Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2002 Nov 12;110(1):11–18. doi: 10.1016/0022-1759(88)90077-4

Measurement of the concentration of murine IgG monoclonal antibody in hybridoma supernatants and ascites in absolute units by sensitive and reliable enzyme-linked immunosorbent assays (ELISA)

JO Fleming 1,∗, LB Pen 1
PMCID: PMC7130540  PMID: 2836513

Abstract

We have investigated an enzyme-linked immunosorbent assay (ELISA) for mouse IgG using affinity-purified goat anti-mouse antibodies for capture and detection. This assay was used to measure the absolute or weight/volume concentration of murine monoclonal antibody in hybridoma supernatants. Bovine or horse serum did not interfere with the assay, which permitted reliable measurement of all murine IgG subclasses except IgG3 in the 1–20 ng/ml range. Antibody capture was essentially complete in the optimized assay. In combination with an antigen-dependent ELISA, the assay allowed estimation of the absolute concentration of specific monoclonal antibody in ascites. These rapid and relatively simple assays may be applicable in many situations in which a practical means of measuring murine monoclonal antibodies in weight/volume units is needed.

Keywords: Monoclonal antibody; IgG, murine; Ascites; Protein determination

Abbreviations: Ig, immunoglobulin; IgG, immunoglobulin G; ELISA, enzyme-linked immunosorbent assay; viral-ELISA, ELISA measuring antibodies to different coronaviruses; C-ELISA, capture ELISA for measuring murine IgG concentration; GAM-HRP, goat anti-mouse IgG conjugated to horseradish peroxidase; GAM-CAP, goat anti-mouse IgG, unconjugated, used in the capture assay; EM, ELISA medium; PBS, phosphate-buffered sal0ine; SPA, Sepharose-protein A

References

  1. Bosworth J.M., Brimfield A.A., Naylor J.A., Hunter K.W. Measurement of monoclonal antibody concentrations in hybridoma cultures: comparisons of competitive inhibition and antigen capture enzyme immunoassays. J. Immunol. Methods. 1983;62:331. doi: 10.1016/0022-1759(83)90177-1. [DOI] [PubMed] [Google Scholar]
  2. Butler J.E., Spradling J.E., Suter M., Dierks S.E., Heyermann H., Peterman J.H. The immunochemistry of sandwich ELISAs. I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates. Mol. Immunol. 1986;23:971. doi: 10.1016/0161-5890(86)90128-8. [DOI] [PubMed] [Google Scholar]
  3. Butler J.E., Peterman J.H., Suter M., Dierks S.E. Vol. 46. 1987. The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays; p. 2548. (Fed. Proc.). [PubMed] [Google Scholar]
  4. Dierks S.E., Butler J.E., Richerson H.B. Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA. Mol. Immunol. 1986;23:403. doi: 10.1016/0161-5890(86)90138-0. [DOI] [PubMed] [Google Scholar]
  5. Ey P.L., Prowse S.J., Jenkin C.R. Isolation of pure IgG1, IgG2a, and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry. 1978;15:429. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  6. Ferrante A., Rowan-Kelly B., Beard L.J., Maxwell G.M. An enzyme-linked immunosorbent assay for the quantitation of human IgG subclasses using monoclonal antibodies. J. Immunol. Methods. 1986;93:207. doi: 10.1016/0022-1759(86)90190-0. [DOI] [PubMed] [Google Scholar]
  7. Fleming J.O., Stohlman S.A., Harmon R.C., Lai M.M.C., Frelinger J.A., Weiner L.P. Antigenic relationships of murine coronaviruses: Analysis using monoclonal antibodies of JHM (MHV-4) virus. Virology. 1983;131:296. doi: 10.1016/0042-6822(83)90498-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galfrè G., Milstein C. Preparation of monoclonal antibodies: Strategies and procedures. Methods Enzymol. 1981;73:3. doi: 10.1016/0076-6879(81)73054-4. [DOI] [PubMed] [Google Scholar]
  9. Gilmore W., Fleming J.O., Stohlman S.A., Weiner L. Vol. 185. 1987. Characterization of the structural proteins of the murine coronavirus strain A59 using monoclonal antibodies; p. 177. (Proc. Soc. Exp. Biol. Med.). [DOI] [PubMed] [Google Scholar]
  10. Herlyn D., Lubeck M., Sears H., Koprowski H. Specific detection of anti-idiotypic immune responses in cancer patients treated with murine monoclonal antibody. J. Immunol. Methods. 1985;85:27. doi: 10.1016/0022-1759(85)90271-6. [DOI] [PubMed] [Google Scholar]
  11. Hunter K.W., Bosworth J.M. Measurement of monoclonal immunoglobulin concentrations in hybridoma cultures by competitive inhibition enzyme immunoassay. Methods Enzymol. 1986;121:541. doi: 10.1016/0076-6879(86)21053-8. [DOI] [PubMed] [Google Scholar]
  12. Kay C., Nix A.B.J., Kemp K.W., Rowlands R.J., Richards G., Groom G.V., Griffiths K., Wilson D.W. Dose-interpolation of immunoassay data: Uncertainties associated with curve-fitting. Stat. Med. 1986;5:183. doi: 10.1002/sim.4780050208. [DOI] [PubMed] [Google Scholar]
  13. Manil L., Motté P., Pernas P., Troalen F., Bohuon C., Bellet D. Evaluation of protocols for purification of mouse monoclonal antibodies. J. Immunol. Methods. 1986;90:25. doi: 10.1016/0022-1759(86)90379-0. [DOI] [PubMed] [Google Scholar]
  14. Mattila P.S. Quantitation of antibody isotypes in solid-phase assays. Comparison of myeloma protein and monoisotypic antibody standards. J. Immunol. Methods. 1985;83:43. doi: 10.1016/0022-1759(85)90056-0. [DOI] [PubMed] [Google Scholar]
  15. Panush R.S., Katz P., Longley S. In vitro immunoglobulin production by mononuclear cells in rheumatoid arthritis. Clin. Immunol. Immunopathol. 1983;28:252. doi: 10.1016/0090-1229(83)90159-9. [DOI] [PubMed] [Google Scholar]
  16. Partridge C.S., Britton D.W., Bagshawe K.D. Use of the direct linear plot to estimate, in an immunoglobulin solution, the proportion which is specific monoclonal antibody against an unknown cell surface antigen. J. Immunol. Methods. 1985;78:95. doi: 10.1016/0022-1759(85)90332-1. [DOI] [PubMed] [Google Scholar]
  17. Pesce A.J., Ford D.J., Makler M.T. Properties of enzyme immunoassays. In: Ishikawa E., Kawai T., Miyai K., editors. Enzyme Immunoassay. Igaku-Shoin; Tokyo: 1981. p. 27. [Google Scholar]
  18. Sancho J., Sánchez-Madrid F., Felipe O., Bernabeu C., Morago G., Carreira J. Quantitative measurement of human immunoglobulin E using monoclonal antibodies to distinct epitopes. J. Immunol. Methods. 1986;90:71. doi: 10.1016/0022-1759(86)90385-6. [DOI] [PubMed] [Google Scholar]
  19. Shields J.G., Turner M.W. The importance of antibody quality in sandwich ELISA systems. Evaluation of selected commercial reagents. J. Immunol. Methods. 1986;87:29. doi: 10.1016/0022-1759(86)90340-6. [DOI] [PubMed] [Google Scholar]
  20. Springer T.A. Quantitation of hybridoma immunoglobulins and selection of light-chain loss variants. Methods Enzymol. 1983;92:147. doi: 10.1016/0076-6879(83)92014-1. [DOI] [PubMed] [Google Scholar]
  21. Steward M.W., Lew A.M. The importance of antibody affinity in the performance of immunoassays for antibody. J. Immunol. Methods. 1985;78:173. doi: 10.1016/0022-1759(85)90074-2. [DOI] [PubMed] [Google Scholar]
  22. Tijssen P. In: Laboratory Techniques in Biochemistry and Molecular Biology. Burdon R.H., van Knippenberg P.H., editors. Vol. 15. Elsevier; Amsterdam: 1985. p. 197. (Practice and Theory of Enzyme Immunoassays). [Google Scholar]; Tijssen P. In: Laboratory Techniques in Biochemistry and Molecular Biology. Burdon R.H., van Knippenberg P.H., editors. Vol. 15. Elsevier; Amsterdam: 1985. p. 385. (Practice and Theory of Enzyme Immunoassays). [Google Scholar]
  23. Underwood P.A. Removal of bovine immunoglobulin from serum in hybridoma culture media using protein A. Methods Enzymol. 1986;121:301. doi: 10.1016/0076-6879(86)21028-9. [DOI] [PubMed] [Google Scholar]
  24. Ziai M.R., Imberti L., Ferrone S. An enzyme-linked double antibody immunoassay to measure murine immunoglobulins - Its application to determine the specific activity of radiolabeled monoclonal antibodies. J. Immunol. Methods. 1985;82:233. doi: 10.1016/0022-1759(85)90355-2. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Immunological Methods are provided here courtesy of Elsevier

RESOURCES