Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1982 Jun;48(3):705–714.

Anti-C1q column: ligand specific purification of immune complexes from human serum or plasma. Analysis of the interaction between C1q and immune complexes.

W Kilgallon, P L Amlot, B D Williams
PMCID: PMC1536616  PMID: 6811171

Abstract

An efficient and reproducible procedure has been developed for the specific isolation of immune complexes. PEG precipitation of EDTA serum or plasma was an essential preliminary step to separate complex-bound from free C1q. PEG had no discernible effect on the molecular weight size of the extracted complexes. Redissolved complexes were incubated with a Sepharose-4B column coated with anti-human C1q antibodies and following removal of unbound material the bound complexes were sequentially eluted with 0.02 M EDTA, 0.5 M NaCl and 1 M propionic acid. Characteristics of the affinity column were established by the purification of 125I-labelled BSA-anti-BSA complexes and heat-aggregated IgG (HAGG) incubated in normal human serum (NHS). EDTA and NaCl eluted complexes were of similar molecular size and contained antigen, specific antibody, as well as human IgM, IgG, albumin, C3, C3c, C3d and C1q. Acid eluted complexes contained the highest yield of specific antigen and antibody and comprised in addition human C1q and C3d. Activation of complement components after C1q made the bond between C1q and immune complexes resistant to 0.5 M NaCl and interfered with the binding between solid phase anti-C1q and complex bound C1q. Using BSA-anti-BSA complexes and HAGG activated in NHS it was apparent that only a minority of the complexed material was isolated via the C1q ligand and this probably applies to the C1q binding assay. Most complexed material could be isolated using an anti-C3 affinity column.

Full text

PDF
705

Images in this article

708Fig. 2
on p.708

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amlot P. L., Pussell B., Slaney J. M., Williams B. D. Correlation between immune complexes and prognostic factors in Hodgkin's disease. Clin Exp Immunol. 1978 Feb;31(2):166–173. [PMC free article] [PubMed] [Google Scholar]
  2. Amlot P. L., Slaney J. M., Williams B. D. Circulating immune complexes and symptoms in Hodgkin's disease. Lancet. 1976 Feb 28;1(7957):449–451. doi: 10.1016/s0140-6736(76)91475-6. [DOI] [PubMed] [Google Scholar]
  3. Casali P., Bossus A., Carpentier N. A., Lambert P. H. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with 125I-labelled Clq binding and Raji-cell RIA tests. Clin Exp Immunol. 1977 Aug;29(2):342–354. [PMC free article] [PubMed] [Google Scholar]
  4. Casali P., Lambert P. H. Purification of soluble immune complexes from serum using polymethylmetacrylate beads coated with conglutinin or C1q. Application to the analysis of the components of in vitro formed immune complexes and of immune complexes occurring in vivo during leishmaniasis. Clin Exp Immunol. 1979 Aug;37(2):295–309. [PMC free article] [PubMed] [Google Scholar]
  5. Chia D., Barnett E. V., Yamagata J., Knutson D., Restivo C., Furst D. Quantitation and characterization of soluble immune complexes precipitated from sera by polyethylene glycol (PEG). Clin Exp Immunol. 1979 Sep;37(3):399–407. [PMC free article] [PubMed] [Google Scholar]
  6. Creighton W. D., Lambert P. H., Miescher P. A. Detection of antibodies and soluble antigen-antibody complexes by precipitation with polyethylene glycol. J Immunol. 1973 Oct;111(4):1219–1227. [PubMed] [Google Scholar]
  7. Czop J., Nussenzweig V. Studies on the mechanism of solubilization of immune precipitates by serum. J Exp Med. 1976 Mar 1;143(3):615–630. doi: 10.1084/jem.143.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Greenwood B. M., Brown J. C., De Jesus D. G., Holborow E. J. Immunosuppression in murine malaria. II. The effect on reticulo-endothelial and germinal centre function. Clin Exp Immunol. 1971 Sep;9(3):345–354. [PMC free article] [PubMed] [Google Scholar]
  9. Hauptman S. P., Sobczak G. Origin of immunoglobulin-albumin complexes. Nature. 1976 Sep 2;263(5572):64–67. doi: 10.1038/263064a0. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Male D., Roitt I. M. Analysis of the components of immune complexes. Mol Immunol. 1979 Mar;16(3):197–203. doi: 10.1016/0161-5890(79)90146-9. [DOI] [PubMed] [Google Scholar]
  13. Mannik M. Binding of albumin to gamma-A-myeloma proteins and Waldenström macroglobulins by disulfide bonds. J Immunol. 1967 Nov;99(5):899–906. [PubMed] [Google Scholar]
  14. Markwell M. A., Fox C. F. Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril. Biochemistry. 1978 Oct 31;17(22):4807–4817. doi: 10.1021/bi00615a031. [DOI] [PubMed] [Google Scholar]
  15. Theofilopoulos A. N., Eisenberg R. A., Dixon F. J. Isolation of circulating immune complexes using Raji cells. Separation of antigens from immune complexes and production of antiserum. J Clin Invest. 1978 Jun;61(6):1570–1581. doi: 10.1172/JCI109077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Volanakis J. E., Stroud R. M. Rabbit C1q: purification, functional and structural studies. J Immunol Methods. 1972 Nov;2(1):25–34. doi: 10.1016/0022-1759(72)90014-2. [DOI] [PubMed] [Google Scholar]
  17. Zubler R. H., Lange G., Lambert P. H., Miescher P. A. Detection of immune complexes in unheated sera by modified 125I-Clq binding test. Effect of heating on the binding of Clq by immune complexes and application of the test to systemic lupus erythematosus. J Immunol. 1976 Jan;116(1):232–235. [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES