Reducing interference from heterophilic antibodies in a two-site immunoassay for carcinoembryonic antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer.
To reduce heterophilic antibody interference in a two-site immunoassay for carcinoembryonic antigen (CEA), we utilized a human/mouse chimeric antibody to CEA as the tracer. One mouse monoclonal antibody (MAb), F82-61, which reacts with an epitope present on the domain N of CEA, was immobilized on 96-well polystyrene microtiter plates. A human/mouse chimeric antibody (Ch F11-39), which recognizes an epitope present on the domain B3 of CEA, was biotinylated for the tracer (Ch F11-39 system). Another MAb F11-39, the parental MAb of Ch F11-39, was also biotinylated and used as the control tracer (F11-39 system). For a fair comparison, the same 503 serum samples from healthy individuals were simultaneously assayed in the present study. When a tentative common reference limit of 5 ng/ml was used, the false positive rate with the Ch F11-39 system was only 2.8% (14/503) and that with the F11-39 system was 29.0% (146/503). Adding normal mouse serum (NMS; 1%) or a mixture of purified mouse IgG subclasses (heterophilic blocking reagent (HBR, 15 micrograms/test)) to the F11-39 system reduced the false positive rate from 29.0% to 6.2% (31/503) or 4.8% (24/503), respectively, suggesting that heterophilic antibodies reactive with mouse IgG gave rise to the high positive rate in normal populations with the F11-39 system. On the other hand, the false positive rate with the Ch F11-39 system was only slightly reduced from 2.8% to 2.6% (13/503) or to 2.0% (10/503) by adding NMS or HBR to the Ch F11-39 system. The false positive rates with two commercially available assay systems, CEA Roche EIA.DM or Abbott IMx CEA, were 5.4% (27/503) and 5.8% (29/503), respectively, which both corresponded roughly to that with the F11-39 system including NMS or HBR. These results indicate that the application of human/mouse chimeric antibodies in two-site immunoassays is more effective for reducing interference from heterophilic antibodies than the adding of NMS or purified mouse IgG in the assay using conventional MAbs.
Kuroki
M
M
First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Matsumoto
Y
Y
Arakawa
F
F
Haruno
M
M
Murakami
M
M
Kuwahara
M
M
Ozaki
H
H
Senba
T
T
Matsuoka
Y
Y
D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology; D000910:Antibodies, Heterophile / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002272:Carcinoembryonic Antigen / Q000032:analysis* / Q000276:immunology; D005189:False Positive Reactions; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D012217:Rheumatoid Factor / Q000276:immunology
D000974:Antibodies, Antinuclear; D000910:Antibodies, Heterophile; D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D011993:Recombinant Fusion Proteins; D012217:Rheumatoid Factor