Premium
Detection of human immunodeficiency virus (HIV) by colorimetric assay for reverse transcriptase activity on magnetic beads
Author(s) -
Suzuki K.,
Craddock BP,
Okamoto N.,
Kano T.,
Steigbigel RT
Publication year - 1993
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1993.tb00254.x
Subject(s) - digoxigenin , reverse transcriptase , microbiology and biotechnology , alkaline phosphatase , chemistry , immunoassay , thymidine , enzyme , dig , biology , virology , antibody , biochemistry , in vitro , polymerase chain reaction , messenger rna , in situ hybridization , immunology , gene
A colorimetric assay for detection of reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using oligodeoxythymidylic acid (oligo‐dT)‐linked magnetic beads and digoxigenin‐deoxyuridine triphosphate (dig‐dUTP). During the RT reaction, dig‐dUTP was incorporated into oligo‐dT which had been hybridized to polyadenylic acid [poly (A)]. At the detection step, an alkaline phosphatase‐conjugated antibody to digoxigenin was added, followed by the addition of a colorimetric substrate for this enzyme. This method showed excellent correlation with the isotopic RT assay, which used tritiated thymidine triphosphate ([3H]dTTP), for detection of purified avian‐myeloblastosis‐virus RT (AMV‐RT). This assay also demonstrated close correlation with the isotopic RT assay using human peripheral‐blood lymphocytes infected in vitro with HIV. This colorimetric RT assay offers important advantages over the conventional radioactive RT assays with respect to its simplicity, safety and cost. The total assay time, including the RT reaction step, was less than 1 h, and therefore provides a reliable rapid assay for detection and quantification of HIV.