| 1. |
- 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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| 3. |
- Ohlin, Mats, et al.
(författare)
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Human monoclonal antibodies against a recombinant HIV envelope-antigen produced by primary in vitro immunization. Characterization and epitope mapping.
- 1989
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Ingår i: Immunology. - 0019-2805. ; 68:3, s. 325-331
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Tidskriftsartikel (refereegranskat)abstract
- Peripheral blood lymphocytes from healthy, HIV sero-negative blood donors have been in vitro immunized using penv9, a recombinant fragment of the envelope of HIV-1. This primary in vitro immunization followed by Epstein-Barr virus (EBV) transformation and somatic cell fusion subsequently gave rise to several specific anti-penv9 monoclonal antibodies (MO28, MO30 and MO43) of μ isotype. The hybridomas have been kept in culture for over 6 months and the antibody productivity for MO30 was measured to 18 μg × (24 hr × 106 cells)-1. The fine specificity of the antibodies was mapped by a peptide inhibition enzyme immunoassay, using overlapping synthetic pentadeca peptides covering the whole penv9. These human monoclonal antibodies exhibited a similar epitope specificity directed against a non-sequential determinant, including the amino acids 632–646, 677–681 and 687–691. This specificity is very rarely found in immune sera from sero-positive patients and presently not reported in human monoclonal antibodies derived from in vivo immunized individuals, indicating that different antibody specificities can be obtained by the in vitro immunization technology. These human monoclonal antibodies did not neutralize HIV. The results presented here demonstrate the feasability of generating human monoclonal antibodies against HIV by primary in vitro immunizations, thereby avoiding the use of lymphocytes derived from infected patients when human monoclonal antibodies for therapeutic purposes are to be produced.
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| 4. |
- Ohlin, Mats, et al.
(författare)
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The effect of leucyl-leucine methyl ester on proliferation and Ig secretion of EBV-transformed B lymphocytes.
- 1989
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Ingår i: Immunology. - 0019-2805. ; 66:4, s. 485-490
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Tidskriftsartikel (refereegranskat)abstract
- The selective cytotoxicity of the lysosomotropic methyl esters of leucine or its lysosomal condensation product leucyl-leucine has been used to investigate the effect of cytolytic cells on the clonal outgrowth, cellular proliferation and antibody secretion of Epstein-Barr virus (EBV)-transformed human B cells. Large granular lymphocytes (LGL), monocytes, and a subset of T cells (CD8/CD11+) were permanently eliminated by the ester treatment. These lysosome-rich cells severely inhibit the clonal outgrowth of EBV-infected B cells, as determined by Poisson distribution calculations. Furthermore, leucyl-leucine methyl ester-treated and EBV-infected lymphocytes showed a significant increase in proliferative capability as well as immunoglobulin (Ig) production (three to 11 times) compared to non-treated but similarly infected lymphocytes. Since the effect of leucyl-leucine methyl ester treatment was also detectable in low-density (100 B cells/well) cultures, the suppression was unlikely to be exerted by EBV-specific T-cell clones, but pointed rather to the natural killer (NK) cells as effectors.
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