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The use of dextran as a blocking agent on nitrocellulose membrane in the analysis of sporozoite antigens of Plasmodium vivax
Journal of Biochemical and Biophysical Methods, 17 (1988) 135-142 Elsevier
135
BBM 00697
The use of dextran as a blocking agent on nitrocellulose membrane in the analysis of sporozoite antigens of Plasmodium vivax Leo F. Y u a n 1, R o b e r t A. Wirtz 2 a n d R i c h a r d L. B e a u d o i n 1 I Naval Medical Research Institute, Bethesda, MD 20014, U.S.A. and 2 Walter ReedArmy Institute of Research, Washington, DC20307, U.S.A.
(Received 5 March 1988) (Accepted 12 July 1988)
Summary Dextran (molecular weight, 71200) has been found to block the unbound sites of the nitrocellulose membrane, to which antigens have been electroblotted from acrylamide gel, for use in assaying monoclonal antibodies. The use of polysaccharide as a blocking agent allows the antigens on the nitrocellulose membrane to be digested with pronase and subsequently reacted with monoclonal antibodies. Sporozoite antigens of Plasmodium vivax, after being digested with pronase, completely lost their antigenicity to bind to the sporozoite-specific monoclonai antibodies, thus suggesting that they are proteins or protein conjugates in nature. The method described here for qualitative determination of protein antigens requires as little as 2000 sporozoites for each assay. Key words: Immunoblotting; Dextran; Pronase
Introduction The development of the Western blotting method for transferring proteins or nucleic acids from electrophoresis gels to a solid phase such as nitrocellulose membrane provides the capacity of using antibody probes to identify antigens of biological interest. In general, the membrane containing the bound antigen is first treated with the blocking agent, 5~ bovine serum albumin (BSA), to saturate the sites on the nitrocellulose membrane that contain no antigen, and then is reacted with specific antibodies. BSA has been known to bind some proteins nonspecifiCorrespondence address." L.F. Yuan, Malarial Branch, Naval Medical Research Institute, Bethesda, M D 20814-5055, U.S.A. 0165-022X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
136 cally. Our early results (unpublished) showed that when sporozoites of Plasmodium berghei isolated by a gradient containing BSA were used as an immunogen for immunizing animals, anamnestic shock to BSA occurred when the animals were boosted with sporozoites following the primary injection. This observation raises the possibility that BSA may be able to bind to sporozoites nonspecifically; consequently, studies were carried out to search for an alternative blocking agent that would not bind to sporozoites and could be used to study the sporozoite antigens on the nitrocellulose membrane. This paper reports the finding that dextran can be used successfully as a blocking agent and furthermore, by using dextran as the blocking agent, pronase can be employed to determine qualitatively if the antigenic epitopes recognized by specific monoclonal antibodies are proteins in nature.
Materials and Methods
Membrane solubilization Sporozoites of Plasmodium vivax [2] were suspended in an equal volume of saline, and the suspension was then subjected to freeze-thawing in an a c e t o n e / d r y ice bath for about 10 cycles. The resulting mixture was adjusted to pH 7.4 with 0.5% N a H C O 3 and centrifuged at 10000 x g for 20 min. The supernatant solution was removed and the sporozoite pellet was washed repeatedly with 0.1 M Tris-HCl buffer, pH 7.4, until the final washing had an absorbance of less than 0.05 at 280 nm. The sporozoite membrane was solubilized by suspending the sporozoite pellet in 1.5 volumes of 0.1 M Tris-HCl buffer, p H 7.4, containing 0.5% Nonidet-40 (NP-40; Particle Data Laboratories, Ltd., Emhurst, IL). The suspension was incubated at room temperature for 30 min with occasional vortexing and was then washed twice with an additional 2 volumes of buffer. The combined supernatant fluid was exhaustively dialyzed against several changes of fresh 0.01 M Tris-HCl buffer, p H 7.4, for two days. The dialyzed solution was lyophilized and reconstituted to 1 / 1 0 of the original volume with distilled water. The resulting supernatant solution, after centrifugation at 10000 x g for 30 min, was clear and appeared light yellow; it was referred to as sporozoite membrane extract. Gel electrophoresis SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli [3] using an 8-12% linear gradient acrylamide slab gel (15 x 15 x 0.15 cm). The stacking gel contained 3% acrylamide/0.1% bisacrylamide. Samples were prepared in glycerol, Tris-HCl, p H 6.8, SDS, and bromophenol blue, and heated at 1 0 0 ° C for 3 min. Radioactive labeled molecular weight standards (Bethesda Research Laboratory, Bethesda, Maryland) containing 0.03/~Ci of 14C in 20 ffl were used in each sample well and run together with experimental samples'.' Electrophoresis was carried out at 25 mA until the tracking dye reached the bottom of the gel. Electrotransfer The blotting procedure was essentially the same as described by Towbin et al. [4].
137 Following electrophoresis, the gels were immersed in buffer containing 0.025 M Tris, 0.192 M glycine, p H 8.3, and 20% methanol for 10 min and then placed on a single nitrocellulose membrane previously equilibrated with the same buffer. The gel-nitrocellulose assembly was placed between pairs of Whatman No. 3 filter paper, Scotch-Brite pads and supports, and immersed in the buffer chamber. Electroblotting was performed under a constant current of 0.1 A at room temperature overnight (approximately 15 h) using an E-C electroblot apparatus (Bio-Rad). i
Detection of antigen with monoclonal antibodies The blots, washed once in phosphate-buffered saline (PBS), p H 7.2, were air dried and then immersed in 5% dextran (molecular weight, 71 200) in PBS for 30 min at room temperature to block the unoccupied sites of the nitrocellulose membrane. After blocking, the membranes were rinsed twice in PBS at 10 min intervals with gentle shaking and soaked in antibody solution (Ascitic fluid having an immunofluorescent assay [5] titer of 50). Time of incubation was 1 h at room temperature and then overnight at 4 °C. After incubation the membranes were repeatedly washed with PBS containing 0.05% Tween 20 to remove unbound antibodies until the final washing had an absorbance of less than 0.5 at 280 nm. In general, six changes of 100 ml washing buffer at 30 min intervals with agitation is sufficient to render the membrane free of unbound antibodies. After the final rinsing, the membranes were blotted with Whatman No. 3 paper to remove excess washing buffer and then treated with 12~I-goat anti-mouse Ig (New England Nuclear, Boston, MA), specific activity 9.2 /tCi/#g, containing a radioactivity of approximately 40000-50000 c p m / m l in PBS for 3 h at room temperature. The membranes were repeatedly washed with PBS until the final washing had a radioactivity of less than 100 c p m / m l . Membranes were air dried between two sheets of Whatman No. 3 paper, and exposed to K o d a k XAR-5 films at - 7 0 ° C using a Kodak X-ray cassette plus Intensifying Screen. The same experimental procedures were followed when 5% BSA was used as the blocking agent to saturate the binding sites of the nitrocellulose membrane.
Pronase digestion of antigens on nitrocellulose membrane The enzyme solution was prepared by dissolving pronase (Calbiochem, San Diego, CA) from Streptomyces griseus K-1 in 0.1 M Tris-HC1 buffer, p H 7.4, containing 0.01 M CaC12 to give 38.5 units/ml. One enzyme unit was defined as the amount of enzyme that would yield a digestion product equivalent to 25 /tg of tyrosine per min. After blocking with 5% dextran solution was complete, the membranes were rinsed three times with 0.1 M Tris-HCl buffer, p H 7.4, then immersed in the enzyme solution, and incubated for 6 h at 4 0 ° C with gentle agitation. A 100 ml enzyme solution in a tray (15 x 21 x 4 cm) would be sufficient to cover one sheet of nitrocellulose membrane (12 x 16 cm). Controls that had the same compositions of substrate and buffer, except saline substituting for the enzyme, were run simultaneously with the experimental samples to determine if there was any non-enzymatic alteration of the antigens during the incubation period. At the end of incubation, the membranes were washed three times with PBS
138 to completely remove the p r o n a s e a n d then reacted with m o n o c l o n a l a n t i b o d i e s and 125I-goat a n t i - m o u s e Ig as described in the previous section.
Results and Discussion P r e l i m i n a r y experiments to investigate blocking agents were performed by spotting 5 /~1 o v a l b u m i n solution (10 m g / m l in saline) o n t o strips of nitrocellulose m e m b r a n e . The m e m b r a n e s were blocked with dextran solutions u n d e r different e x p e r i m e n t a l conditions, i n c u b a t e d with mouse a n t i - o v a l b u m i n serum, a n d then
M~nf
x 10-:' 20O
18.4
AG
(A) Fig. 1. Comparison of the effectivenessof blocking agents to saturate the unbound sites of nitrocellulose membrane. The nitrocellulose membranes on which the sporozoite membrane antigens (AG) were electrobloned were treated with (A) 5% dextran and (B) 5% BSA. The membranes were reacted with sporozoite-specific monoclonal antibodies and then with 1251-goat anti-mouse Ig. Autoradiography was carried out as described in Materials and Methods.
139
Fig. 1. (continued).
treated with 125I-goat anti-mouse Ig. The background of the membrane was examined by autoradiography to determine the degree of effectiveness of the blocking agents in saturating the unbound sites of the membrane that contained antigens. When nitrocellulose membranes were blotted in 1%, 5%, and 10% dextran solutions in PBS, pH 7.2 for 1 h, no significant differences in background were observed except in one experiment in which a 1% dextran solution gave a slightly darkened background as compared with other strips treated with 5% and 10% dextran solutions. The blocking of unbound sites of the nitrocellulose membrane with 5% dextran solutions at pH 5, 6, 7, 8, and 9 gave a background essentially similar to that at pH 7.2. Thus, the blocking of the unbound sites on the nitrocellulose membrane appeared to be due to the trapping of dextran in the matrix of the membrane rather than the attraction of a charged surface on the nitrocellulose membrane. 30 rain of incubation with 5% dextran solution in PBS, pH 7.2, was found to be sufficient for binding the unoccupied sites on the membrane; incuba-
140 tion for longer than 30 min, e.g., 1 h or 2 h, gave no further improvement of the background on autoradiographs. Sporozoite antigens were separated in an 8-12% polyacrylamide slab gel and then electrobiotted to nitrocellulose membranes. One membrane that contained the antigen was treated with 5% BSA; another with 5% dextran in PBS. Both were treated with sporozoite-specific monoclonal antibodies and reacted with ~251-goat anti-mouse Ig under identical experimental conditions. The autoradiographs of the nitrocellulose membranes treated with either BSA or dextran shown in Fig. 1 indicate that there is no difference in the backgrounds produced by the two; however, membranes blocked with BSA solution occasionally showed a number of small radioactive spots on autoradiographs, suggesting the possibility that BSA aggregates were present in a solution that nonspecifically bound the ~251-goat anti-mouse Ig to the membrane. Pronase from Streptornyces griseus K-1, a mixture of endopeptidases and exopeptidases containing both alkaline and neutral aminopeptidases and carboxypeptidases, has been used to digest glycoproteins in the nitrocellulose membrane [6]. Using dextran as the blocking agent, antigens on the nitrocellulose membrane can be digested with pronase, reacted with specific monoclonal antibodies, and then treated with 125I-goat anti-mouse Ig to determine qualitatively if the antigens are proteins in nature. When sporozoite antigens of P. vivax were electrophoretically separated and electroblotted to nitrocellulose membranes, a strip for each sample was cut from the membrane (2 × 10 cm), and then blocked with 5% dextran in PBS, p H 7.2. One strip was treated with pronase solution, while the replica was treated with saline under the same experimental conditions. Fig. 2 shows the effect of pronase on the capacity of P. vivax antigens to bind sporozoite-specific monoclonal antibodies (H-4, H-3, H-l). All the antigenic properties of sporozoite antigens were destroyed by the treatment with pronase; under the same experimental conditions. controls treated with saline had no effect on the antigens to bind to the antibodies. The effectiveness of pronase digestion is also evident from results showing that radioactive labeled protein molecular weight markers were completely destroyed with pronase under the same experimental conditions as tested samples. The susceptibility of P. viuax sporozoite antigens towards pronase digestion suggests that the antigens which reacted with the monoclonal antibodies were proteins in nature, but whether or not they are solely proteins or they are protein conjugates such as glycoproteins remains to be determined. The method recently reported by Woodward et al. [7] using periodate oxidation for detecting carbohydrate epitopes on the nitrocellulose membrane, along with the method described here, could be applicable for determining qualitatively if the antigen is a carbohydrate or glycoprotein conjugate. The use of BSA instead of dextran as the blocking agent on the nitrocellulose membrane in the pronase treatment experiment resulted in a high background of the final X-ray film, suggesting that the pronase may digest BSA in the matrix on the nitrocellulose membrane thus diminishing the blocking capacity of BSA. Our studies on the sporozoite antigens showed that as little as 2000 sporozoites were sufficient for the assay described. In addition to the advantage that a very
141
Fig. 2. Effect of pronase on the antigenicity of sporozoite membrane antigens to bind to monoclonal antibodies. After having been separated on an 8-12% polyacrylamide gel, membrane antigens were electroblotted to nitrocellulose membrane. The membrane was cut for each sample and then immersed in 5% dextran. One strip was treated with pronase (P); the replica strip with saline as a control. After incubation, the strips were reacted with monoclonal antibodies as indicated and treated with 125I-goat anti-mouse Ig. Autoradiography was carried out as described in Materials and Methods.
m i n u t e a m o u n t of a n t i g e n is required for each assay, the use of d e x t r a n as a b l o c k i n g agent o n the nitrocellulose m e m b r a n e would be helpful in studying other biological substances that m a y have non-specific affinity towards BSA. I n practice, the d e x t r a n solution when kept at 4 ° C can be used repeatedly at least 5 - 1 0 times w i t h o u t a n y detectable change in its blocking capacity.
Simplified description of the method and its advantages The m e t h o d of using dextran as a blocking agent o n nitrocellulose m e m b r a n e provides a m e a n s of studying biological materials which non-specifically b i n d to b o v i n e serum a l b u m i n that is c o n v e n t i o n a l l y used to block the u n o c c u p i e d sites on nitrocellulose m e m b r a n e . I n addition, the use of dextran, a n o n - p r o t e i n a c e o u s
142 substance, allows the antigens o n the nitrocellulose m e m b r a n e to be digested with p r o n a s e a n d s u b s e q u e n t l y reacted with specific a n t i b o d y to d e t e r m i n e qualitatively if the a n t i g e n is p r o t e i n in nature. The a d v a n t a g e of this technique is the ability to analyze a very small a m o u n t of a n t i g e n by a u t o r a d i o g r a p h y or enzyme such as peroxidase-linked antibody. This m e t h o d could be extended to analyze antigens by e m p l o y i n g various enzymes of k n o w n specificity.
Acknowledgements This work was s u p p o r t e d by Naval Medical Research a n d D e v e l o p m e n t C o m m a n d W o r k U n i t No. 2M161102BS10AF427. The o p i n i o n s a n d assertions c o n t a i n e d herein are the private ones of the authors a n d are not to be c o n s t r u e d as official or as reflecting the views of the U.S. N a v y D e p a r t m e n t of the naval service at large.
References 1 Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. 2 Wirtz, R.A., Burkot, T.R., Andre, R.G., Rosenberg, R., Collins, W.E. and Roberts, D.R. (1985) Am. J. Trop. Med. Hyg. 34, 1048-1054. 3 Laemmli, U.K. (1970) Nature (London) 227, 680-685. 4 Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 5 Ingram, R.L., Okten, Jr., L.B. and Jumper, J.R. (1961) Proc. Soc. Exp. Biol. and Med. 106, 52-54. 6 Dion, A.S. and Pomenti, A.A. (1985) Anal. Biochem. 147, 525-528. 7 Woodward, M.P., Young, Jr., W.W. and Bloodgood, R.A. (1985) J. lmmunol. Methods 78, 143 153.
135
BBM 00697
The use of dextran as a blocking agent on nitrocellulose membrane in the analysis of sporozoite antigens of Plasmodium vivax Leo F. Y u a n 1, R o b e r t A. Wirtz 2 a n d R i c h a r d L. B e a u d o i n 1 I Naval Medical Research Institute, Bethesda, MD 20014, U.S.A. and 2 Walter ReedArmy Institute of Research, Washington, DC20307, U.S.A.
(Received 5 March 1988) (Accepted 12 July 1988)
Summary Dextran (molecular weight, 71200) has been found to block the unbound sites of the nitrocellulose membrane, to which antigens have been electroblotted from acrylamide gel, for use in assaying monoclonal antibodies. The use of polysaccharide as a blocking agent allows the antigens on the nitrocellulose membrane to be digested with pronase and subsequently reacted with monoclonal antibodies. Sporozoite antigens of Plasmodium vivax, after being digested with pronase, completely lost their antigenicity to bind to the sporozoite-specific monoclonai antibodies, thus suggesting that they are proteins or protein conjugates in nature. The method described here for qualitative determination of protein antigens requires as little as 2000 sporozoites for each assay. Key words: Immunoblotting; Dextran; Pronase
Introduction The development of the Western blotting method for transferring proteins or nucleic acids from electrophoresis gels to a solid phase such as nitrocellulose membrane provides the capacity of using antibody probes to identify antigens of biological interest. In general, the membrane containing the bound antigen is first treated with the blocking agent, 5~ bovine serum albumin (BSA), to saturate the sites on the nitrocellulose membrane that contain no antigen, and then is reacted with specific antibodies. BSA has been known to bind some proteins nonspecifiCorrespondence address." L.F. Yuan, Malarial Branch, Naval Medical Research Institute, Bethesda, M D 20814-5055, U.S.A. 0165-022X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
136 cally. Our early results (unpublished) showed that when sporozoites of Plasmodium berghei isolated by a gradient containing BSA were used as an immunogen for immunizing animals, anamnestic shock to BSA occurred when the animals were boosted with sporozoites following the primary injection. This observation raises the possibility that BSA may be able to bind to sporozoites nonspecifically; consequently, studies were carried out to search for an alternative blocking agent that would not bind to sporozoites and could be used to study the sporozoite antigens on the nitrocellulose membrane. This paper reports the finding that dextran can be used successfully as a blocking agent and furthermore, by using dextran as the blocking agent, pronase can be employed to determine qualitatively if the antigenic epitopes recognized by specific monoclonal antibodies are proteins in nature.
Materials and Methods
Membrane solubilization Sporozoites of Plasmodium vivax [2] were suspended in an equal volume of saline, and the suspension was then subjected to freeze-thawing in an a c e t o n e / d r y ice bath for about 10 cycles. The resulting mixture was adjusted to pH 7.4 with 0.5% N a H C O 3 and centrifuged at 10000 x g for 20 min. The supernatant solution was removed and the sporozoite pellet was washed repeatedly with 0.1 M Tris-HCl buffer, pH 7.4, until the final washing had an absorbance of less than 0.05 at 280 nm. The sporozoite membrane was solubilized by suspending the sporozoite pellet in 1.5 volumes of 0.1 M Tris-HCl buffer, p H 7.4, containing 0.5% Nonidet-40 (NP-40; Particle Data Laboratories, Ltd., Emhurst, IL). The suspension was incubated at room temperature for 30 min with occasional vortexing and was then washed twice with an additional 2 volumes of buffer. The combined supernatant fluid was exhaustively dialyzed against several changes of fresh 0.01 M Tris-HCl buffer, p H 7.4, for two days. The dialyzed solution was lyophilized and reconstituted to 1 / 1 0 of the original volume with distilled water. The resulting supernatant solution, after centrifugation at 10000 x g for 30 min, was clear and appeared light yellow; it was referred to as sporozoite membrane extract. Gel electrophoresis SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli [3] using an 8-12% linear gradient acrylamide slab gel (15 x 15 x 0.15 cm). The stacking gel contained 3% acrylamide/0.1% bisacrylamide. Samples were prepared in glycerol, Tris-HCl, p H 6.8, SDS, and bromophenol blue, and heated at 1 0 0 ° C for 3 min. Radioactive labeled molecular weight standards (Bethesda Research Laboratory, Bethesda, Maryland) containing 0.03/~Ci of 14C in 20 ffl were used in each sample well and run together with experimental samples'.' Electrophoresis was carried out at 25 mA until the tracking dye reached the bottom of the gel. Electrotransfer The blotting procedure was essentially the same as described by Towbin et al. [4].
137 Following electrophoresis, the gels were immersed in buffer containing 0.025 M Tris, 0.192 M glycine, p H 8.3, and 20% methanol for 10 min and then placed on a single nitrocellulose membrane previously equilibrated with the same buffer. The gel-nitrocellulose assembly was placed between pairs of Whatman No. 3 filter paper, Scotch-Brite pads and supports, and immersed in the buffer chamber. Electroblotting was performed under a constant current of 0.1 A at room temperature overnight (approximately 15 h) using an E-C electroblot apparatus (Bio-Rad). i
Detection of antigen with monoclonal antibodies The blots, washed once in phosphate-buffered saline (PBS), p H 7.2, were air dried and then immersed in 5% dextran (molecular weight, 71 200) in PBS for 30 min at room temperature to block the unoccupied sites of the nitrocellulose membrane. After blocking, the membranes were rinsed twice in PBS at 10 min intervals with gentle shaking and soaked in antibody solution (Ascitic fluid having an immunofluorescent assay [5] titer of 50). Time of incubation was 1 h at room temperature and then overnight at 4 °C. After incubation the membranes were repeatedly washed with PBS containing 0.05% Tween 20 to remove unbound antibodies until the final washing had an absorbance of less than 0.5 at 280 nm. In general, six changes of 100 ml washing buffer at 30 min intervals with agitation is sufficient to render the membrane free of unbound antibodies. After the final rinsing, the membranes were blotted with Whatman No. 3 paper to remove excess washing buffer and then treated with 12~I-goat anti-mouse Ig (New England Nuclear, Boston, MA), specific activity 9.2 /tCi/#g, containing a radioactivity of approximately 40000-50000 c p m / m l in PBS for 3 h at room temperature. The membranes were repeatedly washed with PBS until the final washing had a radioactivity of less than 100 c p m / m l . Membranes were air dried between two sheets of Whatman No. 3 paper, and exposed to K o d a k XAR-5 films at - 7 0 ° C using a Kodak X-ray cassette plus Intensifying Screen. The same experimental procedures were followed when 5% BSA was used as the blocking agent to saturate the binding sites of the nitrocellulose membrane.
Pronase digestion of antigens on nitrocellulose membrane The enzyme solution was prepared by dissolving pronase (Calbiochem, San Diego, CA) from Streptomyces griseus K-1 in 0.1 M Tris-HC1 buffer, p H 7.4, containing 0.01 M CaC12 to give 38.5 units/ml. One enzyme unit was defined as the amount of enzyme that would yield a digestion product equivalent to 25 /tg of tyrosine per min. After blocking with 5% dextran solution was complete, the membranes were rinsed three times with 0.1 M Tris-HCl buffer, p H 7.4, then immersed in the enzyme solution, and incubated for 6 h at 4 0 ° C with gentle agitation. A 100 ml enzyme solution in a tray (15 x 21 x 4 cm) would be sufficient to cover one sheet of nitrocellulose membrane (12 x 16 cm). Controls that had the same compositions of substrate and buffer, except saline substituting for the enzyme, were run simultaneously with the experimental samples to determine if there was any non-enzymatic alteration of the antigens during the incubation period. At the end of incubation, the membranes were washed three times with PBS
138 to completely remove the p r o n a s e a n d then reacted with m o n o c l o n a l a n t i b o d i e s and 125I-goat a n t i - m o u s e Ig as described in the previous section.
Results and Discussion P r e l i m i n a r y experiments to investigate blocking agents were performed by spotting 5 /~1 o v a l b u m i n solution (10 m g / m l in saline) o n t o strips of nitrocellulose m e m b r a n e . The m e m b r a n e s were blocked with dextran solutions u n d e r different e x p e r i m e n t a l conditions, i n c u b a t e d with mouse a n t i - o v a l b u m i n serum, a n d then
M~nf
x 10-:' 20O
18.4
AG
(A) Fig. 1. Comparison of the effectivenessof blocking agents to saturate the unbound sites of nitrocellulose membrane. The nitrocellulose membranes on which the sporozoite membrane antigens (AG) were electrobloned were treated with (A) 5% dextran and (B) 5% BSA. The membranes were reacted with sporozoite-specific monoclonal antibodies and then with 1251-goat anti-mouse Ig. Autoradiography was carried out as described in Materials and Methods.
139
Fig. 1. (continued).
treated with 125I-goat anti-mouse Ig. The background of the membrane was examined by autoradiography to determine the degree of effectiveness of the blocking agents in saturating the unbound sites of the membrane that contained antigens. When nitrocellulose membranes were blotted in 1%, 5%, and 10% dextran solutions in PBS, pH 7.2 for 1 h, no significant differences in background were observed except in one experiment in which a 1% dextran solution gave a slightly darkened background as compared with other strips treated with 5% and 10% dextran solutions. The blocking of unbound sites of the nitrocellulose membrane with 5% dextran solutions at pH 5, 6, 7, 8, and 9 gave a background essentially similar to that at pH 7.2. Thus, the blocking of the unbound sites on the nitrocellulose membrane appeared to be due to the trapping of dextran in the matrix of the membrane rather than the attraction of a charged surface on the nitrocellulose membrane. 30 rain of incubation with 5% dextran solution in PBS, pH 7.2, was found to be sufficient for binding the unoccupied sites on the membrane; incuba-
140 tion for longer than 30 min, e.g., 1 h or 2 h, gave no further improvement of the background on autoradiographs. Sporozoite antigens were separated in an 8-12% polyacrylamide slab gel and then electrobiotted to nitrocellulose membranes. One membrane that contained the antigen was treated with 5% BSA; another with 5% dextran in PBS. Both were treated with sporozoite-specific monoclonal antibodies and reacted with ~251-goat anti-mouse Ig under identical experimental conditions. The autoradiographs of the nitrocellulose membranes treated with either BSA or dextran shown in Fig. 1 indicate that there is no difference in the backgrounds produced by the two; however, membranes blocked with BSA solution occasionally showed a number of small radioactive spots on autoradiographs, suggesting the possibility that BSA aggregates were present in a solution that nonspecifically bound the ~251-goat anti-mouse Ig to the membrane. Pronase from Streptornyces griseus K-1, a mixture of endopeptidases and exopeptidases containing both alkaline and neutral aminopeptidases and carboxypeptidases, has been used to digest glycoproteins in the nitrocellulose membrane [6]. Using dextran as the blocking agent, antigens on the nitrocellulose membrane can be digested with pronase, reacted with specific monoclonal antibodies, and then treated with 125I-goat anti-mouse Ig to determine qualitatively if the antigens are proteins in nature. When sporozoite antigens of P. vivax were electrophoretically separated and electroblotted to nitrocellulose membranes, a strip for each sample was cut from the membrane (2 × 10 cm), and then blocked with 5% dextran in PBS, p H 7.2. One strip was treated with pronase solution, while the replica was treated with saline under the same experimental conditions. Fig. 2 shows the effect of pronase on the capacity of P. vivax antigens to bind sporozoite-specific monoclonal antibodies (H-4, H-3, H-l). All the antigenic properties of sporozoite antigens were destroyed by the treatment with pronase; under the same experimental conditions. controls treated with saline had no effect on the antigens to bind to the antibodies. The effectiveness of pronase digestion is also evident from results showing that radioactive labeled protein molecular weight markers were completely destroyed with pronase under the same experimental conditions as tested samples. The susceptibility of P. viuax sporozoite antigens towards pronase digestion suggests that the antigens which reacted with the monoclonal antibodies were proteins in nature, but whether or not they are solely proteins or they are protein conjugates such as glycoproteins remains to be determined. The method recently reported by Woodward et al. [7] using periodate oxidation for detecting carbohydrate epitopes on the nitrocellulose membrane, along with the method described here, could be applicable for determining qualitatively if the antigen is a carbohydrate or glycoprotein conjugate. The use of BSA instead of dextran as the blocking agent on the nitrocellulose membrane in the pronase treatment experiment resulted in a high background of the final X-ray film, suggesting that the pronase may digest BSA in the matrix on the nitrocellulose membrane thus diminishing the blocking capacity of BSA. Our studies on the sporozoite antigens showed that as little as 2000 sporozoites were sufficient for the assay described. In addition to the advantage that a very
141
Fig. 2. Effect of pronase on the antigenicity of sporozoite membrane antigens to bind to monoclonal antibodies. After having been separated on an 8-12% polyacrylamide gel, membrane antigens were electroblotted to nitrocellulose membrane. The membrane was cut for each sample and then immersed in 5% dextran. One strip was treated with pronase (P); the replica strip with saline as a control. After incubation, the strips were reacted with monoclonal antibodies as indicated and treated with 125I-goat anti-mouse Ig. Autoradiography was carried out as described in Materials and Methods.
m i n u t e a m o u n t of a n t i g e n is required for each assay, the use of d e x t r a n as a b l o c k i n g agent o n the nitrocellulose m e m b r a n e would be helpful in studying other biological substances that m a y have non-specific affinity towards BSA. I n practice, the d e x t r a n solution when kept at 4 ° C can be used repeatedly at least 5 - 1 0 times w i t h o u t a n y detectable change in its blocking capacity.
Simplified description of the method and its advantages The m e t h o d of using dextran as a blocking agent o n nitrocellulose m e m b r a n e provides a m e a n s of studying biological materials which non-specifically b i n d to b o v i n e serum a l b u m i n that is c o n v e n t i o n a l l y used to block the u n o c c u p i e d sites on nitrocellulose m e m b r a n e . I n addition, the use of dextran, a n o n - p r o t e i n a c e o u s
142 substance, allows the antigens o n the nitrocellulose m e m b r a n e to be digested with p r o n a s e a n d s u b s e q u e n t l y reacted with specific a n t i b o d y to d e t e r m i n e qualitatively if the a n t i g e n is p r o t e i n in nature. The a d v a n t a g e of this technique is the ability to analyze a very small a m o u n t of a n t i g e n by a u t o r a d i o g r a p h y or enzyme such as peroxidase-linked antibody. This m e t h o d could be extended to analyze antigens by e m p l o y i n g various enzymes of k n o w n specificity.
Acknowledgements This work was s u p p o r t e d by Naval Medical Research a n d D e v e l o p m e n t C o m m a n d W o r k U n i t No. 2M161102BS10AF427. The o p i n i o n s a n d assertions c o n t a i n e d herein are the private ones of the authors a n d are not to be c o n s t r u e d as official or as reflecting the views of the U.S. N a v y D e p a r t m e n t of the naval service at large.
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