Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR).
Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody-antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (< 1.25 micrograms/ml) in a physiologically relevant range (1-40 micrograms/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.
Vikinge
T P
TP
Laboratory of Applied Physics, IFM, Linköping University, Sweden. trv@ifm.liu.se
Askendal
A
A
Liedberg
B
B
Lindahl
T
T
Tengvall
P
P
D000818:Animals; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000097:blood*; D002645:Chickens; D003167:Complement Activation; D003176:Complement C3 / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007136:Immunoglobulins / Q000276:immunology*; D051379:Mice; D012031:Refractometry; D020349:Surface Plasmon Resonance
D000941:Antigens; D003176:Complement C3; C016512:IgY; D007074:Immunoglobulin G; D007136:Immunoglobulins