Designing and Optimizing of Multiplex Polymerase Chain Reaction for Amplification of Human Anti-Tetanus Toxoid Antibody Genes

Antibodies are used in many areas of diagnosis and treatment. Fully human monoclonal antibodies (mAb) have attracted high attention due to not stimulating immune response in the body. This study was carried out with the purpose of designing and optimizing multiplex polymerase chain reaction for amplification human anti-tetanus toxoid antibody genes. After collecting blood samples from a human immunized with tetanus toxoid and lymphocyte separation, total RNA was extracted and cDNA was synthesized. all varieties of light (Kapa) and heavy chains of antibody were amplified by two separated multiplex PCR reactions using 14 pair primers. PCR was performed to link VH and VL together and make a ScFv segment, and semi nested PCR was performed to add cutting sites of restriction enzymes at the two ends of the segment. In two multiplex PCR reactions, VH and VL segments were amplified with the length of 410 and 680 bp, respectively. After gel extraction, VH and VL were linked together as a 1070 bp ScFv segment. Restriction sites were added to the two ends of ScFv. By selection of an appropriate primer set and optimization of effective factors of multiplex PCR, all varieties of antibody genes derived from total human lymphocyte cDNA could be amplified and extracted using two multiplex PCR reactions instead of several uniplex ones. Therefore, using only two Multiplex PCR reactions, all genetic varieties of human antibody could be amplified as VH and VL segments from the blood of a subject immunized with tetanus toxoid.