Matrix interference from Fc-Fc interactions in immunoassays for detecting human IgG4 therapeutics.
BACKGROUND
An assay measuring an IgG4 biotherapeutic in human serum used a drug-specific monoclonal antibody (mAb) capture reagent and an antihuman IgG4 mAb as detection reagent. However, serum IgG4 binding to the capture mAb via Fc-interactions was detected by the anti-IgG4 mAb, causing high background.


RESULTS
Two approaches were developed to minimize background; incorporating a mild acid sample preparation step or using the Fab of the capture antibody. Either strategy improved signal:noise dramatically, increasing assay sensitivity >20-fold. Biophysical analyses of antibody domains indicated that noncovalent Fc oligomers could inhibit the background.


CONCLUSIONS
Matrix interference from human IgG4 binding to the capture mAb was reduced with a Fab fragment of the drug-specific capture antibody or by incorporating a mild acid sample treatment into the assay.
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Michael A
MA
Partridge
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Elif Kabuloglu
EK
Karayusuf
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Gangadhar
G
Dhulipala
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Robert
R
Dreyer
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Thomas
T
Daly
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Giane
G
Sumner
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Erica
E
Pyles
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
Albert
A
Torri
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000276:immunology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000882:Haplorhini; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology; D051379:Mice; D063347:Native Polyacrylamide Gel Electrophoresis; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G