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- Larrick, James W, et al.
(författare)
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Polymemse chain reaction using mixed primers. Cloning of human monoclonal antibody variable region genes from single hybridoma cells
- 1989
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Ingår i: Bio/Technology. - : Springer Science and Business Media LLC. - 0733-222X. ; 7:9, s. 934-938
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Tidskriftsartikel (refereegranskat)abstract
- We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity. It will also facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies.
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| 2. |
- Larrick, James W, et al.
(författare)
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Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction
- 1989
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Ingår i: Biochemical and Biophysical Research Communications. - 1090-2104. ; 160:3, s. 1250-1256
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Tidskriftsartikel (refereegranskat)abstract
- A general method to directly obtain the DNA sequence of the variable regions of any immunoglobulin chain using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5′ signal peptide and a conserved 3′ constant region primer were used for enzymatic amplification of each of the heavy and light chain variable regions of a human hybridoma producing a monoclonal antibody recognizing an epitope of gp120 of the human immunodeficiency virus 1. The amplified DNA segments were cloned and the sequence was determined for the heavy chain variable region. This method will greatly facilitate structural and functional studies of immunoglobulins by reducing the effort to clone and sequence the members of the immunoglobulin as well as other multigene families.
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