Effects of antibody affinity and antigen valence on molecular forms of immune complexes

Masayuki Oda, Susumu Uchiyama, Masanori Noda, Yoshinori Nishi, Maiko Koga, Kouta Mayanagi, Carol V. Robinson, Kiichi Fukui, Yuji Kobayashi, Kosuke Morikawa, Takachika Azuma

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32 Citations (Scopus)


The effect of antibody affinity on molecular forms of immune complexes was investigated by measuring antigen-antibody interactions using surface plasmon resonance (SPR), electrospray ionization time-of-flight mass spectrometry under non-denaturing conditions (MS), analytical ultracentrifugation (AUC), and transmission electron microscopy (TEM). (4-Hydroxy-3-nitrophenyl)acetic acid (NP) of different valences was conjugated to bovine serum albumin (BSA) and these conjugates were used as antigens. In the interaction between N1G9, a low affinity antibody, and NP7-BSA, a 1:1 immune complex was detected as the major product and higher molecular weight complexes were not obtained by any of the methods employed. These results suggested that N1G9 predominantly formed an intramolecular divalent complex with NP7-BSA using the two Fab arms of an antibody. Although complexes of various sizes were detected by MS, AUC, and TEM in the interaction between C6, a high affinity antibody, and NP7-BSA, only 1:1 immune complexes were observed by SPR. These results showed that two NP7-BSA molecules cannot simultaneously bind to an antibody, irrespective of antibody affinity strength, when the Fc region is immobilized to a flexible dextran matrix on sensor chip but are able to do so with high affinity antibodies free in solution. The results also showed that the stoichiometry of the antigen-antibody interaction is altered by restricting the movement of the Fc region. Since immunoglobulins exist as antibodies in solution or as B cell receptors on the cell surface, it is suggested that interactions of B cell receptors with polyvalent antigens such as NP-BSA might be different from those of antibodies free in solution.

Original languageEnglish
Pages (from-to)357-364
Number of pages8
JournalMolecular Immunology
Issue number2-3
Publication statusPublished - Dec 2009
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Immunology
  • Molecular Biology


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