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Nonspecific binding of immunoglobulins to coat proteins of certain plant viruses in immunoblots and indirect ELISA
Journal of Virofogicul Methods,
15 (1987) 159-164
159
Elsevier JVM 00550
Short Communication
Nonspecific binding of immunoglobulins to coat proteins of certain plant viruses in immunoblots and indirect ELBA R.G.
Dietzgen
and R.I.B.
Francki
Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, South Australia, Australia
(Accepted
19 September
1986)
___. Nonspecific binding of immunoglobulins to coat protein of cucumber mosaic virus and several other plant viruses was observed in Western immunoblots, and to a much lesser extent in enzyme-linked immunosorbent assays. The biniling appears to occur between immunoglobulin molecules and the basic domains of viral coat proteins which bind to RNA during encapsidation. In all cases tested, the nonspecific reactions could be prevented by addition of 5 U/ml of heparin to the incubation buffers. Western immunoblotting;
Indirect ELISA; Plant virus protein; Nonspecific reaction; Heparin
Indirect enzyme-linked immunosorbent assays (ELISAs) and Western immunoblotting are frequently used to detect viruses and their specific antibodies and to screen for monoclonal antibodies (Devergne et al., 1981; Towbin and Gordon, 1984; MacKenzie et al., 1986). It is generally assumed that after blocking of unoccupied binding sites on the solid phase (polystyrene or nitrocellulose) reactions detected are due to specific antigen-antibody interactions. In the course of a study regarding a common antigenic determinant detected on the coat protein of tobacco mosaic virus (TMV) and the large subunit of the host plant enzyme ribulose-1,5_bisphosphate carboxylase (Dietzgen and Zaitlin, 1986), several viruses of different taxonomic groups were employed as controls in Western immunoblots. Surprisingly, it was observed that the coat protein of cucumber mosaic virus (CMV) reacted strongly with antisera to TMV (Fig. 1A). Further investigations revealed the reactivity of CMV coat protein with antisera to other serologically unrelated plant viruses and to host cell components (data not shown). Similar nonspecific reactions were also observed with preimmune serum (Fig. 1E). This nonspecific reactivity was common to all 5 CMV isolates tested and to another cucumovirus, Correspondence to: R.G. Dietzgen, Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, South Australia, Australia.
0166-~34/87/$03.50 @ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Analysis of the nonspecific binding of antibodies to basic viral coat proteins in Western immunoblots and its elimination by the addition of heparin. Purified preparations of -20 ug coat protein/lane of southern bean mosaic virus (track I), tomato aspermy virus (tracks 2 and 5), cucumber mosaic virus strain M (tracks 3 and 7) and strain NY (track 6) and tobacco mosaic virus strain Ul (tracks gels (Maizel, 1971). Proteins 4 and 8) were fractionated by electrophoresis in 12 % SDS-polyacrylamidc were transferred electrophoretically to nitrocellulose membrane (Schleicher & Schuell, BA83) at 60 V for 5 h (Towbin and Gordon, 1984) and the residual binding sites were blocked with bovine serum albumin. Immobilized antigens were probed with 1:lOOO diluted anti-U1 TMV antiserum (panels A and B), anti-MCMV antiserum (panels C and D), or rabbit preimmune serum (panels E and F). The immune complexes were reacted with I:2000 diluted goat anti-rabbit affinity-purified antibodies conjugated with alkaline phosphatase (Miles, Illinois). Antibodies and enzyme-conjugated antiglobulins were incubated with the nitrocellulose membrane in buffers without (panels A, C and E) or containing heparin (5 U/ml) (panels B, D and F). Reactive proteins were visualized by incubation with 1 mg Fast Red TR/ml naphtol AS BI phosphate solution. The resulting red bands on the nitrocellulose membrane were photographed through a dark green filter. Molecular weight markers phosphorylase b (M,=97,000), bovine serum albumin (~~=~8,0~), carbonic anhydrase ~~~=29,~) and TMV coat protein (M,=i7.000) are indicated on the side.
tomato aspermy virus (TAV) (Fig. lA, E), which is only distantly related to CMV (Rao et al., 1982). The nonspecific reactions occurred when either enzyme-labelled antiglobulins or “‘I-protein A was used to detect the antigen-antibody complexes. Nonspecific reactions in Western immunoblots have also been reported by Theisen et al. (1986), who observed reactions even when only enzyme-conjugated antibody was incubated with blocked blots. Modifications in their washing and blocking solutions did not solve the problem. A recent report by Pesce et al. (1986) described nonspecific reactions in indirect ELISA associated with positively charged cationic antigens due to nonspecific binding of primary and enzyme conjugated (secondary) antibodies. The nonspecific binding was overcome successfully by the addition of the polyanions, heparin or dextran sulfate.
CMV coat protein has regions of basic amino acids (Gould and Symons, 1982; C. Davies, pers. comm.) and hence it seemed possible that the nonspecific reaction in its detection by Western blotting (Fig. lA, E) was caused by immunoglobulins binding to the positive charge on the antigen. This appears to be so because incorporation of 5 U/ml of heparin into buffers during incubation with both the primary and secondary antibodies prevented the nonspecific reactions (Fig. 1, compare A to 3 and E to Ff. The virus-specific reactions were not impaired by the heparin because TMV coat protein was detected in the presence and absence of the additive with equal efficiency (Fig. fA, B, track 4). Similarly, C%fV coat protein and some contaminating antigens were detected by an antiserum to CMV irrespective whether heparin was included or not in the buffers (Fig. lC, D). The CMV antiserum cross-reacted with TAV but not TMV coat protein (Fig. lC, D). To determine if the nonspecific reactions in immunoblots were peculiar to CMV and TAV (cucumoviruses), we tested homologous and heterologous reactions with a number of other plant viruses. We detected nonspecific reactions similar to those with CMV and TAV, with southern bean mosaic virus (SBMV), sowbane mosaic virus (SoMV), velvet tobacco mottle virus (VTMoV) which are members or tentative members of the sobemovirus group (Francki et al., 198Sb), as well as alfalfa mosaic virus (AMV) (data not shown). All these viruses are dependent on protein-RNA interactions for the integrity of their particle structures and have been shown to have, or would be expected to have, strongly basic amino-terminal protein domains which are involved in binding to the encapsidated RNA (Harrison, 1983; Johnson and Argos, 1985; Tremaine et al., 1980, 1981). However, no nonspecific reactions were detected with coat proteins of TMV ~tobam~virus) cowpea mosaic virus (CPMV, comovirus), tobacco ringspot virus (TRSV, nepovirus) or turnip yellow mosaic virus (TYMV, tymovirus) (data not shown). The coat proteins of these viruses do not interact extensively with their encapsidated RNAs and are capable of forming capsids devoid of RNA (Butler, 1984; Francki et al., 1985a). They do not have, or would not be expected to have, strongly basic domains (Peter et al., 1972; Goeiet et al., 1982; Van Wezenbeek et al., 1983). In ~mmunob~ots with the plant rhabdo~~rus, lettuce necrotic yellows virus (LNYV) which has 5 structural proteins (Dale and Peters, 19$1), we observed that only the N protein reacted nonspeci~cal~y (data not shown), N is the protein which binds to the RNA in the nucleocapsids and in the case of another plant rhabdovirus, potato yellow dwarf virus, has been shown to be strongly basic {Knudson and McLeod, 1972). In all the cases where we encountered nonspecific reactions in immunoblots with viral coat proteins, they could be prevented by addition of 5 U/ml of heparin to the incubation buffers. We also tested whether the nonspecific reaction between CMV coat protein and preimmune serum could be prevented by i~cIuding SDS in the buffer during transfer of the protein from the polyacrylamide gel to the nitrocellulose membrane. However, the nonspecific reaction was observed in these tests and. moreover, the SDS interfered with the recognition of TMV coat protein by its specific antibody (data not shown). The addition of 5 U/ml of heparin to the buffers used in indirect ELTSA during incubation with primary and secondary ~enzyme-conjugated) antibodies also re-
162 TABLE 1 The effect of heparin on the reactivity of cucumber mosaic virus (CMV) and its coat protein in the indirect enzyme-linked immunosorbent assay (ELISA)“. CMV antigen used for coating
Serum
Presence of heparin during incubationh
None Preimmune Anti-CMV virion’ Anti-CMV coat proteing
+ + + +
Untreated’
Stabihsed by glutaraldehyde fixatio&
Dissociated with 0.1% SDS’
0.069 0.000 0.152 0.043 0.846 0.867 0.757 0.725
0.004 0.000 0.108 0.044 0.828 0.845 0.613 0.577
0.006 0.000 0.017 0.014 0.749 0.749 0.668 0.668
a The microtite~lates (Nunc, Immunoplate II) were coated with 10 ugiml of the appropriate antigen for 3 h at 25°C blocked with 1 mgiml BSA for 1 h at 25”C, reacted with serum diluted 1: 1000 for 16 h at 4°C probed with goat anti-rabbit alkaline phosphatase conjugate for 3 h at ZS’C, and incubated for .l h at 25°C with 1 mgiml p-nitrophenyl phosphate. The absorbance at 405 nm was then measured. The buffers used were the same as those recommended by Clark and Adams (1977). ’ Heparin was incorporated as indicated into the buffers for incubation with the sera and enzyme conjugates at a concentration of 5 U/ml. ’ The NY strain of CMV was purified as described by Habili and Francki (1974a). d Purified virus was fixed as described by Francki and Habili (1972) except that 0.25% glutaralde~yde was used instead of formaldehyde. e Purified virus was dissociated by the addition of sodium dodecyt sulphate (SDS) to a finaf concentration of 0.1% in bicarbonate coating buffer, pH 9.6 (Habib and Francki, 1974b). f Antiserum prepared against glutaraldehyde-fixed Z strain of CMV. When tested by immunodiffusion (Francki and Habili, 1972), the antiserum had a titre of l/256 against intact virus particles and 114 against dissociated CMV. 8 Antiserum prepared against the Z strain of CMV dissociated with LiCl (Francki et al., 1966). When tested by immunodiffusion (Francki and Habili, 1972), the antiserum had a titre of 114against intact virus particles and l/2 against dissociated CMV.
duced nonspecific reactions (Table 1) which were not as pronounced as those in the immunoblots (Fig. 1). When the 3 CMV antigens (untreated, glutaraldehydefixed and SDS-dissociated virus) were reacted with only the secondary antibody, all background absorbance was eliminated (Table 1). It was also significantly reduced when preimmune serum was used in the assays as the primary antibody (Table 1). The effect of heparin was only marginal in assays with the anti-CMV sera when untreated and glutaraldehyde-fixed antigen was used but had no effect on reactions with SDS-associated virus (Table 1). In the assays done in the presence of SDS, CMV capsids should be fully dissociated (Habili and Francki, 1974b). However, the presence of the anionic detergent would be expected to neutralise the charge on the basic protein domains responsible for any nonspecific reactions with immunoglobulins. The results of experiments reported here indicate that nonspecific reactions can occur when coat proteins of some viruses are to be detected by immunoblotti~g.
163
These reactions appear to result from nonspecific binding of immunoglobulins with the basic domains of the proteins and can be prevented by the presence of the polyanionic mucopolysaccharide, heparin. Our experience also stresses the importance of including preimmune sera as controls in experiments involving immunoblotting; especially when determining serological relationships among viruses and characterizing monoclonal antibodies. We did not observe significant nonspecific reactions between CMV and immunoglobulins in the indirect ELISA, but the inclusion of heparin in the buffers did reduce the background absorbance. However, it could be expected that nonspecific reactions with this type of virus could become a problem if a significant amount of the reacting antigen was dissociated coat protein with its basic domains exposed. It would seem, nevertheless, that this could be prevented by either heparin or SDS.
Acknowledgements
We thank Drs. J.W. Randles and M. Zaitlin for valuable discussions. R.G. Dietzgen was supported by a Feodor-Lynen-Research Fellowship from the Alexander von Humboldt Foundation. Purified virus preparations were generously supplied by C. Grivell, M.R. Hajimorad and S. Sakay. References Butler, P.J.G. (1984) The current picture of the structure and assembly of tobacco mosaic virus. J. Gen. Viral. 65, 253-279. Clark, M.F. and Adams, A.N. (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34. 475-483. Dale, J.L. and Peters, D. (1981) Protein composition of the virions of five plant rhabdoviruses. Intervirology 16, 86-94. Devergne, J.C., Cardin, L., Burckard, J. and Van Regenmortel, M.H.V. (1981) Comparison of direct and indirect ELISA for detecting antigenically related cucumoviruses. J. Viral. Methods 3, 193-200. Dietzgen, R.G. and Zaitlin, M. (1986) Tobacco mosaic virus coat protein and the large subunit of the host protein ribulose-1,5-bisphosphate carboxylase share a common antigen determinant. Virology. in press. Francki, R.I.B. and Habili, N. (1972) Stabilization of capsid structure and enhancement of immunogenicity of cucumber mosaic virus (Q strain) by formaldehyde. Virology 48, 309315. Francki, R.I.B., Randles, J.W., Chambers, T.C. and Wilson, S.B. (1966) Some properties of purified cucumber mosaic virus (Q strain). Virology 28, 729-741. Francki, R.I.B., Mime, R.G. and Hatta, T. (1985a). In: Atlas of Plant Viruses, Vols. 1 and 2. CRC Press, FL. Francki, R.I.B., Randles, J.W., Chu, P.W.G., Rohozinski, J. and Hatta, T. (1985b) Viroid-like RNAs incorporated in conventional virus capsids. In: Subviral Pathogens of Plants and Animals: Viroids and Prions (Maramorosch, K. and McKelvey, J.J., eds.), pp. 265-297. Academic Press, New York. Goelet, P., Lomonossoff, G.P., Butler, P.J.G., Akam, M.E., Gait, M.J. and Karn, J. (1982) Nucleotide sequence of tobacco mosaic virus RNA. Proc. Natl. Acad. Sci. U.S.A. 79, 5818-5822. Gould, A.R. and Symons, R.H. (1982) Cucumber mosaic virus RNA 3. Determination of the nucleotide sequence provides the amino acid sequences of protein 3A and viral coat protein. Eur. J. Biochem. 126, 217-226.
164 Habili, N. and Francki, R.I.B. (1974a) Comparative studies on tomato aspermy and cucumber mosaic viruses. I. Physical and chemical properties. Virology 57, 392-401. Habili, N. and Francki, R.I.B. (1974b) Comparative studies on tomato aspermy and cucumber mosaic viruses. II. Virus stability. Virology 60, 29-36. Harrison, S.C. (1983) Virus structure: high-resolution perspectives. Adv. Virus Res. 28, 175-240. Johnson, J.E. and Argos, P. (1985) Virus particle stability and structure. In: The Plant Viruses: Polyhedrai Virions with Tripartite Genomes (Francki. R.I.B., ed.), pp. 19-56. Plenum Press, New York. Knudson, D.L. and McLeod, R. (1972) The proteins of potato yellow dwarf virus. Virology 47,285-295. MacKenzie, D.J. and Tremaine, J.H. (1986) The use of a monoclonal antibody specific for the N-terminal region of southern bean mosaic virus as a probe of virus structure. J. Gen. Virol. 67, 727-735. Maizel, J.V. (1971) Polyacrylamide gel electrophoresis of viral proteins. In: Methods in Virology, Vol. 5 (Maramorosch, K. and Koprowski, H., eds.), pp. 179-246. Academic Press, New York. Pesce, A.J., Apple, R., Sawtell, N. and Michael, J.G. (1986) Cationic antigens. Problems associated with measurement by ELISA. J. Immunol. Methods 87, 21-27. Peter, R., Stehelin, D., Reinbolt, J.. Callot, D. and Duranton, H. (1972) Primary structure of turnip yetlow mosaic virus coat protein. Virology 49, 61.5-617. Rao, A.L.N.. Hatta. T. and Francki, R.I.B. (1982) Comparative studies on tomato aspermy and cucumber mosaic viruses VII. Serological relationships reinvestigated. Virology 116, 318-326. Theisen, N., Schulze Lohoff, E., Von Figura, K. and Hasilik, A. (1986) Sequential detection of antigens in Western blots with differently colored products. Anal. Biochem. 152, 211-214. Towbin, H. and Gordon, J. (1984) Immunoblotting and dot immuno-binding - current status and outlook. J. Immunol. Methods 72, 313-340. Tremaine, J.H., Ronald, W.P. and Kelly, E.M. (1980) Chemical and serological properties of a cyanogen bromide peptide of southern bean mosaic virus protein. Can. J. Microbial. 26, 1450-1459. Tremaine. J.H., Ronald. W.P. and Kelly, E.M. (1981) A highly basic cyanogen bromide peptide from sowbane mosaic virus protein. Virotogy 114, 282-285. Van Wezenbeek, P., Verver, J., Harmsen, J.. Vos, P. and Van Kammen, A. (1983) Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus. EMBO J. 2,941-946.
15 (1987) 159-164
159
Elsevier JVM 00550
Short Communication
Nonspecific binding of immunoglobulins to coat proteins of certain plant viruses in immunoblots and indirect ELBA R.G.
Dietzgen
and R.I.B.
Francki
Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, South Australia, Australia
(Accepted
19 September
1986)
___. Nonspecific binding of immunoglobulins to coat protein of cucumber mosaic virus and several other plant viruses was observed in Western immunoblots, and to a much lesser extent in enzyme-linked immunosorbent assays. The biniling appears to occur between immunoglobulin molecules and the basic domains of viral coat proteins which bind to RNA during encapsidation. In all cases tested, the nonspecific reactions could be prevented by addition of 5 U/ml of heparin to the incubation buffers. Western immunoblotting;
Indirect ELISA; Plant virus protein; Nonspecific reaction; Heparin
Indirect enzyme-linked immunosorbent assays (ELISAs) and Western immunoblotting are frequently used to detect viruses and their specific antibodies and to screen for monoclonal antibodies (Devergne et al., 1981; Towbin and Gordon, 1984; MacKenzie et al., 1986). It is generally assumed that after blocking of unoccupied binding sites on the solid phase (polystyrene or nitrocellulose) reactions detected are due to specific antigen-antibody interactions. In the course of a study regarding a common antigenic determinant detected on the coat protein of tobacco mosaic virus (TMV) and the large subunit of the host plant enzyme ribulose-1,5_bisphosphate carboxylase (Dietzgen and Zaitlin, 1986), several viruses of different taxonomic groups were employed as controls in Western immunoblots. Surprisingly, it was observed that the coat protein of cucumber mosaic virus (CMV) reacted strongly with antisera to TMV (Fig. 1A). Further investigations revealed the reactivity of CMV coat protein with antisera to other serologically unrelated plant viruses and to host cell components (data not shown). Similar nonspecific reactions were also observed with preimmune serum (Fig. 1E). This nonspecific reactivity was common to all 5 CMV isolates tested and to another cucumovirus, Correspondence to: R.G. Dietzgen, Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, South Australia, Australia.
0166-~34/87/$03.50 @ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Analysis of the nonspecific binding of antibodies to basic viral coat proteins in Western immunoblots and its elimination by the addition of heparin. Purified preparations of -20 ug coat protein/lane of southern bean mosaic virus (track I), tomato aspermy virus (tracks 2 and 5), cucumber mosaic virus strain M (tracks 3 and 7) and strain NY (track 6) and tobacco mosaic virus strain Ul (tracks gels (Maizel, 1971). Proteins 4 and 8) were fractionated by electrophoresis in 12 % SDS-polyacrylamidc were transferred electrophoretically to nitrocellulose membrane (Schleicher & Schuell, BA83) at 60 V for 5 h (Towbin and Gordon, 1984) and the residual binding sites were blocked with bovine serum albumin. Immobilized antigens were probed with 1:lOOO diluted anti-U1 TMV antiserum (panels A and B), anti-MCMV antiserum (panels C and D), or rabbit preimmune serum (panels E and F). The immune complexes were reacted with I:2000 diluted goat anti-rabbit affinity-purified antibodies conjugated with alkaline phosphatase (Miles, Illinois). Antibodies and enzyme-conjugated antiglobulins were incubated with the nitrocellulose membrane in buffers without (panels A, C and E) or containing heparin (5 U/ml) (panels B, D and F). Reactive proteins were visualized by incubation with 1 mg Fast Red TR/ml naphtol AS BI phosphate solution. The resulting red bands on the nitrocellulose membrane were photographed through a dark green filter. Molecular weight markers phosphorylase b (M,=97,000), bovine serum albumin (~~=~8,0~), carbonic anhydrase ~~~=29,~) and TMV coat protein (M,=i7.000) are indicated on the side.
tomato aspermy virus (TAV) (Fig. lA, E), which is only distantly related to CMV (Rao et al., 1982). The nonspecific reactions occurred when either enzyme-labelled antiglobulins or “‘I-protein A was used to detect the antigen-antibody complexes. Nonspecific reactions in Western immunoblots have also been reported by Theisen et al. (1986), who observed reactions even when only enzyme-conjugated antibody was incubated with blocked blots. Modifications in their washing and blocking solutions did not solve the problem. A recent report by Pesce et al. (1986) described nonspecific reactions in indirect ELISA associated with positively charged cationic antigens due to nonspecific binding of primary and enzyme conjugated (secondary) antibodies. The nonspecific binding was overcome successfully by the addition of the polyanions, heparin or dextran sulfate.
CMV coat protein has regions of basic amino acids (Gould and Symons, 1982; C. Davies, pers. comm.) and hence it seemed possible that the nonspecific reaction in its detection by Western blotting (Fig. lA, E) was caused by immunoglobulins binding to the positive charge on the antigen. This appears to be so because incorporation of 5 U/ml of heparin into buffers during incubation with both the primary and secondary antibodies prevented the nonspecific reactions (Fig. 1, compare A to 3 and E to Ff. The virus-specific reactions were not impaired by the heparin because TMV coat protein was detected in the presence and absence of the additive with equal efficiency (Fig. fA, B, track 4). Similarly, C%fV coat protein and some contaminating antigens were detected by an antiserum to CMV irrespective whether heparin was included or not in the buffers (Fig. lC, D). The CMV antiserum cross-reacted with TAV but not TMV coat protein (Fig. lC, D). To determine if the nonspecific reactions in immunoblots were peculiar to CMV and TAV (cucumoviruses), we tested homologous and heterologous reactions with a number of other plant viruses. We detected nonspecific reactions similar to those with CMV and TAV, with southern bean mosaic virus (SBMV), sowbane mosaic virus (SoMV), velvet tobacco mottle virus (VTMoV) which are members or tentative members of the sobemovirus group (Francki et al., 198Sb), as well as alfalfa mosaic virus (AMV) (data not shown). All these viruses are dependent on protein-RNA interactions for the integrity of their particle structures and have been shown to have, or would be expected to have, strongly basic amino-terminal protein domains which are involved in binding to the encapsidated RNA (Harrison, 1983; Johnson and Argos, 1985; Tremaine et al., 1980, 1981). However, no nonspecific reactions were detected with coat proteins of TMV ~tobam~virus) cowpea mosaic virus (CPMV, comovirus), tobacco ringspot virus (TRSV, nepovirus) or turnip yellow mosaic virus (TYMV, tymovirus) (data not shown). The coat proteins of these viruses do not interact extensively with their encapsidated RNAs and are capable of forming capsids devoid of RNA (Butler, 1984; Francki et al., 1985a). They do not have, or would not be expected to have, strongly basic domains (Peter et al., 1972; Goeiet et al., 1982; Van Wezenbeek et al., 1983). In ~mmunob~ots with the plant rhabdo~~rus, lettuce necrotic yellows virus (LNYV) which has 5 structural proteins (Dale and Peters, 19$1), we observed that only the N protein reacted nonspeci~cal~y (data not shown), N is the protein which binds to the RNA in the nucleocapsids and in the case of another plant rhabdovirus, potato yellow dwarf virus, has been shown to be strongly basic {Knudson and McLeod, 1972). In all the cases where we encountered nonspecific reactions in immunoblots with viral coat proteins, they could be prevented by addition of 5 U/ml of heparin to the incubation buffers. We also tested whether the nonspecific reaction between CMV coat protein and preimmune serum could be prevented by i~cIuding SDS in the buffer during transfer of the protein from the polyacrylamide gel to the nitrocellulose membrane. However, the nonspecific reaction was observed in these tests and. moreover, the SDS interfered with the recognition of TMV coat protein by its specific antibody (data not shown). The addition of 5 U/ml of heparin to the buffers used in indirect ELTSA during incubation with primary and secondary ~enzyme-conjugated) antibodies also re-
162 TABLE 1 The effect of heparin on the reactivity of cucumber mosaic virus (CMV) and its coat protein in the indirect enzyme-linked immunosorbent assay (ELISA)“. CMV antigen used for coating
Serum
Presence of heparin during incubationh
None Preimmune Anti-CMV virion’ Anti-CMV coat proteing
+ + + +
Untreated’
Stabihsed by glutaraldehyde fixatio&
Dissociated with 0.1% SDS’
0.069 0.000 0.152 0.043 0.846 0.867 0.757 0.725
0.004 0.000 0.108 0.044 0.828 0.845 0.613 0.577
0.006 0.000 0.017 0.014 0.749 0.749 0.668 0.668
a The microtite~lates (Nunc, Immunoplate II) were coated with 10 ugiml of the appropriate antigen for 3 h at 25°C blocked with 1 mgiml BSA for 1 h at 25”C, reacted with serum diluted 1: 1000 for 16 h at 4°C probed with goat anti-rabbit alkaline phosphatase conjugate for 3 h at ZS’C, and incubated for .l h at 25°C with 1 mgiml p-nitrophenyl phosphate. The absorbance at 405 nm was then measured. The buffers used were the same as those recommended by Clark and Adams (1977). ’ Heparin was incorporated as indicated into the buffers for incubation with the sera and enzyme conjugates at a concentration of 5 U/ml. ’ The NY strain of CMV was purified as described by Habili and Francki (1974a). d Purified virus was fixed as described by Francki and Habili (1972) except that 0.25% glutaralde~yde was used instead of formaldehyde. e Purified virus was dissociated by the addition of sodium dodecyt sulphate (SDS) to a finaf concentration of 0.1% in bicarbonate coating buffer, pH 9.6 (Habib and Francki, 1974b). f Antiserum prepared against glutaraldehyde-fixed Z strain of CMV. When tested by immunodiffusion (Francki and Habili, 1972), the antiserum had a titre of l/256 against intact virus particles and 114 against dissociated CMV. 8 Antiserum prepared against the Z strain of CMV dissociated with LiCl (Francki et al., 1966). When tested by immunodiffusion (Francki and Habili, 1972), the antiserum had a titre of 114against intact virus particles and l/2 against dissociated CMV.
duced nonspecific reactions (Table 1) which were not as pronounced as those in the immunoblots (Fig. 1). When the 3 CMV antigens (untreated, glutaraldehydefixed and SDS-dissociated virus) were reacted with only the secondary antibody, all background absorbance was eliminated (Table 1). It was also significantly reduced when preimmune serum was used in the assays as the primary antibody (Table 1). The effect of heparin was only marginal in assays with the anti-CMV sera when untreated and glutaraldehyde-fixed antigen was used but had no effect on reactions with SDS-associated virus (Table 1). In the assays done in the presence of SDS, CMV capsids should be fully dissociated (Habili and Francki, 1974b). However, the presence of the anionic detergent would be expected to neutralise the charge on the basic protein domains responsible for any nonspecific reactions with immunoglobulins. The results of experiments reported here indicate that nonspecific reactions can occur when coat proteins of some viruses are to be detected by immunoblotti~g.
163
These reactions appear to result from nonspecific binding of immunoglobulins with the basic domains of the proteins and can be prevented by the presence of the polyanionic mucopolysaccharide, heparin. Our experience also stresses the importance of including preimmune sera as controls in experiments involving immunoblotting; especially when determining serological relationships among viruses and characterizing monoclonal antibodies. We did not observe significant nonspecific reactions between CMV and immunoglobulins in the indirect ELISA, but the inclusion of heparin in the buffers did reduce the background absorbance. However, it could be expected that nonspecific reactions with this type of virus could become a problem if a significant amount of the reacting antigen was dissociated coat protein with its basic domains exposed. It would seem, nevertheless, that this could be prevented by either heparin or SDS.
Acknowledgements
We thank Drs. J.W. Randles and M. Zaitlin for valuable discussions. R.G. Dietzgen was supported by a Feodor-Lynen-Research Fellowship from the Alexander von Humboldt Foundation. Purified virus preparations were generously supplied by C. Grivell, M.R. Hajimorad and S. Sakay. References Butler, P.J.G. (1984) The current picture of the structure and assembly of tobacco mosaic virus. J. Gen. Viral. 65, 253-279. Clark, M.F. and Adams, A.N. (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34. 475-483. Dale, J.L. and Peters, D. (1981) Protein composition of the virions of five plant rhabdoviruses. Intervirology 16, 86-94. Devergne, J.C., Cardin, L., Burckard, J. and Van Regenmortel, M.H.V. (1981) Comparison of direct and indirect ELISA for detecting antigenically related cucumoviruses. J. Viral. Methods 3, 193-200. Dietzgen, R.G. and Zaitlin, M. (1986) Tobacco mosaic virus coat protein and the large subunit of the host protein ribulose-1,5-bisphosphate carboxylase share a common antigen determinant. Virology. in press. Francki, R.I.B. and Habili, N. (1972) Stabilization of capsid structure and enhancement of immunogenicity of cucumber mosaic virus (Q strain) by formaldehyde. Virology 48, 309315. Francki, R.I.B., Randles, J.W., Chambers, T.C. and Wilson, S.B. (1966) Some properties of purified cucumber mosaic virus (Q strain). Virology 28, 729-741. Francki, R.I.B., Mime, R.G. and Hatta, T. (1985a). In: Atlas of Plant Viruses, Vols. 1 and 2. CRC Press, FL. Francki, R.I.B., Randles, J.W., Chu, P.W.G., Rohozinski, J. and Hatta, T. (1985b) Viroid-like RNAs incorporated in conventional virus capsids. In: Subviral Pathogens of Plants and Animals: Viroids and Prions (Maramorosch, K. and McKelvey, J.J., eds.), pp. 265-297. Academic Press, New York. Goelet, P., Lomonossoff, G.P., Butler, P.J.G., Akam, M.E., Gait, M.J. and Karn, J. (1982) Nucleotide sequence of tobacco mosaic virus RNA. Proc. Natl. Acad. Sci. U.S.A. 79, 5818-5822. Gould, A.R. and Symons, R.H. (1982) Cucumber mosaic virus RNA 3. Determination of the nucleotide sequence provides the amino acid sequences of protein 3A and viral coat protein. Eur. J. Biochem. 126, 217-226.
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