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Luminescent visualization of antigens on blots
Journal of Immunological Methods, 92 (1986) 161-165 Elsevier
161
JIM 04023
Luminescent visualization of antigens on blots Peter L a i n g Department of Pathology, Universityof Bristol, The Medical School, University Walk, Bristol BS8 1TD, U.K. (Received 14 February 1986, accepted 7 April 1986)
Proteins separated by SDS gel electrophoresis and transferred to a nitrocellulose sheet can be visualised by 'probing' with peroxidase-linked reagents which are detected by luminescence. A modified luminescent substrate is described containing 4-methylumbelliferone which enhances light emission four-fold. Using the modified substrate, luminescent detection was found to be more sensitive than chromogenic detection of peroxidase using 4-chloro-l-napthol. The new technique was used in conjunction with the immunoblot method to demonstrate antigenic differences between rat and mouse erythrocytes. Key words: Luminescence;Immunoblot; Membrane protein
Introduction
Materials and methods
The use of enzymes instead of radioisotopes as labels has a number of practical advantages: exposure to radiation can be avoided, enzymelabelled reagents have a long 'shelf life' and results can be obtained quickly. These virtues have led to the widespread adoption of enzyme labels in immunoassay and blotting techniques. However, 125I-labelled reagents generally offer the highest attainable sensitivity for the detection of antigens on nitrocellulose blots (reviewed by Towbin and Gordon, 1983). Recently, Whitehead et al. (1983) developed a luciferin-enhanced luminescent procedure for immunoassay which allows highly sensitive determination of peroxidase activity. This prompted us to investigate whether this tube based method could be adapted to the immunoblotting technique of Towbin et al. (1979) thereby obtaining images of electrophoretically separated antigens on X-ray film exposed to nitrocellulose blots.
Reagents Except where indicated, reagents were obtained from Sigma (Poole, U.K.).
Abbreviations: SDS, sodium dodecyl sulphate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MUB, 4-methylumbelliferone;PAS, periodic acid Schiff.
Immunization procedure C B A / H mice were immunised with Leeds strain (Wistar-derived) rat erythrocytes as described by Naysmith et al. (1980). Briefly, 2 x 10 8 erythrocytes in 0.9% w / v NaC1 were injected intraperitoneally, without adjuvant, four times at weekly intervals. Pooled sera taken 1 month after the first immunisation were used. A control serum pool was prepared from unimmunised C B A / H mice. Preparation of erythrocyte membranes Erythrocyte ghosts were prepared according to Dodge et al. (1963). S D S polyacrylamide gel electrophoresis Electrophoresis was performed in 10% w / v polyacrylamide gels according to Laemmli (1970) except that sample buffer contained 8 M urea (Koch-Light, Puriss.), 5% w / v sodium dodecyl
0022-1759/86/$03.50 © 1986 Elsevier SciencePublishers B.V. (Biomedical Division)
162 sulphate (SDS, BDH) and 5% v / v 2-mercaptoethanol. Samples were not heated. Some erythrocyte proteins do not dissolve in the absence of 8 M urea (Moore et al. 1982). Gels were silverstained according to Burk et al. (1983) or were stained for sialoglycoproteins using the N a O H periodic acid-Schiff method of Sarris and Palade (1979). Molecular weights were estimated using Sigma high molecular weight protein standard mixture SDS-6H for calibration. This was used at 1 / 1 0 of the recommended concentration. Acrylamide and N, N'-methylenebisacrylamide were Electran grade from BDH.
Electrophoretic transfer of proteins to nitrocellulose Electrophoretic blotting was performed by a method based on those described by Towbin et al. (1979) and by Burnette (1981). Transfer was performed in an apparatus similar to that of Bittner et al. (1980) in 192 mM glycine 25 mM Tris containing 20% v / v methanol. Two Shandon electrophoretic destain power-packs were connected in series to give a current of about 600 mA for 2.5 h with the transfer cell standing in an iced water bath. The contents of the cell were stirred magnetically to assist cooling. Blots were stained for protein using amido-black according to Schaffner and Weissmann (1973).
Development of blots with antisera and peroxidaselinked reagents Blots of SDS gels were immersed in phosphate-buffered saline containing 5% w / v bovine serum albumin (PBS-BSA) for 30 min or overnight (to saturate non-specific binding capacity). Strips corresponding to sample lanes were cut and incubated in antiserum at a dilution of 1/100 in PBS-BSA for 1 h followed by sheep anti-mouse IgG-peroxidase conjugate at 1 /~g m1-1 in PBSBSA for 1 h. After each incubation, blots were washed (6 x 5 rain) in PBS containing 0.05% v / v Tween 20. Incubations and washes were performed at room temperature on a rocking platform. To compare the sensitivity of chromogenic and luminescent detection methods, biotin-labelled flgalactosidase was used. Dilutions of biotin-labelled fl-galactosidase in carrier-free PBS were spotted onto nitrocellulose at 1 cm intervals and were
allowed to air-dry. The sheet was blocked in PBSBSA, then incubated in avidin-peroxidase at 5 ~tg m1-1 in PBS-BSA for 1 h at 45°C. The sheet was washed (6 x 5 min) in PBS-Tween at room temperature and strips were cut and developed in chromogenic or luminescent substrate solutions.
Substrate solutions for peroxidase The purple/blue chromogenic substrate 4chloro-l-napthol was used as described by Hawkes et al. (1982). For luminescent detection, the luciferin-enhanced substrate described by Whitehead et al. (1983) was used. This was prepared by dissolving luminol (4.4 mg) in 1.5 ml of 1 M Tris to which was added 0.5 ml of 1 M Tris-HCl and 18 ml of H 2 0 giving a solution of pH 8.5. (Luminol does not dissolve readily at pH 8.5). 25 /~1 of 10 mg m1-1 synthetic firefly luciferin (sodium salt, stored in aliquots at - 7 0 ° C ) was then added. Where stated, this was followed by 10 mg of 4-methylumbelliferone (MUB, sodium salt). H202 (6 /tl of 30%) was added just before use. Luminescent substrates were prepared in the light.
Exposure of luminescent samples to X-ray film Blots probed with peroxidase-linked reagents were rocked in luminescent substrate solution for 30 s or more (up to 5 min), excess substrate was allowed to drain off, and the wet blot was sandwiched between polyethylene sheets and exposed to Fuji RX X-ray film. In early experiments the sandwich was left unsealed so that the blot could be easily retrieved for re-exposure in modified substrate solutions. In the method finally adopted, samples were heat-sealed with a bag-sealer and MUB-containing substrate was used. Exposure was performed in an X-ray cassette (Genetic Research Instrumentation, Bishops Stortford, U.K.) with the sample taped to the back of the cassette. Exposure times were varied (5 s-1 h) depending on the brightness of the sample. All exposures were at room temperature.
Determination of emission spectra of peroxidasecatalysed luminescence Emission spectra were determined in an Aminco-Bowman spectrofluorometer with a slit width of 1 mm and with the excitation lamp turned off. A 1 cm path-length quartz cuvette was
163
used with a sample volume of 1 ml. Reaction was initiated by the addition of 20 /tl of 1 mg m1-1 sheep anti-mouse IgG-peroxidase conjugate.
Results
Comparison of chromogenic and luminescent procedures for the detection of peroxidase activity on nitrocellulose In principle, the simplest way to compare chromogenic and luminescent procedures would be to spot peroxidase directly onto nitrocellulose at various concentrations and test strips in each of the substrates. However, it was found that peroxidase is denatured by contact with nitrocellulose. For this reason, avidin-peroxidase was attached to nitrocellulose via biotin-labelled flgalactosidase. Fig. 1 shows that nitrocellulosebound peroxidase activity was readily detected by exposure to blue-sensitive X-ray film. In terms of the minimum amount of biotin-labelled fl-galac-
tosidase detectable, the luminescent substrate of Whitehead et al. (1983) was found to be marginally inferior to chromogenic detection with 4chloro-l-napthol. However, the dose-response characteristics of the luminescent substrate were much 'steeper' and maximum contrast was attained with much lower amounts of biotin-labelled fl-galactosidase. Further, whereas the reaction had completely ceased after 30 min or less in the case of the chromogenic substrate, the luminescent reaction continued for several hours (not shown) resulting in increased sensitivity which was eventually limited by non-specific binding of the peroxidase conjugate. The modified luminescent substrate containing MUB was more sensitive than the original luminescent substrate or the chromogenie substrate. Fig. 2 shows that the spectral output of each of the luminescent substrates was in the blue region and therefore corresponded well with the sensitivity spectrum of the X-ray film.
Enhancement of peroxidase-catalysed luminescence by 4-methylumbelliferone Enhancement by MUB was first observed in auto-graphic experiments of the type shown in Fig. 1. Fig. 2 confirms that this effect was also demonstrable in solution. Further, it was also evident that MUB affects the quality of the light. A small red-shift of about 10 nm was evident in the )~-max values of the emission spectra, giving a 433nm
445nm
200
too .C
Fig. 1. Direct comparison of the sensitivity of detection of peroxidase by chromogenic and luminescent methods. Serial 3-fold dilutions of biotin-labelled fl-galactosidase were spotted onto nitrocellulose and treated with avidin-peroxidase as described in the materials and methods section. Strips cut from the sheet were developed in the chromogenic substrate 4chloro-l-napthol (a), luminescent substrate (b), and modified luminescent substrate (c) containing 4-methylumbelliferone at 0.5 mg m l - i . Development in 4-chloro-l-napthol was allowed to proceed until colour development had ceased (30 rain). Luminescent strips were exposed for 30 min. The minimal detectable quantity in pg is indicated in each case by an arrow.
0
400
450
500
550
wavelength nm
Fig. 2. Enhancement of peroxidase-catalysed luminescence by methylumbelliferone. Emission spectra of the luminescent reaction were determined in the presence ( + MUB) and absence ( - MUB) of 0.5 mg m l - ] 4-methylumbelliferone. Light emission was undetectable in the absence of H202 or peroxidase. Methylumbelliferone caused an approximately 4-fold enhancement in light emission.
164
h-max of 445 nm characteristic of MUB fluorescence. MUB also acted as a substrate for the luminescent reaction in its own right, but was about 80-fold weaker than when combined with luminol and luciferin.
Visualisation of rat erythrocyte membrane proteins by immunoblotting Fig. 3 shows the application of luminescent detection to the immunoblot technique. Development in mouse anti-rat erythrocyte serum gave a single band of 37 kDa after 1 min of exposure. Further development showed the presence of vari-
7R
ous minor bands. Traces of the 28 kDa and 22 kDa components were present in the lane developed with normal mouse serum. The 76 kDa and 37 kDa components were probably glycophorins since they correspond to the major PAS-positive bands of rat erythrocyte membranes (unpublished observations). Rat glycophorins are known to differ from their mouse counterparts in the O-acetylation status of their terminal sialic acid residues (Sarris and Palade, 1979). These differences in carbohydrate structure could be responsible for triggering the immune response against rat erythrocytes and might also be involved in the induction of erythrocyte autoimmunity which occurs concomitantly (Naysmith et al., 1980). Conspicuously absent from the set of bands recognised by mouse anti-rat erythrocyte serum was 'band 3' (nomenclature of Fairbanks et al., 1971) the major erythrocyte membrane protein. This illustrates the high degree of specificity attained with this technique.
Discussion
Fig. 3. Luminescent immunoblot of rat erythrocyte membrane proteins. A 10% polyacrylamide SDS eleetrophoresis gel was loaded with rat erythrocyte ghosts equivalent to 10 ~1 of packed ceils in 50 #1 of sample buffer (lanes a - c ) or at 1 / 1 0 of this concentration (lane d). The gel was run and two lanes containing ghosts (d) and molecular weight standards (e) were cut from the end of the gel and silver stained. The contents of the remainder of the gel were transferred electrophoretically to nitrocellulose. Strips from the blot were developed using sheep anti-mouse lgG-peroxidase conjugate and the luminescent substrate with normal mouse serum (a) or with mouse anti-rat erythrocyte serum (b). Exposure time was 20 rain. Strip 'c' was stained with amido-black. The numbers are molecular weights × 1 0 - 3 and refer to lanes b and e.
X-ray film is more sensitive to light than it is to gamma rays. This is why intensifying screens are used in the fluorographic detection of t25I (Laskey and Mills, 1977). It seemed therefore that a technique such as luminescence, where light is emitted directly by the sample, would lend itself readily to detection with X-ray film. The results confirmed this. Indeed, sensitive detection was possible with brief exposures at room temperature. In fluorography, the light emitted by the fluor is of low intensity, and low temperature exposure ( - 7 0 ° C ) is used to increase the sensitivity of the film to light (Laskey and Mills, 1977). In 'luminography', the intensity of the light is probably much higher since intense bands are often visible to the naked eye. This high light output can be attributed to the capacity of a single molecule of peroxidase to catalyse numerous light-producing events, in contrast to a single atom of a radioisotope which decays once only. Although luminography has not been compared directly to fluorography in this study, it seems likely that its sensitivity will be as good. Surprisingly, despite the small amount of
165 luminescent substrate solution present in the sample during exposure, the luminescent reaction lasted longer than its chromogenic counterpart. The tendency of highly active samples to give a diffuse image compromised resolution but gave a subjective impression of quantity which was more realistic than with chromogenic detection. When the luminescent reaction was observed in the dark in a quartz cuvette, the light emitted appeared violet/blue. Thus it seemed possible that some of the light emitted was in the ultra-violet and perhaps escaped detection by the film in autographic exposures. This suggested that a fluorescent molecule absorbing in the ultra-violet and emitting in the blue region might enhance detection by the blue-sensitive film. With this rationale, methylumbelliferone was added. However, the light emission of the luminescent substrate was found to be weak in the ultra-violet region (Fig. 2) and insufficient to explain the observed enhancement. It would appear therefore that methylumbelliferone may enhance light output by 'rescuing' light which is otherwise lost through quenching or self-absorption. Luminography is a sensitive technique which is likely to find wide application in the field of immunology, and with minor modifications should also be applicable to molecular biology.
Acknowledgements I wish to thank Dr. B. Parkar for help with luminescence measurements, Dr. C.J. Elson for
helpful discussion and the Medical Council for financial support.
Research
References Bittner, N., P. Kupferer and C.F. Morris, 1980, Anal. Biochem. 102, 459. Burk, R.R., M. Eschenbruch, P. Leuthard and G. Steck, 1983, Methods Enzymol.91,247. Burnette, W.N., 1981, Anal. Biochem. 112, 195. Dodge, J.T., C. Mitchell and D. Hanahan, 1963, Arch. Biochem. Biophys, 100, 119. Fairbanks, G., T.L. Steck and D.F.H. Wallach, 1971, Biochemistry 10, 2606. Hawkes, R., E. Niday and J. Gordon, 1982, Anal. Biochem. 119, 142. Laemmli, U.K., 1970, Nature 227, 680. Laskey, R.A. and A.D. Mills, 1977, FEBS Lett. 82, 314. Moore, S., C.F. Woodrow and D.B.C. McLelland, 1982, Nature 295, 529. Naysmith, J.D., C.J. Elson, M. Dallman, E. Fletcher and M.G. Ortega-Pierres, 1980, Immunology39, 469. Sarris, A.H. and G.E. Palade, 1979, J. Biol. Chem. 254, 6724. Schaffner, W. and C. Weissman, 1973, Anal. Biochem. 56, 502. Towbin, H. and J. Gordon, 1984, J. lmmunol. Methods 72, 313. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Whitehead, T.P., G.H.G. Thorpe, T.J.N. Carter, C. Groucutt and L.J. Kricka, 1983, Nature 305, 158.
161
JIM 04023
Luminescent visualization of antigens on blots Peter L a i n g Department of Pathology, Universityof Bristol, The Medical School, University Walk, Bristol BS8 1TD, U.K. (Received 14 February 1986, accepted 7 April 1986)
Proteins separated by SDS gel electrophoresis and transferred to a nitrocellulose sheet can be visualised by 'probing' with peroxidase-linked reagents which are detected by luminescence. A modified luminescent substrate is described containing 4-methylumbelliferone which enhances light emission four-fold. Using the modified substrate, luminescent detection was found to be more sensitive than chromogenic detection of peroxidase using 4-chloro-l-napthol. The new technique was used in conjunction with the immunoblot method to demonstrate antigenic differences between rat and mouse erythrocytes. Key words: Luminescence;Immunoblot; Membrane protein
Introduction
Materials and methods
The use of enzymes instead of radioisotopes as labels has a number of practical advantages: exposure to radiation can be avoided, enzymelabelled reagents have a long 'shelf life' and results can be obtained quickly. These virtues have led to the widespread adoption of enzyme labels in immunoassay and blotting techniques. However, 125I-labelled reagents generally offer the highest attainable sensitivity for the detection of antigens on nitrocellulose blots (reviewed by Towbin and Gordon, 1983). Recently, Whitehead et al. (1983) developed a luciferin-enhanced luminescent procedure for immunoassay which allows highly sensitive determination of peroxidase activity. This prompted us to investigate whether this tube based method could be adapted to the immunoblotting technique of Towbin et al. (1979) thereby obtaining images of electrophoretically separated antigens on X-ray film exposed to nitrocellulose blots.
Reagents Except where indicated, reagents were obtained from Sigma (Poole, U.K.).
Abbreviations: SDS, sodium dodecyl sulphate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MUB, 4-methylumbelliferone;PAS, periodic acid Schiff.
Immunization procedure C B A / H mice were immunised with Leeds strain (Wistar-derived) rat erythrocytes as described by Naysmith et al. (1980). Briefly, 2 x 10 8 erythrocytes in 0.9% w / v NaC1 were injected intraperitoneally, without adjuvant, four times at weekly intervals. Pooled sera taken 1 month after the first immunisation were used. A control serum pool was prepared from unimmunised C B A / H mice. Preparation of erythrocyte membranes Erythrocyte ghosts were prepared according to Dodge et al. (1963). S D S polyacrylamide gel electrophoresis Electrophoresis was performed in 10% w / v polyacrylamide gels according to Laemmli (1970) except that sample buffer contained 8 M urea (Koch-Light, Puriss.), 5% w / v sodium dodecyl
0022-1759/86/$03.50 © 1986 Elsevier SciencePublishers B.V. (Biomedical Division)
162 sulphate (SDS, BDH) and 5% v / v 2-mercaptoethanol. Samples were not heated. Some erythrocyte proteins do not dissolve in the absence of 8 M urea (Moore et al. 1982). Gels were silverstained according to Burk et al. (1983) or were stained for sialoglycoproteins using the N a O H periodic acid-Schiff method of Sarris and Palade (1979). Molecular weights were estimated using Sigma high molecular weight protein standard mixture SDS-6H for calibration. This was used at 1 / 1 0 of the recommended concentration. Acrylamide and N, N'-methylenebisacrylamide were Electran grade from BDH.
Electrophoretic transfer of proteins to nitrocellulose Electrophoretic blotting was performed by a method based on those described by Towbin et al. (1979) and by Burnette (1981). Transfer was performed in an apparatus similar to that of Bittner et al. (1980) in 192 mM glycine 25 mM Tris containing 20% v / v methanol. Two Shandon electrophoretic destain power-packs were connected in series to give a current of about 600 mA for 2.5 h with the transfer cell standing in an iced water bath. The contents of the cell were stirred magnetically to assist cooling. Blots were stained for protein using amido-black according to Schaffner and Weissmann (1973).
Development of blots with antisera and peroxidaselinked reagents Blots of SDS gels were immersed in phosphate-buffered saline containing 5% w / v bovine serum albumin (PBS-BSA) for 30 min or overnight (to saturate non-specific binding capacity). Strips corresponding to sample lanes were cut and incubated in antiserum at a dilution of 1/100 in PBS-BSA for 1 h followed by sheep anti-mouse IgG-peroxidase conjugate at 1 /~g m1-1 in PBSBSA for 1 h. After each incubation, blots were washed (6 x 5 rain) in PBS containing 0.05% v / v Tween 20. Incubations and washes were performed at room temperature on a rocking platform. To compare the sensitivity of chromogenic and luminescent detection methods, biotin-labelled flgalactosidase was used. Dilutions of biotin-labelled fl-galactosidase in carrier-free PBS were spotted onto nitrocellulose at 1 cm intervals and were
allowed to air-dry. The sheet was blocked in PBSBSA, then incubated in avidin-peroxidase at 5 ~tg m1-1 in PBS-BSA for 1 h at 45°C. The sheet was washed (6 x 5 min) in PBS-Tween at room temperature and strips were cut and developed in chromogenic or luminescent substrate solutions.
Substrate solutions for peroxidase The purple/blue chromogenic substrate 4chloro-l-napthol was used as described by Hawkes et al. (1982). For luminescent detection, the luciferin-enhanced substrate described by Whitehead et al. (1983) was used. This was prepared by dissolving luminol (4.4 mg) in 1.5 ml of 1 M Tris to which was added 0.5 ml of 1 M Tris-HCl and 18 ml of H 2 0 giving a solution of pH 8.5. (Luminol does not dissolve readily at pH 8.5). 25 /~1 of 10 mg m1-1 synthetic firefly luciferin (sodium salt, stored in aliquots at - 7 0 ° C ) was then added. Where stated, this was followed by 10 mg of 4-methylumbelliferone (MUB, sodium salt). H202 (6 /tl of 30%) was added just before use. Luminescent substrates were prepared in the light.
Exposure of luminescent samples to X-ray film Blots probed with peroxidase-linked reagents were rocked in luminescent substrate solution for 30 s or more (up to 5 min), excess substrate was allowed to drain off, and the wet blot was sandwiched between polyethylene sheets and exposed to Fuji RX X-ray film. In early experiments the sandwich was left unsealed so that the blot could be easily retrieved for re-exposure in modified substrate solutions. In the method finally adopted, samples were heat-sealed with a bag-sealer and MUB-containing substrate was used. Exposure was performed in an X-ray cassette (Genetic Research Instrumentation, Bishops Stortford, U.K.) with the sample taped to the back of the cassette. Exposure times were varied (5 s-1 h) depending on the brightness of the sample. All exposures were at room temperature.
Determination of emission spectra of peroxidasecatalysed luminescence Emission spectra were determined in an Aminco-Bowman spectrofluorometer with a slit width of 1 mm and with the excitation lamp turned off. A 1 cm path-length quartz cuvette was
163
used with a sample volume of 1 ml. Reaction was initiated by the addition of 20 /tl of 1 mg m1-1 sheep anti-mouse IgG-peroxidase conjugate.
Results
Comparison of chromogenic and luminescent procedures for the detection of peroxidase activity on nitrocellulose In principle, the simplest way to compare chromogenic and luminescent procedures would be to spot peroxidase directly onto nitrocellulose at various concentrations and test strips in each of the substrates. However, it was found that peroxidase is denatured by contact with nitrocellulose. For this reason, avidin-peroxidase was attached to nitrocellulose via biotin-labelled flgalactosidase. Fig. 1 shows that nitrocellulosebound peroxidase activity was readily detected by exposure to blue-sensitive X-ray film. In terms of the minimum amount of biotin-labelled fl-galac-
tosidase detectable, the luminescent substrate of Whitehead et al. (1983) was found to be marginally inferior to chromogenic detection with 4chloro-l-napthol. However, the dose-response characteristics of the luminescent substrate were much 'steeper' and maximum contrast was attained with much lower amounts of biotin-labelled fl-galactosidase. Further, whereas the reaction had completely ceased after 30 min or less in the case of the chromogenic substrate, the luminescent reaction continued for several hours (not shown) resulting in increased sensitivity which was eventually limited by non-specific binding of the peroxidase conjugate. The modified luminescent substrate containing MUB was more sensitive than the original luminescent substrate or the chromogenie substrate. Fig. 2 shows that the spectral output of each of the luminescent substrates was in the blue region and therefore corresponded well with the sensitivity spectrum of the X-ray film.
Enhancement of peroxidase-catalysed luminescence by 4-methylumbelliferone Enhancement by MUB was first observed in auto-graphic experiments of the type shown in Fig. 1. Fig. 2 confirms that this effect was also demonstrable in solution. Further, it was also evident that MUB affects the quality of the light. A small red-shift of about 10 nm was evident in the )~-max values of the emission spectra, giving a 433nm
445nm
200
too .C
Fig. 1. Direct comparison of the sensitivity of detection of peroxidase by chromogenic and luminescent methods. Serial 3-fold dilutions of biotin-labelled fl-galactosidase were spotted onto nitrocellulose and treated with avidin-peroxidase as described in the materials and methods section. Strips cut from the sheet were developed in the chromogenic substrate 4chloro-l-napthol (a), luminescent substrate (b), and modified luminescent substrate (c) containing 4-methylumbelliferone at 0.5 mg m l - i . Development in 4-chloro-l-napthol was allowed to proceed until colour development had ceased (30 rain). Luminescent strips were exposed for 30 min. The minimal detectable quantity in pg is indicated in each case by an arrow.
0
400
450
500
550
wavelength nm
Fig. 2. Enhancement of peroxidase-catalysed luminescence by methylumbelliferone. Emission spectra of the luminescent reaction were determined in the presence ( + MUB) and absence ( - MUB) of 0.5 mg m l - ] 4-methylumbelliferone. Light emission was undetectable in the absence of H202 or peroxidase. Methylumbelliferone caused an approximately 4-fold enhancement in light emission.
164
h-max of 445 nm characteristic of MUB fluorescence. MUB also acted as a substrate for the luminescent reaction in its own right, but was about 80-fold weaker than when combined with luminol and luciferin.
Visualisation of rat erythrocyte membrane proteins by immunoblotting Fig. 3 shows the application of luminescent detection to the immunoblot technique. Development in mouse anti-rat erythrocyte serum gave a single band of 37 kDa after 1 min of exposure. Further development showed the presence of vari-
7R
ous minor bands. Traces of the 28 kDa and 22 kDa components were present in the lane developed with normal mouse serum. The 76 kDa and 37 kDa components were probably glycophorins since they correspond to the major PAS-positive bands of rat erythrocyte membranes (unpublished observations). Rat glycophorins are known to differ from their mouse counterparts in the O-acetylation status of their terminal sialic acid residues (Sarris and Palade, 1979). These differences in carbohydrate structure could be responsible for triggering the immune response against rat erythrocytes and might also be involved in the induction of erythrocyte autoimmunity which occurs concomitantly (Naysmith et al., 1980). Conspicuously absent from the set of bands recognised by mouse anti-rat erythrocyte serum was 'band 3' (nomenclature of Fairbanks et al., 1971) the major erythrocyte membrane protein. This illustrates the high degree of specificity attained with this technique.
Discussion
Fig. 3. Luminescent immunoblot of rat erythrocyte membrane proteins. A 10% polyacrylamide SDS eleetrophoresis gel was loaded with rat erythrocyte ghosts equivalent to 10 ~1 of packed ceils in 50 #1 of sample buffer (lanes a - c ) or at 1 / 1 0 of this concentration (lane d). The gel was run and two lanes containing ghosts (d) and molecular weight standards (e) were cut from the end of the gel and silver stained. The contents of the remainder of the gel were transferred electrophoretically to nitrocellulose. Strips from the blot were developed using sheep anti-mouse lgG-peroxidase conjugate and the luminescent substrate with normal mouse serum (a) or with mouse anti-rat erythrocyte serum (b). Exposure time was 20 rain. Strip 'c' was stained with amido-black. The numbers are molecular weights × 1 0 - 3 and refer to lanes b and e.
X-ray film is more sensitive to light than it is to gamma rays. This is why intensifying screens are used in the fluorographic detection of t25I (Laskey and Mills, 1977). It seemed therefore that a technique such as luminescence, where light is emitted directly by the sample, would lend itself readily to detection with X-ray film. The results confirmed this. Indeed, sensitive detection was possible with brief exposures at room temperature. In fluorography, the light emitted by the fluor is of low intensity, and low temperature exposure ( - 7 0 ° C ) is used to increase the sensitivity of the film to light (Laskey and Mills, 1977). In 'luminography', the intensity of the light is probably much higher since intense bands are often visible to the naked eye. This high light output can be attributed to the capacity of a single molecule of peroxidase to catalyse numerous light-producing events, in contrast to a single atom of a radioisotope which decays once only. Although luminography has not been compared directly to fluorography in this study, it seems likely that its sensitivity will be as good. Surprisingly, despite the small amount of
165 luminescent substrate solution present in the sample during exposure, the luminescent reaction lasted longer than its chromogenic counterpart. The tendency of highly active samples to give a diffuse image compromised resolution but gave a subjective impression of quantity which was more realistic than with chromogenic detection. When the luminescent reaction was observed in the dark in a quartz cuvette, the light emitted appeared violet/blue. Thus it seemed possible that some of the light emitted was in the ultra-violet and perhaps escaped detection by the film in autographic exposures. This suggested that a fluorescent molecule absorbing in the ultra-violet and emitting in the blue region might enhance detection by the blue-sensitive film. With this rationale, methylumbelliferone was added. However, the light emission of the luminescent substrate was found to be weak in the ultra-violet region (Fig. 2) and insufficient to explain the observed enhancement. It would appear therefore that methylumbelliferone may enhance light output by 'rescuing' light which is otherwise lost through quenching or self-absorption. Luminography is a sensitive technique which is likely to find wide application in the field of immunology, and with minor modifications should also be applicable to molecular biology.
Acknowledgements I wish to thank Dr. B. Parkar for help with luminescence measurements, Dr. C.J. Elson for
helpful discussion and the Medical Council for financial support.
Research
References Bittner, N., P. Kupferer and C.F. Morris, 1980, Anal. Biochem. 102, 459. Burk, R.R., M. Eschenbruch, P. Leuthard and G. Steck, 1983, Methods Enzymol.91,247. Burnette, W.N., 1981, Anal. Biochem. 112, 195. Dodge, J.T., C. Mitchell and D. Hanahan, 1963, Arch. Biochem. Biophys, 100, 119. Fairbanks, G., T.L. Steck and D.F.H. Wallach, 1971, Biochemistry 10, 2606. Hawkes, R., E. Niday and J. Gordon, 1982, Anal. Biochem. 119, 142. Laemmli, U.K., 1970, Nature 227, 680. Laskey, R.A. and A.D. Mills, 1977, FEBS Lett. 82, 314. Moore, S., C.F. Woodrow and D.B.C. McLelland, 1982, Nature 295, 529. Naysmith, J.D., C.J. Elson, M. Dallman, E. Fletcher and M.G. Ortega-Pierres, 1980, Immunology39, 469. Sarris, A.H. and G.E. Palade, 1979, J. Biol. Chem. 254, 6724. Schaffner, W. and C. Weissman, 1973, Anal. Biochem. 56, 502. Towbin, H. and J. Gordon, 1984, J. lmmunol. Methods 72, 313. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Whitehead, T.P., G.H.G. Thorpe, T.J.N. Carter, C. Groucutt and L.J. Kricka, 1983, Nature 305, 158.