High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction

• Published in: Nucleic acids research 1995-02, Vol.23 (3), p.522-529
• Main Authors: Hendrickson, Edwin R., Truby, Tina M. Hatfield, Joerger, Rolf D., Majarian, William R., Ebersole, Richard C.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - chemistry; Base Sequence; beta-Galactosidase - analysis; Chorionic Gonadotropin - analysis; DNA, Single-Stranded - chemistry; Enzyme-Linked Immunosorbent Assay; Immunoassay - methods; Indicators and Reagents; Molecular Sequence Data; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Thyrotropin - analysis
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/23.3.522

A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and βGal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specitic monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate co-amplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme Immunoassays by approximately three orders of magnitude. Detection limits for hTSH, β-Gal and hCG were respect Ively 1 × 10−19, 1 × 10−17 and 1 × 10−17 mol. Given the enormous amplification afforded by PCR and the exist ing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays.