JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1985, p. 808-814 Vol. 22, No. 50095-1137/85/110808-07$02.00/0Copyright C) 1985, American Society for Microbiology

Determination of Antibodies to Pneumococcal C Polysaccharide inPatients with Community-Acquired PneumoniaHANS HOLMBERG,'* AUD KROOK,2 AND ANN-MARGRET SJOGREN3

Department of Clinical Microbiology and Immunology' and Department of Infectious Diseases,2 Orebro Medical CenterHospital, S-701 85 Orebro; and Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm,3 Sweden

Received 1 April 1985/Accepted 11 July 1985

The pneumococcal C polysaccharide (PnC) is species specific and believed to be a cell wall component of allcapsular types. Antibodies against PnC in human sera have been demonstrated previously, but the question ofwhether a rise in these antibodies occurs during pneumococcal infections has not been investigated. We usedan indirect enzyme-linked immunosorbent assay (ELISA) for the estimation of PnC antibodies in 124hospital-treated patients with pneumonia. In 3 of 6 patients with pneumococcal bacteremia and in 17 of 44patients with S. pneumoniae isolated in the blood, sputum, or nasopharynx, a significant rise in antibody levelswas recorded, accounting for a sensitivity of 38.6%. Of 35 patients with pneumonia of other known orsuspected etiology, 1 gave a positive result, corresponding to a specificity of 97.1%. In addition, 3 of 8 patientswith PnC antigen in the sputum as the only etiological finding and 5 of 37 patients with unknown etiology gavepositive results. The PnC antibodies did not seem to have any protective capacity against pneumonia caused bypneumococci. The ELISA, in which only one antigen preparation was used, was more simple than other testsin which traditional capsular antigen preparations are used. It might therefore be used as a supplementalmethod in the diagnosis of pneumococcal pneumonia. The problems involved in expressing serum titersobtained with the ELISA are discussed.

Streptococcus pneumoniae is a common cause of pneu-monia, although the exact incidence and mortality is notknown. In the United States, the attack rate for pneumococ-cal pneumonia is estimated to be 1 to 5 per 1,000 persons perannum, with mortality between 4 and 30%, depending onbacteremia, age, and underlying diseases (2). Difficulties indetermining the frequency of pulmonary infections of pneu-mococcal etiology in hospitalized adult patients are evidentin view of reported figures, which range from 11.5 to 76%(20, 29). Schreiner et al. (25) isolated pneumococci in 28.9%of pulmonary infections by cultivation from transtrachealaspirates. Swedish studies have shown frequencies from 34to 45% (10, 17). We cultured pneumococci from blood,sputum, and nasopharynx samples in 35.4% of 147 hospital-treated patients with community-acquired pneumonia. Anadditional 5.4% of patients gave positive results in an antigentest for pneumococci with an enzyme-linked immunosorbentassay (ELISA) (15).A microbiological diagnosis is often difficult to establish in

pneumonia, and different diagnostic procedures have beenused. The use of different populations and criteria fordiagnosis makes it difficult to compare the results of differentinvestigations. A serological diagnosis of pneumococcalpneumonia would be a valuable method for verifying posi-tive culture or antigen findings. Antibodies against type-specific (capsular) antigens have been measured after naturalinfection and after vaccination with pneumococcal polysac-charide vaccines. Current methods include indirect hemag-glutinati6n (1, 8), the radioimmunoassay (7, 9, 22, 24), andthe ELISA (3, 7).A species-specific antigen of S. pneumoniae, C poly-

saccharide (PnC), is believed to be a cell wall component ofpnetmococcal strains of all capsular types. The structure ofPnC was recently established by Jennings et al. (16).PnC contains phosphorylcholine (6, 16). Antibodies

* Corresponding author.

against phosphorylcholine found in normal mouse serumwere shown to be protective against intravenous infectionwith type 3 S. pneuimoniae (5). Subsequent studies byYother et al. (30) and Szu et al. (26) showed thatphosphorylcholine hybridoma antibodies protect mice fromfatal infections with certain, but not all, tested pneumococ-cal types. The existence of PnC antibodies and phos-phorylcholine antibodies in human sera has been establishedpreviously (12, 14, 21), but the question of whether theseantibodies have any protective capacity or could be used fordiagnostic purposes has to our knowledge not been previ-ously studied.

In our efforts to improve the process of laboratory diag-nosis of pneumococcal pneumonia, we used an indirectELISA to study PnC antibodies in 124 hospital-treatedpatients with pneumonia.

MATERIALS AND METHODS

Patients and sera. A study of hospital-treated, community-acquired pneumonia was performed in the Department ofInfectious Diseases, Orebro Medical Center Hospital. A totalof 147 patients with clinical and radiologically verifiedpneumonia were accepted for the study. Diagnostic proce-dures have been described elsewhere (15). Altogether, acuteand convalescent sera from 124 patients were obtained. Acutesera were taken at the time of arrival on the ward, andconvalescent sera were taketl 4 weeks later. All sera werestored at -20°C until assayed.

Antigen preparation. The C polysaccharide was preparedfrom a pneumococcal C mutant strain, CSR-SCS-2 clone 1(kindly provided by J. Henrichsen, Statens Seruminstitut,Copenhagen, Denmark). This strain has been reported tocarry a small capsule consisting of C polysaccharide (4, 23).The pneumococci were grown with slow magnetic stirringfor 11 h at 37°C in 1-liter portions of brain heart infusionmedium (Difco Laboratories, Detroit, Mich.). Each culturewas tested for bacterial contamination, and the supernatants

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ANTIBODIES TO PnC IN PNEUMONIA 809

were removed by centrifugation. The bacteria were sus-pended in distilled water at a concentration of 20 g (dryweight) per liter and extracted with an equal volume of 90%phenol at 68°C for 15 min (28). The polysaccharide prepara-tion was dialyzed against running tap water for 24 h andlyophilized. The protein content of the C-polysaccharidepreparation was 17% as measured by the method of Lowryet al. (19). The protein fraction could be removed withprotease treatment, but then the C polysaccharide did not fixsufficiently to the polystyrene in the micro-ELISA platesused in the assay. The C-polysaccharide antigen used hasbeen studied with nuclear magnetic resonance by P. E.Jansson of the Department of Organic Chemistry, ArrheniusLaboratory, University of Stockholm, Stockholm, Sweden(personal communication). A similar pattern in its essentialsin comparison with the C preparation (7FC5; 1983) of G.Schiffman of the Department of Microbiology, DownstateMedical Center, Brooklyn, N.Y., was demonstrated.ELISA procedures. Micro-ELISA polystyrene plates (Dy-

natech Laboratories, Inc., Alexandria, Va.) were coatedwith 100 [lI of the antigen preparation (75 mg/liter) overnightat 37°C in a moist chamber and then at 4°C for at least 3 days.The plates were stored for up to 6 weeks at 4°C. During thistime, the activity was unchanged. Before use, the plateswere washed three times with phosphate-buffered saline (pH7.2) containing 0.05% Tween 20 (Pharmacia, Uppsala, Swe-den) and 0.5% bovine serum albumin (United States Bio-chemical Corp., Cleveland, Ohio). Twofold dilutions of serain phosphate-buffered saline-Tween-bovine serum albuminwere prepared, starting at a dilution of 1:100. All sera fromone patient were put into the same microtiter plate. The testswere performed in duplicate. Samples (100 ,ul) of the serumdilutions were added to the sensitized plates and incubatedin a moist chamber for 1 h at 37°C. After the plates had beenwashed three times as before with phosphate-buffered sa-line-Tween-bovine serum albumin, 100 pLI of swine anti-human immunoglobulin M (IgM) or IgG conjugated withalkaline phosphatase and diluted 1:250 (Orion Diagnostica,Helsinki, Finland) was added and incubated in a moistchamber for 1 h at 37°C. A substrate solution was preparedimmediately before use by dissolving p-nitrophenyl-phosphate (1 g/liter; Sigma 104; Sigma Chemical Co., St.Louis, Mo.) in a 1 M diethanolamine buffer (pH 9.8) with 0.5mM MgCl. After the plates were washed as described above,100 pul of the substrate solution was added, and the plateswere incubated at 37°C for 60 min. The A405 was measuredwith a spectrophotometer (Titertek Multiskan; Flow Labo-ratories, Inc., McLean, Va.), without stopping the reaction.The mean value for the duplicates was used to calculate theresults. A positive control with a monoclonal phos-phorylcholine antibody, HPC M2 (11), was added to eachplate. In this case, a rabbit anti-mouse immunoglobulinconjugate was used. For blanking purposes, phosphate-buffered saline-Tween-bovine serum albumin with conjugateand substrate as described above was put into the wellswhich had not been coated with the PnC antigen.With the anti-human IgM conjugate, we obtained low

absorbance values, rendering calculations unreliable. De-spite the use of serum dilutions from 1:25, 1:50, etc., pairedsera from only 55 patients could be evaluated, showing a lowsensitivity and specificity for pneumococcal etiology. There-fore, these results will not be further discussed.

Expression of results. In preliminary tests, S-shaped dilu-tion curves were obtained in this ELISA system by using aline-log diagram. The value of net absorbance (absorbance inantigen-coated wells minus background absorbance in anti-

gen- and serum-free wells) was linearly related to the log ofthe serum dilution in the steepest part of every serumdilution curve (Fig. 1). For most sera, this occurred aroundthe absorbance value of 1.0. To minimize the effect ofabsorbance variability within and between each serum dilu-tion, we chose 1.0 as the read-off value. ELISA titers werethus defined as the reciprocal serum dilution which gave anabsorbance value of 1.0. The fold increase in antibodycontent from acute to convalescent serum was calculated bydividing the convalescent titer by the acute titer. Altogetherin a dilution of 1:100, acute and convalescent sera from 30patients gave absorbance values above 0.5 but not 1.0. Forthese 30 patients, we chose 0.5 as the read-off value in ourcalculation of the ELISA titers. Absorbance values below0.5 were obtained with sera from 14 patients, renderingmeaningful calculations not possible in our opinion. Thesera from these 14 patients were regarded as negative in thetest.

RESULTS

The increase in antibody levels from acute to convalescentsera in the groups with different etiology and with regard tothe location from which the pneumococci were isolated isshown in Fig. 2. With a fold increase of 1.5 or more, i.e., a50% higher ELISA titer in convalescent serum than in acuteserum, as the minimum level for a positive test, the resultswere as follows. The sera of 3 of 6 patients with S. pneumo-

2.0

1.5

E

0

z1o.0

0

3200 1600 800

RECIPROCAL SERUM TITER LOG

400 200 100

FlG. 1. Absorbance of acute and convalescent sera (4 weeks)from one patient with pneumonia and S. pneumoniae isolated in thesputum. ELISA titers are defined as the reciprocal serum dilutionwhich would give an absorbance of 1.0. The fold increase ofantibody content is calculated by dividing the convalescent titer bythe acute titer: 1,050/160 = 6.56.

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Group no lb

S. pneumoniaeisolated in sputum

Group no lc

S. pneumoniaeisolated in naso-

pharynx only

Group no ld

PnC - antigenin sputumCulture negative

Group no 2

No S. pneumoniaeisolatedUnknown etiology

Group no 3

No S. pneumoniaeisolatedOther etiology

0

0*@

0

0

w

C/,ui

z

0-j

IJ

IL

2.0 -

1.5

1.3

1.0

0.5

0 0

0O

*~ * r

Estimationnot possible

0

n=6 n=32 n=6 n=8 n=37 n=35

FIG. 2. Fold increase of PnC antibodies obtained by acute and convalescent sera from 124 patients with pneumonia. These are groupedaccording to etiology and with regard to the location of pneumococcal isolation.

niae bacteremia (group la) and of 13 of 32 (40.6%) patientswith S. pneumoniae isolated in the sputum (group lb) were

positive. In the latter group, 10 patients with double infec-tion (5 with S. pneumoniae and Haemophilus influenzae and5 with S. pneumoniae and influenza A) were included. Theserum from one of six patients with S. pneumoniae isolatedin the nasopharynx only was positive (group 1c). In addition,the sera from 3 of 8 patients with PnC antigen in the sputumas the only etiological finding (group ld) (15) and the sera

from 5 of 37 patients with unknown etiology (group 2) were

positive. Of 35 patients with other known or suspectedetiology to their pneumonia (group 3), 1 gave a positiveresult, for a specificity of 97.1%.

DISCUSSION

The problems involved in expressing serum titers obtainedwith an ELISA are not yet solved. One method is plainly tocompare the different absorbance values for acute andconvalescent serum obtained with the same serum dilution.This will give contradictory results, as shown in Fig. 3 (thesame serum dilution curves as in Fig. 1). Using the absor-bance values for the two sera in dilution 1:1,600, 1:400, and

1:100, we obtained a fold increase in absorbance of 3.67,2.51, and 1.37, respectively, leaving the question of whichserum dilution to choose when evaluating the result.With one serum dilution only, another method is to

calculate the absorbance increase between different serum

dilutions from a number of sera and extrapolate these resultsto all sera in the test. For example (Fig. 4), the increase inabsorbance for the acute sera in dilutions of 1:400 to 1:200and 1:200 to 1:100 is near 0.35, which could be interpreted toimply that such an increase in absorbance corresponds to an

increase in the ELISA titer of 100%. The difference inabsorbance for the convalescent and acute serum in dilution1:400 was 0.80 (1.33 to 0.53), resulting in an antibodyincrease of 0.80/0.35 = 2.29. However, with the same

calculation for the two sera in dilution 1:100, we get an

antibody increase of 1.37. Obviously, this method demandslinear and parallel serum dilution curves for all patients if itis to be used.

In a third method, a serial dilution is done to obtain an

endpoint titer often defined as twice the background value.This often means absorbance values around 0.2, and at leastin our experience, the variation in absorbance values ob-tained in this interval from the same serum in duplicate is

Group no la

S. pneumoniaeisolated in blood

2.5 -

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ANTIBODIES TO PnC IN PNEUMONIA 811

2.0

1.5 -

E

UJ

z

m 10-00n

0.5 -

2.0

I I I I 13200 1600 800 400 200 100

RECIPROCAL SERUM TITER LOG

FIG. 3. Absorbance of acute and cotivalescent sera (4 weeks)

from one patient with pneumonia and S. pnelimoniae isolated in thesputum. The absorbance increase is 0.77/0.21 = 3.67, 1.33/0.53 =

2.51, and 1.73/1.26 = 1.37 for reciprocal serum dilutions 1,600, 400,and 100, respectively.

15

E

UJ

z< 1.0co

0

0.5

CONVALESCENT

3200 1600 800

RECIPROCAL SERUM TITER LOG

1.5 -

E

ui1.

1.0

0~~~~~

ACUTE

0.5 ... ..................

1 3000 1 1 650 4501 1ioT

3200 1600 800 400 200 100

RECIPROCAL SERUM TITER LOG

FIG. 5. Absorbance of acute and convalescent sera (4 weeks)from one patient with pneumdnia and S. pneiumoniae isolated in thesputum. The antibody increase is calculated from the reciprQcalserum dilutions which would give absorbances of 0.5 and 1.2. Thefold increase of antibody content is calculated by dividing theconvalescent titer by the acute titer: 3,000/450 = 6.67 and 650/110 =

5.90, respectively.

significantly higher than that obtained with an absorbancevalue between 0.5 and 1.5.As described in Materials and Methods, our dilution

curves often had an S shape with a linear steep gradient in

the middle. Often this linear part crossed the absorbancevalue of 1.0, and this was the reason we chose 1.0 as theread-off value: to minimize the effect of absorbance variabil-ity within and between each serum. This method has theadvantage of resulting in almost the same increase indepen-dent of the read-off line wheni comparing only absorbancevalues within the linear part of the two serum curves. Forexample (Fig. 5), choosing 0.5 as the read-off line will resultin a fold increase of 6.67, choosing 1.2 will result in a

5.90-fold increase, and 1.0 will result in a 6.56-fold increase(Fig. 1). Because of the S-curved lines, any calculation withabsorbance values lower than 0.5 showed increased variabil-ity; we therefore preferred not to draw any conclusions onthe 14 patient sera involved, and they were calculated as

FIG. 4. Absorbance of acute and convalescent sera (4 weeks)ftom one patient with pneumonia and S. pneuinoniae isolated in thesputum. The antibody inctease is calculated by dividing the differ-ence between the absorbances of cohvalescent and acute sera by atheoretical absorbance difference of 0.35 for every dilution step. Forthe reciprocal serum dilutions of 400 and 100, this gives antibody

400 200 100 increases of 1.33-0.53/0.35 = 2.29 and 1.73-1.26/0.35 = 1.37,respectively.

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812 HOLMBERG ET AL.

Group no lb

S. pneumoniaeisolated in sputum

r

0

0*n = 32

Group no lc

S. pneutnoniaeisolated in naso-pharynx only

Group no id

PnC - antigenin sputumCulture negative

0f

00

n = 6

.

n = 8

Group no 2

No S. pneumoniaeisolatedUnknown etiology

S:0

:0

n = 37

Group no a

No S. pneumoniaeisolatedOther etiology

I

t

:-0

n = 35

FIG. 6. Absorbance values obtained from 124 acute sera jn dilution 1:400. The sera and patients are grouped according to etiology and withregard to the location of pneumococcal isolation.

negative. Making serum dilution curves for all sera is timeconsuming. We think that this will be necessary to obtainreliable results from this study. However, different methodsmay be justified for different tests.

1.0*

c.0

0

0.0

m10 2[:3

A

Convalescent

FIG. 7. Absorbance in the ELISA of acute and convalescentsera (final dilution, 1:100) from a pneumonia patient with pneumo-

coccal bacteremia. The sera were absorbed with two C polysaccha-ride preparations. (A) Schiffman's preparation 7FC5 (see the text).(B) Protease-treated phenol-water extract. Concentrations used: 1,unabsorbed; 2, 1 ,ug of C polysaccharide per ml; 3, 50 ,ug of Cpolysaccharide per ml.

In 3 of 6 patients with pneumococcal bacteremia (groupla), the only patients with unquestionable etiology, and in 13of 32 patients (40.6%) with pneumococci isolated in sputa(group lb), a convincing rise in antibody level was obtaiped.The nurnber of patients was small, but only one of sixpatients with S. pnelumoniae isolated in the nasopharynxonly (group lc) showed the sathe increase, which mayindicate that some of these six patients were carriers of S.pneumoniae and that their pneumonia was not caused bypneumococci. Three of eight patients (37.5%) with PnCantigen in their sputa as the only etiological finding (15) hada positive serological test, which in our opinion confirmedthe assumption of a pneumococcal etiology for their pneu-monia. Of the 37 patients with unknown etiology, 5 had a

positive serology. These five patients were all successfullytreated with penicillin and were afebrile within 2 days(median value; range, 1 to 5 days), indicating a possiblepneumococcal etiology. One patient of 35 with other knownor suspected etiologies of their pneumonia sl4owed an anti-body rise >50%. This one patient had a positive serology fprLegionella pneumophila with an increase of IgM (<1/16 to1/32) and IgG antibodies (1/32 to 1/256) in immunofluores-cence (Ingegerd Kallings, Statens BakteriologiskaLaboratorium, Stockholm, Swedeb).With the criteria used for a po§itive test, we got low

sensitivity but a good specificity of 97.1%. Changing thecriteria from a 50 to a 30% intrease in ELISA titer (a foldincrease of 1.30 or more; Fig. 2) for a positive test gaveresults as follows. Three of six patients with S. pneumoniaebacteremia and 25 of 44 patients (56.81%) with S. pneumoniaeisolated in the blood, sputum, or nasopharynx were pQsitive.Of 35 patients with other etiology, 32 still had negativeresults, for a specificity of 91.4%. The number of patientsgiving positive results in the test with PnC antigen in thesputum was unchanged, and 9 instead of 5 of 37 patients with

Group no la

S. pneumoniaeisolated in blood

2.0

UJ

z

o 1.0-0n

a0

0

0

S

0

2.0

1.0

0

n = 6

IRI

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ANTIBODIES TO PnC IN PNEUMONIA 813

unknown etiology gave positive results. These 4 additionalpatients were successfully treated with penicillin and wereafebrile within 2.5 days (median value; range, 2 to 4 days).Of the two additional patients with false-positive results, onehad Branhamella catarrhalis isolated in the nasopharynx asthe only finding of possibly pathogenic bacteria, and theother h4d a positive compleinent-fixation test' for Chlamydiapsittaci, 1/40 to 1/160. Changing the criteria for a positivetest as described above improved the sensitivity markedly,but the resulting decrease of the specificity to 91.4% isprobably not acceptable.The low sensitivity of our test was disappointing. Bernts-

son et al. (3), using a tube ELISA and a mixture of sixcapsular polysapcharide antigens, fouhd a >100% increasein IgG in 14 of 17 patients with pneumococcal pneumonia.Also studying the IgM antibodies, they showed a positivetest in two additional patients. However, these results werenot confirmed by others. Coonrod and Drennan, usingindirect heinagglutination (8), Riley and Douglas, using aradioitnmunoassay (22), and Kalin and Lindberg, using anELISA (18), have shown that many patients fail to increasetheir antibody levels against homologous capsular polysac-charide, although they are monitored for several weeks. Thereason may be a release of soluble antigen from the infectedtissue into the circulation, producing antibody neutralizationor antigen excess (9). Another reason coUld be that an initialantigen excess elicits an immunological "paralysis" (13).With regard to the number pf days with fever, our patientswith S. pneumoniae isolated in the blood or sput4m or bothshowed no difference between the group with a positiveserological test and thy group with a rlegative test,The absorbance values obtained from acute %ra varied

much from patient to patient. The absorbance values of the124 acute sera in dilutiqn 1:400 are shown in Fig. 6. Thepatients were divided as in Fig. 2 into etiological groups andWith regard to the location from which pneUtirococci wereisolated. The duration of Illness before admission to the ward,had no influence on aptibody levels in the acute sera. Thepatients in group la, b, c, and d with high levels of antibodiesshowed a significant rise in levels as often as patients withlow levels. There were no differences in the absorbancevalues obtained by group lb with S. pneumoniae isolated inthe sputum compared with values obtained by group 3 withother known or suspecteol etiology (0.95 and 0.98, respec-tively, median values), indicating that the PnC antibodies didnot afford protection against pneUmonia caused by pneumo-cocci. However, t4h six patients in group la, with S.pneumoniae bacteremia, obtained a low absorbance value,0.38 (median value), which, contradictorily might jmply thatthe PnC antibodies have some protective capacity or that forthese patients with bacteremia they were neutralized bycirculating antigen.The antigenic determinants of the PnC antibodies mea-

sured by us are not now known. Protease treatment of our Cpolysaccharide resulted in a preparation containing approx-imately 1.5% protein. Inhibition tests with this preparationand the Schiffman preparation described above showed anabsorbance deVrease of about 60 and 50%, respectively, withthe two preparations in a concentration of 1 ,ug/ml of serum.Inhibition of more than 80% was obtained with 50 jig/ml qfserum (Fig. 7). Human C-reactive protein (CRP) binds to thephosphorylcholine of the PnC (27). The addition ofphosphorylcholine (Sigma) in a concentration of 200 mg/literto patient sera caused no change in absorption values. Wealso added human CRP (Calbiochem-Behring, La Jolla,Calif.), in a concentration of 50 mg/liter of sera, to 5

CRP-negative sera (radial immunodiffusion; BehringInstitut, Marburg, Federal Republic of Germany) withoutany change in absorption values. These results are confirmedby the fact that the mean absorbapce value for all 35 patientswith known or suspected etiology other than S. pneumoniaewas unchanged from acute to convalescent sera despite highCRP values in acute sera and low CRP values in convales-cent sera (>90 and 0 mg/liter, respectively, median values).We presume that the PnC antibodies bind to one or morestructures of the five saccharides in each PnC repeating unitbut not to the phosphorylcholine only.The ELISA described showed a good specificity. Because

only one antigen preparation is used, this test is more simnplethan the traditional capsular antigen preparations and mighttherefore be a supplemental nmethod in the diagnosis ofpneumococcal pneumonia.

ACKNOWIFDGMENTSThis work was supported by the Swedish Board for Technical

Development and LKB Producter AB, Bromma, Sweden.We thank G. Schiffman for permission to use his C-poly-

saccharide preparation as a referenice. We also thank ElisabethMansfeldt for skillful laboratory assistance.

LITERATURE CITED

1. Ammann, A. J., and R. J. Pelger. 1972. Determination ofantibody to pneumococcal polysaccharides with chromic chlo-ride-treated human red blood cplls and indirect hemagglutina-tion. Appl. Microbiol. 24:679-683.

2. Austrian, R. 1974. Pneumococcal infection. Prev. Med.3:443-445.

3. Elerntsson, E., K.-A. llroholm, and B. Kaijser. 1978. Serologicaldiagnosis of pneumococcal disease with enzyme-linked im-munosorbent assay (ELISA). Scand. J. Infect. Dis. 10:177-181.

4. Borhstein, D. L., G. Schiffman, H. P. Bernheimer, and R.Austrian. 1968. Capsulation of pneumococcus with solubleC-like (Cs) polysaccharide. J, Exp. Med. 128:1385-1400.

5. Bfiles, D. E., M. Nahm, K. Schroer, J. Davie, P. Baker, J.Kearney, and R. Barletta. 1981. Antiphosphocholine antibodiesfound in normal mouse serum are protective against intravenousinfection with type 3 Streptococcus pngumoniae. J. Exp. Med.153:694-705.

6. Brundish, D. E., and J. Badoliley. 1968. Pneumococc4l C-s4bstance, a ribitol teichoic acid containing choline phosphate.Biochem. J. 110:573-581.

7. CSallahan, L. T., II1, A. F. WQodhour, J. B. Meeker, and M. R.Hilleman. 1979. Enzyme-linked immunosorbent assay for mea-surement of antibodies against pneumococcal polysaccharideantigens: comparison with radio immunoassay. J. Clin. Micro-biol. 10:459-463.

8. Coonrod, J. D., and D. P. Drennan. 1976. Pneumococcal pneu-monia. Capsular polysaccharide antigenemia and antibody re-sponses. Ann. Intern. Med. 84:252-260.

9. Dee, T. H., G. Schiffman, M. I. Sottile, and M. W. Rytel. 1977.Immunologic studies in pneumococcal disease. J. Lab. Clin.Med. 89:1198-1207.

10. Franzen, H., and S. Wqloutis. 1q69. Infections with virus,Mycoplasma pneumoniae and bacteria in acute respifatoryillness. Scand. J. Infect. Dis. 1:31-37.

11. Gerhart, P. J., N. D. Johnson, R. Douglas, a'nd L. Hood. 1981.IgG antibodies to phosphorylcholine exhibit more diversity thantheir IgM counterparts. Nature (Lon%Ion) 291:29-34.

12. Gray, B. M., H. C. Dillon, Jr., and D. E. Briles. 1983. Lpide-miological studies of Streptococcus pneumoniae in infants:development of antibody to phosphocholine. J. Clin. Microbiol.18:1102-1107.

13. Halliday, W. J. 1971. Immunological paralysis of mice withpneumococcal polysaccharide antigens. Bacteriol. Rev.35:267-289.

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14. Heidelberg, M., and D. G. Anderson. 1944. The immune re-sponse of human beings to brief infections with pneumococcus.J. Clin. Invest. 23:607-612.

15. Holmberg, H., T. Holme, A. Krook, T. Olsson, L. Sjoberg, andA.-M. Sjogren. 1985. Detection of C polysaccharide in Strepto-coccus pneumoniae in the sppta of pneumonia patients by anenzyme-linked immunosorbent assay. J. Clin. Microbiol.22:111-115.

16. Jennings, H. J., C. Lugowski, and N. M. Young. 1980. Structureof the complex polysaccharide C-spbstance from Streptococcuspneumoniae type 1. Biochemistry 19:4712-4719.

17. Kalin, M., and A. Lindberg. 1983. Diagnosis of pneumococcalpneumonia;' a comparison between microscopic examination ofexpectorate, antigen detection and cultural procedures. Scand.J. Infect. Dis. 15:247-255.

18. Kalip, M., and A. Lindberg. 1985. Antibody response againstthe type specific capsular polysaccharide in pneumococcalpneumonia measured by enzyme linked immunosorbent assay.Scand. J. Infect. Dis. 17:25-32.

19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

20. Macfarlane, J. T., R. G.< Finc,, M. J. Ward, and A. D. Macrae.1982. Hospital study of adult community'-acquired pneumonia.Lancet ii:255-258.

21. Pederseq, F. K., J. Henrichsen, U. S. Sorensen, and J. L.Nielsen. 1982. Anti-C carbohydrate antibodies after pneumococ-cal vaccination. Acta Pathol. Microbiol. Scand. Sect. C90:353-355.

22. Riley, I. D., and R. M. Douglas. 1981. An epidemiological

approach to pneumococcal disease. Rev. Infect. Dis. 3:233-245.23. Schiffman, G., D. L. Bornstein, and R. Austrian. 1971. Capsula-

tion of pneumococcus with soluble cell wall-like polysaccha-ride. J. Exp. Med. 134:600-617.

24. Schiffman, G., R. M. Douglas, M. J. Donner, M. Robbins, and R.Austrian. 1980. A radioimmunoassay for immunologic phenom-ena in pneumococcal disease and for the antibody response topneumococcal vaccines. I. Method for the radioimmunoassay ofanticapsular antibodies and comparison with other techniques.J. Immunol. Methods 33:133-144.

25. Schreiner, A., A. Digranes, 0. Myking, and C. Solberg. 1973.Wert der transtrachealer Aspiration bei bronchopulmonalenInfektion im Erwachsenenalter. Infection 1:137-143.

26. Szu, S. C., S. Clarke, and J. B. Robbins. 1983. Protection againstpneumococcal infection in mice conferred by phosphocholine-binding antibodies; specificity of the phosphocholine-bindingand relation to several types. Infect. Immun. 39:993-999.

27. Volanakis, J. E., and M. H. lKaplan. 1971. Specificity of C-reactive protein for choline phosphate residues of pneumococ-cal C'polysaccharide. Proc. Soc. Exp. Biol. Med. 136:612-614.

28. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide.Extraction' with phenol-water and further applications of theprocedure. Methods Carbohydr. Chem. 5:83-87.

29. White, R. J., A. D. Blainey, K. J. Harrison, and S. K. R. Clarke.1981. Causes of pneumonia presenting to a district generalhospital. Thorax 36:566-570.

30. Yother, J., C. Forman, B. M. Gray, and D. E. Briles. 1982.Protection'of mice from infection with Streptococcus pneumo-niae by anti-phosphocholine antibody. Infect. Immun.36:184-188.

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