Role of rheumatoid factor in complement fixation and indirect hemagglutination tests for immunoglobulin M antibody to cytomegalovirus

(1)

0095-1137/78/0002-0160$02.00/0

Role of Rheumatoid Factor

in

Complement Fixation and

Indirect

Hemagglutination Tests for Immunoglobulin M

Antibody to

Cytomegalovirus

NATALIE E. CREMER, MARJORIE HOFFMAN, AND EDWIN H. LENNETTE*

Viral andRickettsialDisease Laboratory, California State Department ofHealth,Berkeley,

California94704

Received for publication 2 June 1978

Absorption of immunoglobulin M (IgM)-rheumatoid factor (RF) from serum samples byreaction withinsolubilized gamma globulin reduced the

complement-fixing(CF) antibodytiter to cytomegalovirus (CMV) antigen to less than 1:2 in

the IgM fraction of some,but not all, sera. Thus, IgM-CF activity in some sera

appeared to be due to specific IgM anti-CMV antibody and in other sera to

complexes of IgM-RF withantiviral IgG antibody. Prozones were present in the

CF testonIgMfractions. Increasingthe concentration of antigen from 2 to 4 U

reduced the prozone titer by one or two doubling dilutions. This observation

suggested thatacompetition for antigen may beoperating at low dilutions of IgM

antibodyfractions. Removal of RF hadlittleor no effect on the reaction of the

IgM fraction of sera with CMV by the indirecthemagglutination test.

Incytomegalovirus (CMV) infection aberrant

immunological reactions may occur (3, 17, 23,

29, 34). One such reaction is the formation of

rheumatoid factor (RF), an antibody reactive

withimmunoglobulin G (IgG) which isusually

of the IgMclass but may also be of theIgGor

IgA class (1, 15). Complexing ofIgM-RF with

viral antibody of the IgGclass has ledtofalse

positivereactions forIgM viralantibodyin the

indirectfluorescent-antibody test(10, 29).

Analysis of antibody production in human

CMV infection in a previous study indicated

that IgMreactivitywithCMVantigen detected

by indirect hemagglutination (IHA) usually

aroseearlier than did that detected by

comple-mentfixation (CF) (8a). Thedifferencein

tem-poral appearance of these two reactions

sug-gested that different populations ofIgM

mole-culeswere involved. The present report, as an

extension ofourprevious study (8a), investigates

the contribution of RF to the IHA and CF reactivityseen between IgM fractionsof serum

andCMV antigen.

MATERIALS AND MBEHODS

Serum samples. Eight sera in which CMV anti-body was found in the isolated IgM fractions by both IHA and CF were selected for study.

Antibodyassays. IHA tests were doneaccording tothe method ofBernsteinandStewart (5) with slight

modification.Antigens extracted by freezing and thaw-ing infected celLs and noninfected cells (9) were used to coatsheep erythrocytes treated withtannicacid.

The concentration ofantigenforuse wasdetermined

byblocktitration withapositivereferenceserumthat containedIgM anti-CMV antibody. Complete or al-most complete agglutination by antibody of cells coated with theoptimaldilution ofantigenwastaken asthe endpoint.Apositiveserumof known titer and anegativeserum wereincluded in eachrun.CF tests wereperformedaspreviouslydescribed(9),using an-tigen extracted with0.1Mglycine(pH9.5buffer)from

human fetal diploid lung cells infected with CMV

strainAD169. Two units ofantigen,asdeterminedby

blocktitration,wereused in the test. A similar prep-arationwasmade from noninfectedcellsforuse asa controlantigen.

RF.Sera and fractionswerechecked for RF with

Hyland Laboratories' latex globulin reagent in the

slidetest.

Absorptionof RF. Gammaglobulin (immune se-rum globulin, Armour Pharmaceutical Co., Chicago,

Ill., or Pentex human gamma globulin, fraction II,

MilesLaboratories, Inc.,Elkhart, Ind.) was insolubi-lized withglutaraldehyde asdescribed byAvrameas (2). Afterhomogenization and washing, the insolubi-lizedglobulinwasfurther reacted with0.3Mglycine

to block any unreacted glutaraldehyde bonds (23).

After additional washing, the insolubilized material was made to afinal 50% concentration (vol/vol) in phosphate-buffered saline (0.01M phosphatebuffer, pH 7.2).

To determine the amount ofinsolubilized gamma globulin necessary for removal of RF from the test sera, 0.05-mlsamples (i.e., 0.2ml ofa 1:4dilution) of areferenceserum strongly reactive for RFwere ab-sorbed for1hat37°Cwithincreasingamountsof the 50%slurry, i.e., with thepelleted material from 0.05, 0.1, 0.2, 0.4, 0.6, and0.8ml. Theamountfound

satis-factoryfor removal of RF in the referenceserumwas L60

on February 7, 2020 by guest

http://jcm.asm.org/

(2)

then used for absorption of all sera. After absorption, the sera were retested for RF and for IgM, IgG, and IgA byradial immunodiffuion1 usingplatesspecific

for human heavychains(Oxford Laboratories, Foster City,Calif., orKallestad Laboratories, Chaska, Minn.).

Determination ofantibody clase. Serum sam-ples, diluted1:4withphosphate-bufferedsaline, were

centrifugedbeforeand after absorption in a 10 to 40% linear sucrose gradient as previously described (9). The total volume consisted of 5 mlcomprisedof a 4.5-mlgradient and a 0.5-mlsample. Fractions (0.5 ml)

werecollected from the bottom of the gradient. Each fraction was tested by the Ouchterlony method for immunoglobulin class, using antisera to

p,

y, and a

chains (Behring Diagnostics, Somerville,N.J.) andfor IHA and CF antibody to CMV. Fractions from the unabsorbed sera were also checked for RF. Sera and fractionsfrom any one patient were evaluated in the same run toavoiddifferencesbetweenrmns.

RESULTS

Absorption of RF. Absorptionof the

refer-enceserumwith0.2 ml of insolubilizedgamma globulinper0.05 mlofserumremoved the reac-tivityfor RFat a1:20dilution of theserum,the

recommended dilution for testingRFinthe

Hy-land Laboratories latex test. There wasalso a

reduction in the concentration of all ofthe

im-munoglobulins, with the greatest reduction in

IgM (Fig. 1). Absorption of thetest sera atthis

ratio ofinsolubilized gammaglobulinto serum

similarlyremoved RF activity and reducedthe

concentration of theimmunoglobulins (Table 1).

The greatest reduction of IgM occurred with

serumC, theserunwith thegreatestRF activ-ity.

Aweakreaction for RFwasobservedat a 1:5

dilution infraction 3 (the IgMfraction) of the unabsorbed sera from patients B and C. This dilutionrepresents a 1:20dilution of the

original

c o5

t) u

a,

O-70

60

50

40 30 20

10

v

O05.1

.2 4 .6

.8

Insolubilized

IgG (mi

of 50%

slurry)

FIG. 1. Reduction in classes ofimmunoglobulûns inareferenceserumcontainingRFafter absorption with insolubilized gammaglobulin.

TABLE 1. Percentreductioninimmunoglobulins

afterabsorption ofserumwithinsolubilizedgamma

globulin

%Reduction

Patient RF

IgG IgM IgA

A 28 45 27 j+a

Bb 35 55 37 2+

Bc 48 69 39 3+

C 53 81 41 4+

D 52 47 37 2+

E 46 42 39 1+

F 48 45 35 Negative

G 38 53 34 2+

aReaction of serum at a 1:20 dilution before

absorp-tion.Afterabsorption allserawerenegativeat a 1:20 dilution.

b,cAcute-phase(b) and convalescent-phase (c) sera

from the samepatient.

serumsample. RFactivity was notdetected in

anyof the fractions of theunabsorbedserafrom

the otherpatients.Apparentlythe concentration

of RF in these specimenswastoolow for

detec-tion afterserumfractionation.

CF and IRA antibody titers iII serum.

Table2showsthe antibody titers of thewhole

serum before andafter absorption for RF and

gives the RF reaction of the sera prior to

ab-sorption. The usual reduction in CF and IHA

antibody titers afterserumabsorptionwas

two-tofourfold.

Antibodytiters of fractions from sucrose density gradient centrifugation. Table 3 gives the CF and IHA titers of the IgM and IgG

fractions showing the greatest reaction with

CMVantigen, i.e.,fraction3and fraction5or6, respectively. Depending uponthe patient's

an-tibody response, the time of

sampling

of the

serum, the nature of the antigen understudy,

and thetype ofassayprocedure, bothIgMand

IgG classes ofserumantibodiesmaybeseen, or

eitherone orthe other.Thus,intheacute-phase

serumofpatientB,bothIgMandIgGantibodies

were detected by CF (Fig. 2), but only IgM

antibodies were found by IHA (Fig. 3). Good

separationof the

immunoglobulin

classes of

an-tibodieswas

achieved,

asshownbythebimodal

distribution of the CF antibodies in

Fig.

2.

CF antibody. With the

exception

of the CF antibodyinserafrompatientsB

(Fig. 2)

andE, theCMVantibodytiterintheIgMfractionswas

reduced to less than 1:2 by

absorption

of the

sera with insolubilized gamma

globulin.

Of in-terestwasthepresenceofaprozone inthe

IgM

fraction ofseven ofthe

eight

unabsorbed sera

andin theabsorbedserafrom

patients

BandE

thatvaried from 1:2to 1:16

(Table

3).

No

pro-zones were seenin any oftheCF testson the

http://jcm.asm.org/

(3)

TABLE 2. Serum antibody titers to CMV before andafterabsorptionwithinsolubilizedgammaglobulin

Time(days) of Antibodytiter

Patient seru collec- CF IHA RF

tion fromonset _____________

of disease Not absorbed Absorbed Notabsorbed Absorbed

A 26 2,048- 512 4,096 4,096 j+b

B 6 4,096 2,048 2,048 1,024 2+

B 29 512 256 4,096 2,048 3+

C 17 256 128 8,192 2,048 4+

D 16 512 128 4,096 2,048 2+

E 19 2,048 1,024 16,384 4,096 1+

F 22 256 64 1,024 512 Negative

G 33 512 128 16,384 8,192 2+

aTiter=reciprocalof thedilution.

bReactionbefore absorptionata 1:20dilution of serum.Afterabsorption

ail

serawere

negative

at a1:20

dilution.

TABLE

3. Antibodytiterof

isolated

immunoglobulin

toCMVbeforeandafter absorption ofserumwith insolubilized gamma globulin

CF IHA

Patient IgM IgG Prozone IgM IgG

Before After Before After (IgM) Before After Before After

A 64<z <2 128 32 4 1,024 256 16 4

Bb 256 z 256z 128 128" 8 512 256 2 <2

Bc 64 16z 32 8 4 1,024 256 64 32

C 32 z <2 8 2 8 512 512 64 16

D 128 <2 32 8 0 1,024 512 32 16

E 256 z 64 64 32 16 2,048 1,024 16 <4

F 32 z <2 8 4 2 128 64 32 32

G 64 z <2 16 8 4 2,048 2,048 32 16

aTiter=

reciprocal

ofthe

dilution

ofthegradient

fraction.

A1:4dilution oftheserum

samples

was

placed

on thegradient.z,Prozone.

b,CAcute-phase(b)and

convalescent-phase

(c) serafromthesamepatient.

wholesera. The CFtestsonthe IgM fractions wererepeated with 2 and4Uof antigen.

Increas-ing theconcentrationofantigento4Ucauseda

two- to fourfold reduction in the titer of the

prozone. A concentration higher than 4 U of antigen could not be used because at greater

concentrations theantigen became

anticomple-mentary.

CFreactivity in the IgG fractions of the ab-sorbedsera was eithernot reduced orwas

re-duced two- to fourfold from that of the unab-sorbedsera.

IHA antibody. With the exception of two

serawhichshowedafourfold reduction in IHA

titer inthe IgM fraction afterserumabsorption, none of the other absorbedsera showed more

thanatwofold reduction in IgM titer (Table3).

Withregard toIgG antibody, there was either

noreduction in titer (serum frompatient F) or

atwo- tofourfold reduction afterserum

absorp-tion.

DISCUSSION

Absorption ofserumwithparticulate material

usually results in some nonspecific as well as

specificloss ofserumproteins, depending upon the nature of the absorbing material. Thus,

Staphylococcus aureus protein A reduces IgG

concentrationspecifically, but thereis also some

nonspecificlossof IgM and IgA (7, 14, 20, 24).

Similarly,inthepresentstudy,insolubilizedIgG

specificallyremoved RF through bindingtothe antibody-combining site on the RF molecule,

but it also causedsome nonspecificloss ofIgG,

IgA, andpossiblyalso ofnon-RF-IgM.

Absorptionwithinsolubilizedgamma globulin

reduced theIgM-CF antibodytiter of five of the

eightsera(Table3) to less than 1:2 (a 32- to

128-foldreduction). However, there was no change

in IgM-CF antibody titer of the first serum of

patient B (Fig. 2) and only a partial reduction

(fourfold) in that of the second serum from

patientBand in that of the serum frompatient

E.

The antibody titers remaining after absorp-tion are considered to bespecific. The absorbed

sera werenegative for RF,and the nonspecific

controls in theCF test were negative. 162 CREMER, HOFFMAN, AND LENNETTE

http://jcm.asm.org/

(4)

163

256

128 @

64

4-~32

0

16

à-o

F-.D

*_

c

rc

Fraction

No.

IgG

--- - + + + + +

IgA

-

+

- -

-IgM

- +

+---

-FIG. 2. Biphasic distribution of CMV antibodies. Sucrosedensitygradient centrifugationpatternofCF

antibodies in the acute-phase serum of patient B.

Symbols: (0) notabsorbed; (0) absorbed; (U) pro-zone, notabsorbed; ([0)prozone,absorbed.

There arereportsofserumspecimens which

donotreactin theRFtestandyetcontain the factor (21, 29). The serumfrom patient F

ap-pearstobesuchaseemingly negative specimen,

since absorption completely removed the IgM-CF activity. The reduction in serumtiter was

32-fold greater than would be anticipated for

nonspecificabsorption, andparticularlyin

rela-tion tothe loss in IgG antibody titers. CF-IgG titers were reduced byabsorptiontwo- to

fourfold. Since aspecific loss of IgGwouldnot

beexpected unless RFwaspresentinthe insol-ubilized gammaglobulin, this level of titer

re-ductionappearstobe duetononspecific absorp-tion. (Ourgammaglobulinpreparation didnot react in the RF test.) Twofold reductions are

also considered within theerrorofanydoubling dilutiontest.

Two conclusions may be drawn from the above results. First, IgM-RF mayleadtofalse positive reactions in the CFtest forIgM viral antibody, and,second,somepatients producean

IgM antibody capable of fixing complement. With regard to the first point, anti-CMV IgG antibodycompletedwithIgM-RFand therefore present in the IgM fraction would specifically

reactwiththeCMVantigenand fixcomplement. Such activity should then be obviated by

re-moval of the IgM-RF, and such wasthe case.

Whether CF would be enhanced in the unab-sorbed whole serum because of the combined

reaction ofthe CMVantibodywithCMV

anti-512

256

128

64

32

16

8

4

2 *Not

absorbed

o

Absorbed

\-.

0--I

-'

1

2

3

4

5

6 Fraction

No.

IgG

---+

+

IgA

-

- -

+

IgM

- 4

++

--7

8

9

FIG. 3. Monophasic distribution of CMV antibod-ies.Sucrose density gradientcentrifugationpattern

ofIHA antibodies in theacute-phase serum ofpatient B.

genandwith IgM-RF isnotknown. IgM-RFin

its reaction with IgGwasconsideredat onetime

incapable of binding complement. More recent

reports indicate the IgM-RF can lyse sheep erythrocytessensitized with reduced and

alkyl-atedIgGantibody (31, 32).Inthepresentstudy

complexingIgM-RF with IgG in the absence of

CMV antigen did not

result

in CF, since the

reactions with the control antigenwerenegative.

Withregardtothe secondpoint, IgM antibody

in its reaction with viral antigen has usually

beenfoundincapable of fixingcomplement(4, 6, 8,12, 13, 16,18,19, 25, 26, 28,30). Thereareonly

afew studiestothecontrary,and in thesestudies

thepresenceofRFwas notdetermined (11, 27,

33). Therefore, the contribution ofRF to the

results isnotknown. Inthe present study, CF

activitywasnotremovedbyremoval of RF from theseraoftwoindividuals, suggesting that CF-IgM antibodycan atleastoccasionallyoccur.

Theoccurrenceofaprozone in theIgM

frac-tionof all of the unabsorbedseraaswellasthe

absorbedserathatretainedCF-IgMactivity

sug-geststhe additional presence ofa

non-comple-ment-fixingIgMantibodytoCMV inlowtiter.

The non-complement-fixing antibody would have to be ofhigh avidityto compete

success-fullyforCMVantigenand thusblock CFatlow

dilutions.Thistheoryhassomesupport sinceby

increasingthe concentration ofantigen from 2

to4U theprozonewasreduced,

although

itwas

http://jcm.asm.org/

(5)

164 CREMER, HOFFMAN, AND LENNETTE

noteliminated in allcases.Ifahigh

concentra-tion ofsuch an antibodywerethe usualresponse

in viral infections, it could completely mask

CF-IgM activity.Competing antibodyof this nature

could explain the inability ofmostinvestigators

to demonstrate CF-IgM antibody toviral

anti-gens.

Other explanationsarepossible. Binding sites

for complement become available on the Fc

portion of the antibodymoleculethroughspatial

reorientation of the moleculeinits reaction with

antigen. Human IgM has ten binding sites for

antigen (22); IgG has two binding sites. For

effectivebinding of thefirstcomponentof

com-plement (Clq) by IgM antibody, antigen-binding

sites presentontheFabportionoftwoadjacent

subunitsof the IgM moleculemustbecomplexed

with antigen. Therefore,theremay be

competi-tion for antigen at the intramolecular level in

the case ofIgM antibody,necessitating

concen-trationsof antigen greater than thatrequiredfor

IgGantibody. Usually in diagnostic virus

labo-ratories,block titrations fordetermination of the

antigen unit are done with convalescent-phase

serawhereIgG antibody is predominant.

ContrarytotheCFresults,IgM-RF appeared

not tobe significantly involved in the IHA

re-action ofIgMantibody. IgM titers with most of

the sera afterabsorptionwereeither unchanged

orreduced by one doubling dilution. Only two

of theeight serashowed a fourfold reduction in

antibody titer. IHA-IgG antibody titers were

similarlyreduced. SinceIgM-RF may cause false

positive reactions for IgM by the indirect

flu-orescent-antibody method (10, 29), the same

problem will no doubt occur with testsutilizing

newertechniques for labelingantibody such as

the enzyme-linked immunosorbent assay and

radioimmunoassay. Although more

time-con-suming, IHA performed on isolated IgM

frac-tionsprovidesanalternate and sensitive method

for determinationofIgM antibodyto CMV in

which RF appears not to participate to any

significant degree. IgM-RF-IgG antibody com-plexesmaynotbe ofoptimal sizeor

configura-tion forbindingtoantigen-coatederythrocytes.

Also,the IHAtestisreported to be more

sensi-tive to IgMantibody than to IgG antibody (8),

sothatany IgG antibody in theIgM-CF complex

maynotbe in a verycompetitive position for the

viralantigen.

ACKNOWLEDGMENIS

Thisinvestigation was supported by Public Health Service grant AI-01475 from the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED

1. Abraham, G.N.,R. A.Clark, andJ. H.Vaughan.

1972. Characterization of an IgA rheumatoid factor: bindingpropertiesandreactivitywith thesubclasses of human yG globulin. Immunochemistry9:301-315. 2. Avrameas,8. 1969.Coupling ofenzymestoproteins with

glutaraldehyde.Useoftheconjugatesfor thedetection ofantigens andantibodies.Immunochemistry6:43-52. 3. Bartfield, H. 1969. Distribution ofrheumatoid factor activity innon-rheumatoidstates.Ann.N.Y. Acad.Sci. 168:30-40.

4. Bellanti,J.A.,S. B.Ruse, G.E.Holmes, andE.L Buescher. 1965. The nature of antibodies following experimental arbovirus infectioninguinea pigs.J. Im-munol.94:1-11.

5. Bernstein, M. T., andJ. A. Stewart. 1971. Indirect hemagglutination test for detection of antibodiesto

cytomegalovirus.Appl. Microbiol.21:84-89.

6. Best,J. M.,J. E.Banatvala,and D. Watson. 1969. Serum IgG and IgMresponses inpostnatally acquired rubella.Lancet ii:65-68.

7. Chantler,S., E. Devries,P. R. Allen, andB. A.L Hurn.1976. Arapidimmunofluorescentprocedurefor thedetectionofspecificIgGandIgM antibodyinsera

usingStaphylococcusaureusandlatex-IgGas absorb-enta.J. Immunol. Methods 13:367-380.

8. Cowan,K. M.1973.Antibodyresponsetoviralantigens. Adv.Immunol.17:195-253.

8a.Cremer, N.E.,M.Hoffman,andE. H. Lennette. 1978. Analysis ofantibodyassaymethodsandclassesof viral antibodies in serodiagnosis ofcytomegalovirus infec-tion. J. Clin. Microbiol. 8:153-159.

9. Cremer,N.E., N. J.Schmidt,F. Jensen, M.Hoffman, LS.Oshiro,and E. H.Lennette.1975. Complement-fixing antibodyinhuman sera reactive withviral and solubleantigens ofcytomegalovirus.J.Clin. Microbiol. 1:262-267.

10. Fraser,K.B.,P.V.Shirodaria, andC. F.Stanford. 1971.Fluorescent staining and human IgM. Br. Med. J. 3:707.

11. Gerna,G., andR.W. Chambers.1977.Rapid detection of human cytomegalovirus and herpesvirus hominis IgMantibody bytheimmunoperoxidasetechnique. In-tervirology8:257-271.

12. Hanshaw,J. B.1969.Congenitalcytomegalovirus infec-tion. Laboratory methods of detection. J. Pediatr. 75:1179-1185.

13. Hanshaw,J.B.,J.Harvey,R.J.Steinfield,and C. J. White. 1968. Fluorescent antibody test for cytomega-lovirusmacroglobulin. N. Engl. J. Med.279:566-570. 14.Harboe, M., and I. Folling. 1974. Recognitionof two distinct groups of humanIgM and IgA based on differ-ent binding to staphylococci. Scand. J. Immunol. 3:471-482.

15. Heimer, R., and F. M. Levin. 1966. On the distribution ofrheumatoid factors among theimmunoglobulins. Im-munochemistry 3:1-10.

16. Ishii, K., Y. Mataunaga, and R.Kono.1968. Immuno-globulinsproduced in response to Japanese encephalitis virusinfections in man. J. Immunol.101:770-775. 17. Kantor,G. L., L. S. Goldberg, B. LJohnson,M. M.

Derechin, and E. V. Barnett. 1970. Immunologic abnormalities induced bypost-perfusion cytomegalovi-rusinfection. Ann.Intern. Med. 73:553-558.

18. Lang,D. J.,and J. B.Hanshaw. 1969.Cytomegalovirus infection andthepostperfusion syndrome. Recognition ofprimary infections in fourpatients. N.Engl.J. Med. 280:1145-1149.

19.Lang, D. J., and B. Noren. 1968. Cytomegalovirus fol-lowing congenital infection. J. Pediatr. 73:812-819.

20.Lind,I.,M.Harboe, and I. Folling. 1975. A reactivity of

twodistinct groups ofhuman monoclonal IgM. Scand. J. Immunol.4:843-848.

21.Nikoskelainen, J., and P.H11nninen. 1975. Antibody response to Epstein-Barr virus in infections

http://jcm.asm.org/

(6)

Osi. Infect.Immun. 11:42-60.

22. Nisonoff,A.,J. E.Hopper,andS. B.Spring.1975.The antibodymolecule. AcademicPress Inc., New York. 23. Reimer, C. B., C. M.Black, D. J. PhilHpo, L C. Logan,

E. F. Hunter, B.J.Pender, andB.E. McGrew.1975. Thespecificity of fetal IgM: antibody or anti-antibody? Ann.N.Y. Acad.Sci.2U4:77-93.

24. Roggendorf,M., K. E.Schneweis,and M. H. Wolff. 1976. Zum Nachweisrotein-spezifiacher IgM in Hae-magglutinationshemmungtest. Vergleichende Untersu-chungen mit derAbsorptionvonIgG durch Protein-A-haltige Staphylokokken und der dichtegradienten-Ul-trazentifugation.Zentralbl.Bakteriol.Parasitenkd. In-fektionskr.Hyg. Abt. 1Orig.Reihe A235:363-372. 25. Schmidt, N. J., and E. H. Lennette. 1975.Neutralizing

antibodyresponses to varicella-zoster virus. Infect. Im-mun. 12:606-613.

26. Schmidt, N. J., E. H. Lennette, and J. Dennis. 1968. Characterization of antibodies produced in natural and experimental coxsackievirus infections. J. Immunol. 100:99-106.

27. Schmitz,H., and R.Haas. 1972. Determination of dif-ferent cytomegalovirus immunoglobulin (IgG, IgA, IgM) by immunofluorescence. Arch. GesamteVirus. forsch.37:131-140.

28. Schneweis,K. E., M. IL Wolff, G. Marklein, and G.

Stifter. 1974. Untersuchungenmit derIgM-Fraktion von Patienten-Seren bei verschiedenen Viruserkran-kungen. Z. Immunitaetsforsch. Exp. Klin. Immunol. 147:236-249.

29. Shirodaria,P.V.,K. B.Fraser, andF.Stanford.1973. Secondary fluorescent staining of viral antigens by rheumatoid factor andfluorescein-conjugated anti-IgM. Ann.Rheum. Dis. 32:53-57.

30. Stiehm,E. R.,H. J.Hammann, and J. D.Cherry. 1966.Elevated cordmacroglobulins in diagnosis of in-trauterineinfections. N.Engl.J.Med. 275:971-977. 31.Tanimoto, K., N. R. Cooper, J. S. Johnson, and J. H.

Vaughan.1975. Complement fixationby rheumatoid factor.J. Clin.Invest.55:437-445.

32.Taylor-Upsahl,M.M.,P. M.Johnson,O. J.MeIlbye andJ. B.Natvig.1977. Astudyofcomplementfixation byrheumatoid factor using ahaemolyticassay system. Clin.Exp. Immunol.28:204-211.

33. Tokumaru,T.1966.Apossiblerole of IgA-immunoglobu-lin inherpessimplexvirusinfection inman.J.Immunol. 97:248-259.

34.Wager,O.,J.Rasanen,A.Hagman,and E.Klemola. 1968. Mixed cryoimmunoglobulinaemia in infectious mononucleosis and cytomegalovirus mononucleosis. Int.Arch.Allergy34:345-361.

http://jcm.asm.org/