Quantitation of hepatitis B virus DNA in serum by ammonium sulfate precipitation and molecular hybridization.
We developed a method to quantitate hepatitis B virus (HBV) DNA in serum by ammonium sulfate fractionation and DNA hybridization. Serum samples were precipitated with 45% saturated ammonium sulfate, resuspended in buffer, and spotted on a nylon membrane. Following denaturation in alkali, HBV DNA sequences on the membrane were detected by hybridization with a 32P-labeled DNA probe of the entire HBV genome. Bound radioactivity was measured with liquid scintillation counting. Ammonium sulfate fractionation of positive samples increased assay sensitivity by 10-30-fold compared to no treatment. Sensitivity for detection of cloned HBV DNA added to negative serum was 0.2 pg. Recovery of cloned HBV DNA added to negative serum before fractionation was equivalent to direct spotting of DNA onto the membrane in the absence of serum. This method enhanced HBV DNA recovery from serum into small volumes, thereby expanding the potential analytic range of spot hybridization assays.
Wen
L T
LT
Scripps Immunology Reference Laboratory, Scripps Research Institute, La Jolla, California.
Henneberger
M
M
Nguyen
N
N
McPherson
R A
RA
D000328:Adult; D000368:Aged; D000645:Ammonium Sulfate; D004279:DNA, Viral / Q000097:blood*; D005260:Female; D006509:Hepatitis B / Q000097:blood* / Q000628:therapy; D006801:Humans; D006824:Hybridization, Genetic; D016898:Interferon-alpha / Q000008:administration & dosage; D008297:Male; D008875:Middle Aged
D004279:DNA, Viral; D016898:Interferon-alpha; D000645:Ammonium Sulfate