelets. Do not disturb the white mononuclear cell layer. Diluted plasma in the upper layer can be stored and tested to determine the titre of antibodies against the target antigen. Antibodies can also be purified from diluted plasma if required. 7. Make use of a transfer pipette in a slow, circular motion to cautiously suck up mononuclear cells (-)-Gallocatechin gallate (the white layer like a white cloud on top of Ficoll-Paque layer). Transfer the cells into an empty sterile 50?mL conical centrifuge tube. 8. Add at least 2 volumes of chilly DPBS-2?mM EDTA (4CC8C) to the transferred mononuclear cells. 9. Resuspend the cells softly and horizontally centrifuge at 325??for 8?min at 4C (brake: off; acceleration: slow). If it is important to get rid of platelets, centrifuge the cells at low velocity (100C200??for 8?min at 4C (brake: off; acceleration: slow). Discard the supernatant. 12. Resuspend the cells in 3C4?mL 0.83% ammonium chloride and incubate for 5?min on ice. 13. Wash the cells with 20?mL chilly DPBS-2?mM (-)-Gallocatechin gallate EDTA and centrifuge at 150??for 8?min at 4C (brake: on; acceleration: high). 14. Discard the supernatant and repeat the wash step two more occasions. Resuspend the cells in chilly DPBS-2?mM EDTA and count the cells. 15. Centrifuge at GCN5 150??for 8?min at 4C (brake: on; acceleration: high). 16. Discard the supernatant and resuspend the cells in freezing media containing 90% horse serum (Sigma Aldrich) and 10% dimethylsulfoxide (DMSO). Freeze 10C20 million cells per 1?mL freezing media. Label the microtubes before aliquoting the cells as it is not recommended to keep the cell in cryopreservation medium at (-)-Gallocatechin gallate 20CC25C. Place the cryovials in the (-)-Gallocatechin gallate freezing container and store them at ?80C for 24?h (for long-term storage, transfer the vials to.