Patent ID: 8399200

Claim:
A process for determining an antigen-specific V gene repertoire of immunoglobulin classes of an organism in vitro, comprising: (a) isolating a fraction containing B cells from a body fluid and/or tissue; (b) isolating B cells or their genetic material from the isolated fraction and depositing one or more of the B cells or their genetic material into a reaction vessel; (c) amplification of the genetic material coding variable regions of an antibody expressed by one or more of the B cells, with the amplification providing for amplification of all immunoglobulin classes in the group consisting of IgM, IgG, IgE and IgA, comprising: (i) performing reverse transcription reactions on the genetic material using primers comprising a sequence complementary to constant regions of light and heavy chains to obtain a cDNA amplification product; (ii) obtaining PCR amplificates performing PCR reactions with the cDNA amplification product as a template comprising performing PCR reactions with degenerate primers comprising a sequence complementary to the variable regions in the framework 1 region of the 6 gene families of the heavy chains, and/or performing PCR reactions with degenerate primers for the 4 gene families of the κ chains, and/or performing PCR reactions with degenerate primers for the 3 gene families of the λ chains, and/or performing PCR reactions with degenerate primers for the constant regions of the ε, γ and κ chains; and (iii) performing nested PCR reactions with the PCR amplificates as templates using 5′ primers comprising a sequence complementary to the first 6 codons of the V H region or V L regions and 3′ primers comprising a sequence complementary to nested constant regions of the κ, λ, α1, α2, γ1, γ2, γ3, γ4, μ and ε chains to obtain nested PCR amplification products; (d) cloning the nested PCR amplification products into a vector; (e) preparation of recombinant antibodies by expressing the cloned nested PCR amplification products; and (f) determining binding of the recombinant antibodies to recombinant and/or native antigens.