129
CIinica Chimica Acta, 78 (1977) 129-133 @ Els~vier/North-~~IIand Biomedical Press
CCA 8620
THE RADIOIMMUN~ASSAY
KENT
L. WOODS
Department (Received
and DAVID
of Medicine, December
20th,
OF HUMAN LA~ALB~IN
A. HEATH
University
ofBirmingham,
Birmingham
B15
2TH
(U.K.)
1976)
Summary A radioimmunoassay has been developed for the measurement of human lactalbumin, using the protein isolated in pure form from breast milk. Under disequilibrium conditions with second antibody separation the system will detect 20 pg of lactalbumin. Lactalbumin is detectable in the serum of approximately a third of normal male subjects; among women the proportion having measurable serum levels is age-related. Very much higher levels are present in the serum of lactating women.
Introduction La&albumin functions as the specifier protein of the lactose synthetase system [l]. Its production is thought to be confined to mammary epithelium. In human milk it is quantitatively the most important of the whey proteins. For these reasons accurate measurement of human lactalbumin has potential value as an index of protein synthesis by the human breast and might help to clarify the endocrine factors influencing lactation. It is of particular importance to know whether malignant breast epithelium secretes lactalbumin and, if so, whether this can provide any useful information on the viable cell mass or the degree of differentiation of the carcinoma. We have, therefore, developed a sensitive and specific radioimmunoassay for human lactalbumin as described below. Materials
and methods
Lact~bumin was separated from human breast milk by the method of Phillips and Jenness [2]. This involves precipitation of casein at pH 4.6 followed,by fractionation of the whey proteins on a DEAE-Sepahadex column in a linear concentration gradient of phosphate buffered saline and then on a Sephadex G 100 column. The identity and purity of the final product was assessed by disc electrophoresis and by its lactose synthetase B protein activity.
130
Antibodies to la&albumin were raised in rabbits by serial intradermal immunisation. Radioiodinated lactalbumin was prepared by the chloramine T method [31.
Assays were carried out in triplicate at 4°C. Antibody to lac~bumin at a final dilution of 1:400 000 was incubated with serial dilutions of known stan(“tracer”) was dards or unknown samples; after 24 h ‘251-labelled lactalbumin added. Separation of bound and free tracer was effected after a further 24 h by the second antibody method. Antibody bound radioactivity was expressed as a percentage of the total amount of tracer added (B/T(%)). This function plotted against the concentration of unlabelled lactalbumin provided the standard curve. Displacement of tracer by an unknown sample was considered significant if the mean value of
B/T differed from the initial binding point (no unlabelled lactalbumin present) by more than three standard deviations. Wherever possible serial dilutions of the unknown were assayed to confirm parallelism with the standard curve, a phenomenon which provides necessary but not conclusive proof of identity. The range of serum values for normal subjects was obtained from a healthy population of laboratory workers and office workers undergoing routine medical screening. Results Disc gel electrophoresis was performed by the method of Davis [4 1. The electrophoretic patterns of human whey and the purified la&albumin prepara-
tion are shown in Fig. 1. The electrophoretic mobility of the purified protein was similar to that previously reported by Nagasawa et al. [ 51. The identity of this protein as lac~bumin was further confirmed by enzymatic assay (kindly performed by Dr. NJ. Kuhn). Using a rat mammary membrane preparation as a source of galactosyl transferase, lactose was synthesised from glucose and UDP [14C]galactose at a rate of 280 pm01 of lactose per 20 min per pg of lactalbumin. Specific activities of 200-400 pCi/gg (2.8-5.6pCi/pmol) were attained in the preparation of tracer. The affinity of lactalbumin for antibody was not affected by iodination. 1251-labelled lact~bumin could be stored for up to eight weeks at -20°C before use. Fig. 2 shows a typical standard curve for the assay which will detect 20 pg of lactalbumin per tube. The duration of the first and second incubations is particularly important for the achievement of maximal sensitivity; addition of tracer at 12 h rather than 24 will raise the threshold of detection to around 70 pg per tube, as will prolonged incubation to equilibrium. Using the assay design outlined above, the limit of detection is 0.4 ngfml for serum and 0.2 ng/mI for organ culture supernatants or cytosol fractions of homogenised tissues. Coefficients of variation were 3.1% (within-assay, eighteen replicates) and 6.3% (between-assay, six determinations) for a standard containing 2.5 ng a-la&albumin per ml. No displacement of iracer was producer by the following human proteins at the final concentrations stated: growth hormone, 1 pg/ml; FSH 200 ng/ml; LH
131
40
10
L
LoPg
In9
1OOP9 o(
lAClAlEUMiN
lOW
x-x
Fig. 1. Disc gel electrophoresis of human whey proteins (left) and purified lactalbumin (right) in pol~acr~lamide at pH 8.4. The origin is at the top; la&albumin is the fastest major band in the whey protein preparation. Fig. 2. Standard curve for lactalbumin and serial dilutions of serum from five lactating women (inset scale> studied in the same assay. Each point is the mean of triplicates.
200 ng/ml; chorionic gonadotrophin 1 pg/ml; pro&tin 100 ng/ml; lysozyme 100 pg/ml; serum albumin 40 mg/ml. Serum from eleven lactating women so far examined all contain lactalbumin and serum dilution curves are parallel to the standard curve {Fig. 2). No detectable loss of immu~oreactive la&albumin occurred when the protein was diluted in serum (1.1 ng/ml) and stoiced at room temperature for up to four days. We have found immunoreactive lactalbumin in the serum of 27 (45.8%) of 59 seemingly healthy women; in only one subject did the level exceed 10 ng/ml (Fig. 3). The range of values seen in different age groups did not vary significantly. However, the proportion of subjects with detectable circulating lactalbumin was 21/33 (63.6%) below the age of 50 and 6/26 (23.1%) in those aged 50 or over. This difference is highly significant (p < 0.01) by the x2 test. No difference was noted between subjects taking the contraceptive pill and those who were not.
132 c
FEMALES
I
MALES
.(74)
10.0
9.0
.
8.0
.
1.0
.
6.0 E r z
5.c
ii III 2
.
4.c
P "
:
", 3.1
:
2.c
.
.
.
* .
1.c
. *..
AGE
20-34
POS.
10113
Fig. 3. The results baseline represents
*.*.m*.**
,..a.*...
..,*.I**** l ‘**.*..*.
. ......... .I.
.
.
.
.
.
.
.
.
.
-
*.*..
35-49
50-64
20-34
35-49
50-64
11120
6126
7116
4117
8123
of serum lactdlbumin assays on 115 normal subjects, classified by sex and age. The the limit of detection of the assay. POS., proportion having detectable levels.
Circulating lactalbumin was found in 19 (33.9%) of 56 normal males; the level was below 6 ng/ml in every case. Neither the range nor the proportion with detectable levels showed significant age variations in males. In the eleven lactating subjects studied, serum levels have been in the range 125 ng/ml to 1.76 pg/ml. One healthy, non-lactating female subject had a serum lactalbumin level of 74 ng/ml. A repeat assay on the same sample and a further assay of serum taken two weeks later were of the same order; serial dilutions ran parallel to the standard curve. Three similar subjects have since been found in a hospital population. So far the high levels have not been correlated with any particular breast condition and they remain unexplained. Discussion Radioimmunoassay is capable of detecting much smaller amounts of lactalbumin than enzymatic methods; we believe the system described here to be the
133
most sensitive so far reported. Radioimmunoassays for lactalbumin have recently been described by Rose and McGrath [6] and by Kleinberg [ 71. The former authors incubated all the assay components together for 2 h at 37” C, then added goat anti-rabbit y-globulin antiserum to separate bound from free 1251-labelled lactalbumin. The quoted detection limit of their system was 0.150 ng of lactalbumin per assay. At the other extreme, Kleinberg used a 5-day incubated period at 4”C, which would allow equilibrium to be reached; his detection limit of 0.05 ng lactalbumin per assay is close to the sensitivity we found when using equilibrium conditions. To reduce the detection limit of our assay to 0.02 ng we found it necessary to limit the duration of incubation, adding tracer at 24 ? 3 h and second antibody after a further 24 * 3 h. Kleinberg found immunoreactive lactalbumin in the serum of 9 of 22 normal men and 15 of 33 non-lactating women. Our own results are in broad agreement; the presence of circulating lactalbumin in normal men is rather surprising and merits further investigation. It seems improbable that the rudimentary male mammary glands are the source, yet no tissue other than mammary epithelium has so far been shown to produce lactalbumin. To find the protein circulating in non-lactating females is less surprising and the falling incidence of detection above the age of 50 years may be explained by the gradual atrophy of breast tissue beginning around the menopause. The assay is currently being used to study groups of women with both benign and malignant breast disease. It has also been found satisfactory for organ culture supernatants and tissue cytosol fractions and may therefore shed light on the hormonal regulation and synthetic activity of both normal and neoplastic breast tissue. The assay has proved to be a versatile, reproducible and specific technique with many applications in research on the human mammary gland. Acknowledgements We are grateful to Professor R. Hoffenberg, and Professor T.P. Whitehead for their help and encouragement. K.L.W. began this work as a Sheldon Research Fellow (West Midland Regional Health Authority) and is currently an M.R.C. Training Fellow. Professor W.R. Butt kindly supplied the various human hormones for cross-reactivity studies, Dr.A.R. Bradwell the precipitating antibody, Dr.D.Y. Mason the human lysqzyme and Dr.P. Wilding some of the sera. We wish to thank the Smith Kline and French Foundation for financial support. References 1
Brew.
2
Phillips,
K.,
Vanaman. N.I.
3
Hunter,
4
Davis,
W.M.
5
Nagasawa.
B.J.
T.C.
and Jenness. and
(1964) T.,
and
Greenwood, Ann,
Kiyosawa,
Hill,
R.L.
R. (1971) New
F.C. York
(1968)
Biochim. (1962) Acad.
I.,
Kuwahara,
C.M.
(1975)
Proc.
Natl.
Biophys. Nature
Lond.
Sci.
121,
K.,
Fukuwatari.
Rose,
7
Kleinberg.
H.N.
and D.L.
McGrath, (1975)
Science
190,
Science 276-278
194.
229.
Sci.
U.S.A.
59,
491-497
407-410
495-496
404-427
289-293 6
Acad.
Acta
190,673+75
Y.
and
Suzuki,
T.
(1972)
d. Dairy
Sci.
55.
CIinica Chimica Acta, 78 (1977) 129-133 @ Els~vier/North-~~IIand Biomedical Press
CCA 8620
THE RADIOIMMUN~ASSAY
KENT
L. WOODS
Department (Received
and DAVID
of Medicine, December
20th,
OF HUMAN LA~ALB~IN
A. HEATH
University
ofBirmingham,
Birmingham
B15
2TH
(U.K.)
1976)
Summary A radioimmunoassay has been developed for the measurement of human lactalbumin, using the protein isolated in pure form from breast milk. Under disequilibrium conditions with second antibody separation the system will detect 20 pg of lactalbumin. Lactalbumin is detectable in the serum of approximately a third of normal male subjects; among women the proportion having measurable serum levels is age-related. Very much higher levels are present in the serum of lactating women.
Introduction La&albumin functions as the specifier protein of the lactose synthetase system [l]. Its production is thought to be confined to mammary epithelium. In human milk it is quantitatively the most important of the whey proteins. For these reasons accurate measurement of human lactalbumin has potential value as an index of protein synthesis by the human breast and might help to clarify the endocrine factors influencing lactation. It is of particular importance to know whether malignant breast epithelium secretes lactalbumin and, if so, whether this can provide any useful information on the viable cell mass or the degree of differentiation of the carcinoma. We have, therefore, developed a sensitive and specific radioimmunoassay for human lactalbumin as described below. Materials
and methods
Lact~bumin was separated from human breast milk by the method of Phillips and Jenness [2]. This involves precipitation of casein at pH 4.6 followed,by fractionation of the whey proteins on a DEAE-Sepahadex column in a linear concentration gradient of phosphate buffered saline and then on a Sephadex G 100 column. The identity and purity of the final product was assessed by disc electrophoresis and by its lactose synthetase B protein activity.
130
Antibodies to la&albumin were raised in rabbits by serial intradermal immunisation. Radioiodinated lactalbumin was prepared by the chloramine T method [31.
Assays were carried out in triplicate at 4°C. Antibody to lac~bumin at a final dilution of 1:400 000 was incubated with serial dilutions of known stan(“tracer”) was dards or unknown samples; after 24 h ‘251-labelled lactalbumin added. Separation of bound and free tracer was effected after a further 24 h by the second antibody method. Antibody bound radioactivity was expressed as a percentage of the total amount of tracer added (B/T(%)). This function plotted against the concentration of unlabelled lactalbumin provided the standard curve. Displacement of tracer by an unknown sample was considered significant if the mean value of
B/T differed from the initial binding point (no unlabelled lactalbumin present) by more than three standard deviations. Wherever possible serial dilutions of the unknown were assayed to confirm parallelism with the standard curve, a phenomenon which provides necessary but not conclusive proof of identity. The range of serum values for normal subjects was obtained from a healthy population of laboratory workers and office workers undergoing routine medical screening. Results Disc gel electrophoresis was performed by the method of Davis [4 1. The electrophoretic patterns of human whey and the purified la&albumin prepara-
tion are shown in Fig. 1. The electrophoretic mobility of the purified protein was similar to that previously reported by Nagasawa et al. [ 51. The identity of this protein as lac~bumin was further confirmed by enzymatic assay (kindly performed by Dr. NJ. Kuhn). Using a rat mammary membrane preparation as a source of galactosyl transferase, lactose was synthesised from glucose and UDP [14C]galactose at a rate of 280 pm01 of lactose per 20 min per pg of lactalbumin. Specific activities of 200-400 pCi/gg (2.8-5.6pCi/pmol) were attained in the preparation of tracer. The affinity of lactalbumin for antibody was not affected by iodination. 1251-labelled lact~bumin could be stored for up to eight weeks at -20°C before use. Fig. 2 shows a typical standard curve for the assay which will detect 20 pg of lactalbumin per tube. The duration of the first and second incubations is particularly important for the achievement of maximal sensitivity; addition of tracer at 12 h rather than 24 will raise the threshold of detection to around 70 pg per tube, as will prolonged incubation to equilibrium. Using the assay design outlined above, the limit of detection is 0.4 ngfml for serum and 0.2 ng/mI for organ culture supernatants or cytosol fractions of homogenised tissues. Coefficients of variation were 3.1% (within-assay, eighteen replicates) and 6.3% (between-assay, six determinations) for a standard containing 2.5 ng a-la&albumin per ml. No displacement of iracer was producer by the following human proteins at the final concentrations stated: growth hormone, 1 pg/ml; FSH 200 ng/ml; LH
131
40
10
L
LoPg
In9
1OOP9 o(
lAClAlEUMiN
lOW
x-x
Fig. 1. Disc gel electrophoresis of human whey proteins (left) and purified lactalbumin (right) in pol~acr~lamide at pH 8.4. The origin is at the top; la&albumin is the fastest major band in the whey protein preparation. Fig. 2. Standard curve for lactalbumin and serial dilutions of serum from five lactating women (inset scale> studied in the same assay. Each point is the mean of triplicates.
200 ng/ml; chorionic gonadotrophin 1 pg/ml; pro&tin 100 ng/ml; lysozyme 100 pg/ml; serum albumin 40 mg/ml. Serum from eleven lactating women so far examined all contain lactalbumin and serum dilution curves are parallel to the standard curve {Fig. 2). No detectable loss of immu~oreactive la&albumin occurred when the protein was diluted in serum (1.1 ng/ml) and stoiced at room temperature for up to four days. We have found immunoreactive lactalbumin in the serum of 27 (45.8%) of 59 seemingly healthy women; in only one subject did the level exceed 10 ng/ml (Fig. 3). The range of values seen in different age groups did not vary significantly. However, the proportion of subjects with detectable circulating lactalbumin was 21/33 (63.6%) below the age of 50 and 6/26 (23.1%) in those aged 50 or over. This difference is highly significant (p < 0.01) by the x2 test. No difference was noted between subjects taking the contraceptive pill and those who were not.
132 c
FEMALES
I
MALES
.(74)
10.0
9.0
.
8.0
.
1.0
.
6.0 E r z
5.c
ii III 2
.
4.c
P "
:
", 3.1
:
2.c
.
.
.
* .
1.c
. *..
AGE
20-34
POS.
10113
Fig. 3. The results baseline represents
*.*.m*.**
,..a.*...
..,*.I**** l ‘**.*..*.
. ......... .I.
.
.
.
.
.
.
.
.
.
-
*.*..
35-49
50-64
20-34
35-49
50-64
11120
6126
7116
4117
8123
of serum lactdlbumin assays on 115 normal subjects, classified by sex and age. The the limit of detection of the assay. POS., proportion having detectable levels.
Circulating lactalbumin was found in 19 (33.9%) of 56 normal males; the level was below 6 ng/ml in every case. Neither the range nor the proportion with detectable levels showed significant age variations in males. In the eleven lactating subjects studied, serum levels have been in the range 125 ng/ml to 1.76 pg/ml. One healthy, non-lactating female subject had a serum lactalbumin level of 74 ng/ml. A repeat assay on the same sample and a further assay of serum taken two weeks later were of the same order; serial dilutions ran parallel to the standard curve. Three similar subjects have since been found in a hospital population. So far the high levels have not been correlated with any particular breast condition and they remain unexplained. Discussion Radioimmunoassay is capable of detecting much smaller amounts of lactalbumin than enzymatic methods; we believe the system described here to be the
133
most sensitive so far reported. Radioimmunoassays for lactalbumin have recently been described by Rose and McGrath [6] and by Kleinberg [ 71. The former authors incubated all the assay components together for 2 h at 37” C, then added goat anti-rabbit y-globulin antiserum to separate bound from free 1251-labelled lactalbumin. The quoted detection limit of their system was 0.150 ng of lactalbumin per assay. At the other extreme, Kleinberg used a 5-day incubated period at 4”C, which would allow equilibrium to be reached; his detection limit of 0.05 ng lactalbumin per assay is close to the sensitivity we found when using equilibrium conditions. To reduce the detection limit of our assay to 0.02 ng we found it necessary to limit the duration of incubation, adding tracer at 24 ? 3 h and second antibody after a further 24 * 3 h. Kleinberg found immunoreactive lactalbumin in the serum of 9 of 22 normal men and 15 of 33 non-lactating women. Our own results are in broad agreement; the presence of circulating lactalbumin in normal men is rather surprising and merits further investigation. It seems improbable that the rudimentary male mammary glands are the source, yet no tissue other than mammary epithelium has so far been shown to produce lactalbumin. To find the protein circulating in non-lactating females is less surprising and the falling incidence of detection above the age of 50 years may be explained by the gradual atrophy of breast tissue beginning around the menopause. The assay is currently being used to study groups of women with both benign and malignant breast disease. It has also been found satisfactory for organ culture supernatants and tissue cytosol fractions and may therefore shed light on the hormonal regulation and synthetic activity of both normal and neoplastic breast tissue. The assay has proved to be a versatile, reproducible and specific technique with many applications in research on the human mammary gland. Acknowledgements We are grateful to Professor R. Hoffenberg, and Professor T.P. Whitehead for their help and encouragement. K.L.W. began this work as a Sheldon Research Fellow (West Midland Regional Health Authority) and is currently an M.R.C. Training Fellow. Professor W.R. Butt kindly supplied the various human hormones for cross-reactivity studies, Dr.A.R. Bradwell the precipitating antibody, Dr.D.Y. Mason the human lysqzyme and Dr.P. Wilding some of the sera. We wish to thank the Smith Kline and French Foundation for financial support. References 1
Brew.
2
Phillips,
K.,
Vanaman. N.I.
3
Hunter,
4
Davis,
W.M.
5
Nagasawa.
B.J.
T.C.
and Jenness. and
(1964) T.,
and
Greenwood, Ann,
Kiyosawa,
Hill,
R.L.
R. (1971) New
F.C. York
(1968)
Biochim. (1962) Acad.
I.,
Kuwahara,
C.M.
(1975)
Proc.
Natl.
Biophys. Nature
Lond.
Sci.
121,
K.,
Fukuwatari.
Rose,
7
Kleinberg.
H.N.
and D.L.
McGrath, (1975)
Science
190,
Science 276-278
194.
229.
Sci.
U.S.A.
59,
491-497
407-410
495-496
404-427
289-293 6
Acad.
Acta
190,673+75
Y.
and
Suzuki,
T.
(1972)
d. Dairy
Sci.
55.
