TY - JOUR
T1 - Assay interference caused by antibodies reacting with rat kappa light-chain in human sera
AU - Degn, Søren Egedal
AU - Andersen, Stig Henrik
AU - Jensen, Lisbeth
AU - Thiel, Steffen
AU - Jensenius, Jens C
N1 - Copyright © 2011 Elsevier B.V. All rights reserved.
PY - 2011
Y1 - 2011
N2 - The enzyme-linked immunosorbent assay (ELISA) and its derivatives are powerful tools used in research, in the clinic, and in many other analytical and quality control settings. In general, ELISAs are robust, reproducible and reliable. However, a number of pitfalls of ELISAs have been described over the years. The issue of rheumatoid factor (RF), autoantibodies against the Fc portion of IgG, is well recognized (yet often forgotten), as are problems arising from heterophilic antibodies induced by external antigens that cross-react with self-antigens. A few years ago focus was on human anti-mouse antibodies (HAMA) concomitant with the increased use of mouse monoclonal antibody therapy, a problem that is now diminishing due to development of humanized antibodies. Issues pertaining to food antigens or environmentally encountered antigens are less recognized. We report a recently encountered example of the latter resulting in interference in a solid-phase sandwich assay. Due to the set-up employing a monoclonal rat IgG for capture and a monoclonal rat IgM for development the interference had to be human antibodies reacting with rat light-chain. Out of 102 Danish Caucasian blood donors we found a prevalence of anti-rat kappa light chain antibodies of close to 40% (39/102, defined as at least 2-fold elevated measurements), with around 6% (6/102) having very high levels (defined as at least 4-fold elevated measurements), yielding significantly higher measurements in the assay designed to measure the complement component MAp19 in serum samples. The interference could be blocked by the addition of rat immunoglobulin to the sample buffer. An individual, who had been followed over time, demonstrated a periodic increase of interfering antibodies, highlighting that it is an independently varying parameter and thereby a variable interference in assays. Our results highlight a major pitfall of potential relevance to many sandwich-type assays, as well as an approach to rectify such problems.
AB - The enzyme-linked immunosorbent assay (ELISA) and its derivatives are powerful tools used in research, in the clinic, and in many other analytical and quality control settings. In general, ELISAs are robust, reproducible and reliable. However, a number of pitfalls of ELISAs have been described over the years. The issue of rheumatoid factor (RF), autoantibodies against the Fc portion of IgG, is well recognized (yet often forgotten), as are problems arising from heterophilic antibodies induced by external antigens that cross-react with self-antigens. A few years ago focus was on human anti-mouse antibodies (HAMA) concomitant with the increased use of mouse monoclonal antibody therapy, a problem that is now diminishing due to development of humanized antibodies. Issues pertaining to food antigens or environmentally encountered antigens are less recognized. We report a recently encountered example of the latter resulting in interference in a solid-phase sandwich assay. Due to the set-up employing a monoclonal rat IgG for capture and a monoclonal rat IgM for development the interference had to be human antibodies reacting with rat light-chain. Out of 102 Danish Caucasian blood donors we found a prevalence of anti-rat kappa light chain antibodies of close to 40% (39/102, defined as at least 2-fold elevated measurements), with around 6% (6/102) having very high levels (defined as at least 4-fold elevated measurements), yielding significantly higher measurements in the assay designed to measure the complement component MAp19 in serum samples. The interference could be blocked by the addition of rat immunoglobulin to the sample buffer. An individual, who had been followed over time, demonstrated a periodic increase of interfering antibodies, highlighting that it is an independently varying parameter and thereby a variable interference in assays. Our results highlight a major pitfall of potential relevance to many sandwich-type assays, as well as an approach to rectify such problems.
KW - Animals
KW - Blood Donors
KW - Cohort Studies
KW - Cross Reactions
KW - Denmark
KW - Enzyme-Linked Immunosorbent Assay
KW - False Positive Reactions
KW - Humans
KW - Immunoglobulin G
KW - Immunoglobulin kappa-Chains
KW - Mannose-Binding Protein-Associated Serine Proteases
KW - Rats
U2 - 10.1016/j.jim.2011.06.030
DO - 10.1016/j.jim.2011.06.030
M3 - Journal article
C2 - 21771595
SN - 0022-1759
VL - 372
SP - 204
EP - 208
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -