• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. May 1989; 86(10): 3833–3837.
PMCID: PMC287235

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.

Abstract

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Miller RA, Oseroff AR, Stratte PT, Levy R. Monoclonal antibody therapeutic trials in seven patients with T-cell lymphoma. Blood. 1983 Nov;62(5):988–995. [PubMed]
  • Carson DA, Freimark BD. Human lymphocyte hybridomas and monoclonal antibodies. Adv Immunol. 1986;38:275–311. [PubMed]
  • Morrison SL, Johnson MJ, Herzenberg LA, Oi VT. Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6851–6855. [PMC free article] [PubMed]
  • Boulianne GL, Hozumi N, Shulman MJ. Production of functional chimaeric mouse/human antibody. Nature. 1984 Dec 13;312(5995):643–646. [PubMed]
  • Neuberger MS, Williams GT, Mitchell EB, Jouhal SS, Flanagan JG, Rabbitts TH. A hapten-specific chimaeric IgE antibody with human physiological effector function. Nature. 1985 Mar 21;314(6008):268–270. [PubMed]
  • Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 321(6069):522–525. [PubMed]
  • Verhoeyen M, Milstein C, Winter G. Reshaping human antibodies: grafting an antilysozyme activity. Science. 1988 Mar 25;239(4847):1534–1536. [PubMed]
  • Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature. 1988 Mar 24;332(6162):323–327. [PubMed]
  • Oi VT, Morrison SL, Herzenberg LA, Berg P. Immunoglobulin gene expression in transformed lymphoid cells. Proc Natl Acad Sci U S A. 1983 Feb;80(3):825–829. [PMC free article] [PubMed]
  • Ochi A, Hawley RG, Shulman MJ, Hozumi N. Transfer of a cloned immunoglobulin light-chain gene to mutant hybridoma cells restores specific antibody production. Nature. 1983 Mar 24;302(5906):340–342. [PubMed]
  • Neuberger MS. Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. EMBO J. 1983;2(8):1373–1378. [PMC free article] [PubMed]
  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed]
  • Scharf SJ, Horn GT, Erlich HA. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Lee CC, Wu XW, Gibbs RA, Cook RG, Muzny DM, Caskey CT. Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science. 1988 Mar 11;239(4845):1288–1291. [PubMed]
  • Mènard S, Tagliabue E, Canevari S, Fossati G, Colnaghi MI. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res. 1983 Mar;43(3):1295–1300. [PubMed]
  • Bosslet K, Steinsträsser A, Schwarz A, Harthus HP, Lüben G, Kuhlmann L, Sedlacek HH. Quantitative considerations supporting the irrelevance of circulating serum CEA for the immunoscintigraphic visualization of CEA expressing carcinomas. Eur J Nucl Med. 1988;14(11):523–528. [PubMed]
  • Bosslet K, Kern HF, Kanzy EJ, Steinstraesser A, Schwarz A, Lüben G, Schorlemmer HU, Sedlacek HH. A monoclonal antibody with binding and inhibiting activity towards human pancreatic carcinoma cells. I. Immunohistological and immunochemical characterization of a murine monoclonal antibody selecting for well differentiated adenocarcinomas of the pancreas. Cancer Immunol Immunother. 1986;23(3):185–191. [PubMed]
  • Bosslet K, Lüben G, Schwarz A, Hundt E, Harthus HP, Seiler FR, Muhrer C, Klöppel G, Kayser K, Sedlacek HH. Immunohistochemical localization and molecular characteristics of three monoclonal antibody-defined epitopes detectable on carcinoembryonic antigen (CEA). Int J Cancer. 1985 Jul 15;36(1):75–84. [PubMed]
  • Bremer EG, Levery SB, Sonnino S, Ghidoni R, Canevari S, Kannagi R, Hakomori S. Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland. J Biol Chem. 1984 Dec 10;259(23):14773–14777. [PubMed]
  • Brüggemann M, Williams GT, Bindon CI, Clark MR, Walker MR, Jefferis R, Waldmann H, Neuberger MS. Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J Exp Med. 1987 Nov 1;166(5):1351–1361. [PMC free article] [PubMed]
  • Staden R. The current status and portability of our sequence handling software. Nucleic Acids Res. 1986 Jan 10;14(1):217–231. [PMC free article] [PubMed]
  • Zoller MJ, Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. [PMC free article] [PubMed]
  • Carter P, Bedouelle H, Winter G. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res. 1985 Jun 25;13(12):4431–4443. [PMC free article] [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Kearney JF, Radbruch A, Liesegang B, Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol. 1979 Oct;123(4):1548–1550. [PubMed]
  • Potter H, Weir L, Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161–7165. [PMC free article] [PubMed]
  • Galfrè G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73(Pt B):3–46. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Berek C, Milstein C. The dynamic nature of the antibody repertoire. Immunol Rev. 1988 Oct;105:5–26. [PubMed]
  • Maizels N, Bothwell A. The T-cell-independent immune response to the hapten NP uses a large repertoire of heavy chain genes. Cell. 1985 Dec;43(3 Pt 2):715–720. [PubMed]
  • Innis MA, Myambo KB, Gelfand DH, Brow MA. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. [PMC free article] [PubMed]
  • Gopinathan KP, Weymouth LA, Kunkel TA, Loeb LA. Mutagenesis in vitro by DNA polymerase from an RNA tumour virus. Nature. 1979 Apr 26;278(5707):857–859. [PubMed]
  • Skerra A, Plückthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988 May 20;240(4855):1038–1041. [PubMed]
  • Better M, Chang CP, Robinson RR, Horwitz AH. Escherichia coli secretion of an active chimeric antibody fragment. Science. 1988 May 20;240(4855):1041–1043. [PubMed]
  • Bothwell AL, Paskind M, Reth M, Imanishi-Kari T, Rajewsky K, Baltimore D. Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region. Cell. 1981 Jun;24(3):625–637. [PubMed]
  • Li HH, Gyllensten UB, Cui XF, Saiki RK, Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988 Sep 29;335(6189):414–417. [PubMed]
  • Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...