Detection of the products of polymerase chain reaction by an ELISA system based on an ion sensitive field effect transistor.
An ELISA system was developed using a pH sensitive ISFET (pH-FET) as a detector, a pipette tip as a solid phase, and urease as a detecting enzyme. Double stranded PCR products with digoxigenin and biotin at both terminals were obtained by using digoxigenin- and biotin-labeled primers. 1 microliters of the PCR solution was directly introduced into the end part of a pipette tip coated with anti-digoxigenin antibody. Biotin-labeled PCR products captured at the solid phase were detected with avidin-urease, of which the activity was measured by a pH-FET in a pH-measuring cell containing urea solution. The assay was used to detect HTLV-I provirus gene integrated in the genome of a human MT-1 cell, and it was found that 100 pg of the genomic DNA of MT-1 cell was specifically detectable after 35 cycles of PCR. Also the detection limit of the present ELISA system itself was determined by using known amounts of purified PCR product labeled with digoxigenin and biotin, and it was found that 10 amol of the labeled DNA in 1 microliter of sample was detectable.
Biosensor Research Laboratories Co., Ltd., Okayama, Japan.
H
H
Tsuruta
S
S
Matsui
T
T
Hatanaka
T
T
Namba
K
K
Miyamoto
M
M
Nakamura
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016679:Genome, Viral; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000821:virology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics*; D014173:Transistors, Electronic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004279:DNA, Viral