JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1975, p. 510-515Copyright © 1975 American Society for Microbiology

Vol. 2, No. 6Printed in U.S.A.

Counterimmunoelectrophoresis of Pneumococcal Antigens:Improved Sensitivity for the Detection of Types VII and XIV

JOHN P. ANHALT* AND PAULINE K. W. YU

Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation,Rochester, Minnesota 55901

Received for publication 28 August 1975

Rapid identification of Streptococcus pneumoniae has been reported usingcounterimmunoelectrophoresis for the detection of specific capsular antigens inserum, cerebrospinal fluid, and urine. Previous clinical studies have failed todetect type VII or XIV pneumococcal antigen. These two types, however, ac-count for a significant portion of pneumococcal disease. The incorporation of asulfonated derivative of phenylboronic acid in the buffer system provides amethod for the sensitive detection of these types in artificial mixtures withoutgreatly reducing the sensitivity for the detection of other pneumococcal types. Aproblem with false positives encountered using human serum and barbitalbuffer was reduced by the use of buffer containing sulfonated phenylboronic acid.

The detection of bacterial antigen in clinicalspecimens is gaining widespread acceptance asa sensitive method for the rapid diagnosis ofinfection. Antigen of Streptococcus pneumo-niae has been detected by counterimmunoelec-trophoresis (CIE) in a significant percentage ofinfections due to this organism (1, 4, 5, 9, 14). Inthe combined reports of Coonrod and Rytel (4)and Tugwell and Greenwood (14), representing128 cases of pneumococcal pneumonia, antigenwas detected and the serotype was determinedby CIE in 77 cases. Not a single case of infectiondue to serotype VII or XIV was reported (10).This finding is consistent with the earlier re-port by Kenny et al. (9) that these serotypeswere not detectable by the methods employed.They are, however, among the most commonserotypes responsible for pneumococcal disease(8, 11, 12), and we believe that development of aCIE system capable of detecting these serotypescould increase the sensitivity of the test for thedetection of pneumococcal infection.The type-specific pneumococcal capsular anti-

gens are polysaccharides. The chemical struc-tures of types VII and XIV (17) indicate thatthey are neutral and would probably not showadequate anodal migration under the condi-tions used for CIE. The observed cathodal mobil-ity of types VII and XIV (2, 9) is consistent withthe neutral nature of these polysaccharides andcould result from electroendosmosis. Neutralcarbohydrates can be made to migrate anodallyduring electrophoresis in alkaline boratebuffers owing to anionic complexes formed withthe borate ions (6). Complex formation is de-

pendent on the stereochemistry of the carbohy-drate and the pH of the medium. Utilization ofderivatives of boric acid can modify the specific-ity for the reaction and provide for efficient com-plex formation at lower pH (7, 16).Our studies were designed to evaluate the

effect of borate buffers on improving the sensi-tivity for detection of pneumococcal capsulartypes VII and XIV by CIE.

MATERIALS AND METHODSInfrared spectra were determined on a Beckman

model IR18 spectrophotometer and calibratedagainst polystyrene. Melting points were taken witha Fisher-Johns hot-stage apparatus and are uncor-rected. Elemental analyses were performed by Gal-braith Laboratories (Knoxville, Tenn.). CIE resultswere observed with a 10x hand lens and recordedphotographically using dark-field illumination andKodak high-contrast copy film.

Chemical reagents. Phenylboronic acid (ben-zene boronic acid) was purchased from AldrichChemical Co. (Milwaukee, Wis.) and ColumbiaChemical Co. (Columbia, S.C.). Sodium barbitaland barbital were from Sigma Chemical Co. (St.Louis, Mo.). All other reagents were of reagentgrade, except acetone used for crystallization, whichwas National Formulary grade.

Preparation of SPB. Sulfonated phenylboronicacid (SPB) was prepared by the method of Gareggand Lindberg (7) from 49.2 g (0.40 mol) of phenylbo-ronic acid. Difficulties were encountered during thecrystallization step, and it was found that the addi-tion of an equal volume or greater of acetone to theethanol-water solution increased yield. The yield ofSPB was 33.2 g (0.15 mol, 38%). Physical data havenot been reported for this compound. We thereforereport our data: the compound did not melt below

510

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CIE OF PNEUMOCOCCAL TYPES VII AND XIV 511

300 C; infrared spectra (KBr) were 620, 1040, and1190 cm-'. Data calculated for C6H6BO5SNa were: C,32.19; H, 2.70; B, 4.82. Data actually found were: C,32.42; H, 2.65; B, 4.70.

Antigens and antisera. Purified pneumococcalpolysaccharides for types III, VII-L (American type51), and XIV were kindly provided by D. W. McCoyof Lederle Laboratories (Pearl River, N.Y.). Lotanalyses provided with these polysaccharidesshowed approximately 5% protein and less than 2%nucleic acid impurities. Stock solutions containing50 j.g of each antigen per ml were prepared in 0.001M phosphate-buffered 0.85% saline, pH 7.4 (PBS).All solutions of antigen contained sodium azide(0.05%) as preservative and were stored at 5 C. Nor-mal human serum (NHS) was obtained by venipunc-ture from a healthy laboratory worker. Cerebrospi-nal fluid (CSF) was obtained from a patient inciden-tal to myelography. Serial twofold dilutions of eachantigen were prepared in PBS and NHS from the 50jig/ml antigen stock solutions. Appropriate concen-

trations of antigen in CSF were prepared by theaddition of 50 ,ug/ml antigen stock solutions. Serialtwofold dilutions in CSF were not prepared becauseof the small amount of CSF available.

Antiserum active against the 83 pneumococcaltypes (omniserum) and type-specific antisera were

purchased from the Statens Seruminstitut (Copen-hagen, Denmark). These antisera were stored at 5 Cand used undiluted.

Concentration of pneumococcal antigens addedto urine. Midstream urine was collected from a

healthy laboratory worker. Sodium azide (0.05%)was added as a preservative, and the solution was

filtered through a membrane filter (pore size, 0.45,um). The stock solutions of antigen were added tothis urine to the appropriate concentrations. Urinecontaining added antigen and blank urine were con-

centrated by the method of Coonrod and Rytel (3);however, PBS was used for resuspension of the pre-cipitated material, and undissolved material was

not removed prior to use in CIE.Preparation of buffers. Barbital buffer (0.05 M,

pH 8.6) was prepared from 6.2 g (33.7 mmol) of 5,5'-diethyl-barbituric acid (barbital), 34.2 g (166 mmol)of sodium barbital, 1.6 g (5.1 mmol) of calcium lac-tate pentahydrate, and 4 g (30 mmol) of NaN3 dis-

solved to 4 liters in distilled water. SPB (0.024 M)-barbital (0.05 M) buffer (pH 8.6) was prepared from5.44 g (24.3 mmol) of SPB, 10.3 g (50 mmol) of sodiumbarbital, and 0.4 g (1.2 mmol) of calcium lactatepentahydrate dissolved to 1.0 liter. Adjustment ofpH was usually not necessary. The composition ofother buffers employed is summarized in Table 1. Ingeneral, buffers that contained barbital and deriva-tives of boric acid were prepared by the addition of atriple-strength solution of the boronic acid to a tri-ple-strength solution of sodium barbital containingcalcium lactate (1.2 g/liter); pH was adjusted using 1N NaOH, followed by dilution to the appropriateconcentration with distilled water. Borate bufferswere prepared by titration of a concentrated solutionof sodium borate with boric acid to the proper pH,followed by dilution to the appropriate concentra-tion. Alternatively, for borate buffers of low pH, a

concentrated boric acid solution was titrated with 1N NaOH to the proper pH and diluted. Buffers con-

taining only SPB or phenylboronic acid (PBA) were

prepared by titration of a triple-strength solution ofthe boronic acid containing calcium lactate (1.2g/liter) with 1 N NaOH and diluted.

CIE. The apparatus used for electrophoresis was

constructed from a solid plastic block. Each bufferreservoir contained 80 ml of buffer. Power suppliesfrom Lambda Electronics (Melville, N.Y.) (modelLL905) and Schlumberger Products (St. Joseph,Mich.) (Heathkit model IP17) were used interchange-ably. Agarose from L'Industrie BiologiqueFrancaise (IBF) was purchased through Fisher Sci-entific Co. (Chicago, Ill.) (lot 744181). SeaKem aga-

rose (lot 10645) was purchased from Marine Colloids(Rockland, Me.). A premarketing sample of agaroseisolated from Gracilaria agar (RE7257) was a giftfrom Marine Colloids. A melted solution of the ap-

propriate agarose and buffer (6 ml) was poured ontoa glass slide (50 by 75 mm) on a leveled surface.After the agarose had gelled, the slide was stored ina moist chamber at 5 C and used within 48 h. Aplastic template was used for punching antigen andantibody wells in the agarose. Five pairs of wells, 3mm in diameter, were spaced equally across thewidth of the slide. Antigen and antibody wells were

separated 2 mm edge to edge. The wells were filledto the brim using disposable capillary micropipettes.

TABLE 1. Buffer composition

Concentration range (mM)bBuffer" pH range

BAR BOR PBA SPB

BAR 50 8.2-9.0BOR 50-300 8.2-9.2PBA 20-90 8.6-9.0SPB 20-50 8.2-9.0BAR/BOR 30-50 10-70 8.2-9.0BAR/PBA 30-70 10-50 8.0-9.0BAR/SPB 30-50 5-50 8.0-9.0

BAR, Barbital; BOR, boric acid.b Concentrations are expressed as the total acid plus salt, and for buffers containing derivatives of boron

they represent equivalents of boron. In buffer solutions that resulted in high electroendosmosis, NaCl (10 to25 mM) was added to decrease electroendosmosis and deformation of the agarose gel.

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512 ANHALT AND YU

Approximately 15 ,ul of solution was required to filleach well. Antigen was placed in wells nearest thecathode. Strips of Telfa pad (Kendall Co., Chicago,Ill.) were used as connecting wicks to the buffercompartments. Depending on the buffer system em-ployed, electrophoretic conditions varied slightly. Ingeneral, electrophoresis was performed for 1 h usinga current of 12 to 15 mA for each slide. With 0.024 MSPB-0.05 M barbital (pH 8.6), a current of 14 mA foreach slide resulted in a gradient of about 6 V/cmmeasured in the gel (60 V at the source). Afterelectrophoresis, the slides were observed immedi-ately, after 1 to 4 h at 5 C in a moist chamber, andafter overnight washing with 0.85% saline.

Determination of optimal buffer composition.More than 50 buffer solutions covering the range ofcompositions shown in Table 1 were evaluated fordetection of type VII pneumococcal polysaccharide.Agarose gels (1% wt/vol; IBF) were prepared witheach buffer. For electrophoresis done at pH 9.0 or9.2, the electrolyte compartments contained thesame buffer as used in the agarose gel. Otherwise,electrophoresis was performed with 0.05 M barbitalof the same pH as used for the agarose gel in theelectrolyte compartments. Serial twofold dilutionsof antigen in PBS were run, using each buffer todetermine sensitivity. The optimal compositionswere determined by independently varying the bo-rate or boronate concentration and pH using sodiumbarbital or NaCl to maintain a sodium ion concentra-tion of 50 to 75 mM. Sensitivity for pneumococcalpolysaccharide types III and XIV was determined inthe same manner for selected buffers.

Comparison of agarose from different sources.Three lots of agarose were evaluated: (i) IBF agaroseobtained from a Gracilaria agar; (ii) SeaKem aga-rose (Marine Colloids) isolated from a Gelidiumagar; and (iii) a premarketing sample of agarose(RE7257) obtained from Marine Colloids isolatedfrom Gracilaria agar. Agarose gels containing 1%

(wt/vol) IBF agarose or 0.75% (wt/vol) SeaKem orRE7257 agarose were prepared using barbital (0.05M, pH 8.6) and 0.024 M SPB-0.05 M-barbital (pH 8.6)buffers. Sensitivity for detection of pneumococcalpolysaccharides dissolved in PBS and NHS was de-termined using each agarose.

False positives obtained with NHS. The human

J. CLIN. MICROBIOL.

serum used in this report for preparing solutions ofpneumococcal polysaccharides gave a precipitatewith pneumococcal antisera in the absence of addedantigen. This serum was obtained from a male sub-ject with type 0, Rho-negative blood. A partial phe-notype revealed that he was Lea and Leb negativeand a secretor. Serum from this subject was testedagainst omniserum and types VII and XIV monova-lent antisera using IBF agarose (1% wt/vol) andbarbital (0.05 M, pH 8.6) buffer.

RESULTS AND DISCUSSION

Buffer composition. The minimal detect-able concentrations of type VII polysaccharidefound using IBF agarose and different buffersare shown in Table 2. Sensitivity for detectionof type VII polysaccharide generally increasedin the pH range of 8.2 to 9.2; however, underthe more alkaline conditions, sensitivity for de-tection of type III polysaccharide decreased. Op-timization of buffer conditions, therefore, re-quired a balance of the competing effects ofborate-polysaccharide complex formation (6, 16)with inhibition or dissolution of antigen-anti-body precipitates (13). Buffer systems incorpo-rating SPB gave greater sensitivity than didsystems containing PBA at the same pH andconcentration of boronic acid. This observationis consistent with the assumption that an in-creased negative charge, resulting from ioniza-tion of the sulfonate group in complexes withSPB (7), would result in greater mobility of aneutral polysaccharide at lower ratios of boron-ate to polysaccharide.A major disadvantage of buffer systems con-

taining optimal concentrations of PBA was en-countered using antisera from the StatensSeruminstitut. These antisera are normallysupplied containing methylene blue as a color-ing agent (E. Lund, Statens Seruminstitut,Copenhagen, Denmark; personal communica-tion). With such antisera, a dark blue band

TABLE 2. Optimal buffer compositions for detection of type VII polysaccharide in IBF agarose

Composition (mM)b Minimal detectableBuffer" pH concn of type VII poly-

BAR BOR PBA SPB saccharide (pg/ml)BAR 50 8.2-8.8 NDcBOR 100 9.2 3.2PBA 75 8.8 0.8SPB 37 8.6 1.6BAR/BOR 38 50 9.0 1.6BAR/PBA 40 37 8.8 0.8BAR/SPB 50 24 8.6 0.8

BAR, Barbital; BOR, boric acid.b Concentrations are expressed as the total acid plus salt, and for buffers containing derivatives of boron

they represent equivalents of boron.c ND, Not detectable at up to 25 ,ug of antigen per ml using polyvalent or monospecific antisera.

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CIE OF PNEUMOCOCCAL TYPES VII AND XIV 513

formed midway between the antigen and anti-body wells and obscured detection of a precipi-tate. The blue band could be removed by wash-ing the agarose with saline or water after elec-trophoresis, but this required additional time.Antisera without methylene blue were notevaluated using optimal concentrations of PBAbecause PBA was found to be a potent lacri-mator. SPB did not have this disadvantage,and in subsequent experiments the optimal0.024 M SPB-0.05 M barbital buffer (pH 8.6)was used.Comparison of agarose from different

sources. Representative CIE results for thedetection of type VII polysaccharide in PBS andNHS using SPB-barbital buffer and each of theagarose preparations are shown in Fig. 1. Com-parison of the results obtained with PBS solu-tions of antigen showed only slight differences(one dilution or less) in sensitivity with thedifferent agarose preparations. Greater differ-

a

b

c

FIG. 1. Comparison of agarose from differentsources for detection of type VII polysaccharide inPBS and NHS. Antigen concentrations (left to right):6.4 pAg/ml in NHS; 32 pg/ml in NHS; 1.6 pAglml inNHS; and 1.6 pAglml in PBS. (a) One percent IBFagarose; (b) 0.75% SeaKem agarose; (c) 0.75%RE7257 agarose. Upper well of each pair containedantigen solution.

ences in sensitivity were observed using serumsolutions of antigen. In this case, RE7257 aga-rose gave superior sensitivity to the other aga-rose preparations. In addition, the precipitinlines observed using RE7257 agarose were moredistinct and were located farther from the anti-gen well. In SeaKem agarose, isolated fromGelidium agar, a precipitin arc formed on thecathodal side of the antigen well containingNHS (Fig. 2). The anodal tailing of this arcinterfered with the detection of weak antigen-antibody reactions located close to the anodalside of the antigen well. This phenomenon oc-curred to a lesser extent in IBF and RE7257agarose isolated from Gracilaria agar. SeaKemand RE7257 agarose are purified by similarprocedures (R. B. Cook, Marine Colloids, Rock-land, Me.; personal communication). There-fore, the intensity of this cathodal arc may berelated to the variety of agar used for isolationof the agarose.

False positives. The NHS in these experi-ments gave a precipitin line in the absence ofadded antigen between the antigen and anti-body wells when barbital buffer was used (Fig.3). This effect was independent of the source ofthe agarose; however, various degrees of precip-itate formation were observed with sera fromdifferent individuals. Since soluble blood groupsubstances had been reported to cross-reactwith pneumococcal type XIV antisera (15), weassumed that this effect might be related toblood group substances in serum. In this con-text, NHS reacted with omniserum and mono-valent type XIV antiserum but not with mono-valent type VII antiserum (Fig. 4). A largenumber of sera have not been tested; however,our preliminary results are consistent with theassumption that cross-reactivity with soluble

a b cFIG. 2. Interaction ofNHS with different prepara-

tions of agarose. (a) One percent IBF agarose; (b)0.75% SeaKem agarose; (c) 0.75% RE7257 agarose.Upper well of each pair contained NHS.

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514 ANHALT AND YU

a

FIG. 3. False-positive reactions observed withNHS. (a) Barbital buffer (0.05 M, pH 8.6); (b) SPB-barbital buffer (optimal composition). Upper well ofeach pair contained NHS.

a b

FIG. 4. Reaction of NHS with different pneumo-coccal antisera. (a) Type XIV antiserum; (b) type VIIantiserum; (c) omniserum. Upper well of each paircontained NHS.

blood group substances in serum may lead tofalse-positive reactions for pneumococcal anti-gens. A precipitin line was not observed usingSPB-barbital buffer, although a less distinctprecipitate was sometimes present (Fig. 3).

Concentration of peumococcal antigens inurine. A 20-fold concentration of pneumococcalpolysaccharide in urine using ethanol precipita-tion has been reported (3). Our results con-

firmed the earlier observation of increased sen-

sitivity. The maximal sensitivity of the methodwas not determined using SPB-barbital buffer;however, precipitin lines formed were muchmore intense than for the unconcentrated ur-

ine. No problem with interfering substances inthe concentrated urine was encountered.Comparison of barbital and SPB-barbital

buffers. Comparative results for sensitivity for

the three antigen types tested using barbitaland SPB-barbital buffers are summarized inTable 3. The highest concentration of antigentested in each case was 25 ,ug/ml. Neither typeVII nor XIV was detectable using barbitalbuffer and omniserum. Type XIV antigen, how-ever, could be detected at 6.4 ,g/ml in PBSusing barbital buffer and monovalent antise-rum. In this case, the precipitin line was faintand near the antibody well. Anodal mobility oftype XIV antigen has been reported previouslyby Coonrod and Rytel (3); however, this is inconflict with other reports of cathodal mobilityfor this antigen (2, 9). Our results are consist-ent with the assumption that heterogenicity ordecomposition in the antigen preparation couldresult in the presence of an antigenically simi-lar material that has good anodal mobility andis detectable only with more potent monovalentantiserum.

Conclusions. CIE using barbital buffer wasineffective in detecting pneumococcal types VIIand XIV polysaccharides. The incorporation ofSPB in the buffer greatly increased the sensi-tivity of CIE for these two capsular types with-out greatly reducing the sensitivity for type III.In addition, the optimal buffer composition was0.05 M barbital containing 0.024 M SPB at pH8.6. A problem with false-positive reactionsthat occurred with some sera using a barbitalbuffer was nearly eliminated by the use ofSPB-barbital buffer. An adverse interaction ofserum with certain lots of agarose was observedand may be related to the source of the agarfrom which the agarose is extracted. Best re-sults were obtained using an agarose isolatedfrom Gracilaria agar and provided by MarineColloids.TABLE 3. Comparison of barbital and SPB-barbital

buffers in RE7257 agarose

Minimal detectable anti-

Antigen Solution gen concn (p.g/ml)Atigen Solutiontype Barbital SPB-barbi-

Barbitala talb

III PBS 0.012 0.05III Serum 0.05 0.05III CSF 0.012 0.05VII PBS NDc 0.4VII Serum ND 1.6VII CSF ND 0.4XIV PBS ND 0.8XIV Serum ND 1.6XIV CSF ND 0.4

0.05 M, pH 8.6.b0.024 M SPB, 0.05 M barbital, pH 8.6.e ND, Not detectable at up to 25 lug of antigen per

ml.

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CIE OF PNEUMOCOCCAL TYPES VII AND XIV 515

ADDENDUM

Shortly after the development of this buffer sys-tem, S. pneumoniae was isolated by our laboratoryfrom the CSF of a patient with meningitis. The CSFobtained from this patient was negative by CIEusing barbital buffer but was strongly positive usingSPB-barbital buffer. Capsular typing by the quel-lung method showed that the organism was typeVII.

LITERATURE CITED1. Bartram, C. E., Jr., J. G. Crowder, B. Beeler, and A.

White. 1974. Diagnosis of bacterial diseases by detec-tion of serum antigens by counterimmunoelectropho-resis, sensitivity, and specificity of detecting Pseu-domonas and pneumococcal antigens. J. Lab. Clin.Med. 83:591-598.

2. Coonrod, J. D. 1974. Physical and immunologic proper-

ties of pneumococcal capsular polysaccharide pro-

duced during human infection. J. Immunol.112:2193-2201.

3. Coonrod, J. D., and M. W. Rytel. 1973. Detection oftype-specific pneumococcal antigens by counterimmu-noelectrophoresis. I. Methodology and immunologicproperties of pneumococcal antigens. J. Lab. Clin.Med. 81:77Q-777.

4. Coonrod, J. D., and M. W. Rytel. 1973. Detection oftype-specific pneumococcal antigens by counterimmu-noelectrophoresis. II. Etiologic diagnosis of pneumo-coccal pneumonia. J. Lab. Clin. Med. 81:778-786.

5. Fossieck, B., Jr., R. Craig, and P. Y. Paterson. 1973.Counterimmunoelectrophoresis for rapid diagnosis ofmeningitis due to Diplococcus pneumoniae. J. Infect.Dis. 127:106-109.

6. Foster, A. B. 1957. Zone electrophoresis of carbohy-drates. Adv. Carbohydr. Chem. 12:81-115.

7. Garegg, P. J., and B. Lindberg. 1961. Paper electropho-resis of carbohydrates in sulphonated phenylboronicacid buffer. Acta Chem. Scand. 15:1913-1922.

8. Kaiser, A. B., and W. Schaffner. 1974. Prospectus: theprevention of bacteremic pneumococcal pneumonia; aconservative appraisal of vaccine intervention. J.Am. Med. Assoc. 230:404-408.

9. Kenny, G. E., B. B. Wentworth, R. P. Beasley, and H.M. Foy. 1972. Correlation ofcirculating capsular poly-saccharide with bacteremia in pneumococcal pneu-monia. Infect. Immun. 6:431-437.

10. Lund, E. 1960. Laboratory diagnosis of Pneumococcusinfections. Bull. W.H.O. 23:5-13. (The Danish sys-tem of nomenclature is used in this report.)

11. Lund, E. 1971. Distribution of pneumococcus types atdifferent times in different areas, p. 49-56. In M.Finland, W. Marget, and K. Bartmann (ed.), Bacte-rial infections: changes in their causative agents,trends, and possible basis. Bayer-Symposium III, Co-logne, 1970. Springer-Verlag, New York.

12. Mufson, M. A., D. M. Kruss, R. E. Wasil, and W. I.Metzger. 1974. Capsular types and outcome of bacter-emic pneumococcal disease in the antibiotic era.Arch. Intern. Med. 134:505-510.

13. Svensson, S., S. G. Hammarstr6m, and E. A. Kabat.1970. The effect of borate on polysaccharide-proteinand antigen-antibody reactions and its use for thepurification and fractionation of crossreacting anti-bodies. Immunochemistry 7:413-422.

14. Tugwell, P., and B. M. Greenwood. 1975. Pneumococcalantigen in lobar pneumonia. J. Clin. Pathol. 28:118-123.

15. Watkins, W. M. 1966. Blood-group substances: in theABO system the genes control the arrangement ofsugar residues that determine blood-group specific-ity. Science 152:172-181.

16. Weigel, H. 1963. Paper electrophoresis of carbohy-drates. Adv. Carbohydr. Chem. 18:61-97.

17. Woodside, E. E., and J. B. G. Kwapinski. 1974. Micro-bial polysaccharides, p. 129-184. In J. B. G. Kwapin-ski, S. G. Bradley, E. R. Brown, P. R. Burton, H.Fraenkel-Conrat, N. Kordova, W. M. O'Leary, F. J.Reithel, M. R. J. Salton, R. Storck, and E. E. Wood-side (ed.), Molecular microbiology. John Wiley &Sons, New York.

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