Quantification Analysis of Dot Blot Assays for Human Immunodeficiency Virus Type 1 and 2 Antibodies

Document Type: Original Article


1 Department of Virology, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran

2 Biotechnology Research Center, Pasture Institute of Iran, Tehran, Iran


Dot Blot (DB) assay provides highly specific results, but usually not reliable for quantification of antibody production. The need for a more objective DB assay to provide a better definition of the immune status, against HIV antigens, promoted this study to be done to develop a quantitative DB assay.
Material and Methods
Dot blot (DB) strips for antibodies directed to human immunodeficiency virus (HIV) type 1 and 2 were analyzed by a video densitometer. This method was used to quantify the antibody response to different HIV proteins in infected patients. In order to increase reproducibility, reagents and protocols were accurately standardized and internal controls were added. In the first format, an internal control band consisting of Human IgG was added to each dot to minimize the effects of band intensity variation. In the second format, antibody concentrations were calculated from the ratio of the densities produced by test sera and by positive and negative standard sera.
The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the obtained results were compared with those of the corresponding DB. A statistically significant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titers were compared to corrected DB values (p = 0.001).
Densitometric analysis of DB assays led to quantify the antibodies against HIV-1 and 2 Gag and Env proteins and might be useful to investigate possible humoral immune correlates of production in HIV vaccine studies and antibody production in the early phase of infection.   


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