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.   


1.Allain, J.-P., Y. Laurian, D. A. Paul, F. Verroust, M. Leuther, C. Gazengel, D. Senn, M.-J. Larrieu, and C. Bosser. Longterm evaluation of HIV antigen and antibodies to p24 and gp4l in patients with hemophilia. (1987) N. Engl. J. Med. 317:1114-1121.
2.Chen J, Wang L, Chen JJ, Sahu GK, Tyring S, Ramsey K, Indrikovs AJ, Petersen JR, Paar D, Cloyd MW. Detection of antibodies to human immunodeficiency virus (HIV) that recognize conformational epitopes of glycoproteins 160 and 41 often allows for early diagnosis of HIV infection. (2002) J Infect Dis. 1; 186(3):321-31.
3. Kiptoo MK, Mpoke SS, Ng'ang'a ZW. New indirect immunofluorescence assay as a confirmatory test for human immunodeficiency virus type 1. (2004) East Afr Med J; 81(5):222-5.
4.Consortium for Retrovirus Serology Standardization. Serologic diagnosis of human immunodeficiency virus infection by Western blot testing. (1998) J. Am. Med. Assoc. 260:674-679.
5.Cooper, D. A., J. Gold, P. Maclean, B. Donovan, R. Finlayson, T. G. Barnes, H. M. Michelmore, P. Brooke, and R. Penny. Acute AIDS retrovirus infection. Definition of a clinical illness associated with seroconversion. (1985) Lancet i: 537-540.
6.Ravanshad M, Sabahi F, Mahboudi F, Kazemnejad A. Evaluation of a new dot blot assay for confirmation of human immunodeficiency virus type 1 and 2 infections using recombinant p24, gp41, gp120 and gp36 antigens. (2006) Saudi Med J.; 27(1):31-6.
7.Frosner, G. G., V. Erfle, W. Mellert, and R. Hehlmann. Diagnostic significance of quantitative determination of HIV antibody specific for envelope and core protein. (1997) Lancet i: 159-160.
8.Goudsmit, J., J. M. A. Lange, D. A. Paul, and G. J. Dawson. Antigenemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex, and subclinical human immunodeficiency virus infection. (1987) J. Infect. Dis. 155:558-560.
9.Hofbauer, J. M., T. F. Schulz, P. Hengster, C. Larcher, R. Zangerle, H. Kofler, P. Fritsch, H. Wachter, and M. P. Dierich. Comparison of Western blot based on recombinant-derived p4l with conventional tests for serodiagnosis of human immunodeficiency virus infections. (1988) J. Clin. Microbiol. 26:116-120.
10.Hogervorst  E., Jurriaans S., de Wolf, F., van Wijk, A., Wiersma, A., Valk, M., Roos, M., van Gemen, G.B., Coutinho, R., Miedema, F., Predictors for non- and slow-progression in human immunodeficiency virus (HIV) type 1 infection: low viral RNA copy numbers in serum and maintenance of high HIV-1 p24-specific but not V3-specific antibody levels. (1995) J. Infect. Dis. 171, 811-821.
11.Lange, J., Goudsmit, J., Decline of antibody reactivity to HIV core protein secondary to increase production of HIV antigen. (1997) Lancet i, 448-449.
12.Mayer, K. H., L. A. Falk, D. A. Paul, G. J. Dawson, A. M. Stoddard, J. McCusker, S. P. Saltzmann, M. W. Moon, R. Ferriani, and J. E. Groopman. Correlation of recombinant antigen and antibody to the human immunodeficiency virus (HIV) with subsequent clinical outcomes in a cohort of asymptomatic homosexual males. (1997) Am. J. Med. 83:208-212.
13.Ranki, A., E. Johansson, and K. Krohn. Interpretation of antibodies reacting solely with human retroviral core proteins. (1998) N. Engl. J. Med. 318:448-449.
14.Yerly S, Simon F, Perrin L. Early diagnosis of primary HIV infections: using a combined screening test (p24 antigen and anti-HIV antibodies) (1999) Schweiz Med Wochenschr. 27; 129(8):319-22.
15.Schmidit, G., Amiraian, K., Frey, H., Stevens, R.W., Berns, D.S., Analysis of Western blot assays for human immunodeficiency virus antibodies and correlation with clinical status. (1997) J. Clin. Microbiol. 25, 1993-1998.
16.Shatzman, A., and M. Rosenberg. Expression, identification, and characterization of recombinant gene products in Escherichia coli. Methods Enzymol. (1997) 152:661-673.
17.Strathdee, S.A., Frank, J.W., McLaughlin, J., Leblanc, M., Major, C., O’Shaughnessy, M.V., Read, S.E., Quantitative measure of human immunodeficiency virus-specific antibodies predicts progression to AIDS. (1995) J. Infect. Dis. 172, 1375-1379.
18.Ng VL, Chiang CS, Debouck C, McGrath MS, Grove TH, Mills J. Reliable confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) with an enzyme-linked immunoassay using recombinant antigens derived from the HIV-1 gag, pol, and env genes. (1989) J Clin Microbiol.; 27(5):977-82.
19.Lawoko A, Johansson B, Rabinayaran D, Pipkorn R, Blomberg J. Increased immunoglobulin G, but not M, binding to endogenous retroviral antigens in HIV-1 infected persons. (2000) J Med Virol.; 62(4):435-44.
20.Saville RD, Constantine NT, Cleghorn FR, Jack N, Bartholomew C, Edwards J, Gomez P, Blattner WA. Fourth-generation enzyme-linked immunosorbent assay for the simultaneous detection of human immunodeficiency virus antigen and antibody. (2001) J Clin Microbiol.; 39(7):2518-24.
21.Mahboudi F, Irina NA, Chevalier A, Ghadiri A, Adeli A, Amini-Bavil-Olyaee S, Barkhordari F, Farzamfar B, Alinejad M. A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia (2006), J Biotechnol. 1; 125(2):295-303.
22.Van Der Poel, C. L., H. W. Reesink, T. Tersmette, P. N. Lelie, H. Huisman, and F. Miedema. Blood donations reactive to HIV in Western blot, but non-infective in culture and recipients of blood. (1996) Lancet ii: 752-753.
23.Weiss, S. H., J. J. Goeert, M. G. Sarngadharan, A. J. Bodner, The AIDS Seroepidemiology Collaborative Working Group, R. C. Gallo, and W. A. Blattner. Screening test for HTLV-II (AIDS agent) antibodies. (1985)  J. Am. Med. Assoc. 253:221-225.