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Colorimetric capture assay for human-immunodeficiency-virus-I reverse transcriptase activity.

A S Rytting1, L Akerblom, J S Gronowitz

  • 1Department of Genetics and Pathology, Section of Medical Genetics, Uppsala University, BMC, Box 578, SE-751 23 Uppsala, Sweden.

Biotechnology and Applied Biochemistry
|May 21, 1999
PubMed
Summary

A new colorimetric capture assay accurately detects HIV-1 reverse transcriptase (RT) activity in samples. This sensitive assay is specific for HIV-1 RT and can be used with human serum or plasma.

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Area of Science:

  • Virology
  • Biochemistry
  • Immunology

Background:

  • HIV-1 reverse transcriptase (RT) is a key enzyme in viral replication.
  • Accurate detection of RT activity is crucial for HIV diagnosis and research.
  • Existing assays may have limitations in sensitivity, specificity, or sample compatibility.

Purpose of the Study:

  • To develop and characterize a novel colorimetric capture assay for quantifying HIV-1 RT activity.
  • To establish the sensitivity, specificity, and sample tolerance of the new assay.

Main Methods:

  • A microtitre plate-based assay utilizing specific monoclonal antibodies to capture HIV-1 RT.
  • Enzyme purification, RT reaction, and colorimetric product detection performed in situ.
  • Detection of incorporated bromodeoxyuridine triphosphate (BrdUTP) using alkaline-phosphatase-conjugated antibodies.

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Main Results:

  • The assay demonstrated a detection limit of 1.2 micro-units of RT activity (0.3 pg protein).
  • High specificity was observed, detecting HIV-1 RT from subtypes A, B, and D, but not from non-HIV-1 RTs.
  • The assay tolerated up to 50 microliters of human serum or plasma without compromising recovery.

Conclusions:

  • The developed colorimetric capture assay is a sensitive and specific method for detecting HIV-1 RT activity.
  • This assay is suitable for use with clinical samples like serum and plasma.
  • The assay offers a valuable tool for HIV research and diagnostics.