Limits...
Detection of hepatitis B virus DNA in human serum by dot hybridization using a biotin-labelled probe.

Roe IH, Roe JH, Lee DH - Korean J. Intern. Med. (1988)

View Article: PubMed Central - PubMed

ABSTRACT

A dot blot hybridization technique utilizing a biotin-labelled recombinant DNA probe was used to examine hepatitis B virus (HBV) DNA in serum. The lowest amount of HBV DNA in serum detectable by the color development of an avidin-biotin alkaline phosphatase complex was 40 picogram per 50 microliter. Validity of this method was confirmed by autoradiography using 32P-labelled and 3H-labelled HBV DNA probes. HBV DNA was found in 100% (34/34) of the HBsAg-positive and in 73.5% (25/34) of the HBsAg-negative subjects. In contrast, all nine cases showing negativity in HBV DNA were also HBsAg-negative. Correlation of HBe antigen/antibody with HBV DNA was investigated in 19 sera of which HBsAg was negative but anti-HBc-positive. Of 13 sera with anti-HBe 10 (76.9%) cases revealed HBV DNA positivity, while four (66.7%) of six sera without anti-HBe were positive in HBV DNA.

In conclusion, serum dot hybridization assay utilizing a biotinylated probe proved useful in the detection of a free form of HBV DNA regardless of the presence of HBsAg and irrespective of HBeAg/anti-HBe status. Moreover, it is emphasized that practical advantages in speed, reproducibility, and safety have made this alternative even more attractive than autoradiography using radioisotope-labelled probes.

Show MeSH
Dot bot hybridization assay of patient’s sera using biotin-labelled HBV DNA probe.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4532123&req=5

f2-kjim-3-1-9-2: Dot bot hybridization assay of patient’s sera using biotin-labelled HBV DNA probe.

Mentions: In order to determine the sensitivity of the biotin-labelled HBV DNA probe assay, a control study using a radioisotope-labelled DNA probe was performed. The indicated amounts of pure unlabelled HBV DNA with serial dilutions were spotted directly on nitrocellulose paper in 50 μl volumes, followed by hybridization with labelled DNA and the signal detection by either enzymatic color development of ABC-AP with substrate or autoradiography of 32P or 3H-labelling. As seen in Figure 1, the 32P-labelled probe is the most powerful in resolution and quantitation of the lowest value of 40pg/50 μl The tritiated hybridization assay, however, yields poor in resolution to quantitate that value. By dotting recombinant lambda-HBV DNA to nitrocellulose and hybridizing with our biotinylated probe, a standard pattern was established and used to classify the quantity in serum according to the intensity of staining (Figures 1 and 2). The lowest detection limit of the biotinylated probe hybridization assay was found to be also 40pg/50μl but faint in intensity in comparison to the 32P-labelled DNA hybridization assay. Contrarilly, the detection limit of tritiated probe was 100pg/50μl.


Detection of hepatitis B virus DNA in human serum by dot hybridization using a biotin-labelled probe.

Roe IH, Roe JH, Lee DH - Korean J. Intern. Med. (1988)

Dot bot hybridization assay of patient’s sera using biotin-labelled HBV DNA probe.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4532123&req=5

f2-kjim-3-1-9-2: Dot bot hybridization assay of patient’s sera using biotin-labelled HBV DNA probe.
Mentions: In order to determine the sensitivity of the biotin-labelled HBV DNA probe assay, a control study using a radioisotope-labelled DNA probe was performed. The indicated amounts of pure unlabelled HBV DNA with serial dilutions were spotted directly on nitrocellulose paper in 50 μl volumes, followed by hybridization with labelled DNA and the signal detection by either enzymatic color development of ABC-AP with substrate or autoradiography of 32P or 3H-labelling. As seen in Figure 1, the 32P-labelled probe is the most powerful in resolution and quantitation of the lowest value of 40pg/50 μl The tritiated hybridization assay, however, yields poor in resolution to quantitate that value. By dotting recombinant lambda-HBV DNA to nitrocellulose and hybridizing with our biotinylated probe, a standard pattern was established and used to classify the quantity in serum according to the intensity of staining (Figures 1 and 2). The lowest detection limit of the biotinylated probe hybridization assay was found to be also 40pg/50μl but faint in intensity in comparison to the 32P-labelled DNA hybridization assay. Contrarilly, the detection limit of tritiated probe was 100pg/50μl.

View Article: PubMed Central - PubMed

ABSTRACT

A dot blot hybridization technique utilizing a biotin-labelled recombinant DNA probe was used to examine hepatitis B virus (HBV) DNA in serum. The lowest amount of HBV DNA in serum detectable by the color development of an avidin-biotin alkaline phosphatase complex was 40 picogram per 50 microliter. Validity of this method was confirmed by autoradiography using 32P-labelled and 3H-labelled HBV DNA probes. HBV DNA was found in 100% (34/34) of the HBsAg-positive and in 73.5% (25/34) of the HBsAg-negative subjects. In contrast, all nine cases showing negativity in HBV DNA were also HBsAg-negative. Correlation of HBe antigen/antibody with HBV DNA was investigated in 19 sera of which HBsAg was negative but anti-HBc-positive. Of 13 sera with anti-HBe 10 (76.9%) cases revealed HBV DNA positivity, while four (66.7%) of six sera without anti-HBe were positive in HBV DNA.

In conclusion, serum dot hybridization assay utilizing a biotinylated probe proved useful in the detection of a free form of HBV DNA regardless of the presence of HBsAg and irrespective of HBeAg/anti-HBe status. Moreover, it is emphasized that practical advantages in speed, reproducibility, and safety have made this alternative even more attractive than autoradiography using radioisotope-labelled probes.

Show MeSH