MILK ALLERGY

(Submitted February 23; accepted for publication April 24, 1963.)

ADDRESSES: (S.S.) Research Laboratories, Pharmaceutical Division, The Borden Co., Box 533, Elgin,

illinois; (D.W.A.) Pharmaceutical Division, The Borden Co., 350 Madison Avenue, New York 17, New

York; (A.S.G.) Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas; and

(W.T.K.) Department of Pediatrics, University of Arkansas Medical School, Litfie Rock, Arkansas. Present

address: Scripps Clinic and Research Foundation, 476 Prospect Street, La Jolla, California.

PEDIATRICS, October 1963

580

MILK

ALLERGY

III. Immunological

Studies

with

Sera from

Allergic

and

Normal

Children

S. Saperstein, Ph.D., D. W. Anderson, Jr., Ph.D., A. S. Goldman, M.D.,

and W. T. Kniker, M.D.

Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas

T

HE PRESENCE of nonprecipitating skin

sensitizing antibody (reagin) in allergic

patients has been known for many years.1

Likewise, other types of circulating

anti-bodies have at times been detected to a

variety of antigens in patients with allergic

disorders.2 The significance and

relation-ships of these antibodies remain to be

de-termined.

As early as 1923, Anderson and Schloss

were able to demonstrate precipitins to milk

in sera from a number of cases of milk

allergy. Recently, Heftier et used agar

gel diffusion and immunoelectrophoresis for

detecting precipitins to cow’s milk proteins

in the sera of patients with chronic iron

de-ficiency anemia, recurrent pneumonia,

fail-ure to thrive and other features.

In the study by Holland et aL6 and in

later work by Heiner et al.,7 a

reduc-tion in the number of precipitating bands

formed or the complete disappearance of

precipitating bands was demonstrated after

removal of milk from the diet of these

pa-tients. Concurrently, these patients showed

an amelioration or cessation of symptoms.

Generally, both of these groups used agar

gel diffusion to screen the sera of pediatric

admittances to their respective hospitals. In

those instances where precipitating milk

antibodies were found, an attempt was

made to relate the patients’ symptoms to

the ingestion of cow’s milk. This approach

resulted in describing a group of patients as

having a type of milk hypersensitivity

pre-viously unrecognized.

Recently, Parish et al.8 and Gunther et al.

attempted to relate milk protein

hemag-glutinin titers in sudden unexpected death

in infancy.

During the course of a controlled clinical

investigation of milk gy11 employing

milk and relatively pure isolated milk

pro-teins in provocative oral challenges, a

paral-lel study was made to determine if

circulat-ing antibodies to milk and these proteins

could be detected in the sera of milk

aller-gic individuals.

The purpose of the present investigation

was to evaluate agar gel diffusion, tanned

cell hemagglutination and passive

cutane-ous anaphylaxis in the guinea pig as

labora-tory methods for the confirmation of clinical

milk allergy. Also, we wished to compare

these results with clinical data and

infor-mation relative to the presence of skin

sen-sitizing tibo

METHODS AND MATERIALS

Blood samples were collected from 79 of

the milk allergic infants and children

de-scribeci earlier.bo

For control purposes, sera was collected

from 47 normal, nonallergic children and

from 52 children who were allergic to

sub-stances other than milk. The age

distribu-tion of these children corresponded to that

ARTICLES 581

umbilical cord blood samples were obtained

to investigate the possibility of transplacen-tal passage of milk antibodies.

The antigens consisted of raw skim milk,

colostrum, whole casein, alpha-lactalbumin,

beta-lactoglobulin, and serum albumin. All

of these were of bovine origin and their

preparation has been 12

Double Diffusion in Agar Gel

The method employed was essentially

that described by Crowle.13 Agar (Oxoid

lonagar #2) was used at a concentration

of 1% w/v in three different systems: (1)

glycine, 1M; (2) saline, 0.85% w/v; (3)

phos-phate buffer, 0.05M, pH 7.4.

Each serum was diffused against various

concentrations of skim milk and colostrum.

Dilutions of these antigens were made with

distilled water and ranged from 1 :2 to

1 :500. In most instances, best results were

obtained by over-night incubation of the

serum-inoculated slides before the antigens

were placed in their respective wells. The

slides containing the sera and antigens were

allowed to react for seven days in a moist

atmosphere at approximately 8#{176}C.Before

staining, the slides were cleared of residual

sera and proteins by soaking overnight in

phosphate buffer, pH 7.5-8.0.

The slides were stained for ten minutes

with thiazine red, 0.1% w/v in 0.2% v/v

acetic acid. Then they were soaked

re-peatedly in 1% v/v acetic acid until a clear

background was obtained. Those sera

desig-nated as negative were tested at least 6

times before being so classffied.

Coated Tanned Cell Hemagglutination

(TCH)

The methods of Boyden and Gunther

et al. were used, modified as follows: (1)

sheep erythrocytes were used in place of

human Type 0 erythrocytes, and (2)

dilu-tions of sera were made in 0.2% w/v gelatin

in place of the dilute normal rabbit serum.”

The gelatin was prepared in phosphate

buf-fered saline pH 7.2.

The

sera were tested at dilutions of 1:4

through 1 :65,536. The cord sera were

tested undiluted and through dilutions of

1 :8 if

necessary. Agglutination tests were

performed in 10 X 70 mm tubes using

volumes of 0.2 ml of the serum dilution to

which 0.05 ml of a 2% suspension of coated

cells was added. The agglutination patterns

were read as positive or negative after the

tubes were refrigerated overnight and

warmed to room temperature for thirty

minutes. Where positive results were

ob-tamed, inhibition of hemagglutination was

demonstrated in parallel tests by the

addi-tion of homologous milk antigens to various

dilutions of the sera. Controls were run

by

adding tanned but uncoated cells to the

sera, and coated cells to the diluting

solu-tions.

All sera were previously absorbed with

equal volumes of washed sheep erythrocytes.

Complement was inactivated by holding the

sera for thirty minutes in a 56#{176}C.water

bath.

Passive Cutaneous Anaphylaxis (PCA)

The method used was similar to that

de-scribed by Ovary.” Details of the

modifica-lion are given by Saperstein and Anderson.12

The sera obtained from the patients were

tested by injecting 0.1 ml into the skin of

a guinea pig. A maximum of six sites on the

back of each animal was used. The

anti-body, if present, was allowed to fix to the

tissue during a six hour incubation period.

Serum from each milk allergic patient was

tested for antibodies to alpha-lactalbumin,

beta-lactoglobulin, casein, serum albumin,

and unpasteurized colostrum or pasteurized

milk. It was found that sera from milk

allergic patients, which were positive to one

or more of the four protein antigens used,

were positive to milk and colostrum also.

Therefore, sera from control patients, both

normal and allergic, were screened for

anti-bodies against colostrum and pasteurized

milk, and only positive sera were tested for

antibodies to the four purified proteins.

5ev-eral of the positive sera were heated at

56#{176}C.for 4 hours to test the thermostability of the antibodies.

in-Patients PCA* Tanned Cell Hernagglutinin Titert

Milk Allergicli 79

Positivet 4

Positives 58

Median 82

Range

‘2-2048

Allergic, but not to milk 51 3 4 256 2-4096

Normal 40 ‘2 4 64 2-4096

SPassive cutaneous anaphylaxis in the guinea pig using casein, alpha-lactalbumin, beta-lactoglobulin, serum

albumin, and milk as the antigens.

tTanned sheep cells coated with colotrum protein.

Milk or colostrum was antigen used. § Poeitive to one or more antigens. IIGroup A and Group B patients combined.

582

MILK

ALLERGY

jected intravenously twenty minutes before

the antigen challenge to allow for the

de-tection of non-specific positive reactions.

The

antigen

level

for

the

challenge

dose

was 0.2-0.5 ml of a 1% w/v solution of the

purified protein dissolved in 0.85% w/v

saline, pH 7.0-7.2. When milk or colostrum

was used, the challenge dose was

1.0

ml

of the undiluted substance. Positive sera

gave visible reactions within fifteen minutes

in all cases. All reactions were rechecked;

negative results were confirmed three times in most cases.

Where possible, further serum samples

were obtained after the patient had been

on a milk free diet for several months.

These sera were examined by all three

methods.

RESULTS

The results of the milk antibody studies

for the different patient groups are shown

in Table I. Among the 40 normals, 2 sera

gave positive responses by agar gel

diffu-sion. The median hemagglutinin titer for

this group was 1 :64 with a range of

1:2-1

:4096. Positive responses by

PCA

were

obtained with 4 of these sera when

cobs-trum was used as the antigen.

With the cord sera, no positive agar gel

diffusion tests, no significant hemagglutinin

titers, nor any positive PCA reactions were

observed.

Of

the 51 sera collected from children

allergic to substances other than milk, 3

gave a positive response by agar gel

diffu-sion. The median hemagglutinin titer was

1 :256 with

a range of 1 :2-i :4096. Positive

responses by PCA were obtained with four

of these sera.

There were 79 individual sera collected

from the milk allergic group. Of these, 4

gave a positive response by agar gel

diffu-sion. The median hemagglutinin titer for

this group was 1 :32 with a range of

1:2-1 :2048.

In 53 instances positive PCA tests

were demonstrated. Of these positive tests,

30

were to casein, 27 to beta-lactogbobulin,

19 to serum albumin and 9 to

alpha-lactalbumin. Three patients’ sera were

posi-tive to milk and cobostrum, but not to the

purified proteins, indicating antibodies to

an antigen(s) of milk other than the four

proteins used. In those sera where tests

were made with both the isolated proteins

and milk, all sera positive to one or more

proteins were positive to milk also.

In an earlier

report,1#{176} relative to the oral

challenges of the milk allergic children,

pa-tients were classified as those who were

challenged with both milk and purffied milk

proteins (Group A), and those who were

challenged with milk only (Group B). The

serologic data for each of these patients is

shown in Tables II and III respectively.

Whether compared by hemagglutinin titers,

agar gel diffusion results, or PCA responses

to the individual proteins, no significant

TABLE I

Misx ANTIBODY STUDIES WITH SrnA FROM ALLERGIC AND NORMAL CHILDREN

Agar Gel

No. .

ARTICLES 583

TABLE II

MILK ANTIBODY ItRSULTS ON SERA FROM GROUP A PATIENTS*

Passive Cutaneous Anaphylaxi4

Month8 -

-

- Agar

Hernagglu-Patieni off Alpha- Beta- Gel tinin

Milkt (wejn lactal- loctoglob- Serum .%IilL#{149} Diffusion TiterI

. Albumin

bumin unn

I None + + + + id 128

‘2 None + + - - ml - 16

3 ‘2 - - - - - - 4

4 3 - - - - - - 64

5 4 - - - - - - 4

6 14 - - - - 128

7 nd nd iid ml ud iid iicl nd 8 3 - - - - - - 56

9 - - - - - - 16

to

6 - - - - - - 8

11 ‘2 + - - - id - 16 12 12 - - - - 32

13 - + + - 11(1 - 128 14 34 - - - - + - 16

is None + + + + + - 32 16 1 - - + - id - 8 17 ml lid lid rid rid iid iid nd 18 1 + - - - + - ‘2

24 54 - - - - 32

25 - - + - + + 2,048 26 1 - - - - iid - ml ‘27 Unknown - + - iid iid - 8 ‘28 1 - - - - - - 2

29 24 - - - - - - ‘2

30 l + ml iid + iid - nd 31 14 - - - - - - 16

32 None + - + - lid - ‘2 33 nd 1141 nd nd li(l tid nd nd 34 None + - - - + - 32

35 1 + - - - iid - 8 36 None - - + - lid - 64 37 1 + - - - nd - 128 38 8 - - - - - - 16

39 2 - - + + nd - 16 40 None + - + - nd - 16 41 ‘2 - - + - nd + ‘2,048 42 1 + - + - nd - 32 43 24 + - - - + - 4

44 + - - - +

-45 Unknown - - - + nd - 64

* Challenged orally with milk and purified milk proteins. t Months on milk-free diet before blood was collected.

nd = not done because serum not collected or quantity collected insufficient.

§Double diffusion in agar gel. Milk or colostrum was antigen used. 1!Tanned sheep cells coated with colostrum protein.

584

MILK

ALLERGY

TABLE III

Ssaowoicu RESULTS FOR GROUP B PATIENTS*

Patient

Months

off

Milkt

Panive Cutaneous Anaphylaxi4

Agar Gel

Hemagglu-tinin

TII

Casein

Alpha-

lactal-bumin

Beta-

lactoglob-ulin

Serum

Albumin Milk

I 2 + - - nd

-2 4 - - + - + - 64

3 1 - - - + nd - 64

4 None + - - + nd - 4

5 None - - - - nd - nd

6 nd nd nd nd nd nd rid nd

7 nd iid nd rid nd nd nd nd

8 nd nd nd nd nd nd nd nd

9 1 + - - - + - 64

10 1 - - - - - - 128

11 None + - + + 16

12 1 - - + - nd - 256

13 None + - + 512

14 None - - + - nd + 2,048

15 6 + + + + nd - nd

16 None + + nd + 2,048

17 3 + - + - nd - 128

18 4 - - - - + - 32

19 nd nd nd nd nd nd nd nd

20 None - - - + nd - 16

21 9 - - - - - - 4

22 None - - + - nd - nd

23 11 - - - - - rid 32

24 nd nd nd nd nd nd nd

25 None + - + nd - 16

26 - - + + + - 32

27 3 - - - + nd - 32

28 None - - - - - - 32

29 Unknown - - - - - - 32

30 1 + + + - + 512

31 1 + - - - + - 128

32 - - + - nd - 8

33 None + - - - tid - 32

34 nd nd nd nd nd nd nd ad

35 3 + - + - + - ad

36 8 - - - - - - 32

37 1 - + - + nd - 64

38 ‘2 - - - - - - 16

39 1 + - - - + - 16

40 2 - - - - + - 32

41 ad nd nd nd lid ad ad ad

42 None - - + - + 64

43 4 - + - + nd - 1,024

44 4 + - + - ad - ad

* Challenged orally with milk only.

t Months on milk-free diet before blood was collected.

nd = not done because either serum not collected or quantity collected insufficient.

§Milk or colostrum was antigen used.

TABLE IV

RELATIONSHIP OF RESULTS OF PCA* TESTS TO TIME

THE Miu ALLERGIC PATIENTt WAS ON A MILK-FREE DIET PRIOR TO COLLECTING BLOOD SAMPLE

ARTICLES 585

difference was noted between Groups A

and B.

The results of PCA of sera from milk

allergic patients were compared to

impor-tant features which had been previously

documented for these same patients.1#{176} No

correlation was found between positive

PCA

results and the following: (1) age of

onset of milk allergy; (2) duration of the

allergic state; (3) presenting symptoms; (4)

challenge symptoms; (5) time of onset or

duration of the oral challenge reactions;

(6)

results of oral challenge with each of

the individual proteins; (7) results of skin

testing with each of the isolated proteins;

(8) age of the patient at the time serum was

collected.

An inverse relationship was found

be-tween PCA response and the time interval

a patient had been on a milk-free diet prior

to the collection of the blood sample

(Table IV).

COMMENT

The tanned cell hemagglutination method

and the agar gel diffusion method did not

prove to be of value in our hands as

labo-ratory tools for the confirmation of milk

allergy. The demonstration of

hemagglu-tinins to milk was not indicative of the

presence of milk allergy inasmuch as the

pattern in normal and allergic children was

essentially the same. The premise that

he-magglutinins to milk may be involved in

anaphylaxis in nf9 is not supported

by this work. In several instances in which

an anaphylactic response was obtained on

oral challenge with milk proteins,10 the sera

from these patients revealed very low titers

by the hemagglutination method. On the

other hand, several normal children showed

extremely high titers by this procedure.

High hemagglutinin titers correlated with

the other tests only to the extent that high

titers (1:2000 and up) were found when

precipitins were demonstrated by agar gel

diffusion. In these instances, the PCA tests

were also positive. However, as seen in

Tables III and IV, positive PCA results

Time on

Milk-free Diet

(mo)

&SUltS of PCA

Positives Negative 1 1-2 2-3 3-4 <4 Unknown Total 35 8 3 5 1 2 54 6 4 3 ‘2 9 1 25

* Passive cutaneous anaphylaxis in the guinea pig using casein, alpha-lactalbumin, beta-lactoglobulin,

serum albumin and milk as the antigens.

t Group A and Group B patients combined.

Positive to one or more antigens.

were found in many instances where the

hemagglutinin titer was insignificant.

Our results with the TCH method are in

close agreement with those of Gunther

et al.#{176}relative to the titers found with cord

sera. On the other hand, these results do

not agree with the findings of Wright

et d17 who showed very high titers to

several milk proteins in the cord sera they

examined. The two studies may not be

corn-parable since Wright et al.’ used the

iso-lated proteins as antigens whereas we used

the entire colostrum in the TCH method.

Our results demonstrated that precipitins

could be found with the same frequency in

the sera of normal and non-milk allergic

children as in the sera of milk allergic

chil-dren. We do not look upon our results with

agar gel diffusion as contrary to the

find-ings of Holland et al. and Heiner et

Both groups have found precipitins to milk

in normal children. The general syndrome

with which Heftier et ai were able to

as-sociate a high incidence of milk precipitins

is quite different from the more common

milk allergic symptoms usually observed.

Our study group was different from that of

both Holland and Heiner, as was our

586 MILK ALLERGY

The PCA method proved to be the most

promising of the three serologic methods,

since positive reactions were found in high

frequency only in the milk allergic children.

Paradoxically, the results of this antibody

determination did not correlate with the

results of oral challenge with the same milk

protein. The reason for this discrepancy is

not clear, although in some instances the oral

challenge dose could have been below the

response threshold.

The antibodies concerned with positive

PCA

responses do not appear to be reagins

as they were heat stable and the results did

not correlate with those of skin testing when

the same protein was used. A further point

of difference is that the PCA antibodies

dis-appeared within a few months following

milk elimination whereas the skin

sensitiz-ing antibodies persisted.

To obtain meaningful PCA results when

studying milk allergic children, the sera

samples should be collected before, or very

soon after, dietary milk elimination. It was

observed that the PCA reaction tended to

be negative following a milk elimination

diet of several months, although this was

an individual variation. This was confirmed

by

further serum sampling of the individual

patients. Precipitins in the sera of milk

al-lergic patients also disappeared, a finding

which has been reported by 67

Examination of Table

IV

shows that if

the sera collected from patients who had

been on a milk-free diet for 4 or more months

are eliminated from the calculations, then

approximately 80% of the milk allergic

pa-tients elicited a positive PCA response.

It

was of interest that positive PCA

re-sponses were obtained with sera from several

milk allergic infants under age 3 months.

Much evidence indicates that

gamma-globulin antibodies of 160,000 molecular

weight do not develop in response to

anti-gen administration before 4 to 8 weeks in

the human infant.” In spite of this, it may

be possible that the antibodies being

meas-ured are of the gamma-globulin or

beta-2A-globulin types, because macroglobulins have

been shown” not to give positive PCA

re-actions. Further studies are necessary along

these lines in order to characterize these

an-tibodies and assess their significance to the

diagnosis and understanding of milk allergy.

SUMMARY

The examination of sera from milk

al-lergic children, children allergic to

sub-stances other than milk, and normal

chil-dren, revealed no significant differences

relative to coated tanned cell hemagglutinin

titer or the presence of precipitating

anti-body as measured by gel diffusion for milk

components.

The passive cutaneous anaphylaxis test

revealed a high percentage of positive

re-sponses with sera from milk allergic

mdi-viduals only.

The presence of milk hemagglutmnmns

could not be demonstrated in the cord sera

from 30 newborns.

The results of this study suggest that the

passive cutaneous anaphylaxis technique

de-serves further evaluation as a laboratory

method for the confirmation of milk allergy.

REFERENCES

1. Prausnitz, C., and Kustner, H. : Studien #{252}ber die Veberempfindlichkeit Zbl. Bakt., 86:160, 1921.

2. Boyd, W. C. : Fundamentals of Immunology, Ed. 3. New York, Interscience, 1956. 3. Anderson, A. F., and Schloss, 0. M. : Allergy

to cow’s milk in infants with nutritional

disorders. Amer. J. Dis. Child., 26:451,

1923.

4. Heiner, D. C., Sears, J. W., and Kniker, W. T.:

Multiple precipitins to cow’s milk in chronic

respiratory disease. Amer. J. Dis. Child.,

103:40, 1962.

5. Wilson, J. F., Heiaer, D. C., and Lahey, E.

M. : Studies on iron metabolism: I.

Evi-dence of gastrointestinal dysfunction in

infants with iron deficiency anemia : a

pre-liminary report. J. Pediat., 60:787, 1962.

6. Holland, N. H., et al.: Significance of

pre-cipitating antibodies to milk proteins in

the serum of infants and children. J. Pediat.,

61:181, 1962.

7. Heiner, D. C., at al.: Precipitins to antigens

of wheat and cow’s milk in celiac disease.

J. Pediat., 61:813, 1962.

ARTICLES 587

milk and sudden death in infancy. Lancet,

2:1106, 1960.

9. Gunther, M., at at.: The level of antibodies

to the proteins of cow’s milk in the serum

of normal human infants. Immunology, 3:

296, 1960.

10. Goldman, A. S., et at.: Milk allergy: I. Oral

challenge with milk and isolated milk

pro-teins in allergic children. Pr.ntmics, 32:425, 1963.

11. Goldman, A. S., et al.: Milk allergy: II. Skin

testing of allergic and normal children with

purified milk proteins. PEDIATRICS, 32: 572,

1963.

12. Saperstein, S., and Anderson, D. W. :

Anti-genicity of milk proteins of prepared

formu-las measured by precipitin ring test and passive cutaneous anaphylaxis in the guinea pig. J. Pediat., 61 :196, 1962.

13. Crowle, A. J.: A simple micro

double-diffu-sion agar precipitin technique.

J.

Lab. Clin. Med., 52:784, 1958.

14. Boyden, S. V.: i’he adsorption of proteins on

erythrocytes treated with tannic acid and

subsequent hemagglutination by antiprotein

sera.

J.

Exper. Med., 93: 107, 1951.

15. Kabat, E. A., and Mayer, M. M. : Experimental

Immunochemistry. Springfield, Illinois;

Thomas; 1961, Chap. 3.

16. Ovary, Z. : Immediate reaction in skin of ex-perimental animals provoked by antibody-antigen interaction. Prog. Allergy, 5:459, 1958.

17. Wright, R., et at.: Circulating antibodies to

cow’s milk proteins and gluten in the

new-born. Brit. Med.

J.,

2:513, 1962.

18. Bridges, R. A., et at.: The morphologic basis

of antibody formation development during

the neonatal period. J. Lab. Clin. Med.,

53:331, 1959.

Acknowledgment

We wish to thank Evelyn M. Cary for her

1963;32;580

Pediatrics

S. Saperstein, D. W. Anderson, Jr., A. S. Goldman and W. T. Kniker

Children

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Pediatrics

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Children

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