RUBELLA ANTIBODY DETERMINATIONS

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ARTICLES

RUBELLA

ANTIBODY

DETERMINATIONS

John L. Sever, M.D., Ph.D., David A. Fuccillo, Ph.D., Gary 1. Gitnick, M.D.,

Robert J. Huebner, M.D., Mary Ruth Gilkeson, B.S., Anita C. Ley, M.S.,

Nancy Tzan, and Renee G. Traub, A.B.

Section on Infectious Diseases, Perinatal Research Branch, National Institute of Neurological Diseases

and Blindness, and the Laboratory of Infectious Diseases, National institute of Allergy

and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

(Received March 22; revision accepted for publication June 19, 1967.)

ADDRESS: (J.L.S.) National Institute of Neurological Diseases and Blindness, Department of Health, Education and Welfare, Bethesda, Maryland 20014.

PEDIATRICS, Vol. 40, No. 5, November 1967

F

OUR METHODS are now available for the

detection of rubella antibody:

hemag-glutination inhibition, neutralization,

fluores-cence, and complement fixation.

Determina-tions with these tests show differences in

titer and variations in patterns of

appear-ance and persistence of antibody. These dif-ferences are important for the clinical and

research applications of the tests. The

pres-ent paper compares the antibody findings

with these four tests for patients with

ac-quired and congenital rubella.

MATERIALS AND METHODS

Performance of Tests

HEMAGGLUTINATION INHIBITION ANTIBODY

(HI): Hemagglutination (HA) antigen was

prepared according to a slight modification

of the method recently reported by Stewart, et a!.’ BHK-21 tissue cultures in 32 oz

pre-scription bottles were inoculated with 1.0 ml of RV strain rubella virus and incubated at 35#{176}C.2The virus had been grown in pri-mary African green monkey kidney tissue cultures and had a titer of 4.5 logs10/0.2

ml. The fluids were harvested on the fourth day after inoculation, pooled, and frozen and thawed three times. This antigen had a titer of 16 units. HA and HI tests were per-formed with the microtechnique previously described using a micro procedure similar to the one described by Stewart, et al. Spiral loops and disposable Microtiter “V”#{176}

plates were used.3

For the HA titration 0.025 ml serial dilu-tions of the antigen were made in

dextrose-*Purchased from Cooke Engineering Company,

Alexandria, Virginia.

gelatin-veronal

(

DCV

)

diluent, with the use of the loops. One drop

(

.025 ml) of DCV was added followed by a similar vol-time of an 0.25% suspension of red blood

cells in DCV. The red cells were obtained

from unfed white Leghorn chicks 24 hours after hatching. These cells were allowed to

settle at 4#{176}Covernight. The test was read

at room temperature and the titer of the

an-tigen was the reciprocal of the last dilution

showing complete agglutination.

Specimens of sera and gamma-globulin

were first adsorbed with kaolin and chick RBC, and were then inactivated. Each

serum was mixed with an equal volume of

DCV (.2 ml and .2 ml); 0.6 ml of 25%

(W/\7) acid washed kaolin in DCV was

added, and the mixture was shaken

vigor-ously and incubated for 20 minutes at room

temperature. The kaolin was sedimented by

centrifugation at 4#{176}Cfor 20 minutes at 600

C. To the supernatant a volume of 0.05 ml

of a 50% suspension of chick red cells in

DCV was added. These cells were obtained

from unfed white Leghorn chicks which

were 24 hours old. The mixture was shaken

and refrigerated at 4#{176}Covernight. The sus-pension was centrifuged at 4#{176}Cfor 20

min-utes at 600 C and the supernatant was re-moved and inactivated for 30 minutes at 56#{176}C.The serum treated in this way was

considered a 1:4 dilution of the original. For the HI test 0.025 ml serial dilutions

of the treated sera were made with the spi-ral loops in DCV diluent. Four units of HA antigen were added (0.025 ml) and the

mixture was incubated at room tempera-ture for 1 hour. The suspension of chick

(2)

ployed in the range of 1:8 through 1:64 and

chromatographed. Conjugated goat

anti-human serum was used.

COMPLEMENT FIXING (CF) ANTIBODY:

Rubella CF antigen was prepared with in-fected BHK-21 tissue cultures in a 5% sus-pension according to the methods reported previously. The antigen had a titer of 16 units. CF tests were performed utilizing the

microtechnique.3 Spiral ioops and dispos-able Microtiter “U” plates were used. For the antigen titrations 0.025 ml serial

dilu-tions of antigen were made with the use of

loops. To each antigen dilution 0.025 ml of

serum (appropriately diluted) and two ex-act units of complement (0.025 ml) were

added and the mixtures were incubated overnight at 4#{176}C.The hemolytic system (0.05 ml) was added to each mixture and this was incubated at 37#{176}Cfor 1 hour and afterwards at 4#{176}Cfor 4 hours. For serum titrations, serial dilutions of inactivated serum were made with the loops and these dilutions were titrated with four units of

antigen. volume of 0.025 ml and the red cells were

allowed to settle at 4#{176}Covernight.

Anti-body titer was reported as a highest dilu-tion of serum causing complete inhibition of agglutination.

Five variations in the test procedure were also studied:

(

1

)

HA antigen was

treated with Tween 80 and ether according

to the method of Norrby and compared to

the untreated antigen;4

(

2) HA and HI

tests were performed using RBC with a

concentration of 0.16% and compared to the tests using 0.25% RBC suspensions; (3)

macro tube HA and HI tests using 0.2 ml

volumes of each reagent were compared to the micro method; (4) performance of the test at acid pH (6.35) with the use of pH 6.0 phosphate buffered saline for the sus-pension of red cells was compared to the present method (pH 6.8); and (5) HI tests conducted with the sera treated with 0.4 ml

of 25% kaolin were compared to the pres-ent method where the treatment was with

0.6 ml of the kaolin suspension.

NEUTRALIZING ANTIBODY (NEuT): The

enterovirus interference technique in pri-mary African green monkey kidney tissue culture was used according to the method described previously.5 For these tests, roll-er tube cultures were each inoculated with 0.2 ml of a mixture containing 0.5 log10 of the RV strain of rubella virus and an ap-propriate dilution of heat inactivated serum.

Serial twofold dilutions of serum 1:4 to

1:64 were included. After 8 days the main-tenance media was changed and the cul-tures were inoculated with 100 TCID5O of Coxsackie A-9 virus. The tissue cultures

were examined 4 days later and the

appear-ance of the cytopathic effect due to Cox-sackie A-9 was interpreted as indicating neutralization of the interfering effect of rubella virus. The titer of the serum was reported as a reciprocal of the last dilution which completely neutralized the interfer-ing effect.

FLUORESCENT ANTIBODY (FA):

Chron-ically infected tissue cultures of LLC-MK2

were used according to the method

re-ported by Brown and Maassab.6 Serial

two-fold dilutions of inactivated sera were

em-Serum Specimens

PREGNANT WOMEN WITH CLINICAL Ru.

BELLA: Serial serum samples were obtained

from pregnant women with rubella as part of the longitudinal studies of the Collabora-tive Perinatal Research Investigation.8

INDIVIDUALS wim RUBELLA 10 TO 20 YEARS

PREviousLy: Blood specimens were avail-able from young adults 18 to 30 years of age

throughout the United States who had

ru-bella 10 to 20 years previously.

YOUNG ADULTS WITH RUBELLA: Paired sera from patients who were diagnosed as having rubella were supplied by

collaborat-ing physicians. In each case the first speci-men was obtained prior to the onset of rash but following exposure, and a second

specimen was taken 20 to 60 days later. CONGENITAL RUBELLA: Serial serum spec-imens were obtained from mothers and

their children in studies of congenital

ru-bella. Congenital rubella was diagnosed in

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TABLE I ACQUIRED RUBELLA Age of Individual (yr) Antibody Responses <8 <4 8 tr 16 8 16 16 16t 4

1 yr prior

2nd day of rash

1 Iflo 7 1110

15 1110

I mo prior (lay of rash 14 da

2 1110

4 1110

15 mo

4 mo prior

(lay of rash

1 1150

2 mo prior 1 wk prior ‘2fliO so NA <8 64 128 128 <8 <8 512 1,024 128 256 <8 <8 128 <8 <8 4,096 <4 4 16 ‘32 32 <4 <4 32 32 32 ‘32 <4 4 64 <4 <4 32 <8 32 32 16 16 16t <8 16 16 <8 <8 64 <4 <4 4 8 8 4 <4 <4 4 <4 <4 8 18-39 20) 20 64 19 20 16f 10 20 4

Young Adults wit/i Rubella-Paired Sera Bracketing infection

Number seroconversions 10

Number tested 10

20-34

Mean titer of 512

positive speennens

* Based on five titers, other patients only

-

+ designations.

t Weak fluorescence.

Time Serum Obtained in

Relaiwn to

Onset of Rash

!femagglulination ‘s’eutraliza1ion

Inhibition (III) (Neut)

Pregnant Homen 111th (‘linical Rubella

I1uorese-ent Complement

(F11) Fixation (CF)

individuals with Rubella 10 to 20 Years Previously

Patients Tested

Number positive Number tested

Mean titer of positive specimens

20

16

10 10

OLDER CHILDREN WITH CONGENITAL

Ru-BELLA: Serum specimens were obtained at

Pacific State Hospital, Pomona, California, through the collaboration of Dr. Stanley Wright. In each case the affected individual had characteristic multiple clinical findings of congenital rubella.

SEROLOGICAL SURVEYS: Serum specimens

from 120 young adult volunteers were ob-tained at the Petersburg Federal Reforma-tory, Petersburg, Virginia. These men were between the ages of 21 and 28.

Gamma-Globulin

Preparations of standard commercial

(4)

sever-TABLE II

CONGENITAL RUBELLA

‘Weak fluorescence.

792

al manufacturers in the United States and

Sweden. The reference gamma-globulin for rubeola was obtained from the Division of Biologics Standards. Convalescent rubella gamma-globulin

(

12%), prepared in

Swe-den, was obtained from Kahi Laboratories

through the courtesy of Dr. Rolf

Lund-strom. A special preparation of

convales-cent rubella gamma-globulin was prepared from plasma obtained from military re-cruits 1 month after onset of rubella at the

Chanute Air Force Base, Rantoul, Illinois.

This was processed by Hyland Laborato-ries.

RESULTS

Antibody responses to acquired rubella

obtained with the HI, Neut, FA, and CF methods are summarized in Table I. The pregnant women with clinical rubella de-veloped antibody which was detected with all four tests shortly after the onset of rash

and this antibody persisted for at least 15

months in these patients. The HI, Neut,

and FA determinations showed similar pat-terns of response. The serum specimens taken prior to the onset of rubella had no detectable antibody; at the time of the rash

Neut and FA antibody was usually detect-ed. HI tests become positive subsequent to the second day of rash. Within a few weeks

after rash all three methods showed maxi-mum titers but HI antibody levels usually

began to decrease 4 months to 1 year after rubella and FA showed weakening of fluorescence 15 months after infection. In general, HI titers were four- to sixteenfold higher than Neut and FA levels. CF anti-body was not present at the time of rash but was detected in subsequent specimens. Titers were lower than those obtained with the other three methods and decreased 15

months following rash.

For individuals with rubella 10 to 20 years previously, the HI and Neut tests

de-tected antibody in all of the 20 patients studied. The FA test showed antibody in 19 of the 20. With the CF method, however, only half of the patients had detectable

an-tibody. Mean FII titers were fourfold higher

Putie,iL

Anlibody Responses

Neal rat- Fluores- Complement

flatiOfl

zzation cent Fixation inhibition (Neat) (FA) (CF)

Mother Delivery 8 moo

postpar-turn Baby Cord 8rno l7rno Mother Delivery Baby Srno 7rno lyr 51 56 5i 61 16 18 l8 >56 lOtS 4 4 4 4 4 4 4 16 S 8 8 8 4 <4 <4 >16 16 Baby

Irno 118 4 8

4 ‘nO t.56 4 8

Older Children

iS yr 16 4 3’ <4

iSyr 8 4 16’ <4

l5yr 16 4 <8 <4

11 yr 31 4 3* <4

than Neut and FA. FA titers showed weak fluorescence and the CF titers were

gener-ally only at the lowest level of detection. Young adults with rubella showed

sero-conversions in all 10 cases with HI, Neut,

and CF tests. The FA method detected only 7 out of 10 seroconversions. Mean HI titers were sixteen- to thirty-twofold higher than

Neut or FA. CF titers were near the level of detection.

A comparison of the mean HI titer of

in-dividuals shortly after rubella and those who had experienced infection 10 to 20 years previously showed an eightfold lower

level of HI antibody with the latter group. For congenital rubella, HI, Neut, and FA

tests demonstrated antibody in the sera ob-tained from tile mothers at delivery and in

specimens from affected children (Table II). The HI tests gave the highest antibody

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de-TABLE Ill

SENSITIVITY .tND SPECIFICITY OF 1lEMAGGI1TINATION INHIBITION TESTS

120 Sera Sereened at 1: . Dilution for \eutraliziug

and Ilemagglutination 1 nhibiliou A ntihodies

A ntibody Number %

Neutralizing Antibody Present 113 94.2

I lelnagglutinat iOI1 IIlliil)itiOfl

IIIltil)O(lv prt-sellt 113 94 .‘2

I)istribution of Positive and .Vegatire Tests for the 120 Sera

Test Veut Positive Veut Negative

III positive 113 94.’2% 0) 0.0%

HI negative 0) 0.0% 7 5.8%

crease in titer following birth while for a second child there was an increase in titer for the first year. Neut antibody was pres-ent in all specimens tested. The sera were

screened for Neut antibody at 1:4 dilutions.

A lack of sera did not permit subsequent

titrations. FA tests in two cases showed the persistence of antibody for at least 7

months in both children. The CF tests showed the passive transfer of CF antibody to the child in the first case and then

con-tinued detectable antibody for at least 8 months. In the second case, the mother no

longer had detectable CF antibody at the

time of delivery. The child showed

de-velopment of antibody at 7 months of age. In the third case both the 1-month and

4-month specimens had CF antibody.

For older children with congenital rubel-la, HI and Neut antibody were detected in

each case and FA antibody was present in

three of the four cases. The HI titers were

considerably lower than those noted for the

infants and young children. CF antibody was not found in any of these children.

Tile sensitivity and specificity of the HI

method was compared with the Neut

meth-od in Table III. Both tests gave identical

results and showed that 94.2% of tile mdi-viduals had antibody.

Gamma-globulin antibody titers deter-mined with the HI and Neut methods are summarized in Table IV. Tile standard commercial gamma-globulins gave HI

ti-ters in the range of 2,048 to 4,096 and Neut

titers of 256 to 2,048. The convalescent ru-bella gamma-globulins from Sweden and

similar material prepared in the United

States gave titers which were two- to

four-fold higher than the highest standard

corn-mercial gamma-globulins.

The variations in tile HA and HI test procedures which were studied frequently

produced significant changes in titers:

1. Treatment of HA antigen with Tween

80 and ether was performed with 15

anti-gens. In nine cases the HA titer increased

by twofold while the remaining six antigens

showed no change or a slight decrease in

titer. HI tests with 10 sera using the two

Tween-ether treated antigens showed titers

two- to fourfold higher than with untreated

antigens; however, slipping of the pattern

was noted and this made interpretation

difficult.

2. HA tests for five antigens with 0.16%

RBC gave titers twofold higher than when

0.25% RBC were used. HI tests with five

sera with 0.16% RBC showed no difference in titer when compared to tests with 0.25%

RBC.

3. Macro tube HA tests with 10 antigens

gave titers two- to fourfold higher than the

micro plate method. HI macro tests with 20 sera showed no difference from titers ob-tained with the micro method.

4. Performance of the HA tests at pH 6.35 with five untreated and five Tween-ether treated antigens gave titers two- to

eightfold higher than tests conducted at pH 6.8. HI tests at the lower pH with 10 sera showed titers two- to fourfold higher than

those obtained at the higher pH when the

appropriate amounts of antigens were used,

however, negative sera frequently showed

false inhibition of agglutination.

5. HI tests with 10 paired sera treated

with 0.4 ml of 25% kaolin showed HA

(6)

Inhihi-Gam ma-Globulin

Lot

Source

Population Antibody Titer

Concentration

(%)

Number of Donors

Location of Donors

liemagglu-ti7lOJiOfl

Inhibition

Neutralization

1 Com.* A 16.5 8,000 Japan 2,048 256

2 Corn. A 16.5 8,000 Japan 2,048 512

3 Corn. A 16.5 8,000 Japan 4,096 2,048

4 Corn. B 16.5 13,000 Urban East U.S. 4,096 1,024

5 Corn. C 12.0 _t Sweden 2,048 256

6 Corn. C 12.0 t Sweden 2,048 512

7 DBS 16.5 Rubeo a Reference 2,048 512

8 Convalescent 12.0 t Sweden 8,192 4,096

9 Convalescent 16.5 85 Illinois 8,192 4,096

TABLE IV

GAMMA-GLOBULIN-RUBELLA ANTIBODY TITERS

* Commercial standard gamma-globulin.

t Not available.

tion was not present when these sera were

treated with 0.6 ml of 25% kaolin.

COMMENT

The indirect neutralization test has been

considered the “standard” for rubella anti-body determinations since it was intro-duced in 1962.9,10 Titers determined with

this method correlate well with clinical ru-bella and susceptibility to infection.h1 The

FA test has an advantage in that it can be

performed in a few hours rather than days. In our hands it has not been as reliable for determining seroconversions as the Neut

test. The CF test is rapid and easy to

per-form and detects seroconversions with a

high degree of accuracy. Both the CF and

FA tests, however, show loss of detectable

antibody or weakening of fluorescence with specimens taken more than a few months

after infection. This reduces the value of

these methods for determining past infec-tion and susceptibility. The HI test is the newest method and is gaining wide

acceptance.1 It is rapid to perform and

cor-relates well with clinical infection and Neut

determinations. Some of the laboratory

variables which affect the sensitivity and accuracy of the test warrant special atten tion and some of them will be discussed.

Acquired rubella results in the rapid de-velopment of antibody at the time rash de-velops or within a few days after the rash. Three tests, HI, Neut, and FA show similar patterns of development and persistence of antibody. The present data indicates that

HI antibody appears within a few days

after the rash. This is in agreement with the

report of Stewart, et al.1 Neut and FA

anti-body is first detected within 1 or 2 days after the appearance of rash. With these three tests peak titers were obtained 1 to 2 months after infection. HI titers are usually eight- to sixteenfold higher than Neut or FA. HI antibody levels 10 to 20 years after rubella were eightfold lower than convales-cent levels. The FA test shows some weak-ening of fluorescence, which begins approx-imately 1 year after infection. Neut

anti-body remains essentially constant for life.7 HI titers are usually four- to eightfold higher than Neut or FA. CF titers with ac-quired rubella usually appear several days after the disappearance of rash and reach their peak 1 to 2 months later. CF antibody begins decreasing 8 to 12 months after in-fection. By 10 to 20 years after rubella the

(7)

For the serological diagnosis of acute ac-quired rubella, all four tests are quite use-ful. In our hands the FA test was not as re-liable as the other three methods. Because of the rapidity of performance the HI and CF tests are particularly advantageous. Since CF antibody appears several days later than antibody detected with the other tests, we have usually employed this

meth-od when the first serum specimen was taken at the time of onset of rash or shortly after the appearance of rash. For all four

tests the first specimen should be obtained

as soon as possible after exposure or rash

and the second specimen should be taken 3 weeks later. The occurrence and time of rash should be reported since this will influence the choice of test to be per-formed.

To determine susceptibility of

individ-uals to rubella, the HI, Neut, and FA tests are most useful. Because of the lower levels of HI antibody in these individuals than in patients immediately following rubella,

special attention must be paid to careful treatment of the sera. The effect of

incom-plete treatment of sera for this test will he

discussed.

Children with congenital rubella have antibody in the cord blood which is both

passively acquired from the mother and

ac-tively produced by the fetus.1215 The pas-sively acquired antibody decreases in titer over a period of 3 or 4 months and

speci-mens tested after 4 months of age can be

considered to reflect only the active anti-body produced by the child as part of the congenital rubella syndrome. Uninfected

children do not have detectable antibody at

this time. HI, Neut, and FA antibody was

actively produced by the children we have

studied with congenital rubella. In some

cases we have noted an increase in HI anti-body titer in children at 7 months to 1 year of age. These children also usually have an

increase in CF antibody during this period.

An example of this pattern is shown in

Table II (the second baby). It has been our

impression that these children are usually

the most severely affected and tend to shed

virus from the nasopharynx for the longest

periods of time. Neut and HI antibody are

present in children with congenital rubella and persist for many years. Three of four older children with congenital disease also had FA antibody. Actively produced. CF

antibody develops in approximately half of the affected children and is at maximum titer 8 months to 1 year after birth. It then decreases rapidly so that it was not detect-ed in the children studied 13 to 22 years after birth.

For tile serological diagnosis of congeni tal rubella, HI, Neut, or FA tests may be

used. A positive test with sera from an

older child is consistent with the diagnosis of congenital rubella but the possibility of acquired infection at some time after birth

must be considered.

The sensitivity and specificity of the HI test with 120 sera showed complete agree-ment with the Neut test. These determina-tions were repeated a Ilumber of times.

Neut test findings were also confirmed with the use of the RK-1 continuous rabbit kid-ney system)’ Therefore, it would appear

that, when properly performed, the HI test produces results identical to the Neut test.

Gamma-globulin antibody titers with tile

HI and Neut tests gave comparable results.

\Vith both methods standard commercial gamma-globulin from the United States and Sweden and the DBS reference ruheola gamma-globulin gave HI titers of 2,048 to 4,096 and Neut titers of 256 to 2,048. These

findings are in general agreement with those reported by Stewart, et al.1 The con-valescent gamma-globulins prepared in Sweden and the United States gave titers with both methods which were two- to fourfold higher than the highest standard commercial gamma-globulins. The higher HI titers in the convalescent gamma-globii-un are consistent with the fact that the

mili-tary recruits were bled 1 month after rubel-la at the time when HI titers were at their peak. This again points to the need for

(8)

Variations in the performance of the HA

and HI tests can produce significant

changes in titer. These changes are of great importance when clinical decisions are

being made with these methods. Tween-ether treated antigens resulted in twofold increases in HA titer and two- to

four-fold higher HI titers. There was greater difficulty in interpreting the tests with

anti-gens treated in this way. An increase in HA titer was noted with the use of 0.16% red

cells in place of 0.25% cells. No HI titer

in-crease occurred with these more dilute

cells. The macro tube HA titers were

two-to fourfold higher than tile micro method. However, this had no effect on the HI titers

of sera performed in two tests. In addition, the performance of the test at pH 6.35 gave an increase in HA titers and false positive

HI titers. Because of these effects, it is

oh-viotis that paired sera should be tested with

the identical method, preferably

simulta-neously, and appropriate controls should he

used for each test.

The most important variation of the HI

test relates to the treatment used for serum

HA inhibitors. These inhibitors are usually

removed by the addition of kaolin. Tests in

which 0.4 ml of 25% kaolin were used

showed that this procedure did not adsorb

inhibitors in antibody negative sera. This

lack of adsorption gave false indication of antibody in the sera. For this reason the tests in the present study were conducted with 0.6 ml of 25% kaolin. Unfortunately,

kaolin also removes some antibody. The 0.6

ml treatment reduces antibody titers by

two- to fourfold compared to adsorption with 0.4 ml. This is particularly important

when sera contain low antibody levels such

as those which occur in individuals who

Ilave had rubella more than 10 years

pre-viously. With these sera, over treatment with more than 0.6 ml of 25% kaolin or

re-treatment can reduce or eliminate

detect-able antibody and result in false negative

results. Particular care and control must he

exercised by all laboratories in performing

these adsorption procedures since both

false positive and false negative

determina-tions can be obtained with the procedures

which are currently available.

SUMMARY

Four metilods were used to determine rubella antibody titers in patients with ac-quired and congenital rubella and in gamma-globulin. These methods include

hemagglutination inhibition

(

HI),

neutrahi-zation

(

Neut

)

, fluorescence

(

FA

)

, and

complement fixation

(

CF

)

. With acquired

rubella, all four methods showed a rapid

development of antibody shortly after the

occurrence of rash. Maximum titers

ap-peared within a few weeks, following

which HI and CF titers began to decrease

and the FA tests showed a weakening of

fluorescence. HI titers were four- to

six-teenfold higher than Neut, FA, and CF

1ev-els. Ten or 20 years after rubella, HI, Neut,

and FA tests continued to show detectable

antibody but half of the patients no longer

had CF antibody. HI titers were still higher

than Neut and FA but were considerably

lower than convalescent levels. There was

complete agreement between HI and Neut tests in the detection of antibody in young adults.

For congenital rubella, HI, Neut, and FA

antibody persisted in tile affected children

at least through adolescence. HI and CF

antibody increased occasionally during the

period 8 to 12 months after birth.

Gamma-globulin titers with the HI and Neut methods were in close agreement.

Convalescent rubella gamma-globulin gave

titers two- to fourfold higher than standard

commercial gamma-globulin.

Variations in the HA and HI tests were

caused by modification of the method of treatment of the antigen, the concentration of red cells used, the size of the test (macro versus micro), the pH of the test, and the

treatment of sera. The most significant effects were related to the treatment used to eliminate nonspecific inhibitors in the

sera. Improper treatment can give false

negative or false positive tests. When prop-erly performed and appropriately

(9)

identical to the Neut method, although the titers are consistently higher than the Neut test. This new method, then, is rapid and

convenient for obtaining reliable

informa-tion on the antibody status and susceptibili-ty of patients.

REFERENCES

1. Stewart, G. I., Parkman, P. D., Hopps, H. E., Douglas, R. D., Hamilton, J. P., and Meyer, H. M.: Rubella virus hemagglutination-inhi-bition test. New Eng. J. Med., 276:554,

1967.

2. Sever, J. L., Schiff, C. M., and Traub, R. G.:

Rubella virus. J.A.M.A., 182:663, 1962.

3. Sever, J. L.: Application of a microtechnique to viral serological investigations.

J.

Immun., 88:320, 1962.

4. Norrby, E.: Hemagglutination by measles

virus. III.Identification of two different

hem-agglutinins. Virology, 19:147, 1963.

5. Schiff, G. M., Sever, J. L., and Huebner, R. J.:

Rubella virus: neutralizing antibody in

com-mercial gamma globulin. Science, 142:58,

1963.

6. Brown, C. C., Maassab, H. F., Veronelli,

J.

A.,

and Francis, T. J.: Rubella antibodies in hu-man serum: detection by the indirect fluo-rescent-antibody technique. Science, 144:

943, 1964.

7. Sever, J. L., Huebner, R. J., Fabiyi, A., Monif, C. R., Castellano, G., Cusumano, C. L., Traub, R. C., Ley, A. C., Gilkeson, NI. R.,

and Roberts, J. M.: Antibody responses in acute and chronic rubella. Proc. Soc. Exp. Biol. Med., 122:513, 1966.

8. Sever, J. L., Nelson, K. B., and Cilkeson, M.

R.: Rubella epidemic, 1964: effect on 6,000 pregnancies. Amer. J. Dis. Child., 110:395, 1965.

9. Parkman, P. D., Buescher, E. L., and

Arten-stein, M. S.: Recovery of rubella virus from

army recruits. Proc. Soc. Exp. Biol. Med.,

111:225, 1962.

10. Weller, T. H., and Neva, F. A.: Propagation in tissue culture of cytopathic agents from patients with rubella-like illness. Proc. Soc.

Exp. Biol. Med., 111:215, 1962.

11. Schiff, C. NI., Sever, J. L., and Huebner, R. J.: Experimental rubella. Clinical and

labora-tory findings. Arch. Intern. Med., 116:537, 1965.

12. Alford, C. A.: Studies on antibody in congeni-tal rubella infections. Amer. J. Dis. Child.,

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13. Bellanti, J. A., Artenstein, M. S., Olson, L. C.,

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Pediatrics

Anita C. Ley, Nancy Tzan and Renee G. Traub

John L. Sever, David A. Fuccillo, Gary L. Gitnick, Robert J. Huebner, Mary Ruth Gilkeson,