Patent ID: 8722587

Claim:
A method for preparing nucleotides of single-chain variable fragments (scFv) encoding an antigen-specific binding domain, comprising the steps of: a) isolating RNA encoding an antibody from splenocytes or lymphocytes of a non-immunized canine, or a canine immunized with a specific antigen; b) generating cDNAs of the isolated RNA; c) amplifying the variable regions of the antibody's heavy chain and the lambda and kappa light chains to generate V L and V H amplicons using PCR with a set of primers designed for the variable regions, wherein the set of primers include at least one pair of V H chain primers, at least one pair of V L lambda chain primers and at least one pair of V L kappa chain primers, wherein the at least one pair of V H chain primers comprises at least one VH chain forward primer and one V H chain reverse primer, wherein the at least one pair of V L lambda chain primers comprises at least one V L lambda chain forward primer and at least one V L lambda chain reverse primer, wherein the at least one pair of V L kappa chain primers comprises at least one V L kappa chain forward primer and at least one V L kappa chain reverse primer, wherein the at least one V H chain forward primer is selected from the group consisting of SEQ ID Nos. 1-7, wherein the one V H chain reverse primer is SEQ ID No. 8, wherein the at least one V L lambda chain forward primer is selected from the group consisting of SEQ ID Nos. 9-21, wherein the at least one V L lambda chain reverse primer is selected from the group consisting of SEQ ID Nos. 22-23, wherein the at least one V L kappa chain forward primer is selected from the group consisting of SEQ ID Nos. 24-27, wherein the at least one V L kappa chain reverse primer is selected from the group consisting of SEQ ID Nos. 28-31, wherein the primers are designed to incorporate secondary primer binding sites into the 5′ end of the V L amplicons and the 3′ end of the V H amplicons and a flexible linker into the 3′ end of the V L amplicons and the 5′ end of the V H amplicons; and d) using the flexible linker, randomly linking the V H and V L amplicons, thereby generating nucleotides encoding single-chain variable fragments.