Effects of antibody affinity and antigen valence on molecular forms of immune complexes.
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 NP(7)-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 NP(7)-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 NP(7)-BSA, only 1:1 immune complexes were observed by SPR. These results showed that two NP(7)-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.
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
Masayuki
M
Oda
Susumu
S
Uchiyama
Masanori
M
Noda
Yoshinori
Y
Nishi
Maiko
M
Koga
Kouta
K
Mayanagi
Carol V
CV
Robinson
Kiichi
K
Fukui
Yuji
Y
Kobayashi
Kosuke
K
Morikawa
Takachika
T
Azuma
D000818:Animals; D000915:Antibody Affinity / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology*; D000941:Antigens / Q000737:chemistry / Q000276:immunology*; D002417:Cattle; D055767:Immobilized Proteins / Q000737:chemistry; D013058:Mass Spectrometry; D018448:Models, Immunological; D009596:Nitrophenols / Q000276:immunology; D010648:Phenylacetates / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000648:ultrastructure
D000936:Antigen-Antibody Complex; D000941:Antigens; D055767:Immobilized Proteins; D009596:Nitrophenols; D010648:Phenylacetates; C026242:4-hydroxy-5-nitrophenyl acetic acid; D012710:Serum Albumin, Bovine