Transfusion Medicine, 1992. 2, 249-250
CORRESPONDENCE
Western blotting for HIV-antibody confirmation in blood donors: an improved approach by recombinant immunoblots
(p24, p17, gp41, gp31) and for HIV-2, one antigen (gp36) was applied. In addition, each strip had four reference lines, representing two cut-off points: one strong positive and a sample application control. True blot positives have activity against a t least two gene products of the virus (em, gag or pol). Sera with ELISA positivity but showing only one band were considered to be indeterminate reactions. Using the conventional WB (Dupont, U.S.A.) we investigated samples in parallel with LiaTek. Table 1 summarizes our experience with the two types of blot, showing 100% concordance in four HIV-I positive donor sera (found during 1989 screening and kept frozen). The two French/African sera (gifts from Dr L. Nod, CTS, Versailles), were clearly identified as HIV-2 in LiaTek. Eight repeatably ELISA (Wellcome) reactive sera recently obtained in our centre showed no reaction by LiaTek while 4/8 showed indeterminate bands by the conventional WB; the latter seems to be due to non-specific reactivity because all four sera were negative by two additional tests including a sensitive gelatin particle agglutination (Serodia, Fujirebio) assay (Barbara et af., 1989) and Abbott ELISA. Furthermore, all four donors showing non-specific single bands were followed-up for between 5 and 15 months. During this period they remained healthy and second samples were tested, when 2/4 were completely negative in WB, while two donors had maintained
SIR,It is sensible and customary to retest ELISA HIV antibody-positive donors by independent methods, and for the last 5 years the virus lysate-derived Western blots (WB) have been mostly used for this purpose. However, the interpretation of these results, especially in the presence of indeterminate bands, continues to be a problem; there seems to be no easy solution as reflected by the complex flow-chart devised by the French group (Noel et af., 1988) to interpret such WBbands. There are two possible strategies to circumvent the problem: firstly, there is a case for introducing modern ELISA tests for confirmatory purpose, as pleaded recently by Mortimer (1991); or secondly, for using less complex but refined WB recombinant/ synthetic antigens as has been recently described by an American group (Busch et af., 1991). Similar recombinant/synthetically derived immuno blot assays (RIBA) are commercially available in Europe, that can not only validate ELISA-positive reactions with high specificity but are also capable of differentiating between HIV-I and HIV-2 antibody reactivities. Over a period of 6 months we have had experience with a RIBA assay called LiaTek (Organon, The Netherlands). The assay was performed according to the manufacturer’s instructions. Briefly, recombinant proteins/peptides of HIV-I and HIV-2 were coated as discrete antigen lines on nylon strip with plastic backings provided in the kit. For HIV-I, four antigens Table 1. LiaTek (RIBA) versus
conventional Western Blot (WB) for HIV antibody confirmation
Samples description
LiaTek characteristics
Dupont WB characteristics
4 HIV-I positive sera 2 HIV-2 positive sera
4 sera HIV-I positive 2 sera HIV-2 positive
4 sera HIV-I positive ND
8 sera repeatably ELISA positive*
8 sera negative
1 Serum with P l 8 t 2 sera with P24t 1 serum with GP160t 4 sera negative
N D = not done. Found during 1990 and IY91 donor screening. t T w o additional tests (Serodia and Abbott ELISA) were negative.
249
250
Correspondence
their single non-specific bands (p24, gp160) but were negative by additional tests, including ELISA (Wellcome, Abbott), Serodia and LiaTek. In our experience LiaTek assays are easy to perform with clear-cut differentiation of bands leading to a reduction in nonspecific results. In addition, there was excellent concordance in the interpretation of the LiaTek immunoblots when three technologists reported blindly LiaTek blot patterns on 15 sera. Thus, this type of recombinant synthetic protein and peptide-derived immunoblot is convenient for routine application in the Blood Bank laboratory to confirm HIV-antibody-positive ELISA results. When fully validated, with its added specificity, it could be of considerable value especially in reducing the follow-up testing of blood donors. The small number of donors examined from a single centre with a low prevalence of anti-HIV reactive donors, probably do not themselves justify a change from Western blot to RIBA; however, we hope this will encourage colleagues to review their practice and report further series of LiaTek or other RIBA investigations of reactive donors.
REFERENCES Barbara, J.A.J., Salker, R., Challis, P. & Contreras, M. (1989) Gealtin particle agglutination assay for HIV antibodies: a rapid, economical modification with increased sensitivity. Medicaland Laboratory Sciences, 46, 135-140. Busch, M.P.,Amad, Z.E., McHugh, T.M.,Chen, D. & Polite, A.J. (1991) Reliableconfirmation and quantitation of human immunodeficiencyvirus type 1 antibody using a recombinant antigen immunoblot assay. Transfwion,31, 129-1 37. Mortimer, P.P. (1991) The fallibility of HIV Western Blot. Lancet, 331,286-287. N&l, L., Courouct. A.M.et le groupe de travail retrovirus de la SociCtC Nationale de Transfusion Sanguine (1988) Le Western Blot HIV-I, qualitcs et difficultis d'un test de confirmation. Spectra Biologie, 5, 31-35.
P. C. Das, A. H. de Vries, R. L. McShine and C. Th. Smit Sibinga Regional Red Cross Blood Bank Groningen-Drenthe. Prof. Rankestraat 42-44, 9713 GG Groningen The Netherlands
CORRESPONDENCE
Western blotting for HIV-antibody confirmation in blood donors: an improved approach by recombinant immunoblots
(p24, p17, gp41, gp31) and for HIV-2, one antigen (gp36) was applied. In addition, each strip had four reference lines, representing two cut-off points: one strong positive and a sample application control. True blot positives have activity against a t least two gene products of the virus (em, gag or pol). Sera with ELISA positivity but showing only one band were considered to be indeterminate reactions. Using the conventional WB (Dupont, U.S.A.) we investigated samples in parallel with LiaTek. Table 1 summarizes our experience with the two types of blot, showing 100% concordance in four HIV-I positive donor sera (found during 1989 screening and kept frozen). The two French/African sera (gifts from Dr L. Nod, CTS, Versailles), were clearly identified as HIV-2 in LiaTek. Eight repeatably ELISA (Wellcome) reactive sera recently obtained in our centre showed no reaction by LiaTek while 4/8 showed indeterminate bands by the conventional WB; the latter seems to be due to non-specific reactivity because all four sera were negative by two additional tests including a sensitive gelatin particle agglutination (Serodia, Fujirebio) assay (Barbara et af., 1989) and Abbott ELISA. Furthermore, all four donors showing non-specific single bands were followed-up for between 5 and 15 months. During this period they remained healthy and second samples were tested, when 2/4 were completely negative in WB, while two donors had maintained
SIR,It is sensible and customary to retest ELISA HIV antibody-positive donors by independent methods, and for the last 5 years the virus lysate-derived Western blots (WB) have been mostly used for this purpose. However, the interpretation of these results, especially in the presence of indeterminate bands, continues to be a problem; there seems to be no easy solution as reflected by the complex flow-chart devised by the French group (Noel et af., 1988) to interpret such WBbands. There are two possible strategies to circumvent the problem: firstly, there is a case for introducing modern ELISA tests for confirmatory purpose, as pleaded recently by Mortimer (1991); or secondly, for using less complex but refined WB recombinant/ synthetic antigens as has been recently described by an American group (Busch et af., 1991). Similar recombinant/synthetically derived immuno blot assays (RIBA) are commercially available in Europe, that can not only validate ELISA-positive reactions with high specificity but are also capable of differentiating between HIV-I and HIV-2 antibody reactivities. Over a period of 6 months we have had experience with a RIBA assay called LiaTek (Organon, The Netherlands). The assay was performed according to the manufacturer’s instructions. Briefly, recombinant proteins/peptides of HIV-I and HIV-2 were coated as discrete antigen lines on nylon strip with plastic backings provided in the kit. For HIV-I, four antigens Table 1. LiaTek (RIBA) versus
conventional Western Blot (WB) for HIV antibody confirmation
Samples description
LiaTek characteristics
Dupont WB characteristics
4 HIV-I positive sera 2 HIV-2 positive sera
4 sera HIV-I positive 2 sera HIV-2 positive
4 sera HIV-I positive ND
8 sera repeatably ELISA positive*
8 sera negative
1 Serum with P l 8 t 2 sera with P24t 1 serum with GP160t 4 sera negative
N D = not done. Found during 1990 and IY91 donor screening. t T w o additional tests (Serodia and Abbott ELISA) were negative.
249
250
Correspondence
their single non-specific bands (p24, gp160) but were negative by additional tests, including ELISA (Wellcome, Abbott), Serodia and LiaTek. In our experience LiaTek assays are easy to perform with clear-cut differentiation of bands leading to a reduction in nonspecific results. In addition, there was excellent concordance in the interpretation of the LiaTek immunoblots when three technologists reported blindly LiaTek blot patterns on 15 sera. Thus, this type of recombinant synthetic protein and peptide-derived immunoblot is convenient for routine application in the Blood Bank laboratory to confirm HIV-antibody-positive ELISA results. When fully validated, with its added specificity, it could be of considerable value especially in reducing the follow-up testing of blood donors. The small number of donors examined from a single centre with a low prevalence of anti-HIV reactive donors, probably do not themselves justify a change from Western blot to RIBA; however, we hope this will encourage colleagues to review their practice and report further series of LiaTek or other RIBA investigations of reactive donors.
REFERENCES Barbara, J.A.J., Salker, R., Challis, P. & Contreras, M. (1989) Gealtin particle agglutination assay for HIV antibodies: a rapid, economical modification with increased sensitivity. Medicaland Laboratory Sciences, 46, 135-140. Busch, M.P.,Amad, Z.E., McHugh, T.M.,Chen, D. & Polite, A.J. (1991) Reliableconfirmation and quantitation of human immunodeficiencyvirus type 1 antibody using a recombinant antigen immunoblot assay. Transfwion,31, 129-1 37. Mortimer, P.P. (1991) The fallibility of HIV Western Blot. Lancet, 331,286-287. N&l, L., Courouct. A.M.et le groupe de travail retrovirus de la SociCtC Nationale de Transfusion Sanguine (1988) Le Western Blot HIV-I, qualitcs et difficultis d'un test de confirmation. Spectra Biologie, 5, 31-35.
P. C. Das, A. H. de Vries, R. L. McShine and C. Th. Smit Sibinga Regional Red Cross Blood Bank Groningen-Drenthe. Prof. Rankestraat 42-44, 9713 GG Groningen The Netherlands
