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. 1984 Apr;51(4):809–814.

Immune complex formation analysed by high-performance size exclusion chromatography (HPLC-SEC) using either 125I-labelled antigen or enzyme-linked immunosorbent assay (ELISA) for detection.

U Holmskov-Nielsen, J C Jensenius, K Erb, S Husby
PMCID: PMC1454548  PMID: 6423527

Abstract

A physical/immunochemical method has been developed for the analysis of soluble immune complexes. Human serum with high amount of antibody against bovine serum albumin (BSA) was incubated at 37 degrees with a wide range of 125I-labelled BSA and separated according to size by high-performance size exclusion chromatography (HPLC-SEC) on a TSK G 6000 PW column. Fractions were collected onto microplates and analysed either by gamma counting or by an enzyme-linked immunosorbent assay (ELISA) technique detecting anti-BSA antibody. The size of the immune complexes were the same when analysed by the two methods. The heaviest immune complexes were seen in moderate antibody excess where the majority of the complexes were eluted in a broad peak around Mr of 7 X 10(6). In large antibody excess the immune complexes eluted at around 3 X 10(6). Around equilibrium two peaks were seen corresponding to 3 X 10(6) and 5 X 10(5). In antigen excess the peak at 3 X 10(6) is diminished and free antigen appears. No changes in the distribution of the complexes were observed when the serum was made 20 mM in EDTA before incubation with BSA.

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Selected References

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

  1. Folkersen J., Teisner B., Svendsen P., Svehag S. E. An immunoprecipitation-dissociation technique for large scale antibody purification and an antigen consumption electroimmunoassay for antibody quantitation. A model study with antibodies to pregnancy zone protein. J Immunol Methods. 1978;23(1-2):127–135. doi: 10.1016/0022-1759(78)90116-3. [DOI] [PubMed] [Google Scholar]
  2. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  3. Kijlstra A., Knutson D. W., van der Lelij A., van Es L. A. Characteristics of soluble immune complexes prepared from oligovalent DNP conjugates and anti-DNP antibodies. J Immunol Methods. 1977;17(3-4):263–277. doi: 10.1016/0022-1759(77)90109-0. [DOI] [PubMed] [Google Scholar]
  4. Kroll J. Line immunoelectrophoresis. Scand J Immunol Suppl. 1973;1:61–67. [PubMed] [Google Scholar]
  5. Regnier F. E., Gooding K. M. High-performance liquid chromatography of proteins. Anal Biochem. 1980 Mar 15;103(1):1–25. doi: 10.1007/978-1-4615-6728-8_1. [DOI] [PubMed] [Google Scholar]
  6. Steensgaard J., Johansen A. S. Biochemical aspects of immune complex formation and immune complex diseases. Allergy. 1980 Sep;35(6):457–472. doi: 10.1111/j.1398-9995.1980.tb01794.x. [DOI] [PubMed] [Google Scholar]
  7. Takahashi M., Takahashi S., Hirose S. Solubilization of antigen-antibody complexes: a new function of complement as a regulator of immune reactions. Prog Allergy. 1980;27:134–166. [PubMed] [Google Scholar]
  8. Theofilopoulos A. N., Dixon F. J. Immune complexes in human diseases: a review. Am J Pathol. 1980 Aug;100(2):529–594. [PMC free article] [PubMed] [Google Scholar]
  9. Voller A., Bidwell D. E., Bartlett A. Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull World Health Organ. 1976;53(1):55–65. [PMC free article] [PubMed] [Google Scholar]

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