Abstract
Antigen Used for Immunization
The SKBr3 cell line was cultured routinely in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and the antibiotics penicillin and streptomycin. The tumor cells were harvested and homogenized and used for immunization.(1,2)
Method of Immunization
Two adult male camelids (Camelus dromedaries) purchased from local sources were immunized intramuscularly with the mechanically homogenized SKBr3 cell line, or 30 days for several months. The protocol of immunization and bleeding has been described in a previous report.(1,2)
Parental Cell Line Used for Fusion
N/A
Selection and Cloning Procedure
Total RNA was isolated from 107 lymphocytes. mRNA was purified using the Nucleotrap mRNA kit (Macherey-Nagel, Düren, Germany). One microgram mRNA was used for preparing first-strand cDNA after an oligo(dt) priming using enzymes (Fermentas, Utrecht, Netherlands). DNA fragments encoding VHH fragments were amplified by PCR. A VHH library was constructed in the phagmid vector using electrical transformation of the ligation product to TG1 bacterial strain. Phages were subjected to four rounds of affinity selection on the specific cell line. Pooled phages from each round of panning were screened by immunocytochemistry for positive binding, as described in a previous study.(1)
Heavy and Light Chains of Immunoglobulin
N/A
Specificity
The positive pooled phages from the fourth round of panning of the two VHH libraries were used to infect a new bacterial strain (HB 2151) and subsequently plated for separation of individual clones. The specificity and reactivity of the VHH toward the SKBr3 cell line were determined using immunocytochemistry.(2,3)
Specific Antigen Identified
N/A