- Email: [email protected]
Submicrogram quantitation of an acidic polysaccharide by rocket immunoelectrophoresis and rocket affinoelectrophoresis
ANALYTICAL
BIOCHEMISTRY
73, 20-26 (1976)
Submicrogram Quantitation of an Acidic Polysaccharide by Rocket lmmunoelectrophoresis and Rocket Affinoelectrophoresis PETEROWENAND Department
of Microbiology, New
MILTON
R.J.
New York University York, New York 10016
SALTON School
of Medicine
Received October 22, 1975; accepted December 4. 1975 Rocket immunoelectrophoresis has been used to quantitate an acidic polysaccharide (a succinylated lipomannan) isolated from the membranes ofthe bacterium, Micrococcus lysodeikticcrs. This procedure is superior in sensitivity to standard calorimetric assays for carbohydrate and allows the detection of as little as 10 ng of mannan. The observed relationships between rocket height and both antibody and antigen concentrations follow those described by Weeke for protein antigens [Weeke, B. (1973) Stand. J. Immunol. 2, Suppl. 1, 37-461. Furthermore, the lectin concanavalin A could be used as an alternative to immune serum in the quantitation of this polysaccharide by rocket electrophoresis. However, this modification (rocket affinoelectrophoresis) appeared to be less sensitive than rocket immunoelectrophoresis, allowing the detection of about 2.5ng of mannan.
The concentration of an individual polysaccharide is usually estimated indirectly by the selection of a calorimetric reaction appropriate for its sugar composition (l-5) or by gas chromatographic analysis of hydrolyzed preparations. These methods are based upon the measurement of the constituent monosaccharides such as hexose (2), pentose (3), amino sugars (4), and uranic acids (5) rather than on the native polysaccharide itself. Moreover, calorimetric assays are generally sensitive only to the microgram level, and gas chromatographic analysis is time consuming. A similar criticism also applies to the estimation of reducing equivalents (6). Furthermore, these indirect methods of polysaccharide quantitation can often give misleading results due to the presence of other contaminating species in the preparations. Direct and sensitive biological assays have been developed for lipopolysaccharides such as endotoxins (7), but these methods have not been generally applicable to other polysaccharide preparations. Heidelberger and his colleagues have shown that antigen-antibody precipitation reactions can be used to quantitate both intact polysaccharides and proteins (8,9). More recent developments in quantitative immunoassay have been the techniques of one dimensional and two dimensional (crossed) immunoelectrophoresis (IO- 12). These methods have been widely used for a number of years for the rapid quantitation of 20 Copyright All nghtr
0 1976 by Academx Pre\\. Inc. of reproduction in any form reelved.
POLYSACCHARIDE
QUANTlTATlON
21
proteins, glycoproteins and lipoproteins (for review see (13)). With the routine staining procedures available (e.g., Coomassie Brilliant blue) onedimensional immunoelectrophoresis (rocket immunoelectrophoresis) can detect several nanograms of sample (14). However, the possibility of using this method for the detection and quantitation of small amounts of polysaccharide has, to the authors’ knowledge, not been investigated. Accordingly, we wish to report that the technique of rocket immunoelectrophoresis can be adapted to detect as little as 10 ng of a bacterial succinylated lipomannan isolated from the membranes of the grampositive organism, Micrococcus lysodeikticus. Furthermore, we have investigated the potentials of using the lectin concanavalin A, as a substitute for immune sera in the quantitation of this polysaccharide. MATERIALS
Antigen
and Antibody
AND METHODS
Preparations
Succinylated lipomannan was isolated from washed membranes of Micrococcus lysodeikticus (NCTC 2665) (1.5) and purified as previously described (16). Antiserum to washed membrane fractions from this organism was raised in the rabbit (17) and fractionated immunoglobulins (18) were concentrated tenfold with respect to the original serum volume (final concentration 150 mg protein/ml). Human transferrin was purchased from Behring Diagnostics and carbamylated as described by Weeke (19). Rabbit antihuman transferrin was purchased from Dakopatts AIS, Denmark. Electrophoretic
Techniques
General. One percent (w/v) agarose gels containing barbital/HCl buffer (1 = 0.02, pH 8.6) and 1% (v/v) Triton X-100 were used throughout these studies and cast on glass plates to give a volume to surface area ratio of 0.132 ml/cm2. Electrophoresis was performed in a Behring Diagnostic water-cooled immunoelectrophoresis cell. Following completion of electrophoresis, gels were either pressed, washed and dried and stained with Coomassie Brilliant blue (20) or immersed in 75% (v/v) ethanol for precipitation of dextran used as an indicator of electroendoosmosis. Peak heights of the rockets were determined on millimeter ruled graph paper by measuring the distance from the top of the application well to the tip of the precipitin rocket. Rocket immunoelectrophoresis. Gels containing antibody were cast on glass plates (102 x 82 x 0.8 mm) and antigen (total volume 2.0 ~1) was applied to wells 1.5 mm in diameter with the current on. Electrophoresis was continued for 18 hr at 4.5 V/cm with water coolant at ambient temperature. Rocket af$noelectrophoresis. This procedure was performed essentially
22
OWEN AND SALTON
t3
FIG. 1. Crossed immunoelectrophoresis of the succinylated lipomannan isolated from M. (A) Mannan (600 ng) together with the acidic marker protein carbamylated transfertin (350 ng) and (B) carbamylated transferrin (350 ng) alone were subjected to crossed immunoelectrophoresis as described in Materials and Methods. Antibody gels contained 7.5 ~1 of antimembrane serum and 7.5 ~1 of antitransferrin serum per ml. The position to which neutral dextran (M, = 20,000) migrates in the first dimension of electrophoresis is indicated thus (0). The application well is identified thus, (0). lysodeikticus.
as described for rocket immunoelectrophoresis, but with concanavalin A incorporated into the gel in place of antimembrane serum as an “affinity” precipitin. Crossed immunoelectrophoresis . Crossed immunoelectrophoresis was based on a modification (12) of Laurell’s technique (IO). Agarose gels were cast on glass plates (50 x 50 x 0.6 mm) and samples applied to wells (1.5 mm i.d.) centrally located in the gel. Electrophoresis was continued in the first direction for 55 min at 5 V/cm. The central agarose strip (10 x 50 mm) containing antigen(s) was retained on the plate after removal of the rest of the gel which was replaced by two adjacent gels (20 x 50 mm) containing antibody. Electrophoresis in the second dimension was performed at 2 V/cm for 12-18 hr. Materials Concanavalin A and agarose were purchased from Miles Labs. and Triton X-100 was obtained from Baker Chemical Co. Dextran (M, = 20,000), which was used as a marker for electroendoosmosis, was obtained from Sigma Chemical Co. RESULTS AND DISCUSSION
Quantitation of an antigen by rocket immunoelectrophoresis is simplified if a single precipitating species is present. This can be achieved either by
POLYSACCHARIDE
23
QUANTITATION
use of a highly purified antigen or, alternatively, a monovalent antiserum (21,22). Fig. 1 (A and B) shows the crossed immunoelectrophoresis pattern obtained for purified M. lysodeikticus mannan tested against antimembrane serum. The results suggest the presence of a single anionic antigen possessing charge heterogeneity, probably due to varying degrees of succinylation (23,24). The broadness of the mannan precipitin band is unlikely to reflect a heterogeneity in size since it has previously been shown that this polymer has a relatively low molecular weight (about 10,000 (16,23)), and resolution of molecular sizes would probably not occur by molecular sieving in the agarose gel. Rocket immunoelectrophoresis of mannan preparations into antimembrane serum resulted in precipitin rockets of reproducible height (Fig. 2A). For various antibody concentrations, the height of the rocket exhibited an almost linear relationship to the quantity of antigen added (Fig. 3A) provided the operating voltage was held at 4.5 V/cm. At lower
+ t
A
R lP j
2
2
3
3
4
4 5
5
~+ I<*
it22334455 FIG. 2. (A) Rocket immunoelectrophoresis and (B) rocket affinoelectrophoresis of M. ~ysodeikticus mannan. 300, 600, 900,450, and 150 ng of mannan were added to wells numbered 1-5, respectively, in both A and B. Gel A contained antimembrane serum (12.5 ~llml) and gel B, concanavalin A (75 &ml).
24
OWEN AND SALTON
25. 20-
‘5. ,. *
//
..’ .:’ ...’ / o..’
// o y ,. ...’ 0‘/ ,Y @.: 0
/
.:’
5*
0
200
400
600
800
1000
4 1200 0 Mannan
200 (ng)
400
600
800
4 1000 1200
FIG. 3. Standard curves for M. lysodeikticus mannan by (A) rocket immunoelectrophoresis and (B) rocket affinoelectrophoresis. Gels in A contained anti-membrane serum at the following concentrations n . . . .m. 21.4 ~liml; n n , 10.7 PI/ml; l . . . .O, 7.1 pi/ml; 0 0, 3.75 @ml. Gels in B contained concanavalin A at the following concentrations 0 0, 100 p&ml; O- 1. .O. 75 &ml; q 0, 50 p&/ml.
operating voltages the rockets had a much more rounded shape and linear relationship between peak height and antigen concentration was found to apply over a reduced concentration range. This is attributed either to the relatively high diffusion coefficient of this polymer (16) or to the formation of antigen-antibody complexes of low precipitability (21). As has been observed by other workers, linearity is also lost as the peak height approaches zero (22). In agreement with results obtained for protein antigens (22), alteration of both polysaccharide and antibody concentrations by a similar factor resulted in precipitin rockets of unchanged peak height (Fig. 3A). However, rockets were not detected by Coomassie Brilliant blue staining for gels containing less then 3.75 ~1 antibody/ml. Thus at the sensitivity limit of the method, a precipitin rocket of height 2 mm represents about 10 ng of mannan. Slightly lower detection limits (about 3 ng) have been reported for some protein antigens (14). However, it should be remembered that, in the present instance, the antibody protein in the precipitin rocket is the sole contributor to staining with Coomassie Brilliant blue. We have shown previously that M. lysodeikticus mannan can coprecipitate up to 15 times its own weight of concanavalin A, and that it gives discrete precipitin bands in agargel diffusion experiments against this lectin (16,24). Bog-Hansen et al. have indicated that glycoproteins can give precipitin rockets upon electrophoresis into concanavalin A, a technique they termed rocket affinoelectrophoresis (25,26). However, the procedure has not been put on a quantitative basis. It was thus of interest to determine whether concanavalin A could be used as an alternative to
POLYSACCHARIDE
QUANTITATION
25
immune sera in the quantitation of the mannan. Rocket affinoelectrophoresis of mannan into concanavalinA resulted in distinct precipitin rockets of reproducible height (Fig. 2B) which showed similar relationships between peak height and both polysaccharide and lectin concentrations as described for immune sera (Fig. 3B). The precipitin rockets formed in the presence of lectin were more diffuse than those formed in the presence of antibody (see Figs. 2A and B) thus making the estimation of peak height slightly more difficult. Furthermore, no stainable precipitin peaks were observed at a lectin concentration of 25 pg/ml or below. Thus the limit of sensitivity of this method for this particular polysaccharide is about 25 ng. We have thus shown that the sensitive technique of rocket immunoelectrophoresis can be successfully applied to the quantitation of an acidic polysaccharide. The method is simple, rapid, specific, and has the advantage of not being affected by the presence of other noninteracting molecular species. Thus, individual antigens in a complex mixture of immunogens can be quantitated if either monovalent antisera are available (21,22) or if the antigens in question can be identified in the pattern of rockets obtained following electrophoresis into polyvalent antiserum (27). The application of this procedure to the assay of other acidic polysaccharides of medical importance, such as capsular polysaccharides, teichoic acids, lipoteichoic acids, and lipopolysaccharides should be obvious. Furthermore, we have also shown that the lectin concanavalinA can substitute for immune serum in the quantitation of M. lysodeikticus mannan by rocket electrophoresis. Recent work in this laboratory performed in collaboration with Dr. G. D. Shockman, Temple University School of Medicine, Philadelphia, Pennsylvania, has shown that this technique can also be used to quantitate streptococcal lipoteichoic acid possessing kojibiose residues. Further experimentation will be necessary, however, to determine whether rocket affinoelectrophoresis can be applied to the quantitation of polysaccharides containing sugar residues in conformations allowing interaction with other lectins. ACKNOWLEDGMENT This research was supported Foundation.
by Grant BMS 7503934 from the National
Science
REFERENCES 1. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28,350-356. 2. Morris, D. L. (1948) Science 107, 254-255. 3. Dische, Z., and Schwarz, K. (1937) Mikrochim. Acra 2, 13-19. 4. Elson, L. A., and Morgan, W. T. J. (1933) Biochem. J. 27, 1824-1828. 5. Dische, Z. (1947)J. Biol. Chem. 167, 189-198. 6. McLean, C., Werner, D. A., and Aminoff, D. (1973) Anal. Biochem. 55, 72-84.
26
OWEN AND SALTON
7. Yin, E. T., Galanos, C., Kinsky, S., Bradshaw, R. A., Wessler, S., Luderitz, 0.. and Sarmiento, M. E. (1972) Eiochim. Biophys. Acta 261, 284-289. 8. Heidelberger, M., and Kendall, F. E. (1929) J. Exp. Med. 50, 809-823. 9. Heidelberger, M. (1939) Bacterial. Rev. 3, 49-95. 10. Laurel], C.-B. (1%5) Anal. Biochem. 10, 358-361. 11. Laurel], C.-B. (l%6) Anal. Biochem. 15, 54-52. 12. Clarke, H. G. M., and Freeman, T. (1966) in Prot. Biol. Fluids (Peeters, H., ed.), Vol. 14, pp. 503-509, Elsevier, Amsterdam. 13. Verbruggen, R. (1975) Clin. Chem. 21, 5-43. 14. Johansson, B. G., and Malmquist, J. (197l)Scund. J. Clin. Lob. Invest. 27,255-261. IS. Owen, P., and Freer, J. H. (l972) Biochem. J. 129, 907-917. 16. Owen, P., and Salton, M. R. J. (1975) Biochim. Biophys. Acta 406, 214-234. 17. Owen, P., and Salton, M. R. J. (1975) Proc. Nat. Acad. Sci. USA 72, 371 l-3715. 18. Harboe, N., and Ingild, A. (1973) Stand. J. Immunol. 2, Suppl. I, 161-164. 19. Weeke, B. (1970) &and. J. Clin. Lab. Invest. 25, 161-163. 20. Weeke, B. (1973) &and. J. Immunol. 2, Suppl. 1, 15-35. 21. Laurel], C.-B. (1972) Stand. J. Clin. Lab. Invest. 29, Suppl. 124, 21-37. 22. Weeke, B. (1973) Scund. J. Immunol. 2, Suppl. I, 37-46. 23. Pless, D. D., Schmit, A. S., and Lennarz, W. J. (1975)5. Biol. Chem. 250, 1313-1327. 24. Owen, P., and Salton, M. R. J. (1975) Biochem. Biophys. Res. Commun. 63,875-880. 25. Bog-Hansen, T. C., Brogren, C.-H., and McMurrough, I. (1974) J. Inst. Brew. 80, 443-446. 26. Bog-Hansen, T. C., and Brogren, C.-H. (1975)Scnnd.J. Immunol. 4, Suppl. 2,135-139. 27. Muriel, J. (1971) in Methods in Immunology and immunochemistry (Williams, C. A. and Chase, M. W., eds.), Vol. 3, pp. 294-321, Academic Press, New York.
BIOCHEMISTRY
73, 20-26 (1976)
Submicrogram Quantitation of an Acidic Polysaccharide by Rocket lmmunoelectrophoresis and Rocket Affinoelectrophoresis PETEROWENAND Department
of Microbiology, New
MILTON
R.J.
New York University York, New York 10016
SALTON School
of Medicine
Received October 22, 1975; accepted December 4. 1975 Rocket immunoelectrophoresis has been used to quantitate an acidic polysaccharide (a succinylated lipomannan) isolated from the membranes ofthe bacterium, Micrococcus lysodeikticcrs. This procedure is superior in sensitivity to standard calorimetric assays for carbohydrate and allows the detection of as little as 10 ng of mannan. The observed relationships between rocket height and both antibody and antigen concentrations follow those described by Weeke for protein antigens [Weeke, B. (1973) Stand. J. Immunol. 2, Suppl. 1, 37-461. Furthermore, the lectin concanavalin A could be used as an alternative to immune serum in the quantitation of this polysaccharide by rocket electrophoresis. However, this modification (rocket affinoelectrophoresis) appeared to be less sensitive than rocket immunoelectrophoresis, allowing the detection of about 2.5ng of mannan.
The concentration of an individual polysaccharide is usually estimated indirectly by the selection of a calorimetric reaction appropriate for its sugar composition (l-5) or by gas chromatographic analysis of hydrolyzed preparations. These methods are based upon the measurement of the constituent monosaccharides such as hexose (2), pentose (3), amino sugars (4), and uranic acids (5) rather than on the native polysaccharide itself. Moreover, calorimetric assays are generally sensitive only to the microgram level, and gas chromatographic analysis is time consuming. A similar criticism also applies to the estimation of reducing equivalents (6). Furthermore, these indirect methods of polysaccharide quantitation can often give misleading results due to the presence of other contaminating species in the preparations. Direct and sensitive biological assays have been developed for lipopolysaccharides such as endotoxins (7), but these methods have not been generally applicable to other polysaccharide preparations. Heidelberger and his colleagues have shown that antigen-antibody precipitation reactions can be used to quantitate both intact polysaccharides and proteins (8,9). More recent developments in quantitative immunoassay have been the techniques of one dimensional and two dimensional (crossed) immunoelectrophoresis (IO- 12). These methods have been widely used for a number of years for the rapid quantitation of 20 Copyright All nghtr
0 1976 by Academx Pre\\. Inc. of reproduction in any form reelved.
POLYSACCHARIDE
QUANTlTATlON
21
proteins, glycoproteins and lipoproteins (for review see (13)). With the routine staining procedures available (e.g., Coomassie Brilliant blue) onedimensional immunoelectrophoresis (rocket immunoelectrophoresis) can detect several nanograms of sample (14). However, the possibility of using this method for the detection and quantitation of small amounts of polysaccharide has, to the authors’ knowledge, not been investigated. Accordingly, we wish to report that the technique of rocket immunoelectrophoresis can be adapted to detect as little as 10 ng of a bacterial succinylated lipomannan isolated from the membranes of the grampositive organism, Micrococcus lysodeikticus. Furthermore, we have investigated the potentials of using the lectin concanavalin A, as a substitute for immune sera in the quantitation of this polysaccharide. MATERIALS
Antigen
and Antibody
AND METHODS
Preparations
Succinylated lipomannan was isolated from washed membranes of Micrococcus lysodeikticus (NCTC 2665) (1.5) and purified as previously described (16). Antiserum to washed membrane fractions from this organism was raised in the rabbit (17) and fractionated immunoglobulins (18) were concentrated tenfold with respect to the original serum volume (final concentration 150 mg protein/ml). Human transferrin was purchased from Behring Diagnostics and carbamylated as described by Weeke (19). Rabbit antihuman transferrin was purchased from Dakopatts AIS, Denmark. Electrophoretic
Techniques
General. One percent (w/v) agarose gels containing barbital/HCl buffer (1 = 0.02, pH 8.6) and 1% (v/v) Triton X-100 were used throughout these studies and cast on glass plates to give a volume to surface area ratio of 0.132 ml/cm2. Electrophoresis was performed in a Behring Diagnostic water-cooled immunoelectrophoresis cell. Following completion of electrophoresis, gels were either pressed, washed and dried and stained with Coomassie Brilliant blue (20) or immersed in 75% (v/v) ethanol for precipitation of dextran used as an indicator of electroendoosmosis. Peak heights of the rockets were determined on millimeter ruled graph paper by measuring the distance from the top of the application well to the tip of the precipitin rocket. Rocket immunoelectrophoresis. Gels containing antibody were cast on glass plates (102 x 82 x 0.8 mm) and antigen (total volume 2.0 ~1) was applied to wells 1.5 mm in diameter with the current on. Electrophoresis was continued for 18 hr at 4.5 V/cm with water coolant at ambient temperature. Rocket af$noelectrophoresis. This procedure was performed essentially
22
OWEN AND SALTON
t3
FIG. 1. Crossed immunoelectrophoresis of the succinylated lipomannan isolated from M. (A) Mannan (600 ng) together with the acidic marker protein carbamylated transfertin (350 ng) and (B) carbamylated transferrin (350 ng) alone were subjected to crossed immunoelectrophoresis as described in Materials and Methods. Antibody gels contained 7.5 ~1 of antimembrane serum and 7.5 ~1 of antitransferrin serum per ml. The position to which neutral dextran (M, = 20,000) migrates in the first dimension of electrophoresis is indicated thus (0). The application well is identified thus, (0). lysodeikticus.
as described for rocket immunoelectrophoresis, but with concanavalin A incorporated into the gel in place of antimembrane serum as an “affinity” precipitin. Crossed immunoelectrophoresis . Crossed immunoelectrophoresis was based on a modification (12) of Laurell’s technique (IO). Agarose gels were cast on glass plates (50 x 50 x 0.6 mm) and samples applied to wells (1.5 mm i.d.) centrally located in the gel. Electrophoresis was continued in the first direction for 55 min at 5 V/cm. The central agarose strip (10 x 50 mm) containing antigen(s) was retained on the plate after removal of the rest of the gel which was replaced by two adjacent gels (20 x 50 mm) containing antibody. Electrophoresis in the second dimension was performed at 2 V/cm for 12-18 hr. Materials Concanavalin A and agarose were purchased from Miles Labs. and Triton X-100 was obtained from Baker Chemical Co. Dextran (M, = 20,000), which was used as a marker for electroendoosmosis, was obtained from Sigma Chemical Co. RESULTS AND DISCUSSION
Quantitation of an antigen by rocket immunoelectrophoresis is simplified if a single precipitating species is present. This can be achieved either by
POLYSACCHARIDE
23
QUANTITATION
use of a highly purified antigen or, alternatively, a monovalent antiserum (21,22). Fig. 1 (A and B) shows the crossed immunoelectrophoresis pattern obtained for purified M. lysodeikticus mannan tested against antimembrane serum. The results suggest the presence of a single anionic antigen possessing charge heterogeneity, probably due to varying degrees of succinylation (23,24). The broadness of the mannan precipitin band is unlikely to reflect a heterogeneity in size since it has previously been shown that this polymer has a relatively low molecular weight (about 10,000 (16,23)), and resolution of molecular sizes would probably not occur by molecular sieving in the agarose gel. Rocket immunoelectrophoresis of mannan preparations into antimembrane serum resulted in precipitin rockets of reproducible height (Fig. 2A). For various antibody concentrations, the height of the rocket exhibited an almost linear relationship to the quantity of antigen added (Fig. 3A) provided the operating voltage was held at 4.5 V/cm. At lower
+ t
A
R lP j
2
2
3
3
4
4 5
5
~+ I<*
it22334455 FIG. 2. (A) Rocket immunoelectrophoresis and (B) rocket affinoelectrophoresis of M. ~ysodeikticus mannan. 300, 600, 900,450, and 150 ng of mannan were added to wells numbered 1-5, respectively, in both A and B. Gel A contained antimembrane serum (12.5 ~llml) and gel B, concanavalin A (75 &ml).
24
OWEN AND SALTON
25. 20-
‘5. ,. *
//
..’ .:’ ...’ / o..’
// o y ,. ...’ 0‘/ ,Y @.: 0
/
.:’
5*
0
200
400
600
800
1000
4 1200 0 Mannan
200 (ng)
400
600
800
4 1000 1200
FIG. 3. Standard curves for M. lysodeikticus mannan by (A) rocket immunoelectrophoresis and (B) rocket affinoelectrophoresis. Gels in A contained anti-membrane serum at the following concentrations n . . . .m. 21.4 ~liml; n n , 10.7 PI/ml; l . . . .O, 7.1 pi/ml; 0 0, 3.75 @ml. Gels in B contained concanavalin A at the following concentrations 0 0, 100 p&ml; O- 1. .O. 75 &ml; q 0, 50 p&/ml.
operating voltages the rockets had a much more rounded shape and linear relationship between peak height and antigen concentration was found to apply over a reduced concentration range. This is attributed either to the relatively high diffusion coefficient of this polymer (16) or to the formation of antigen-antibody complexes of low precipitability (21). As has been observed by other workers, linearity is also lost as the peak height approaches zero (22). In agreement with results obtained for protein antigens (22), alteration of both polysaccharide and antibody concentrations by a similar factor resulted in precipitin rockets of unchanged peak height (Fig. 3A). However, rockets were not detected by Coomassie Brilliant blue staining for gels containing less then 3.75 ~1 antibody/ml. Thus at the sensitivity limit of the method, a precipitin rocket of height 2 mm represents about 10 ng of mannan. Slightly lower detection limits (about 3 ng) have been reported for some protein antigens (14). However, it should be remembered that, in the present instance, the antibody protein in the precipitin rocket is the sole contributor to staining with Coomassie Brilliant blue. We have shown previously that M. lysodeikticus mannan can coprecipitate up to 15 times its own weight of concanavalin A, and that it gives discrete precipitin bands in agargel diffusion experiments against this lectin (16,24). Bog-Hansen et al. have indicated that glycoproteins can give precipitin rockets upon electrophoresis into concanavalin A, a technique they termed rocket affinoelectrophoresis (25,26). However, the procedure has not been put on a quantitative basis. It was thus of interest to determine whether concanavalin A could be used as an alternative to
POLYSACCHARIDE
QUANTITATION
25
immune sera in the quantitation of the mannan. Rocket affinoelectrophoresis of mannan into concanavalinA resulted in distinct precipitin rockets of reproducible height (Fig. 2B) which showed similar relationships between peak height and both polysaccharide and lectin concentrations as described for immune sera (Fig. 3B). The precipitin rockets formed in the presence of lectin were more diffuse than those formed in the presence of antibody (see Figs. 2A and B) thus making the estimation of peak height slightly more difficult. Furthermore, no stainable precipitin peaks were observed at a lectin concentration of 25 pg/ml or below. Thus the limit of sensitivity of this method for this particular polysaccharide is about 25 ng. We have thus shown that the sensitive technique of rocket immunoelectrophoresis can be successfully applied to the quantitation of an acidic polysaccharide. The method is simple, rapid, specific, and has the advantage of not being affected by the presence of other noninteracting molecular species. Thus, individual antigens in a complex mixture of immunogens can be quantitated if either monovalent antisera are available (21,22) or if the antigens in question can be identified in the pattern of rockets obtained following electrophoresis into polyvalent antiserum (27). The application of this procedure to the assay of other acidic polysaccharides of medical importance, such as capsular polysaccharides, teichoic acids, lipoteichoic acids, and lipopolysaccharides should be obvious. Furthermore, we have also shown that the lectin concanavalinA can substitute for immune serum in the quantitation of M. lysodeikticus mannan by rocket electrophoresis. Recent work in this laboratory performed in collaboration with Dr. G. D. Shockman, Temple University School of Medicine, Philadelphia, Pennsylvania, has shown that this technique can also be used to quantitate streptococcal lipoteichoic acid possessing kojibiose residues. Further experimentation will be necessary, however, to determine whether rocket affinoelectrophoresis can be applied to the quantitation of polysaccharides containing sugar residues in conformations allowing interaction with other lectins. ACKNOWLEDGMENT This research was supported Foundation.
by Grant BMS 7503934 from the National
Science
REFERENCES 1. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28,350-356. 2. Morris, D. L. (1948) Science 107, 254-255. 3. Dische, Z., and Schwarz, K. (1937) Mikrochim. Acra 2, 13-19. 4. Elson, L. A., and Morgan, W. T. J. (1933) Biochem. J. 27, 1824-1828. 5. Dische, Z. (1947)J. Biol. Chem. 167, 189-198. 6. McLean, C., Werner, D. A., and Aminoff, D. (1973) Anal. Biochem. 55, 72-84.
26
OWEN AND SALTON
7. Yin, E. T., Galanos, C., Kinsky, S., Bradshaw, R. A., Wessler, S., Luderitz, 0.. and Sarmiento, M. E. (1972) Eiochim. Biophys. Acta 261, 284-289. 8. Heidelberger, M., and Kendall, F. E. (1929) J. Exp. Med. 50, 809-823. 9. Heidelberger, M. (1939) Bacterial. Rev. 3, 49-95. 10. Laurel], C.-B. (1%5) Anal. Biochem. 10, 358-361. 11. Laurel], C.-B. (l%6) Anal. Biochem. 15, 54-52. 12. Clarke, H. G. M., and Freeman, T. (1966) in Prot. Biol. Fluids (Peeters, H., ed.), Vol. 14, pp. 503-509, Elsevier, Amsterdam. 13. Verbruggen, R. (1975) Clin. Chem. 21, 5-43. 14. Johansson, B. G., and Malmquist, J. (197l)Scund. J. Clin. Lob. Invest. 27,255-261. IS. Owen, P., and Freer, J. H. (l972) Biochem. J. 129, 907-917. 16. Owen, P., and Salton, M. R. J. (1975) Biochim. Biophys. Acta 406, 214-234. 17. Owen, P., and Salton, M. R. J. (1975) Proc. Nat. Acad. Sci. USA 72, 371 l-3715. 18. Harboe, N., and Ingild, A. (1973) Stand. J. Immunol. 2, Suppl. I, 161-164. 19. Weeke, B. (1970) &and. J. Clin. Lab. Invest. 25, 161-163. 20. Weeke, B. (1973) &and. J. Immunol. 2, Suppl. 1, 15-35. 21. Laurel], C.-B. (1972) Stand. J. Clin. Lab. Invest. 29, Suppl. 124, 21-37. 22. Weeke, B. (1973) Scund. J. Immunol. 2, Suppl. I, 37-46. 23. Pless, D. D., Schmit, A. S., and Lennarz, W. J. (1975)5. Biol. Chem. 250, 1313-1327. 24. Owen, P., and Salton, M. R. J. (1975) Biochem. Biophys. Res. Commun. 63,875-880. 25. Bog-Hansen, T. C., Brogren, C.-H., and McMurrough, I. (1974) J. Inst. Brew. 80, 443-446. 26. Bog-Hansen, T. C., and Brogren, C.-H. (1975)Scnnd.J. Immunol. 4, Suppl. 2,135-139. 27. Muriel, J. (1971) in Methods in Immunology and immunochemistry (Williams, C. A. and Chase, M. W., eds.), Vol. 3, pp. 294-321, Academic Press, New York.