Biopanning Against Colorectal Cell Lines LS147T cell line which expresses high levels of the AgSK1 protein was used for the selection of VHH nanobodies, whereas HeLa cell line lacking AgSK1 was used in a counter-selection step to remove non-specific VHH nanobodies. Briefly, cells were cultured in the complete DMEM medium (10% FBS 2 mM L-glutamine, 100 unit.mL-1 penicillin and 100 g.mL-1Streptomycin) at 37 oC supplied with 5% CO2 for 24 hours. expressing different levels of AgSK1 tumor associated marker. The high affinity binders were selected and subcloned for higher expression levels of the VHH. The affinity and specificity of the isolated VHH were tested using ELISA. The reactivity of the VHH toward cancer cells was analyzed by competitive ELISA applying sera isolated from colorectal cancer patients. Results: Results show that this isolated VHH recognizes and binds to the colorectal cancer cells with a high affinity. Moreover, the isolated nanobody is able to compete with the antibodies in the patient sera for the binding to the cancer cells. Conclusions: Results suggest that this nanobody has a specific reaction toward colorectal cells and can be used for further investigation around the tumor associated antigens or production of mimotopes useful for immunotherapy. BL21(DE3) and TG1 were obtained from Shahed University bacterial collection and were cultured in LuriaCBertani (LB) medium (1% NaCl, 1% Peptone, and 0.5% yeast extract). HT29, Ls174T, and HeLa cell lines were purchased from Pasture Institute of Iran (Tehran, Iran) and were cultured in the DMEM (Dulbeccos Modified Eagles Medium) medium made up of 10% FBS at 37oC and 5% CO2. 3.2. Preparation of the VHH Phage Library The naive VHH phage library was from our previous studies (18). Briefly, peripheral blood lymphocytes were isolated from a and used for total RNA extraction, cDNA was generated, and VHH genes were amplified using nested PCR. The library was created by cloning all the VHH genes into pComb3X phagemid vector. After transformation of TG1 bacteria with the library the phage TAK 259 particles harboring the VHH gene were propagated by infecting the bacteria with the helper Rabbit polyclonal to COT.This gene was identified by its oncogenic transforming activity in cells.The encoded protein is a member of the serine/threonine protein kinase family.This kinase can activate both the MAP kinase and JNK kinase pathways. phage (M13K07, Amersham, USA), as explained before (18). VHH coding phage particles were isolated from supernatant following to the TAK 259 removal of bacteria by centrifugation and were used for bio-panning. 3.3. Biopanning Against Colorectal Cell Lines LS147T cell line which expresses high levels of the AgSK1 protein was used for the selection of VHH nanobodies, whereas HeLa cell line lacking AgSK1 was used in a counter-selection step to remove non-specific VHH nanobodies. Briefly, cells were cultured in the complete DMEM medium (10% FBS 2 mM L-glutamine, 100 unit.mL-1 penicillin and 100 g.mL-1Streptomycin) at 37 oC supplied with 5% CO2 for 24 hours. A total number of 5104 cells from each cell line were counted and transferred into individual 1.5 mL microtubes made up of 1 mL complete DMEM medium and incubated for 2 hours. Cells were sedimented at 1200 and washed twice with 0.5 mL of cold PBS. The total amount of 150 L of the phage library was added to the microtube made up of HeLa cell line and was incubated for 2 hours. After centrifugation at 1200 at 4 oC for 5 minutes, the supernatant made up of unbound phages was removed and added to the microtube made up of Ls174T cell line. After 2 hours of TAK 259 incubation, cells were centrifuged as before, washed three times with cold PBS, followed by three times of washing with PBS-T (PBS + 0.05% Tween-20). The supernatant was removed and the bound phages were eluted by addition of 100 L Glycine-HCl answer (2M HCl, pH was adjusted to 2.2 with Glycine). After 10 minutes of incubation, the eluted phages were harvested by centrifugation and were neutralized to pH 7.2 by the addition of 1M Tris buffer, pH 9. The eluted phages were used to infect TG1 at mid-log phase. The phage particles were propagated as described before (21) and were used for the subsequent panning rounds. A total of.