An improved immunoblotting procedure for the detection of antibodies against HIV

of Virologicai

Journal

Methods, 16 (1987) 87-96

87

Elsevier JVM 00583

An improved immunoblotting procedure for the detection of antibodies against HIV R. Thorpe,

Maryvonne D.R. Brasher, C.R. Bird, A.J. Garrett, J.P. Jacobs, P.D. Minor and G.C. Schild

National Institute for Biological Standards and Control, Potters Bar, Hertfordshire,

UK

(Accepted 9 January 1987)

Summary Immunoblotting (‘Western blotting’) is routinely used for detection of antibodies against HIV in the diagnosis of HIV infection. We describe an improved procedure, which does not require virus purification and is easy to control for ‘falsepositive’ results. The technique also does not produce erroneous results due to reactivity of the developing system with residual cellular proteins or viral antigens and does not give high nonspecific background staining. The technique can be applied to the detection of antibodies to HIV in serum, plasma, and blood products. Human immunodeficiency clonal antibody

virus; AIDS; Immunoblotting;

Blood product; Mono-

Introduction Reliable and sensitive immunochemical methods for the detection of antibodies to human immunodeficiency virus (HIV) play a critically important role in screening human plasma for diagnostic purposes (Sarngadharan et al., 1984) and for seroepidemiological investigations (Gocke et al., 1986). In the absence of sensitive virus isolation techniques appropriate to large scale routine application, procedures for ensuring the safety of blood and blood products rely heavily on such methods. Among the methods available ELISA techniques have been most extensively used owing to their simplicity and convenience of use particularly as semCorrespondence to: R. Thorpe, National institute for Biological Standards and Control, Blanche Lane, South Nimms, Potters Bar, Hertfordshire EN6 3QG, UK. Old-0934/87/$03.50

0

1987 Elsevier Science Pubfishers B.V. (Biomedical Division)

88

iautomated systems as well as being readily mass-produced as kits (Ward et al., 1986; Reesink et al., 1986). However, even the most sophisticated versions of this methodology have disadvantages especially with the occurrence of false-positive reactions (Morgan et al., 1986; Katchaki et al., 1986; Watson Martin et al., 1986; Marlink et al., 1986). It has therefore been proposed that ELISA-positive results are ‘confirmed’ using an alternative technique, and immunoblotting (Esteban et al., 1986; Ulstrup et al., 1986), radioimmunoprecipitation (Kitchen et al., 1984; Allan et al., 3985), or immuno~ytochemistry (Karpas et al., 1985) have all been proposed for this purpose. The immunoblotting procedure has been particularly favoured for confirmatory testing. Viral antigens are resolved by SIX-PAGE, electrophoretically transferred to nitrocellulose membranes and then exposed to the test serum or blood product. A developing system, usually a second antibody conjugated to an enzyme or radioisotope, is then used to reveal the presence of antigen-antibody complexes. Immunoblotting is particularly valuable as it provides information as to which viral polypeptide~s) are recognized by antibodies as well as being sensitive and easy to control. However most of the immunoblotting procedures previously described require purification of the virus prior to blotting, and also can produce erroneous results due to reactivity of the developing system (either second antibody or enzyme substrate) with residual cellular proteins, or with viral proteins. This can be a particular problem with the screening of therapeutic immunoglobulin preparations with which false-positive bands and high nonspecific backgrounds are a considerable problem. We describe here an improved procedure which can be performed using pelleted cell culture harvests as antigen, does not require purified virus and involves the use of a radioiodinated monoclonal anti-human IgG antibody as a standard reagent. The method does not produce spurious false-positive results nor high non-specific background staining, which are often associated with previously reported techniques, and it has been applied successfully to the detection of antibody in blood products produced from plasma pools from large numbers of individual donations.

Materials

and Methods

Production of virus and preparation of whole cell !ysate H9 or GEM cells chronically infected with HTLV-III strain B were cultured serially at 36-37°C to effect concomitant propagation of the virus. The culture medium was RPM1 1640 supplemented with 10% foetal calf serum (heated at 56°C for 30 min), 200 U/ml benzyl penicillin, and 100 (*g/ml streptomycin sulphate. Infected or uninfected cells at approx. 10” cells/ml were collected by centrifugation at 600 x g for 8 min, washed once with phosphate-buffered saline and suspended at approx. 2 x lo7 cells/ml in lysis buffer containing 0.1 M Tris-HCI (pH 7.0), 4% sodium dodecyl suphate, 4% 2-mercaptoethanol, 20% (w/v) sucrose, and 0.001% bromophenol blue. This was heated in a boiling water bath for 4 min and either used immediately, stored at -20°C or lyophilized.

89

~~epff$ation, puri~cation, man IgG

and iodinatio~ of ~onoclonaI

antibody specific for hu-

Monoclonal antibody Gl, which is a mouse IgG, hybridoma-derived antibody reacting specifically with all four subclasses of human IgG was purified from ascitic fluid by a combination of precipitation with ammonium sulphate (45% saturation) and HPLC using a mono Q (Pharmacia) ion-exchange column (Clezardin et al., 1985). Iodination was carried out using aliquots of monoclonal antibody (2 mg/ml) which had been equilibrated with 0.1 M sodium phosphate buffer (pH 7.4). For this 30 ~1 of antibody solution was mixed with 150 l.~lof 0.1 M sodium phosphate buffer (pH 7.4) containing 150 @i Na “‘1 ( SA 100 mCi/ml; Amersham International PLC) and then 10 ~1 of chloramine T (5 mg/ml in water) added. After 45 s the reaction was terminated by the addition of 50 p-1tyrosine (0.4 mg/ml). Nonprotein-bound lzsI was removed by adsorption to a column of Dowex l-X8 (Cl) ion-exchange resin (bed volume 1 ml) equiiibrated with 0.1 M sodium phosphate buffer (pH 7.4) containing 2% bovine serum aibumin, and 1251-labelled monoclonal antibody recovered by washing through with 2 ml of this solution. Radiolabelled monoclonal antibody was stored at 4°C after the addition of 0.02% sodium azide. Inmunoblotting

Cell lysate was electrophoresed using 12.5% total ac~lamide slab gels overlaid with 4% total acrylamide stacking gels and the SDS containing buffer system described by Laemlli and Favre (1973). After electrophoresis the separated proteins were transferred to nitrocellulose sheets essentially as previously described (Towbin et al., 1979). For this the slab gels were covered with wet nitrocellulose sheets (0.45pm pore size; Schleicher & Schtill) and then packed into the transfer cassette of either an Eiectroblot system (EC Corp., Boston, U.S.A.) or a transblot apparatus (BioRAd Laboratories); electrophoretic transfer was then carried out for 2 h at 0.45-0.5 A. During transfer the tank buffer was cooled to IO-14°C. After transfer, the unoccupied protein binding sites on the nitrocellulose sheets were blocked by incubation for 30 min with phosphate-buffered saline containing 3% bovine haemoglobin (Hb-PBS). Blots were then incubated overnight at room temperature with antisera or blood product diluted in 5-25 ml Hb-PBS. Prior to dilution the antisera or blood product was centrifuged for 5 min in a microfuge and a total of 25-50 ~1 antisera or 100 ~1 blood product was used (for details, see results section). The blots were then washed for 30 min (6 changes 20-100 ml each) in Hb-PBS and incubated at room temperature for H h with ‘“51-labelIed antihuman IgG monoclonal antibody diluted in 20 ml Hb-PBS (5 x 10’ cpmltrack). Blots were finally washed for 30 min (6 changes 20-100 ml each) with PBS, then dried and exposed to preflashed (Laskey and Mills, 1977) Kodak X-OMAT S film for 1-S d at -70°C in a cassette equipped with one fast tungstate intensifying screen.

Fig, I, Immunoblots of cell lysates using serum from an ~IV-sero~sitive individua1. (A) Lysate from uninfected H9 cells; (B) lysate from H9 cells infected with HIV. The approx. M, (X 10m3)of immunolabelled bands is indicated.

Results Antibodies with specificity for retroviral protein components could be readily detected by imm~noblotting SDS polyacrylamide gel electrophoretogrammes of lysates of HIV-infected H9 cells. Using this system antisera from individuals were shown to contain antibodies against a number of viral proteins, the most commonly found antigens being the 24k gag viral core protein and its 55k precursor. In no cases did antibodies present in such sera bind to proteins of similar molecular weight found in uninfected cells (Fig. 1) and sera from healthy, uninfected laboratory workers, failed to react with proteins from infected cells even on prolonged exposure to X-ray film. Immunoblots using infected CEM cells as antigen produced similar binding patterns.

55 41

24

Fig. 2. Immunoblots of HIV-infected H9 cell lysate using sera from individuals showing differing degrees of seropositivity. Approx. M, values x lo-” are indicated. All sera produced no bands when blotted against uninfected H9 cell lysate.

A considerable variation in the pattern of immunoreactive proteins was noted when using sera from different infected individuals (Fig. 2). Although antibodies were frequently observed against the 41k glycoprotein envelope antigen, these were not always present. Other frequently detected antigens were proteins of approx. molecular weight 33k, 36k, 43k, 60k, and 140/160k, the latter presumably being the high molecular weight end gene products (Fig. 2). Occasionally other bands were seen, especially a 17k antigen. In nearly all cases antibodies present in the sera from positive individuals did not bind to antigens from uninfected cells. However in one serum antibodies were present which bound to a number of high molecular weight proteins clearly unrelated to any viral antigen. Inclusion of the appropriate control (lysate from uninfected cells) clearly demonstrated that these antibodies were of nonviral origin (Fig. 3). The sensitivity of the immunoblotting procedure was difficult to assess directly as it depends on the amount of viral protein(s) loaded on the polyacrylamide gel and also the specific activity of the 1251labelfed monoclonal antibody. However a moderately potent positive serum sample gave a detectable reaction for the strongest bands (24k and 55k) at a final dilution of 5 x 10e6. However less potent sera could not be diluted to this extent and still give an unambiguous positive result. The technique could also be used to detect antibodies to HIV in blood products. Several therapeutic immunoglobulin preparations (intended for either intramuscular or intravenous use) were found to contain such antibodies (Fig. 4). As with serum samples the 24k gag protein and/or the 55k gag precursor were the most

92

24

Fig. 3. Immunoblots of cell lysates using antiserum Ed3. (A) Lysate from uninfected H9 cell lysate; (B) lysate from H9 cells infected with HIV. See text for details. Approx. M, values x lo-’ of HIVderived antigens are indicated.

common antigens detected although one intravenous immunoglobulin sample also contained antibodies which bound to an antigen with an approx. M, of 50k (Fig.4D). One therapeutic factor VIII concentrate was also found to contain antibodies directed against HIV antigens (Fig. 5). In all cases the reactivity could be definitely attributed to viral antigens as no reactivity was seen on the control (uninfected cell lysate) immunoblots. One intramuscular immunoglobulin preparation showed very strong reactivity with a 65k antigen but this could be easily shown to be a cell-derived antigen as an identical band was produced on an immunoblot of uninfected cell lysate (Fig. 4A, B).

93

ABC

DE

F

Fig. 4. Immunoblots of cell lysates using therapeutic imunoglobulin preparations. (A) Using an intramuscular preparation and HIV infected H9 cell lysate. (B) as A but using uninfected H9 cell. lysatc; (D) and (F) using HIV-infected H9 cell lysate and two different intravenous immunoglobulin preparations; (C) and (E) using HIV-infected H9 cell lysate and serum from an IIIV-positive individual. See text for details. Numbers represent M, x 10W3.

Discussion

The procedure described allows the simple and reproducible detection of antibodies against HIV in sera or in blood products. The principal differences between this procedure and those described previously are that whole infected cell lysate is used as antigen and that a radioiodinated monoclonal antibody is used to detect antigen-antibody complexes.

Fig. 5. Immunoblots of cell lysates using a therapeutic factor VIII preparation. (A) Blot using HIVinfected H9 cells; (B) using uninfected H9 cells. Approx. M, (X lo-“) of immunoreactive bands are indicated.

The use of whole infected cell lysate as antigen has clear advantages over the use of purified virus. Firstly, it is less laborious and avoids potentially hazardous operations. Secondly, no post-cell-lysis loss or proteolysis of viral proteins is possible as antigen is prepared for electrophoresis directly from viable cells. Thirdly, the procedure is easily controlled for antibodies recognising cell-derived proteins by including an uninfected cell lysate control, which contains almost as many protein species as the infected cell preparation. Fourthly, nonspecific binding of some antibodies to electrophoretically separated protein(s) does not appear as an apparently positive result as the cell lysate antigen contains a very large number of protein species. Such samples are easily discounted as being positive as they appear as an overall high background rather than as positive individual bands. This latter attribute is particularly important when analysing therapeutic immunoglobulin preparations and ‘sticky’ serum samples. The advantages of the use of the monoclonal anti-IgG antibody for detection of antibody-antigen complexes is that this reagent is highly specific and avid, is easily

95

purified and radioiodinated and is available in potentially unlimited quantities. The use of radioiodinated rather than the more commonly used enzyme-labelled antibody avoids the occurrence of spurious false-positive bands due to enzymic activity of antigen components or to ‘stickiness’ of the enzyme-antibody label. The use of a well-characterized reference serum preparation reactive against all the major HIV antigens provides an important internal control of immunoblots and should be included in all assays. Such a preparation is currently being examined in a WHO collaborative study (Garrett et al., in preparation). As well as providing valuable information as to antigens recognised by individual antisera and being of use as a confirmatory test after ELISA analysis, the immunoblotting technique can be used to assess the similarities or differences between different strains of HIVs. We have successfully used the technique described to confirm the identity of HIVs and have detected antigenic differences in retroviruses obtained from different clinical isolates. There has been much discussion as to the ‘best’ immunochemical procedure/strategy for detection of antibodies against HIV. Often the use of poorly developed or badly controlled procedures gives a false impression as to the applicability of a general method for this purpose. We propose that the simple procedure described in this report can be successfully used for the detection of anti-HIV antibodies and avoids the problems often associated with some previously described immunoblotting methodologies (Biberfeld et al., 1986; Courouce et al., 1986).

Acknowledgements

We are grateful to Drs. D.P. Thomas and T. Barrowcliffe for helpful and constructive discussion. We would like to thank Dr. R.C. Gallo for HTLV III strain B, Dr. E.M. Supran for serum samples, Susan Creswell and Alison Sykes for carrying out the HPLC and Ms. V. Watson for typing the manuscript.

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96 Garrett. A.J.: Seagroatt, V., Supran, M. and Schild, G.C. Measurement of antibodies to human immunodeficiency virus: an international collaborative study to evaluate WHO reference sera. In preparation. Gocke, D.J., Raska, Jr. K., Pollack, W. and Schwartzer, T. (1986) HTLV-III antibody in commercial immunoglobulins. Lancet i, 37-38. Karpas, A., Bevan, P.C., Gillson, W. and Oates, J.K. (1985) Lytic infection by British AIDS virus and development of rapid cell test for antiviral antibodies. Lancet ii, 695-697. Katchaki, J.N., Siem, T.H.. Brouwer, R. and Nieste, H.L.J. (1986) Anti-HTLV-III screening and false positivity. Lancet i, 448. Kitchen, L.W., Barin, F., Sullivan, J.L., McLane. M.F., Brettler, D.B., Levine, P.H. and Essex, M. (1984) Aetiology of AIDS - antibodies to human T-ceil leukaemia virus (type III) in haemophiliacs. Nature (London) 312, 367-369. Laskey, R.A. and Mills, A.D. (1977) Enhanced autoradiographic detection of 32P and lzsI using intensifying screens and hypersensitized film. FEBS Lett. 82, 314-320. Laemlli, U.K. and Favre, M.F. (1973) Maturation of the head of bacteriophage T4. Part 1 - DNA packaging. J. Mol. Biol. 80, 575-599. Marhnk, R., Allan, J. and Essex, M. (1986) Unusual serotogical profiles in AIDS. Lancet i, 1389. Morgan, J.. Tate, R., Farr, A.D. and Urbaniak, S.J. (1986) Potential source of error in HTLV-III antibody testing. Lancet i, 739-740. Reesink, H.W., Huisman, J.G., Gonsalves. M., Winkel, I.N., Hekker, A.C., Lelie, P.N.. Schaasberg, W., Aaij, C., Van Der Does, J.A., Desmyter, J. and Goudsmit, J. (1986) Evaluation of six enzyme immunoassays for antibody against human immunodeticiency virus. Lancet ii, 483-486. Sarngadharan, M.C., Popovic, M., Bruch, L., Schiipbach, J. and Gallo, R.C. (1984) Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224. 50&508. 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. U.S.A. 76, 4350-4354. Ulstrup, J.C., Skaug, K., Figenschau. K.J.. Osrtavik, I., Brun, J.N. and Petersen, G. (1986) Sensitivity of Western blotting (compared with ELISA and immunofluorescence) during seroconversion after HTLV-III infection. Lancet i, 1151-1152. Ward, J.W., Grindon. A.J., Feorino. P.M., Schable, C., Parvin, M. and Allen, J.R. (1986) Laboratory and epidemiologic evaluation of an enzyme immunoassay for antibodies to HTLV-III. J. Am. Med. Assoc. 256, 357-361. Watson Martin. P.. Burger, D.R., Caquette, S. and Goldstein, A.S. (1986) Importance of confirmatory results after strongly positive HTLV-III screening tests. N. Engl. J. Med. 314, 1577.