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[5] Immunological methods for characterizing polysaccharides
[5]
IMMUNOLOGICAL METHODS FOR POLYSACCHARIDES
79
[5] I m m u n o l o g i c a l M e t h o d s for C h a r a c t e r i z i n g Polysaccharides
By
GERALD SCHIFFMAN
I. General Considerations Introduction Immunological methods are used extensively in systems in which simple and complex polysaccharides are the major antigens. The beststudied systems include the bacterial polysaccharides, lipopolysaccharides of gram negative microorganisms, and the ABH blood group substances. Specific antisera are used for serotyping and identification of organisms. Another major use is the characterization of polysaccharide antigens. It is this latter application which will be treated in this chapter. Immunochemical methods are especially useful as an aid in purification of polysaccharides as well as in studies on the structure of these complex macromolecules. These applications will be discussed in the following two parts of Section I. All immunologic work is dependent on antisera, the production of which is described in Section II. A modification of the quantitative precipitin and oligosaccharide inhibition techniques which can use sera containing as little as 1 ~g of precipitating nitrogen per milliliter and which requires much less time than previously described quantitative precipitin techniques will be presented in Section III. Other very useful methods, e.g., agglutination, immunodiffusion, complement fixation, and immunofluorescence will not be covered in great detail here since they are treated in great length in recently published books. 1-6 1E. A. Kabat, in Kabat and Mayer's "Experimental Immunochemistry," 2nd ed. Thomas, Springfield, Illinois, 1961. "Immunological Methods" (J. F. Ackroyd, ed.). Blackwell, Oxford, 1964. 3D. It. Campbell, J. S. Garvey, N. E. Cremer, and D. It. Sussdorf, "Methods in Immunology." W. A. Benjamin, New York, 1963. 4A. J. Crowle, "Immunodiffusion." Academic Press, New York, 1961. "Clinical Aspects of Immunology" (P. G. H. Gell and R. R. A. Coombs, eds.). F. A. Davis, Philadelphia, Pennsylvania, 1963. ~"Modern Trends in Immunology" (R. Cruickshank, ed.), Vol. 1. Butterworth, London, 1963.
80
ANALYTICAL METHODS
[S]
Immune Techniques as an Aid in the Purification of Polysaccharides It is often difficult to determine when a polysaccharide is pure. In addition to chemical composition, optical rotation, viscosity, and solubility properties, etc., immunochemical characterization is often desired. If a polysaccharide is pure, fractionation with alcohol, Sephadex chromatography, or other means will give samples equally potent in precipitating with specific antiserum. If the starting material is impure, the precipitin curves obtained with the different fractions will show which fraction is the purest. A washed specific precipitate can be extracted with cold trichloroacetic acid, digested with pronase, or treated in any way not deleterious to the polysaccharide and the antigen recovered. Analyses of the recovered antigen when compared with the originally added antigen will often indicate the state of purity of that material. This procedure was recently used by Torii et a l J in separating a and fl teichoic acids. Immunodiffusion in agar gels against antiserum made to crude source material of the polysaccharide will often give multiple bands in the early stages of purification. As contaminants are eliminated, the number of immune bands decreases, hopefully to one. A great array of methods, grouped under the heading immunodiffusion, have been developed: Oudin single diffusion, Ouchterlony-Elek and Oakley-Fulthorpe-Preer double diffusion, and Grabar-Scheidegger immunoelectrophoretic analysis are the most used.4 These methods are deceptively simple, and hence great care must be taken to ensure reliable results. The antiserum used must contain precipitins not only for the polysaccharide being purified, but also to all the likely contaminants. In double diffusion and in immunoelectrophoretic analysis, the concentration of antigen should be varied to allow the reaction to take place in the area between antigen and antibody. The temperature must not be varied, as changes will cause artifacts. Refilling wells will also cause artifacts. Difference in molecular weight of a single antigen can cause double bands. Immune Techniques as an Aid to Polysaccharide Structural Studies Heidelberger and co-workers have studied many gums and other polysaccharides2,9 From been able to make many predictions about the later verified. Substantial leads as to structure
the cross-reactions of these Heidelberger has structures which were can be uncovered if a
' M. Torii, E. A. Kabat, and A. E. Bezer, J. Exptl. Med. 120, 13 (1964). M. Heidelberger, Proc. Chem. Soc., p. 153 (1961). M. Heidelberger, "Lectures in Immunochemistry." Academic Press, New York, 1956.
[5]
IMMUNOLOGICAL METHODS FOR POLYSACCHARIDES
81
sufficiently large number of antisera to characterized polysaccharides is available. As more is learned about the fine structure of polysaccharides, this approach will become more valuable. A more direct approach is the partial depolymerization of complex macromolecules and isolation of the oligosaccharides. Here the tools of immunochemistry can greatly facilitate the difficult work. In a recent review of the chemistry of the ABH blood group substances, the results obtained by this approach have been summarized. 1° II. Production of Antiserum Although raising antiserum is inherently a simple procedure, it can be the most difficult aspect of utilizing immunochemical procedures in polysaccharide research. This anomaly results from the fact that purified polysaccharides are not satisfactory antigens in rabbits, the laboratory animal most ideally suited for the production of antisera. When the polysaccharide is on the surface of a bacterial cell or erythrocyte, excellent antibody responses are usually obtained. In some cases treatment with trypsin leads to a greater anticarbohydrate response. The production of antiserum to a purified polysaccharide antigen generally is not an easy matter. Humans will produce antibody to purified polysaccharides when 0.050-1.0 mg in 1 ml saline is injected intramuscularly and the recipients are bled 1 month later. 1 The titers obtained are usually quite low. Polysaccharides have been absorbed onto living group A streptococci, 11 the complex heated to inactivate the organisms and " f i x " the added polysaccharide, and injected into rabbits. Polysaccharides have also been coated onto erythrocytes and used for immunization with only little success. Rebers e t al. ~2 exposed type VI to a small quantity of periodate. The resulting product could then be bound to erythrocytes, washed, and injected. Provided the periodate does not destroy many of the antigenic determinants of interest, this has two advantages: (1) Red cells so coated can be used for passive hemagglutination; (2) if a preimmunization bleeding is taken, the erythrocytes of the same animal can be coated with polysaccharide and used for immunization. This procedure should minimize the number of added antigenic determinants. Coating of polysaccharides onto latex, bentonite, or polyacrylamide gel should be feasible if sufficient polysaccharide can be bound and if the resultant suspension is not toxic. io G. Schiffman E. B. Brown, " L . E. Glynn, (1954). 1~.p. A. Rebers,
and D. M. Marcus, in "Progress in Hematology" (C. V. Moore and eds.), Vol. 4, p. 97. Grune & Stratton, New York, 1964. E. J. Holborow, and G. D. Johnson, J. Pclthol. Bacteriol. 68, 205 S. Estrada-Parra, and M. Heidelberger, J. Bacteriol. 86, 882 (1963).
82
ANALYTICAL METHODS
[S]
Other interesting suggestions have been proposed. WestphaP 3 recommended the use of epiehlorohydrin to polymerize a saccharide hapten and convert it into an antigen. He also suggested that esterification of a saccharide hapten with a long-chain fatty acid should behave similarly. This latter possibility is analogous to the situation described by GoebeP 4 with the pneumococcal C and F polysaccharides. Procedure for Production of Rabbit Antipneumococcal Serum A vaccine of pneumococci is prepared from heat (60 °, 30 minutes) or formaldehyde (1 ml formalin per 100 ml culture, neutralized with alkali, and allowed to stand 1 hour at 37 °) inactivated bacteria. The collected cells are washed with sterile saline and suspended in sufficient saline to give a final concentration of 100 #g N per milliliter. The N analysis can be performed by the method given in Section III. The vaccine should still be predominantly gram positive and, if made to a cap~ sulated strain, should give a Neufeld quellung reaction in the presence of a specific antiserum. The vaccine should be tested for sterility by culturing 0.1 ml on a blood agar plate. Rabbits are conveniently immunized via the marginal ear vein with a 26-gauge disposable needle. A course of 4 injections per week for 2 weeks with bleeding 6 days after the last injection yields antisera with about 200-500 /~g precipitable N per milliliter. Continued courses of immunization lead to increased titers--as high as 5 mg N per milliliter has been achieved by this regimen. Bleeding is best performed via cardiac puncture using a 50 ml syringe with a 19-gauge disposable needle. As much as 50 ml per day for 3 successive days can be withdrawn without harm to the animal. The blood is collected into sterile 250 ml centrifuge tubes and allowed to clot at 37 ° for 1 hour; the serum is collected after the clot has retracted overnight at 0-4 ° . Sterile technique should be employed throughout, and the serum stored at 4 ° . Serum collected under nonsterile conditions should be frozen or protected by the addition of merthiolate (1:10,000) and phenol (0.25%).1 Notes. 1. Preimmunization bleeding should always be taken. If only small quantities are desired, this can be accomplished by ear bleeding. 2. The timing of bleeding following the last injection is very important. Unlike the persistence of antipolysaccharide antibody in the human, in the system described the titer of antibody on the 6th day after the last injection was usually twice that found on the 4th day. The titers on the 8th day and later were likewise significantly lower. ~30. Westphal, see Chem. Abstr. 61, 6200f (1964). ~4W. F. Goebel and M. H. Adams, J. Exptl. Med. 77, 435 (1943).
[5]
IMMUNOLOGICAL METHODS FOR POLYSACCHARIDES
83
3. The mortality rate in the cardiac puncture technique, in the hand of the author, has been 5%. III. Quantitative Precipitin and Oligosaccharide Inhibition Analysis via the Ninhydrin Procedure
Step 1. Aliquots of antigen, 0.5-10 ~g in 0.2 ml saline, are added to 3 ml conical centrifuge tubes. 15 Step 2. Serum, estimated to contain 1-15 ~g precipitable N, is added. The contents are mixed, capped, and placed in a refrigerator for 18 hours or longer. The recommended incubation at 37 ° for 1 hour has been found to be unnecessary in systems used by the author, but it is recommended that this omission be evaluated for other systems. Similarly, in systems tested, 18 hours at 4 ° gave complete precipitation. Other systems may require a longer time of standing at 0-4 °. Step 3. The specific precipitates are washed with ice-cold saline after centrifugation (5 °, 10,000 rpm, 20 minutes) in a Servall. Rubber adaptor No. 316 allows 16 tubes to be spun in SS-34 rotor. After centrifugation, the supernatant is decanted and the tubes are allowed to drain in an inverted position on a clean absorbent surface. Saline, 0.5 ml, is added in two portions with mixing after each addition. A Vortex, Jr. (Scientific Industries, Queens Village, New York) resuspends the precipitate well. The washing is repeated a second time with an additional 0.5 ml saline. Step 4. The washed specific precipitates are digested in a sand bath with 20 ~l H2SO~ (1:10 dilution with water). A 400-ml steel crucible containing sea sand is capable of supporting about 20 tubes. The digestion is performed on a LABCONC0 (Kansas City, Missouri) electric Kjeldahl digestion rack. The lowest setting (No. 1) is usually sufficient to allow the acid to char the precipitate. This requires about 1 hour. Step 5. The char is cleared by the addition of 10 ~l 30% H~02 (superoxol). Heating is continued until all traces of superoxol are removed (about 1~ to 1 hour longer). Step 6. The tubes containing the digested samples are allowed to cool. Water, 0.4 ml, is added to dissolve the sample. ~inhydrin reagent, 0.2 ml, is added, the tubes are mixed and heated at 95 ° for 20 minutes. Magna-whirl Blue M (Blue Island, Illinois) water bath has been found to be very satisfactory. The samples are then transferred to 10-ml volumetric flasks with 50% ethanol and mixed; the optical density is read at 570 m~. 1~Available from Bellco Glass, Inc., Vineland, New Jersey. These tubes are gently tapered and beaded at the top.
84
ANALYTICAL METHODS
[S]
Reagent. The ninhydrin reagent is made as follows: Solution 1: 4% ninhydrin in Methyl Cellosolve (ethylene glycol monomethyl ether) Solution 2: 4 M sodium acetate containing 1% glacial acetic acid Solution 3:0.01 M KCN in water Solution mixture: Four milliliters of solution 1 is mixed with 1 ml of solution 2. To 4 ml of the resultant solution is added 0.10 ml of solution 3.
Notes. 1. The volume of serum for one experiment is removed sterilely from the stock and is centrifuged for 1 hour at 10,000 rpm at 4 °. If there is any lipid it is removed by suction with a capillary pipette. The clear serum is then decanted from any insoluble debris. 2. The volume in which the antigen is added should be kept small to minimize the loss of precipitate through solubility. The volume of serum used depends on the concentration of antibody. F o r very low titer sera, 1-ml aliquots of undiluted serum have been used; for very high titer sera 0.1 ml of 1:100 dilution with saline has been used. 3. All pipetting should be performed with to-deliver micropipettes. Lang-Levy type has been used exclusively in these laboratories. 4. Each precipitin curve should be set up with control tubes containing no added antigen. 5. Analytical grade ammonium sulfate is used as a standard with each series of determinations. A solution of ammonium sulfate of 40 t~g N per milliliter is used; 2 and 4 t~g N aliquots are taken when the amount of precipitate will be 1-10 #g N. 6. The volume of all reagents can be doubled, extending the range to 30 ~g N. Standards should be increased proportionately. 7. This method of N analysis has been used (a) to determine % N of polysaccharides during purification; (b) in determining N : P ratio of polysaccharides during chromatography; and (c) in determining N content of vaccines. 8. When oligosaccharide~ are added for inhibition, the addition is made before the addition of antigen (step 1 of procedure just described) and all other steps are the same. The 1 hour incubation of inhibitor with antibody has been found to be unnecessary in the systems studied by the author. 9. The washed specific precipitate can also be dissolved in 0.25 M acetic acid 1~ and read at 277 mt~ or assayed by the micro-Folin procedure. 17 1~D. Gitlin, J. Immunol. 62, 437 (1949). 17E. A. Kabat and G. Schiffman, J. Immunol. 88, 782 (1962).
[6]
NEW COLORIMETRIC METHODS OF SUGAR ANALYSIS
85
10. The procedure for N determination is based on one described by Rosevear and Smith is and subsequently modified. ~,19 11. An extremely sensitive procedure for measuring complement fixation and complement fixation inhibition has been described. 2° This procedure has been used by many laboratories with great success. l~j. w. Rosevear and E. I. Smith, J. Biol. Chem. 236, 425 (1961). 19G. Schiffman, E. A. Kabat, and W. Thompson, J. Am. Chem. Soc. 84, 463 (1962). _~oE. Wasserman and L. Levine, Y. Immunol. 87, 290 (1961).
[6]
New
Colorimetric
Methods
of Sugar Analysis
B y GILBERT ASHWELL
This chapter is designed to supplement the more complete description of colorimetric methods of sugar analysis which appeared in an earlier volume of this series (Vol. III [12]). Emphasis has been placed upon the inclusion of new and specific reactions for monosaccharides of biological significance. Several improved modifications, currently employed in the determination of uronic acids, amino sugars, and carbohydrate-protein complexes have not been included here since they are described in detail elsewhere in this volume. I. Determination of Tetroses 1 Principle. In general, the development of specific color reactions for sugars has been based upon the formation of furfural derivatives upon heating an aqueous solution of the sugar with a strong acid. Subsequent complexing of the derivative with an appropriate organic developer provides a basis for the formation of a color which is characteristic for a given class of sugars. The inability of the 4-carbon sugars to form furan derivatives under these conditions retarded the development of a specific assay procedure. The method described here 1 is based upon the observation that tetroses can combine with the breakdown products of higher sugars to yield a chromophore with a characteristic absorption spectrum. Reagents
H~S04. Six parts of concentrated, reagent grade H2S04 are added slowly, and with chilling, to one part of water. Fructose, prepared daily as a 0.1% aqueous solution L-Cysteine hydrochloride monohydrate, freshly prepared as a 3% aqueous solution ' Z. Disehe and M. R. Dische, Biochim. Biophys. Acla 27, 184 (1958).
IMMUNOLOGICAL METHODS FOR POLYSACCHARIDES
79
[5] I m m u n o l o g i c a l M e t h o d s for C h a r a c t e r i z i n g Polysaccharides
By
GERALD SCHIFFMAN
I. General Considerations Introduction Immunological methods are used extensively in systems in which simple and complex polysaccharides are the major antigens. The beststudied systems include the bacterial polysaccharides, lipopolysaccharides of gram negative microorganisms, and the ABH blood group substances. Specific antisera are used for serotyping and identification of organisms. Another major use is the characterization of polysaccharide antigens. It is this latter application which will be treated in this chapter. Immunochemical methods are especially useful as an aid in purification of polysaccharides as well as in studies on the structure of these complex macromolecules. These applications will be discussed in the following two parts of Section I. All immunologic work is dependent on antisera, the production of which is described in Section II. A modification of the quantitative precipitin and oligosaccharide inhibition techniques which can use sera containing as little as 1 ~g of precipitating nitrogen per milliliter and which requires much less time than previously described quantitative precipitin techniques will be presented in Section III. Other very useful methods, e.g., agglutination, immunodiffusion, complement fixation, and immunofluorescence will not be covered in great detail here since they are treated in great length in recently published books. 1-6 1E. A. Kabat, in Kabat and Mayer's "Experimental Immunochemistry," 2nd ed. Thomas, Springfield, Illinois, 1961. "Immunological Methods" (J. F. Ackroyd, ed.). Blackwell, Oxford, 1964. 3D. It. Campbell, J. S. Garvey, N. E. Cremer, and D. It. Sussdorf, "Methods in Immunology." W. A. Benjamin, New York, 1963. 4A. J. Crowle, "Immunodiffusion." Academic Press, New York, 1961. "Clinical Aspects of Immunology" (P. G. H. Gell and R. R. A. Coombs, eds.). F. A. Davis, Philadelphia, Pennsylvania, 1963. ~"Modern Trends in Immunology" (R. Cruickshank, ed.), Vol. 1. Butterworth, London, 1963.
80
ANALYTICAL METHODS
[S]
Immune Techniques as an Aid in the Purification of Polysaccharides It is often difficult to determine when a polysaccharide is pure. In addition to chemical composition, optical rotation, viscosity, and solubility properties, etc., immunochemical characterization is often desired. If a polysaccharide is pure, fractionation with alcohol, Sephadex chromatography, or other means will give samples equally potent in precipitating with specific antiserum. If the starting material is impure, the precipitin curves obtained with the different fractions will show which fraction is the purest. A washed specific precipitate can be extracted with cold trichloroacetic acid, digested with pronase, or treated in any way not deleterious to the polysaccharide and the antigen recovered. Analyses of the recovered antigen when compared with the originally added antigen will often indicate the state of purity of that material. This procedure was recently used by Torii et a l J in separating a and fl teichoic acids. Immunodiffusion in agar gels against antiserum made to crude source material of the polysaccharide will often give multiple bands in the early stages of purification. As contaminants are eliminated, the number of immune bands decreases, hopefully to one. A great array of methods, grouped under the heading immunodiffusion, have been developed: Oudin single diffusion, Ouchterlony-Elek and Oakley-Fulthorpe-Preer double diffusion, and Grabar-Scheidegger immunoelectrophoretic analysis are the most used.4 These methods are deceptively simple, and hence great care must be taken to ensure reliable results. The antiserum used must contain precipitins not only for the polysaccharide being purified, but also to all the likely contaminants. In double diffusion and in immunoelectrophoretic analysis, the concentration of antigen should be varied to allow the reaction to take place in the area between antigen and antibody. The temperature must not be varied, as changes will cause artifacts. Refilling wells will also cause artifacts. Difference in molecular weight of a single antigen can cause double bands. Immune Techniques as an Aid to Polysaccharide Structural Studies Heidelberger and co-workers have studied many gums and other polysaccharides2,9 From been able to make many predictions about the later verified. Substantial leads as to structure
the cross-reactions of these Heidelberger has structures which were can be uncovered if a
' M. Torii, E. A. Kabat, and A. E. Bezer, J. Exptl. Med. 120, 13 (1964). M. Heidelberger, Proc. Chem. Soc., p. 153 (1961). M. Heidelberger, "Lectures in Immunochemistry." Academic Press, New York, 1956.
[5]
IMMUNOLOGICAL METHODS FOR POLYSACCHARIDES
81
sufficiently large number of antisera to characterized polysaccharides is available. As more is learned about the fine structure of polysaccharides, this approach will become more valuable. A more direct approach is the partial depolymerization of complex macromolecules and isolation of the oligosaccharides. Here the tools of immunochemistry can greatly facilitate the difficult work. In a recent review of the chemistry of the ABH blood group substances, the results obtained by this approach have been summarized. 1° II. Production of Antiserum Although raising antiserum is inherently a simple procedure, it can be the most difficult aspect of utilizing immunochemical procedures in polysaccharide research. This anomaly results from the fact that purified polysaccharides are not satisfactory antigens in rabbits, the laboratory animal most ideally suited for the production of antisera. When the polysaccharide is on the surface of a bacterial cell or erythrocyte, excellent antibody responses are usually obtained. In some cases treatment with trypsin leads to a greater anticarbohydrate response. The production of antiserum to a purified polysaccharide antigen generally is not an easy matter. Humans will produce antibody to purified polysaccharides when 0.050-1.0 mg in 1 ml saline is injected intramuscularly and the recipients are bled 1 month later. 1 The titers obtained are usually quite low. Polysaccharides have been absorbed onto living group A streptococci, 11 the complex heated to inactivate the organisms and " f i x " the added polysaccharide, and injected into rabbits. Polysaccharides have also been coated onto erythrocytes and used for immunization with only little success. Rebers e t al. ~2 exposed type VI to a small quantity of periodate. The resulting product could then be bound to erythrocytes, washed, and injected. Provided the periodate does not destroy many of the antigenic determinants of interest, this has two advantages: (1) Red cells so coated can be used for passive hemagglutination; (2) if a preimmunization bleeding is taken, the erythrocytes of the same animal can be coated with polysaccharide and used for immunization. This procedure should minimize the number of added antigenic determinants. Coating of polysaccharides onto latex, bentonite, or polyacrylamide gel should be feasible if sufficient polysaccharide can be bound and if the resultant suspension is not toxic. io G. Schiffman E. B. Brown, " L . E. Glynn, (1954). 1~.p. A. Rebers,
and D. M. Marcus, in "Progress in Hematology" (C. V. Moore and eds.), Vol. 4, p. 97. Grune & Stratton, New York, 1964. E. J. Holborow, and G. D. Johnson, J. Pclthol. Bacteriol. 68, 205 S. Estrada-Parra, and M. Heidelberger, J. Bacteriol. 86, 882 (1963).
82
ANALYTICAL METHODS
[S]
Other interesting suggestions have been proposed. WestphaP 3 recommended the use of epiehlorohydrin to polymerize a saccharide hapten and convert it into an antigen. He also suggested that esterification of a saccharide hapten with a long-chain fatty acid should behave similarly. This latter possibility is analogous to the situation described by GoebeP 4 with the pneumococcal C and F polysaccharides. Procedure for Production of Rabbit Antipneumococcal Serum A vaccine of pneumococci is prepared from heat (60 °, 30 minutes) or formaldehyde (1 ml formalin per 100 ml culture, neutralized with alkali, and allowed to stand 1 hour at 37 °) inactivated bacteria. The collected cells are washed with sterile saline and suspended in sufficient saline to give a final concentration of 100 #g N per milliliter. The N analysis can be performed by the method given in Section III. The vaccine should still be predominantly gram positive and, if made to a cap~ sulated strain, should give a Neufeld quellung reaction in the presence of a specific antiserum. The vaccine should be tested for sterility by culturing 0.1 ml on a blood agar plate. Rabbits are conveniently immunized via the marginal ear vein with a 26-gauge disposable needle. A course of 4 injections per week for 2 weeks with bleeding 6 days after the last injection yields antisera with about 200-500 /~g precipitable N per milliliter. Continued courses of immunization lead to increased titers--as high as 5 mg N per milliliter has been achieved by this regimen. Bleeding is best performed via cardiac puncture using a 50 ml syringe with a 19-gauge disposable needle. As much as 50 ml per day for 3 successive days can be withdrawn without harm to the animal. The blood is collected into sterile 250 ml centrifuge tubes and allowed to clot at 37 ° for 1 hour; the serum is collected after the clot has retracted overnight at 0-4 ° . Sterile technique should be employed throughout, and the serum stored at 4 ° . Serum collected under nonsterile conditions should be frozen or protected by the addition of merthiolate (1:10,000) and phenol (0.25%).1 Notes. 1. Preimmunization bleeding should always be taken. If only small quantities are desired, this can be accomplished by ear bleeding. 2. The timing of bleeding following the last injection is very important. Unlike the persistence of antipolysaccharide antibody in the human, in the system described the titer of antibody on the 6th day after the last injection was usually twice that found on the 4th day. The titers on the 8th day and later were likewise significantly lower. ~30. Westphal, see Chem. Abstr. 61, 6200f (1964). ~4W. F. Goebel and M. H. Adams, J. Exptl. Med. 77, 435 (1943).
[5]
IMMUNOLOGICAL METHODS FOR POLYSACCHARIDES
83
3. The mortality rate in the cardiac puncture technique, in the hand of the author, has been 5%. III. Quantitative Precipitin and Oligosaccharide Inhibition Analysis via the Ninhydrin Procedure
Step 1. Aliquots of antigen, 0.5-10 ~g in 0.2 ml saline, are added to 3 ml conical centrifuge tubes. 15 Step 2. Serum, estimated to contain 1-15 ~g precipitable N, is added. The contents are mixed, capped, and placed in a refrigerator for 18 hours or longer. The recommended incubation at 37 ° for 1 hour has been found to be unnecessary in systems used by the author, but it is recommended that this omission be evaluated for other systems. Similarly, in systems tested, 18 hours at 4 ° gave complete precipitation. Other systems may require a longer time of standing at 0-4 °. Step 3. The specific precipitates are washed with ice-cold saline after centrifugation (5 °, 10,000 rpm, 20 minutes) in a Servall. Rubber adaptor No. 316 allows 16 tubes to be spun in SS-34 rotor. After centrifugation, the supernatant is decanted and the tubes are allowed to drain in an inverted position on a clean absorbent surface. Saline, 0.5 ml, is added in two portions with mixing after each addition. A Vortex, Jr. (Scientific Industries, Queens Village, New York) resuspends the precipitate well. The washing is repeated a second time with an additional 0.5 ml saline. Step 4. The washed specific precipitates are digested in a sand bath with 20 ~l H2SO~ (1:10 dilution with water). A 400-ml steel crucible containing sea sand is capable of supporting about 20 tubes. The digestion is performed on a LABCONC0 (Kansas City, Missouri) electric Kjeldahl digestion rack. The lowest setting (No. 1) is usually sufficient to allow the acid to char the precipitate. This requires about 1 hour. Step 5. The char is cleared by the addition of 10 ~l 30% H~02 (superoxol). Heating is continued until all traces of superoxol are removed (about 1~ to 1 hour longer). Step 6. The tubes containing the digested samples are allowed to cool. Water, 0.4 ml, is added to dissolve the sample. ~inhydrin reagent, 0.2 ml, is added, the tubes are mixed and heated at 95 ° for 20 minutes. Magna-whirl Blue M (Blue Island, Illinois) water bath has been found to be very satisfactory. The samples are then transferred to 10-ml volumetric flasks with 50% ethanol and mixed; the optical density is read at 570 m~. 1~Available from Bellco Glass, Inc., Vineland, New Jersey. These tubes are gently tapered and beaded at the top.
84
ANALYTICAL METHODS
[S]
Reagent. The ninhydrin reagent is made as follows: Solution 1: 4% ninhydrin in Methyl Cellosolve (ethylene glycol monomethyl ether) Solution 2: 4 M sodium acetate containing 1% glacial acetic acid Solution 3:0.01 M KCN in water Solution mixture: Four milliliters of solution 1 is mixed with 1 ml of solution 2. To 4 ml of the resultant solution is added 0.10 ml of solution 3.
Notes. 1. The volume of serum for one experiment is removed sterilely from the stock and is centrifuged for 1 hour at 10,000 rpm at 4 °. If there is any lipid it is removed by suction with a capillary pipette. The clear serum is then decanted from any insoluble debris. 2. The volume in which the antigen is added should be kept small to minimize the loss of precipitate through solubility. The volume of serum used depends on the concentration of antibody. F o r very low titer sera, 1-ml aliquots of undiluted serum have been used; for very high titer sera 0.1 ml of 1:100 dilution with saline has been used. 3. All pipetting should be performed with to-deliver micropipettes. Lang-Levy type has been used exclusively in these laboratories. 4. Each precipitin curve should be set up with control tubes containing no added antigen. 5. Analytical grade ammonium sulfate is used as a standard with each series of determinations. A solution of ammonium sulfate of 40 t~g N per milliliter is used; 2 and 4 t~g N aliquots are taken when the amount of precipitate will be 1-10 #g N. 6. The volume of all reagents can be doubled, extending the range to 30 ~g N. Standards should be increased proportionately. 7. This method of N analysis has been used (a) to determine % N of polysaccharides during purification; (b) in determining N : P ratio of polysaccharides during chromatography; and (c) in determining N content of vaccines. 8. When oligosaccharide~ are added for inhibition, the addition is made before the addition of antigen (step 1 of procedure just described) and all other steps are the same. The 1 hour incubation of inhibitor with antibody has been found to be unnecessary in the systems studied by the author. 9. The washed specific precipitate can also be dissolved in 0.25 M acetic acid 1~ and read at 277 mt~ or assayed by the micro-Folin procedure. 17 1~D. Gitlin, J. Immunol. 62, 437 (1949). 17E. A. Kabat and G. Schiffman, J. Immunol. 88, 782 (1962).
[6]
NEW COLORIMETRIC METHODS OF SUGAR ANALYSIS
85
10. The procedure for N determination is based on one described by Rosevear and Smith is and subsequently modified. ~,19 11. An extremely sensitive procedure for measuring complement fixation and complement fixation inhibition has been described. 2° This procedure has been used by many laboratories with great success. l~j. w. Rosevear and E. I. Smith, J. Biol. Chem. 236, 425 (1961). 19G. Schiffman, E. A. Kabat, and W. Thompson, J. Am. Chem. Soc. 84, 463 (1962). _~oE. Wasserman and L. Levine, Y. Immunol. 87, 290 (1961).
[6]
New
Colorimetric
Methods
of Sugar Analysis
B y GILBERT ASHWELL
This chapter is designed to supplement the more complete description of colorimetric methods of sugar analysis which appeared in an earlier volume of this series (Vol. III [12]). Emphasis has been placed upon the inclusion of new and specific reactions for monosaccharides of biological significance. Several improved modifications, currently employed in the determination of uronic acids, amino sugars, and carbohydrate-protein complexes have not been included here since they are described in detail elsewhere in this volume. I. Determination of Tetroses 1 Principle. In general, the development of specific color reactions for sugars has been based upon the formation of furfural derivatives upon heating an aqueous solution of the sugar with a strong acid. Subsequent complexing of the derivative with an appropriate organic developer provides a basis for the formation of a color which is characteristic for a given class of sugars. The inability of the 4-carbon sugars to form furan derivatives under these conditions retarded the development of a specific assay procedure. The method described here 1 is based upon the observation that tetroses can combine with the breakdown products of higher sugars to yield a chromophore with a characteristic absorption spectrum. Reagents
H~S04. Six parts of concentrated, reagent grade H2S04 are added slowly, and with chilling, to one part of water. Fructose, prepared daily as a 0.1% aqueous solution L-Cysteine hydrochloride monohydrate, freshly prepared as a 3% aqueous solution ' Z. Disehe and M. R. Dische, Biochim. Biophys. Acla 27, 184 (1958).