271
Clinica Chimica Acta, 65 (1975) 271-282 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 7 289
IMMUNOLOGICAL METHODS FOR HUMAN PLACENTAL PHOSPHATASE (REGAN ISOENZYME)
FRANK-GtiNTER
ALKALINE
LEHMANN
Department of Medicine, University of Marburg/Lahn, E. Mannkopffstrasse 1, D-355 Marburg/Lahn (G.F.R.)
(Received
May 9,1975)
Summary Four different immunological methods for the determination of the placental isoenzyme of alkaline phosphatase (Regan isoenzyme) were compared in 64 normal blood donors, 23 healthy laboratory and medical staff workers and 68 pregnant women: a. Inhibition by soluble antibodies to the placental enzyme. b. Precipitation with soluble antibodies. c. Precipitation with immobilized antibodies. d. Measurement of the activity of the placental alkaline phosphatase following binding to an immunoabsorbent that has been obtained by polymerization of anti-placental-alkaline phosphatase y-globulin using glutaraldehyde. The immunoabsorbent method yielded the best results. The optimal conditions were evaluated for the measurement of the activity of immunoabsorbent-fixed placental (tumoral) alkaline phosphatase activity. This method was applied to 209 normal blood donors and to 239 patients with different malignant tumors: 25.5 percent of the cancer patients exhibited an elevated Regan -isoenzyme activity in the serum.
Introduction Human placental alkaline phosphatase (P-AP) contributes in minor degree to the total activity of alkaline phosphatases in normal serum. P-AP activity rises under some pathological conditions, especially in pregnancy and in some tumor patients with neosynthesis of placental-type alkaline phosphastase (Regan isoenzyme). Therefore, a specific and sensitive determination of P-AP is required for the detection of the small portion of P-AP within the total alkaline
212
phosphatase activity. Heat stability [l] , electrophoresis of the antibody-bound P-AP (showing a retarded electrophoretic migration) and staining with a fluorogenie substrate [ 21, radioimmunoassays [ 3,4] and an immunochemical method [ 51 have been described for the measurement of P-AP in cancer patients and in pathological pregnancies. We developed and compared different immunological methods for the placental isoenzyme of alkaline phosphatase as a first step to an immunochemical differentiation of alkaline phosphatases in human serum. Material and methods 1. A Ekaline phosphatases Human placental (P-AP), intestinal (I-AP) and liver (L-AP) alkaline phosphatases were isolated as described in ref. 6. The specific activity was 196 U/mg for P-AP, 436 U/mg for I-AP and 41.3 U/mg for L-AP. Purified human kidney and bone phosphatases were preparations of Professor Dr. G. Pfleiderer (Department of Chemistry, University of Bochum) [7], as well as I-AP until we were able to isolate our own enzyme. 2. Antisera Anti-P-AP sera were obtained by immunizing rabbits with crystalline material in complete Freund’s adjuvant according to the scheme described previously [ 81. Anti-P-AP-y-globulin immunoabsorbents were prepared by precipitation of the antisera first with ammonium sulfate up to 40 percent and second up to 35 percent saturation and by polymerization at pH 7.0 according to the method of Avrameas and Ternynck [ 91. 3. Other substances Human serum albumin and complete Freund’s adjuvant were purchased from Behring, Marburg (G.F.R.), p-nitrophenylphosphate from Boehringer, Mannheim (G.F.R.), glutaraldehyde and all other chemicals were obtained from Merck, Darmstadt (G.F.R.). 4. Methods Alkaline phosphatase was measured at 25°C according to the method of Bessey et al. [lo] in 0.05 M glycine buffer containing 0.5 mM MgClz, pH 10.5, with p-nitrophenylphosphate as substrate.The protein concentration was determined as described by Beisenherz et al. [ll] . Each 1.2 ml of serum of an individual patient was subjected to five different immunological methods (heat stable activity, precipitation techniques with immunoabsorbent and with antiserum, inhibition test with antiserum and measurement of the immunoabsorbent-fixed activity) and the determination of the total alkaline phosphatase activity (Fig. 1): 0.2 ml serum was used for the measurement of the total alkaline phosphatase activity, the remaining 1.0 ml was divided into two equal parts both of which were heat inactivated for 10 minutes at 65°C cooled in an ice bath and centrifuged at 12 000 X g. In the first tube 0.2 ml were used for the determination of the heat stable activity, the remaining 0.3 ml were incubated with 100 ~1 anti-P-AP immunoabsorbent for 60 minutes at 25°C centrifuged at 20 000 X g and 0.2 ml of the supernatant was taken
213
for the measurement of activity. The immunoabsorbent was washed once in phosphate buffered saline, centrifuged at 20 000 X g and incubated at 25°C for 19 minutes and 30 seconds in the reaction mixture containing substrate (final volume 1.0 ml). Then the mixture was centrifuged in a micro-centrifuge (Eppendorf-microliter system) for 30 s and the absorbance of the supernatant was measured at 405 nm. The incubation mixture before addition of the immunoabsorbent served as blank. 10 ~1 of anti-P-AP serum (charge 321) was added to the second heat inactivated serum specimen. The mixture was incubated for 60 min at 25°C and 0.2 ml were used for enzyme activity determination. The remaining 0.3 ml were centrifuged at 20 000 X g and 0.2 ml of the supernatant was taken for the last measurement of enzyme activity (Fig. 1). Results 1. Reaction of anti-P-AP serum with different human alkaline phosphatases Increasing amounts of anti-P-AP serum were incubated with P-AP, intestinal-AP, liver-AP, kidney-AP or bone-AP for 60 min at 25°C in 0.01 M Tris buffer containing 0.002 M MgClz and 1 percent pure human albumin, pH 7.5, then centrifuged at 20 000 X g and the activity of the supernatant was determined. P-AP antibodies totally precipitate P-AP and in higher concentrations I-AP too. The I-AP-anti-P-AP complex is unstable in antibody excess (Fig. 2),
0.2rnl
supornotant
sediment
washed
oncewith
131
--octlvlty
IL
0.1 ml
0.2 ml solution
ammunobsorbent actwnty
15) 0.2 ml
.lO,uI
0,5ml
III
total
(2)
heatstable
(2-31
dtfforonco
serum
olkallne
Fig. 1. Measurement ods. PBS. phosphate
Ant,-P-AP-
60 ml”,
0.51 ml solution
phosphatase
actwty
octwty test
wth
lmmunobsorbont
of the total AP activity buffered saline.
25’C
supanatont
--actwty
0.51 ml s01utnn
0.31 ml solution
IL)
ommunabsorbent
12-51
mhibntlon
test
woth
antlstrum
1 dofftrrnco
test
wth
ontarrum
(2-6
1
PBS
lmmunobsotbont supernotont -
act,wty
and of the placentaLAP
- fIxed
octwty
activity
by five different
meth-
161
274
Fig. 2. Precipitation serum 321. see text).
of different
Pure
human
alkaline
Pbosphatases
by anti-placentaLAP
serum
(anti-
whereas the P-AP-anti-P-AP complex is not dissolved even in high antibody excess. There is no detectable reaction with liver-, kidney- and bone-AP in the range from 0.1 to 500 ~1 antiserum. 2. Comparison of different immunological methods Using the arrangement demonstrated in Fig. 1 the total alkaline phosphatase activity [l] and the P-AP activity measured by five different procedures can be performed using 1.2 ml serum. The heat stable activity [2], the inhibition with soluble antibodies [2--51, the precipitation with an immunoabsorbent [ 2,3] and with soluble antibodies [2-6] and the immunoabsorbent-bound P-AP activity [4] can be calculated. The application of this system to 64 normal blood donors and to 68 pregnant women is shown in Fig. 3. Antiserum 321 (antibody titer: 0.78 mg P-AP/ml) was used for all experiments (inhibition method, precipitation techniques, preparation of the immunoabsorbent). To compare different P-AP measurements, sera of pregnant women were chosen because of the selective increase of P-AP activity during pregnancy. Normal values can be observed up to the 20th week of pregnancy, slightly elevated activities can be observed between the 20th and 30th week and raised activities in the last trimester. The results elucidate the sensitivity of different techniques for P-AP activity under clinical conditions. On the other hand, the specificity for P-AP with regard to other alkaline phosphatases in the serum cannot be investigated for these methods. For the five different methods, mean, standard deviation and normal range of 64 healthy blood donors are listed in Table I. Twenty-three healthy laboratory and medical staff workers showed the same statistical values as the blood donors in a second series which was measured independently. The results in Fig. 3 demonstrate that the inhibition technique gives unsatisfactory results. The determination of the immuno-absorbent-fixed activity surpassed the results of heat stable activity and of precipitated activity: there is no difference between the results obtained with heat stable activity, precipitated activity (with soluble and with insoluble antibodies) and immunoabsorbent-fixed activity in sera with high P-AP activity, but slightly elevated P-AP activities can be better determined with the immunoabsorbent method. The inhibition of the polymer-fixed P-AP activity
Total AP-activity
lmmunoabsorbent fixed AP-activity
-
-
i.*.
.
..
.‘.
8
.
l
1
LO
IM
mUlml
1
dJ/Kll
100 AP-activity
Antiserum-precipitated AP- activity
Heatstable
l*
1
LO
SO
IOa
mUlml
1
mU/ml
loo
0..
.
_
l
l
.
Immunoabsorbent-precipitated AP - activity _ _
AntIserum - inhibited AP-activity
l
*
.
.
antiserum-precipitated Fig. 3. Placental-AP activity in 68 pregnant women measured by heat stable, antiserum-inhibited, immune-absorbent-fixed. absorbent-precipitated activity. Dashed line, mean value; solid line. Pa-standard deviation of 64 healthy blood donors (see Table 1).
0.6
I.5
@.E/2Ormn
!
mlJ~mI
loo
and immuno-
216 TABLE
I
MEAN VALUE, STANDARD DEVIATION AND SURED WITH DIFFERENT METHODS (U/l) The independently results.
measured
values of 23 healthy
NORMAL laboratory
n
x
Total activity
64
Heat-stable
64
Inhibited activity (soluble antibodies) Precipitated (soluble
activity
activity antibodies)
RANGE
OF 64 BLOOD
and medical
staff workers
DONORS
MEA-
showed the same
cl
Normal
25.52
8.55
Clinica Chimica Acta, 65 (1975) 271-282 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 7 289
IMMUNOLOGICAL METHODS FOR HUMAN PLACENTAL PHOSPHATASE (REGAN ISOENZYME)
FRANK-GtiNTER
ALKALINE
LEHMANN
Department of Medicine, University of Marburg/Lahn, E. Mannkopffstrasse 1, D-355 Marburg/Lahn (G.F.R.)
(Received
May 9,1975)
Summary Four different immunological methods for the determination of the placental isoenzyme of alkaline phosphatase (Regan isoenzyme) were compared in 64 normal blood donors, 23 healthy laboratory and medical staff workers and 68 pregnant women: a. Inhibition by soluble antibodies to the placental enzyme. b. Precipitation with soluble antibodies. c. Precipitation with immobilized antibodies. d. Measurement of the activity of the placental alkaline phosphatase following binding to an immunoabsorbent that has been obtained by polymerization of anti-placental-alkaline phosphatase y-globulin using glutaraldehyde. The immunoabsorbent method yielded the best results. The optimal conditions were evaluated for the measurement of the activity of immunoabsorbent-fixed placental (tumoral) alkaline phosphatase activity. This method was applied to 209 normal blood donors and to 239 patients with different malignant tumors: 25.5 percent of the cancer patients exhibited an elevated Regan -isoenzyme activity in the serum.
Introduction Human placental alkaline phosphatase (P-AP) contributes in minor degree to the total activity of alkaline phosphatases in normal serum. P-AP activity rises under some pathological conditions, especially in pregnancy and in some tumor patients with neosynthesis of placental-type alkaline phosphastase (Regan isoenzyme). Therefore, a specific and sensitive determination of P-AP is required for the detection of the small portion of P-AP within the total alkaline
212
phosphatase activity. Heat stability [l] , electrophoresis of the antibody-bound P-AP (showing a retarded electrophoretic migration) and staining with a fluorogenie substrate [ 21, radioimmunoassays [ 3,4] and an immunochemical method [ 51 have been described for the measurement of P-AP in cancer patients and in pathological pregnancies. We developed and compared different immunological methods for the placental isoenzyme of alkaline phosphatase as a first step to an immunochemical differentiation of alkaline phosphatases in human serum. Material and methods 1. A Ekaline phosphatases Human placental (P-AP), intestinal (I-AP) and liver (L-AP) alkaline phosphatases were isolated as described in ref. 6. The specific activity was 196 U/mg for P-AP, 436 U/mg for I-AP and 41.3 U/mg for L-AP. Purified human kidney and bone phosphatases were preparations of Professor Dr. G. Pfleiderer (Department of Chemistry, University of Bochum) [7], as well as I-AP until we were able to isolate our own enzyme. 2. Antisera Anti-P-AP sera were obtained by immunizing rabbits with crystalline material in complete Freund’s adjuvant according to the scheme described previously [ 81. Anti-P-AP-y-globulin immunoabsorbents were prepared by precipitation of the antisera first with ammonium sulfate up to 40 percent and second up to 35 percent saturation and by polymerization at pH 7.0 according to the method of Avrameas and Ternynck [ 91. 3. Other substances Human serum albumin and complete Freund’s adjuvant were purchased from Behring, Marburg (G.F.R.), p-nitrophenylphosphate from Boehringer, Mannheim (G.F.R.), glutaraldehyde and all other chemicals were obtained from Merck, Darmstadt (G.F.R.). 4. Methods Alkaline phosphatase was measured at 25°C according to the method of Bessey et al. [lo] in 0.05 M glycine buffer containing 0.5 mM MgClz, pH 10.5, with p-nitrophenylphosphate as substrate.The protein concentration was determined as described by Beisenherz et al. [ll] . Each 1.2 ml of serum of an individual patient was subjected to five different immunological methods (heat stable activity, precipitation techniques with immunoabsorbent and with antiserum, inhibition test with antiserum and measurement of the immunoabsorbent-fixed activity) and the determination of the total alkaline phosphatase activity (Fig. 1): 0.2 ml serum was used for the measurement of the total alkaline phosphatase activity, the remaining 1.0 ml was divided into two equal parts both of which were heat inactivated for 10 minutes at 65°C cooled in an ice bath and centrifuged at 12 000 X g. In the first tube 0.2 ml were used for the determination of the heat stable activity, the remaining 0.3 ml were incubated with 100 ~1 anti-P-AP immunoabsorbent for 60 minutes at 25°C centrifuged at 20 000 X g and 0.2 ml of the supernatant was taken
213
for the measurement of activity. The immunoabsorbent was washed once in phosphate buffered saline, centrifuged at 20 000 X g and incubated at 25°C for 19 minutes and 30 seconds in the reaction mixture containing substrate (final volume 1.0 ml). Then the mixture was centrifuged in a micro-centrifuge (Eppendorf-microliter system) for 30 s and the absorbance of the supernatant was measured at 405 nm. The incubation mixture before addition of the immunoabsorbent served as blank. 10 ~1 of anti-P-AP serum (charge 321) was added to the second heat inactivated serum specimen. The mixture was incubated for 60 min at 25°C and 0.2 ml were used for enzyme activity determination. The remaining 0.3 ml were centrifuged at 20 000 X g and 0.2 ml of the supernatant was taken for the last measurement of enzyme activity (Fig. 1). Results 1. Reaction of anti-P-AP serum with different human alkaline phosphatases Increasing amounts of anti-P-AP serum were incubated with P-AP, intestinal-AP, liver-AP, kidney-AP or bone-AP for 60 min at 25°C in 0.01 M Tris buffer containing 0.002 M MgClz and 1 percent pure human albumin, pH 7.5, then centrifuged at 20 000 X g and the activity of the supernatant was determined. P-AP antibodies totally precipitate P-AP and in higher concentrations I-AP too. The I-AP-anti-P-AP complex is unstable in antibody excess (Fig. 2),
0.2rnl
supornotant
sediment
washed
oncewith
131
--octlvlty
IL
0.1 ml
0.2 ml solution
ammunobsorbent actwnty
15) 0.2 ml
.lO,uI
0,5ml
III
total
(2)
heatstable
(2-31
dtfforonco
serum
olkallne
Fig. 1. Measurement ods. PBS. phosphate
Ant,-P-AP-
60 ml”,
0.51 ml solution
phosphatase
actwty
octwty test
wth
lmmunobsorbont
of the total AP activity buffered saline.
25’C
supanatont
--actwty
0.51 ml s01utnn
0.31 ml solution
IL)
ommunabsorbent
12-51
mhibntlon
test
woth
antlstrum
1 dofftrrnco
test
wth
ontarrum
(2-6
1
PBS
lmmunobsotbont supernotont -
act,wty
and of the placentaLAP
- fIxed
octwty
activity
by five different
meth-
161
274
Fig. 2. Precipitation serum 321. see text).
of different
Pure
human
alkaline
Pbosphatases
by anti-placentaLAP
serum
(anti-
whereas the P-AP-anti-P-AP complex is not dissolved even in high antibody excess. There is no detectable reaction with liver-, kidney- and bone-AP in the range from 0.1 to 500 ~1 antiserum. 2. Comparison of different immunological methods Using the arrangement demonstrated in Fig. 1 the total alkaline phosphatase activity [l] and the P-AP activity measured by five different procedures can be performed using 1.2 ml serum. The heat stable activity [2], the inhibition with soluble antibodies [2--51, the precipitation with an immunoabsorbent [ 2,3] and with soluble antibodies [2-6] and the immunoabsorbent-bound P-AP activity [4] can be calculated. The application of this system to 64 normal blood donors and to 68 pregnant women is shown in Fig. 3. Antiserum 321 (antibody titer: 0.78 mg P-AP/ml) was used for all experiments (inhibition method, precipitation techniques, preparation of the immunoabsorbent). To compare different P-AP measurements, sera of pregnant women were chosen because of the selective increase of P-AP activity during pregnancy. Normal values can be observed up to the 20th week of pregnancy, slightly elevated activities can be observed between the 20th and 30th week and raised activities in the last trimester. The results elucidate the sensitivity of different techniques for P-AP activity under clinical conditions. On the other hand, the specificity for P-AP with regard to other alkaline phosphatases in the serum cannot be investigated for these methods. For the five different methods, mean, standard deviation and normal range of 64 healthy blood donors are listed in Table I. Twenty-three healthy laboratory and medical staff workers showed the same statistical values as the blood donors in a second series which was measured independently. The results in Fig. 3 demonstrate that the inhibition technique gives unsatisfactory results. The determination of the immuno-absorbent-fixed activity surpassed the results of heat stable activity and of precipitated activity: there is no difference between the results obtained with heat stable activity, precipitated activity (with soluble and with insoluble antibodies) and immunoabsorbent-fixed activity in sera with high P-AP activity, but slightly elevated P-AP activities can be better determined with the immunoabsorbent method. The inhibition of the polymer-fixed P-AP activity
Total AP-activity
lmmunoabsorbent fixed AP-activity
-
-
i.*.
.
..
.‘.
8
.
l
1
LO
IM
mUlml
1
dJ/Kll
100 AP-activity
Antiserum-precipitated AP- activity
Heatstable
l*
1
LO
SO
IOa
mUlml
1
mU/ml
loo
0..
.
_
l
l
.
Immunoabsorbent-precipitated AP - activity _ _
AntIserum - inhibited AP-activity
l
*
.
.
antiserum-precipitated Fig. 3. Placental-AP activity in 68 pregnant women measured by heat stable, antiserum-inhibited, immune-absorbent-fixed. absorbent-precipitated activity. Dashed line, mean value; solid line. Pa-standard deviation of 64 healthy blood donors (see Table 1).
0.6
I.5
@.E/2Ormn
!
mlJ~mI
loo
and immuno-
216 TABLE
I
MEAN VALUE, STANDARD DEVIATION AND SURED WITH DIFFERENT METHODS (U/l) The independently results.
measured
values of 23 healthy
NORMAL laboratory
n
x
Total activity
64
Heat-stable
64
Inhibited activity (soluble antibodies) Precipitated (soluble
activity
activity antibodies)
RANGE
OF 64 BLOOD
and medical
staff workers
DONORS
MEA-
showed the same
cl
Normal
25.52
8.55
