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Performance characteristics of a rapid western blot assay system for antibody to human immunodeficiency virus type 1
Jaw& of Virological Methods, 33 (1991) 291-298 0 1991 Elsevier Science Publishers B.V. / 0168-8510/91/$03.50 ADONIS 01688510100252A
291
VIRMET 01191
Performance characteristics of a rapid Western blot assay system for antibody to human immunode~ciency virus type 1 Olajide Agbede, Syed Wahab, Donna L. Schutt and Helen L. Lucia Department of Pathology, University of Texas Medical Branch, Galveston, Texas, U.S.A. (Accepted
19 March 1991)
Summary
One hundred and twenty reactive sera were selected from specimens studied by enzyme immunoassay (EIA, Abbott Laboratories, Abbott Park, North Chicago, IL) for antibodies against human immunode~ciency virus (HIV-l). Using these sera, the ‘WesPage’ system (American Bionetics, Inc., Haywood, CA), was compared to the Western blot evaluation performed by a commercial reference laboratory (Abbott Laboratories, Abbott Park, North Chicago, IL). Using criteria established by the Food and Drug Administration, all major bands representing specific antigens of HIV-l and their corresponding antibodies were identified on the immunoblot membrane when the strongly reactive control serum was used in the assay. The weakly reactive control serum demonstrated antibodies to the p24 core antigen and the gp120/160 envelope antigen of the virus in addition to others. The non-reactive control serum did not react with any antigen on the immunoblot sheet. All results obtained by our evaluation agreed with the reference laboratory results. The WesPage assay offers a combination of advantages which include rapid turn around time, less direct contact with potentially infectious materials, good resolution of bands and high reproducibility of results.
Correspo~~nce to: Helen L. Lucia, Dept. of Pathology, Clinical Mi~obiology, Medical Branch at Galveston, Galveston, TX 77550, U.S.A.
University of Texas
292
Introduction With the recognition of the human immunodeliciency virus type 1 (HIV-l) as the causative agent of acquired immunodeliciency syndrome (AIDS) (Levy et al., 1984; Montagnier et al., 1984; Shupbach et al., 1984), sensitive serologic procedures have been developed for the detection of antibodies specific to the virus (Centers for Disease Control, 1985; Davey and Lane, 1990; Sarngadharan et al., 1984; Steckelberg and Cockerill, 1988). An enzyme-linked immunosorbent assay (ELISA) for HIV-l has been developed for use by clinical laboratories engaged in screening of potential blood donors (Centers for Disease Control, 1985). Western blot assay was developed as a confirmatory assay for HIV-l testing as a means of providing increased specificity, to confer greater reliability on the entire antibody detecting system (Centers for Disease Control, 1989; Shorr et al., 1985; Gallo et al., 1986; Steckelberg and Cockerill, 1988). The application of the Western blot assay has grown beyond the initial aim of defining the antibody status of potential blood donors. It now serves as a confirmatory test for the diagnosis of HIV infection. With the recent establishment of the criteria for interpretation of test results, it is beginning to find wider applicability in providing useful information in patient care situations. Western blot assay detects the profile of specific antibodies to major antigens of the virus in the patient’s sera. Although we are still uncertain as to how to apply this information, a number of studies have suggested that the loss of specific antibody bands heralds the onset of clinical illness (Forster et al., 1989; Goudsmit et al., 1987; Lindhart et al., 1989; Mehta et al., 1990; Sei et al., 1989; Weber et al., 1987). Rapid diagnosis, however, is essential so that the result of the test can be meaningfully applied to a patient’s situation. A rapid Western blot assay, the ‘WesPage’ HIV-l immunoblot system (W.B.) (American Bionetics Inc., Hayward, CA), has been introduced recently. The Western blot assay can be carried out with a rapid turn around time, while at the same time maintaining a high degree of specificity and reproducibility when this system is employed. In order to determine the usefulness of this system, we evaluated the WesPage HIV-1 Western blot assay under routine laboratory conditions using sera submitted to our clinical laboratory for enzyme immunoassay (EIA-Abbott Laboratories, Abbott Park, North Chicago, IL) for antibodies against HIV- 1.
Materials and Methods The WesPage
system
The WesPage system consists of an automated incubator-rocker-washer and reagents. The manufacturer’s instructions were followed. The antigen preparation is supplied as disrupted HIV-l particles (grown in H-9 cells)
293
which have been electrophoretically resolved and blotted onto nitrocellulose sheets. The nitrocellulose sheets are placed in a jig, a device which defines 24 lanes on the nitrocellulose sheet. The nitrocellulose sheet is wetted with buffer, the buffer is removed, and the specimen is added. Patient sera are diluted 1:100 in the reagent buffer and 200 &lane are inoculated through a set of ports in the jig. Following incubation at 25°C for 50 min, the serum is washed away with two buffer washes of 5 min, after which 300 PI/lane of alkaline phosphataseconjugated goat anti-human immunoglobulin set-a is introduced through a second set of ports, and the incubation is continued for 50 min more. After a second rinse step, 300 pi/lane of chromogen (BCIP-NBT) is added through the third set of ports, incubated for 10 min, rinsed with distilled water, and the nitrocellulose sheet is partially air dried. The nitrocellulose sheet is then removed from the jig, completely dried and read by visual inspection. Highpositive-, low-positive- and negative-control sera are run on every nitrocellulose sheet. Study procedure
One hundred and twenty sera which were repeatedly reactive in EIA antibody testing for HIV-l were split; a portion of each serum sample was sent to a reference laboratory (Abbott Laboratories), for Western blot assay and the remaining portion was used in the evaluation of the WesPage immunoblot instrument. One hundred sera which were repeatedly EIA-negative served as negative controls. All sera were run in duplicate in the W.B. procedure. In order to evaluate the sensitivity of the WesPage W.B. procedure, serum samples obtained from two patients who had recently seroconverted, as determined by the HIV-l EIA test, were evaluated. The sera, along with the positive and negative control sera supplied by the manufacturer, were tested at dilutions of 1:100, 1:200, 1:400, 1:800, and 1:1000. In order to examine the specificity of the assay, 5 samples reactive to the EIA procedure, but negative by Western blot procedure performed by the reference laboratory, and two samples positive for HIV-l by virus isolation, but negative by EIA for antibody were examined. To determine how much variation in banding patterns can be seen with different lots of reagent, 5 samples which have been repeatedly positive for HIV-1 EIA and immunofluorescence but indeterminate by Western blot assay were tested with three consecutive lots of reagents.
Results
Fig. 1 shows the bands corresponding to the electroblotted antigens of HIV1 on the ,WesPage immunoblot membrane. All major bands, representing specific antigens of HIV-l and their corresponding antibodies, were identified on the immunoblot membrane when the strongly reactive control was used in
294
gP 9P gP
P
P
P P
Fig. 1. Antigenic Profile of HIV-I on WesPage immunoblot. Lane 1 represents the strongly positive control, lane 2, the weakly positive control and lane 3 the negative control. Serum was diluted 1:100, and 200 pi/lane was applied to the membrane.
the assay at a dilution of 1: 100 as recommended by the manufacturer. The weakly reactive control demonstrated antibodies to the p24 core antigen and the gp120/160 envelope antigen precursors of the virus in addition to others. The non-reactive control did not react with any antigen on the immunoblot sheet. Table 1 summarizes the interpretation of the results of the Western blot assay. Using the Federal Drug Administration-licensed DuPont criteria for interpretation of results (Centers for Disease Control, 1989) (Table 2) 115 of the TABLE 1 Interpretation No. tested
of results of Western blot of HIV-l EIA-positive Reactivity Positive
120
sera
115
Indeterminate 4
Negative 1
295 TABLE 2 Criteria for positive interpretation
of Western blot tests
Organization
Criteria
Association of State and Territorial Directors/CDC FDA-licensed
Public Health Laboratory
p24 and p31 and gp41 or gpl20/160
DuPont test
American Red Cross
Consortium
for Retrovirus
Any two of: ~24, gp41, gpl20/160
Three or more bands one from each gene-product group: GAG and POL and ENV Serological Standardization
Two or more bands p24 or p3 1, plus gp41 or gpl20/160
Adapted from the CDC (2).
120 serum samples were positive for HIV- 1,4 were indeterminate and one serum sample was consistently negative. These results agreed completely with those obtained from the reference laboratory. Antibody to the envelope and core antigens of the virus were demonstrated most often in the EIA-positive sera.
Fig. 2. Western blot patterns of HIV-l EIA-positive samples. Lane 1 is the positive control serum, Lanes 2 and 3 are sera from patients who recently seroconverted by HIV-l EIA test. A is 1:100 dilution, B is 1:200 dilution, C is 1400 dilution, D is 1:800 dilution and E is 1:lOOOdilution.
296
Fig. 2 shows the bands obtained using the WesPage immunoblot instrument for serum samples from two patients who had recently seroconverted as determined by HIV-l EIA test. These sera, along with the positive and negative controls supplied by the manufacturer, were used at dilutions of l:lOO, 1:200, 1:400, 1800, and 1:lOOO.The positive control {lane l), reacted with all major antigens of the virus. This reactivity was retained even at a dilution of 1:lOOO. The converting sera from each patient (lanes 2 and 3 respectively) reacted only with the envelope glycoprotein precursors (gpl20/140) and the p24 antigen of the virus although the gp120,/160 band was only barely discernible at a serum dilution of 1:1000 in the case of sample 2 (lane 3). None of 100 serum samples from the control population which were negative for antibody to HIV-l in EIA test produced any band on the WesPage immunoblot. Neither did the five samples which were reactive to the EIA procedure but negative by Western blot procedure of the reference laboratory. The sera from which HTLV-I had been isolated which were negative by EIA procedure were also negative by immunoblot. When different batches of reagents were tested using the same set of sera, there was no change either in the number or the, position of the bands. However, there was variation in the intensity of the staining of the experimental sera relative to the intensity of the positive control sera.
Discussion Western blot is a technique which harmonizes the high resolving power offered by gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins blotted onto nitrocellulose sheets (Towbin et al., 19’79)with the highly sensitive enzyme-linked immunosorbent assay to produce a system for analyzing antigen-antibody complexes (Tsang et al., 1983). Originally introduced to blood banks as one of the confirmatory tests for the screening of potential blood donors for HIV-l infection (Centers for Disease Control, 1985; Shorr et al., 1985; Ulstrup et al., 1986), it is increasingly finding wider applicability in the diagnosis of active infection and AIDS. The WesPage system was evaluated for its performance characteristics under routine laboratory conditions. The detection of bands corresponding to all the major gene products of HIV-l in the assay is a function of the affinity spectrum of the viral antibodies in the positive-control serum and a measure of the efficiency of the assay system. In addition, the system eliminates the appearance of unusual bands previously ascribed to the cellular sources in which the virus was grown (Drabick and Baker, 1989). We found that it was easy to use, and that the results were consistent. The procedure required three hours, but only about 45 minutes of technologist time were necessary to run it (See Table 2). Because of the semi-automated nature of the procedure with the enclosed system, the technologist and the environment have relatively little contact with infected sera. This is in contrast to the manual
297 TABLE 3 Comparison
of Western blot kits
Manufacturer Manual methods goF;;t (FDA-licensed
Automated method WesPage Reflbct;
kit)
Price/test strip
Maximum No. of strips per run
To>al time (h)
$49.07 $20.00
0 10
16-20
$15.52*
48
3 l/2
3 l/2
Laboratory $49.00 plus shipping
Variable
* With high volume discount. Machine available through reagent rental ~ang~ent.
procedure where open trays were filled with diluted sera, strips of nitrocellulose were placed in trays which were then incubated on an open rocker. In addition, the rinse steps of the automated procedure are more thorough than the manual rinsing, which results in better definition of the individual bands. The cost of the procedure compares favorably with the cost of preimpregnated nitrocellulose strips available from other manufacturers and with the fee charged by the reference laboratory (Table 3). The logistical problems of shipping large numbers of EIA-positive sera to the reference laboratory were also eliminated. Patient care improved markedly because an EIA-positive serum could be honeyed within days. Additionally, the fact that we can observe and evaluate the intensity of the individual antibody bands has enabled us to increase the amount of information we can give the clinician. Changes in the relative intensity of the various bands has been said to herald the onset of clinical disease (Forster et al., 1989; Linhart et al., 1989; Mehta et al.,. 1990; Weber et al., 1987). Because of the ease of the procedure, we can perform the test on many sera, and can easily follow individual patients as their disease evolves. This gives us an additional tool with which to study the pathogenesis of HIV-l infection in man.
References Centers for Disease Control (1985) Provisional Public Health Service Inter-Agency Recommendations for screening donated blood and plasma antibody to the virus causing Acquired Imm~ode~cien~y Syndrome. MMWR 34, l-16. Centers for Disease Control (1989) Interpretation and Use of the Western Blot Assay for Serodiagnosis of Human Immunodeliciency Virus Type I Infections. MMWR 38 (S-7). Davey, R.T. and Lane, H.C. (1990) Laboratory methods in the diagnosis and prognostic staging of infection with Human immunodeficiency Virus Type I. Rev. Infect. Dis. 12,912-930. Drabick, J.J. and Baker, J.R. (1989) HLA antigen contamination of commercial Western Blot strips for detecting Human Immunodeficiency Virus. J. Inf. Dis. 159, 357-358.
298 Forster, SM., Osborne, L.M., Cheingsong-Popov, R., Kenny, C., Burnell, R., Jeffries, D.J., Pinching, A.J., Harris, J.R. and Weber, J.N. (1989) Decline of anti-p24 antibody precedes antigenaemia as a correlate of prognosis in HIV-1 infection, AIDS 1, 235-240. Goudsmit, J., Lange, J.M.A., Krone, W.J.A., Teunissen, M.B.M., Epstein, L.G., Danner, S.A., van den Berg, H., Breederveld, C., Smit, L., Baker, M., de Wolfe, F., Coutinho, R.A. and van der Noordaa, J. (1987) Pathogenesis of HIV and its implications for serodiagnosis and monitoring of antiviral therapy. J. Virol. Methods 17, 19-34. Levy, J.A., Hoffman, A.D., Kramer, SM., Landis, J.A. and Shimabukuro, J.M. (1984) Isolation of Lymphocytopathic retroviruses from San Francisco Patients with AIDS. Science 225, 840-842. Lindhardt, B.O., Gerstoft, J., Hofmann, B., Pallesen, G., Mathiesen, L., Dickmeiss, E. and Ulrich, K. (1989) Antibodies against the major core protein p24 of human immunode~ciency virus: relation to immunolo~cal, clinical and prognostic findings. Eur. J. Clin. Microbial. Infect. Dis. 8, 614-619. Mehta, S.U., Rupprecht, K.R., Hunt, J.C., Kramer, D.E., McRae, B.J., Allen, R.G., Dawson, G.J. and Devare, S.G. (1990) Prevalence of antibodies to the Core Protein ~17, a serological marker during HIV-l infection. AIDS Res. Hum. Retroviruses 6, 443454. Montagnier, L., Chermann, J.C. and Barre-Sinoussi, F. (1984) A new human T-lymphotropic retrovirus: characterization and possible role in l~phadenopathy and acquired immune deficiency syndromes. In: R.C. Gallo, M. Essex and L. Gross (Eds), Human T-cell leukemia/ lymphoma viruses. Cold Spring Harbor Laboratories, New York, pp. 363-379. Sarngadharan, M.G., Popovic, M., Bruch, L., Schupbach, J. and Gallo, R.C. (1984) Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224, 506508. Schorr, J.B., Berkowitz, A., Cumming, P.D., Katz, A.J. and Sandler, S.G. (1985) Prevalence of HTLV-III antibody in American blood donors. N. Engl. J. Med. 313, 384-385. Schupbach, J., Popovic, M., Gilden, R.V., Gonda, M.A., Sarngadharan, M.G. and Gallo, R.C. (1984) Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497-505. Sei, Y., Tsang, P.H., Chu, F.N., Wallace, I., Roboz, J.P., Sarin, P.S. and Bekesi, J.G. (1989) Inverse relationship between HIV-l p24 antigenemia, anti-p24 antibody and neutralizing antibody response in all stages of HIV-l infection. Immunol. I.&t. 20, 223-230. Steckelberg, J.M. and Cockerill, F.R. (1988) Serologic testing for Human Immunode~ciency Virus Antibodies. Mayo Clin. Proc. 63, 373-380. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Tsang, V.C.W., Peralta, J.M. and Simons, A.R. (1983) Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 92, 377-391. Ulstrup, J.C., Skaug, K., Figenschau, K.J., Orstavik, I., Bruun, J.N. and Petersen, G. (1986) Sensitivity of Western blotting (compared with ELISA and immunofluorescence) during seroconversion after HTLV-III infection. Lancet 1, 115 l-l 152. Weber, J.N., Clapham, P.R., Weiss, R.A., Parker, D., Roberts, C., Duncan, J., Weller, I., Carne, C., Tedder, R.S., Pinching, A.J. and Cheingsong-Popov, R. (1987) Human immunodeficiency virus infection in two cohorts of homosexual men: neutralizing sera and association of anti-gag antibody with prognosis. Lancet 1, 119.-122.
291
VIRMET 01191
Performance characteristics of a rapid Western blot assay system for antibody to human immunode~ciency virus type 1 Olajide Agbede, Syed Wahab, Donna L. Schutt and Helen L. Lucia Department of Pathology, University of Texas Medical Branch, Galveston, Texas, U.S.A. (Accepted
19 March 1991)
Summary
One hundred and twenty reactive sera were selected from specimens studied by enzyme immunoassay (EIA, Abbott Laboratories, Abbott Park, North Chicago, IL) for antibodies against human immunode~ciency virus (HIV-l). Using these sera, the ‘WesPage’ system (American Bionetics, Inc., Haywood, CA), was compared to the Western blot evaluation performed by a commercial reference laboratory (Abbott Laboratories, Abbott Park, North Chicago, IL). Using criteria established by the Food and Drug Administration, all major bands representing specific antigens of HIV-l and their corresponding antibodies were identified on the immunoblot membrane when the strongly reactive control serum was used in the assay. The weakly reactive control serum demonstrated antibodies to the p24 core antigen and the gp120/160 envelope antigen of the virus in addition to others. The non-reactive control serum did not react with any antigen on the immunoblot sheet. All results obtained by our evaluation agreed with the reference laboratory results. The WesPage assay offers a combination of advantages which include rapid turn around time, less direct contact with potentially infectious materials, good resolution of bands and high reproducibility of results.
Correspo~~nce to: Helen L. Lucia, Dept. of Pathology, Clinical Mi~obiology, Medical Branch at Galveston, Galveston, TX 77550, U.S.A.
University of Texas
292
Introduction With the recognition of the human immunodeliciency virus type 1 (HIV-l) as the causative agent of acquired immunodeliciency syndrome (AIDS) (Levy et al., 1984; Montagnier et al., 1984; Shupbach et al., 1984), sensitive serologic procedures have been developed for the detection of antibodies specific to the virus (Centers for Disease Control, 1985; Davey and Lane, 1990; Sarngadharan et al., 1984; Steckelberg and Cockerill, 1988). An enzyme-linked immunosorbent assay (ELISA) for HIV-l has been developed for use by clinical laboratories engaged in screening of potential blood donors (Centers for Disease Control, 1985). Western blot assay was developed as a confirmatory assay for HIV-l testing as a means of providing increased specificity, to confer greater reliability on the entire antibody detecting system (Centers for Disease Control, 1989; Shorr et al., 1985; Gallo et al., 1986; Steckelberg and Cockerill, 1988). The application of the Western blot assay has grown beyond the initial aim of defining the antibody status of potential blood donors. It now serves as a confirmatory test for the diagnosis of HIV infection. With the recent establishment of the criteria for interpretation of test results, it is beginning to find wider applicability in providing useful information in patient care situations. Western blot assay detects the profile of specific antibodies to major antigens of the virus in the patient’s sera. Although we are still uncertain as to how to apply this information, a number of studies have suggested that the loss of specific antibody bands heralds the onset of clinical illness (Forster et al., 1989; Goudsmit et al., 1987; Lindhart et al., 1989; Mehta et al., 1990; Sei et al., 1989; Weber et al., 1987). Rapid diagnosis, however, is essential so that the result of the test can be meaningfully applied to a patient’s situation. A rapid Western blot assay, the ‘WesPage’ HIV-l immunoblot system (W.B.) (American Bionetics Inc., Hayward, CA), has been introduced recently. The Western blot assay can be carried out with a rapid turn around time, while at the same time maintaining a high degree of specificity and reproducibility when this system is employed. In order to determine the usefulness of this system, we evaluated the WesPage HIV-1 Western blot assay under routine laboratory conditions using sera submitted to our clinical laboratory for enzyme immunoassay (EIA-Abbott Laboratories, Abbott Park, North Chicago, IL) for antibodies against HIV- 1.
Materials and Methods The WesPage
system
The WesPage system consists of an automated incubator-rocker-washer and reagents. The manufacturer’s instructions were followed. The antigen preparation is supplied as disrupted HIV-l particles (grown in H-9 cells)
293
which have been electrophoretically resolved and blotted onto nitrocellulose sheets. The nitrocellulose sheets are placed in a jig, a device which defines 24 lanes on the nitrocellulose sheet. The nitrocellulose sheet is wetted with buffer, the buffer is removed, and the specimen is added. Patient sera are diluted 1:100 in the reagent buffer and 200 &lane are inoculated through a set of ports in the jig. Following incubation at 25°C for 50 min, the serum is washed away with two buffer washes of 5 min, after which 300 PI/lane of alkaline phosphataseconjugated goat anti-human immunoglobulin set-a is introduced through a second set of ports, and the incubation is continued for 50 min more. After a second rinse step, 300 pi/lane of chromogen (BCIP-NBT) is added through the third set of ports, incubated for 10 min, rinsed with distilled water, and the nitrocellulose sheet is partially air dried. The nitrocellulose sheet is then removed from the jig, completely dried and read by visual inspection. Highpositive-, low-positive- and negative-control sera are run on every nitrocellulose sheet. Study procedure
One hundred and twenty sera which were repeatedly reactive in EIA antibody testing for HIV-l were split; a portion of each serum sample was sent to a reference laboratory (Abbott Laboratories), for Western blot assay and the remaining portion was used in the evaluation of the WesPage immunoblot instrument. One hundred sera which were repeatedly EIA-negative served as negative controls. All sera were run in duplicate in the W.B. procedure. In order to evaluate the sensitivity of the WesPage W.B. procedure, serum samples obtained from two patients who had recently seroconverted, as determined by the HIV-l EIA test, were evaluated. The sera, along with the positive and negative control sera supplied by the manufacturer, were tested at dilutions of 1:100, 1:200, 1:400, 1:800, and 1:1000. In order to examine the specificity of the assay, 5 samples reactive to the EIA procedure, but negative by Western blot procedure performed by the reference laboratory, and two samples positive for HIV-l by virus isolation, but negative by EIA for antibody were examined. To determine how much variation in banding patterns can be seen with different lots of reagent, 5 samples which have been repeatedly positive for HIV-1 EIA and immunofluorescence but indeterminate by Western blot assay were tested with three consecutive lots of reagents.
Results
Fig. 1 shows the bands corresponding to the electroblotted antigens of HIV1 on the ,WesPage immunoblot membrane. All major bands, representing specific antigens of HIV-l and their corresponding antibodies, were identified on the immunoblot membrane when the strongly reactive control was used in
294
gP 9P gP
P
P
P P
Fig. 1. Antigenic Profile of HIV-I on WesPage immunoblot. Lane 1 represents the strongly positive control, lane 2, the weakly positive control and lane 3 the negative control. Serum was diluted 1:100, and 200 pi/lane was applied to the membrane.
the assay at a dilution of 1: 100 as recommended by the manufacturer. The weakly reactive control demonstrated antibodies to the p24 core antigen and the gp120/160 envelope antigen precursors of the virus in addition to others. The non-reactive control did not react with any antigen on the immunoblot sheet. Table 1 summarizes the interpretation of the results of the Western blot assay. Using the Federal Drug Administration-licensed DuPont criteria for interpretation of results (Centers for Disease Control, 1989) (Table 2) 115 of the TABLE 1 Interpretation No. tested
of results of Western blot of HIV-l EIA-positive Reactivity Positive
120
sera
115
Indeterminate 4
Negative 1
295 TABLE 2 Criteria for positive interpretation
of Western blot tests
Organization
Criteria
Association of State and Territorial Directors/CDC FDA-licensed
Public Health Laboratory
p24 and p31 and gp41 or gpl20/160
DuPont test
American Red Cross
Consortium
for Retrovirus
Any two of: ~24, gp41, gpl20/160
Three or more bands one from each gene-product group: GAG and POL and ENV Serological Standardization
Two or more bands p24 or p3 1, plus gp41 or gpl20/160
Adapted from the CDC (2).
120 serum samples were positive for HIV- 1,4 were indeterminate and one serum sample was consistently negative. These results agreed completely with those obtained from the reference laboratory. Antibody to the envelope and core antigens of the virus were demonstrated most often in the EIA-positive sera.
Fig. 2. Western blot patterns of HIV-l EIA-positive samples. Lane 1 is the positive control serum, Lanes 2 and 3 are sera from patients who recently seroconverted by HIV-l EIA test. A is 1:100 dilution, B is 1:200 dilution, C is 1400 dilution, D is 1:800 dilution and E is 1:lOOOdilution.
296
Fig. 2 shows the bands obtained using the WesPage immunoblot instrument for serum samples from two patients who had recently seroconverted as determined by HIV-l EIA test. These sera, along with the positive and negative controls supplied by the manufacturer, were used at dilutions of l:lOO, 1:200, 1:400, 1800, and 1:lOOO.The positive control {lane l), reacted with all major antigens of the virus. This reactivity was retained even at a dilution of 1:lOOO. The converting sera from each patient (lanes 2 and 3 respectively) reacted only with the envelope glycoprotein precursors (gpl20/140) and the p24 antigen of the virus although the gp120,/160 band was only barely discernible at a serum dilution of 1:1000 in the case of sample 2 (lane 3). None of 100 serum samples from the control population which were negative for antibody to HIV-l in EIA test produced any band on the WesPage immunoblot. Neither did the five samples which were reactive to the EIA procedure but negative by Western blot procedure of the reference laboratory. The sera from which HTLV-I had been isolated which were negative by EIA procedure were also negative by immunoblot. When different batches of reagents were tested using the same set of sera, there was no change either in the number or the, position of the bands. However, there was variation in the intensity of the staining of the experimental sera relative to the intensity of the positive control sera.
Discussion Western blot is a technique which harmonizes the high resolving power offered by gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins blotted onto nitrocellulose sheets (Towbin et al., 19’79)with the highly sensitive enzyme-linked immunosorbent assay to produce a system for analyzing antigen-antibody complexes (Tsang et al., 1983). Originally introduced to blood banks as one of the confirmatory tests for the screening of potential blood donors for HIV-l infection (Centers for Disease Control, 1985; Shorr et al., 1985; Ulstrup et al., 1986), it is increasingly finding wider applicability in the diagnosis of active infection and AIDS. The WesPage system was evaluated for its performance characteristics under routine laboratory conditions. The detection of bands corresponding to all the major gene products of HIV-l in the assay is a function of the affinity spectrum of the viral antibodies in the positive-control serum and a measure of the efficiency of the assay system. In addition, the system eliminates the appearance of unusual bands previously ascribed to the cellular sources in which the virus was grown (Drabick and Baker, 1989). We found that it was easy to use, and that the results were consistent. The procedure required three hours, but only about 45 minutes of technologist time were necessary to run it (See Table 2). Because of the semi-automated nature of the procedure with the enclosed system, the technologist and the environment have relatively little contact with infected sera. This is in contrast to the manual
297 TABLE 3 Comparison
of Western blot kits
Manufacturer Manual methods goF;;t (FDA-licensed
Automated method WesPage Reflbct;
kit)
Price/test strip
Maximum No. of strips per run
To>al time (h)
$49.07 $20.00
0 10
16-20
$15.52*
48
3 l/2
3 l/2
Laboratory $49.00 plus shipping
Variable
* With high volume discount. Machine available through reagent rental ~ang~ent.
procedure where open trays were filled with diluted sera, strips of nitrocellulose were placed in trays which were then incubated on an open rocker. In addition, the rinse steps of the automated procedure are more thorough than the manual rinsing, which results in better definition of the individual bands. The cost of the procedure compares favorably with the cost of preimpregnated nitrocellulose strips available from other manufacturers and with the fee charged by the reference laboratory (Table 3). The logistical problems of shipping large numbers of EIA-positive sera to the reference laboratory were also eliminated. Patient care improved markedly because an EIA-positive serum could be honeyed within days. Additionally, the fact that we can observe and evaluate the intensity of the individual antibody bands has enabled us to increase the amount of information we can give the clinician. Changes in the relative intensity of the various bands has been said to herald the onset of clinical disease (Forster et al., 1989; Linhart et al., 1989; Mehta et al.,. 1990; Weber et al., 1987). Because of the ease of the procedure, we can perform the test on many sera, and can easily follow individual patients as their disease evolves. This gives us an additional tool with which to study the pathogenesis of HIV-l infection in man.
References Centers for Disease Control (1985) Provisional Public Health Service Inter-Agency Recommendations for screening donated blood and plasma antibody to the virus causing Acquired Imm~ode~cien~y Syndrome. MMWR 34, l-16. Centers for Disease Control (1989) Interpretation and Use of the Western Blot Assay for Serodiagnosis of Human Immunodeliciency Virus Type I Infections. MMWR 38 (S-7). Davey, R.T. and Lane, H.C. (1990) Laboratory methods in the diagnosis and prognostic staging of infection with Human immunodeficiency Virus Type I. Rev. Infect. Dis. 12,912-930. Drabick, J.J. and Baker, J.R. (1989) HLA antigen contamination of commercial Western Blot strips for detecting Human Immunodeficiency Virus. J. Inf. Dis. 159, 357-358.
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