Identifying affinity-matured human antibodies

摘要

Antigen-specific immunoglobulin V-regions are identified from a library of nucleic acids amplified using polymerase chain reaction using leader sequence-specific forward primers. The use of leader sequence primers allows all V-region sequences to be amplified (including those with extensive 5′ end mutations) without loss of the original 5′ V gene segment sequence. A second V-region library is made using a larger than conventional set of 5′ V-region primers. The sequence errors introduced into the amplification products by this method are corrected using sequence information obtained in the products amplified by the V-region primers to screen the library created using the leader sequence primers. Amino acid sequence information from fragments of donor immunoglobulins can be used to assist in the identification of nucleic acids encoding the heavy and light chains of donor antibodies as well as to design primers to amplify such nucleic acids.

主权项

1. A method comprising the steps of:
(a) using a first PCR to amplify human immunoglobulin heavy and light chain nucleotide sequences from a cDNA library made from a sample of human peripheral blood mononuclear cells obtained from a human donor previously determined to possess antibodies specific for target antigens of interest, wherein a set of leader-specific primers that amplify at least 90% of the human immunoglobulin V region genes cDNA library is used in the PCR amplification; (b) isolating the amplification products from the first PCR step; (c) creating a first set of constructs by inserting the isolated amplification products from the first PCR step into expression vectors; and (d) introducing the first set of constructs into a first set of host cells to create a first library of human immunoglobulin nucleotide sequences; wherein the set of leader-specific primers comprises a first polynucleotide which binds under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO: 1, a second polynucleotide which binds under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO: 23, a third polynucleotide which binds under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO 32, a fourth polynucleotide which binds under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO 34, and a fifth polynucleotide which binds under stringent hybridization conditions to the complement of the nucleic acid sequence of SEQ ID NO 57.