Method for obtaining immunoglobulin encoding nucleic acid

A method for producing an immunoglobulin comprising the following steps:

• (a) providing a single immunoglobulin-producing cell,
• (b) obtaining from said cell a nucleic acid encoding an immunoglobulin heavy chain variable domain and
• a nucleic acid encoding an immunoglobulin light chain variable domain selected from a kappa light chain variable domain and a lambda light chain variable domain
• by a process comprising the steps:
• (a) performing a first polymerase chain reaction (PCR) to obtain a first PCR product;
• and (b) performing a second PCR with the first PCR product, whereby the distance of the binding locations of the primers employed in the second PCR is reduced compared to the distance in the first PCR, wherein:
• i) for obtaining the nucleic acid encoding the immunoglobulin heavy chain variable domain,
• the first PCR is performed
• with a set of 5′ primers comprising the nucleic acids of
• at least one of SEQ ID NO:
• 05 and SEQ ID NO:
• 06,
• at least one of SEQ ID NO:
• 07 and SEQ ID NO:
• 08,
• SEQ ID NO:
• 09,
• at least one of SEQ ID NO:
• 10 and SEQ ID NO:
• 11,
• SEQ ID NO:
• 12, and
• SEQ ID NO:
• 13, and
• with a 3′ primer or set of primers comprising at least one of the nucleic acids of SEQ ID NO:
• 104, 105 or 106, and
• the second PCR is performed
• with a set of 5′ primers comprising the nucleic acids of SEQ ID NOs:
• 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, and 141 or
• with a 5′ primer comprising the nucleic acid of SEQ ID NO:
• 142;
• and
• with a 3′ primer or set of primers comprising at least one of the nucleic acids of SEQ ID NO:
• 104, 105, 106 or
• with SEQ ID NO:
• 143,
• ii) for obtaining the nucleic acid encoding the immunoglobulin kappa light chain variable domain,
• the first PCR is performed
• with a set of 5′ primers comprising the nucleic acids of SEQ ID NOs:
• 16, 17, 18, and 19, and
• with a 3′ primer comprising the nucleic acid of SEQ ID NO:
• 115, and the second PCR is performed
• with a set of 5′ primers comprising the nucleic acids of
• at least one of SEQ ID NO:
• 53 or SEQ ID NO:
• 54,
• at least one of SEQ ID NO:
• 55 or SEQ ID NO:
• 56,
• at least one of SEQ ID NO:
• 57 or SEQ ID NO:
• 58,
• SEQ ID NO:
• 59,
• SEQ ID NO:
• 60,
• at least one of SEQ ID NO:
• 61 or SEQ ID NO:
• 62,
• at least one of SEQ ID NO:
• 63 or SEQ ID NO:
• 64,
• SEQ ID NO:
• 65,
• SEQ ID NO:
• 66,
• SEQ ID NO:
• 67,
• SEQ ID NO:
• 68,
• SEQ ID NO:
• 69, and
• SEQ ID NO:
• 70, or
• SEQ ID NO:
• 144, and
• with a 3′ primer comprising the nucleic acid of SEQ ID NO:
• 115 or SEQ ID NO:
• 145, and
• iii) for obtaining the nucleic acid encoding the immunoglobulin lambda light chain variable domain,
• the first PCR is performed
• with a set of 5′ primers comprising the nucleic acids of
• SEQ ID NO:
• 21,
• SEQ ID NO:
• 22, and
• at least one of SEQ ID NO:
• 23, SEQ ID NO:
• 24, SEQ ID NO:
• 25 or SEQ ID NO:
• 26, and
• with a 3′ primer or set of primers comprising at least one of the nucleic acids of SEQ ID NO:
• 120, SEQ ID NO:
• 121, SEQ ID NO:
• 122, SEQ ID NO:
• 123, SEQ ID NO:
• 124 or SEQ ID NO:
• 125, and
• the second PCR is performed
• with a set of 5′ primers comprising the nucleic acids of
• SEQ ID NO:
• 72,
• at least one of SEQ ID NO:
• 73 or SEQ ID NO:
• 74,
• SEQ ID NO:
• 75,
• SEQ ID NO:
• 76,
• at least one of SEQ ID NO:
• 77 or SEQ ID NO:
• 78,
• SEQ ID NO:
• 79,
• SEQ ID NO:
• 80,
• SEQ ID NO:
• 81,
• at least one of SEQ ID NO:
• 82 or SEQ ID NO:
• 83,
• at least one of SEQ ID NO:
• 84 or SEQ ID NO:
• 85,
• SEQ ID NO:
• 86,
• at least one of SEQ ID NO:
• 87 or SEQ ID NO:
• 88,
• SEQ ID NO:
• 89,
• at least one of SEQ ID NO:
• 90 or SEQ ID NO:
• 91, and
• SEQ ID NO:
• 92, and
• with a 3′ primer or set of primers comprising at least one of the nucleic acids of SEQ ID NO:
• 120, SEQ ID NO:
• 121, SEQ ID NO:
• 122, SEQ ID NO:
• 123, SEQ ID NO:
• 124 or SEQ ID NO:
• 125;
• (c) linking in operable form the nucleic acid encoding the kappa or lambda light chain variable domain with a nucleic acid encoding an immunoglobulin light chain constant domain, and linking in operable form the nucleic acid encoding the heavy chain variable domain with a nucleic acid encoding an immunoglobulin heavy chain constant region into an expressible set of combined nucleic acids,
• (d) transfecting a eukaryotic cell with the combined nucleic acids,
• (e) cultivating said transfected cell under conditions suitable for the expression of the immunoglobulin, and
• (f) recovering the immunoglobulin from the cell or the cultivation medium;
• thereby producing the immunoglobulin.