DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction.
We developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids and applied it to the detection of hepatitis B virus (HBV) DNA amplified from serum samples by means of the polymerase chain reaction (PCR) technique. The method is based on the ability of an anti-DNA monoclonal antibody to discriminate between single-stranded and double-stranded DNA. A solid phase was coated with a specific oligonucleotide probe, internal to the amplified region of HBV DNA, via an avidin-biotin bridge. The denatured PCR product was hybridized with the solid-phase probe, and the amplified DNA probe hybrid was then incubated with a monoclonal antibody specific for double- but not single-stranded DNA. Colorimetric detection of the DNA-antibody complex was achieved by adding an anti-mouse Ig antibody labeled with horseradish peroxidase. The combined use of DEIA and PCR can reveal a few HBV genome copies present in a serum sample. This method has several advantages: (a) the sensitivity is adequate for the detection of amplified DNA; (b) the signal is associated with the hybridization event, independently of modifications of the probe or of the amplification primers; and (c) the test is simple and rapid and, most importantly, requires only the standard facilities of a routine clinical laboratory.
Mantero
G
G
Consorzio per le Biotecnologie, Consiglio Nazionale delle Ricerche (CNR), Institute of Chemistry, Medical School, Brescia, Italy.
Zonaro
A
A
Albertini
A
A
Bertolo
P
P
Primi
D
D
D000888:Antibodies, Anti-Idiotypic / Q000302:isolation & purification; D001483:Base Sequence; D015139:Blotting, Southern; D003124:Colorimetry / Q000379:methods; D004247:DNA / Q000097:blood*; D015342:DNA Probes; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D000888:Antibodies, Anti-Idiotypic; D015342:DNA Probes; D004247:DNA