- Email: [email protected]
Isolation of monoiodinated gastrin using deae-sephadex and its characteristics in the gastrin radioimmunoassay
321
Clinica Chimica Acta, 67 (1976) 321-323 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CCA 7665
ISOLATION OF MONOIODINATED GASTRIN USING DEAE-SEPHADEX AND ITS CHARACTERISTICS IN THE GASTRIN RADIOIMMUNOASSAY
T.R. BROWN, N. BAGCHI,
R.E. MACK, E. BOOTH and D.P. JONES
Departments of Pathology and Medicine, Hutzel Hospital Medical Unit and Wayne State University, Detroit, Mich. 48201 (U.S.A.) (Received
September
15, 1975)
Introduction The radioimmunoassay of gastrin is considerably improved when monoiodinated gastrin is used instead of a crude mixture of labeled gatrin which contains mono and diiodinated gastrin as well as other labeled gastrin fragments produced during iodination procedures. Following iodination, monoiodinated gastrin has been isolated satisfactorily by ion exchange chromatography on aminoethyl cellulose (Whatman AE-41) [ 1,2] , but recently the production of this resin has been discontinued. We report here a new method to isolate monoiodinated gastrin using DEAE-Sephadex, and compare the purified product to the unpurified mixture of iodination products in gastrin radioimmtmoassays. Materials Synthetic human gastrin I was obtained from Imperial Chemical Industries, Cheshire, England. ‘*‘I was obtained from New England Nuclear. DEAESephadex A-25 and a 0.9 cm X 15 cm column was purchased from Pharmacia Fine Chemicals. Second antibody was produced in our laboratory from goats immunized with rabbit gamma globulins (Miles Laboratories). Pooled antisera were fractionated three times with 45% saturated ammonium sulfate and stored at -70” C. Methods Iodination was carried out according to Walsh [2] excepting that the G-10 column was equilibrated with 0.05 M imidazole buffer, pH 7.5. An aliquot of the pooled tubes from the labeled gastrin peak was dispensed into five vials and
32%
stored at -70°C. The remaining portion was applied to a DEAE-Sephadex column (0.9 cm X 15 cm) equilibrated with imidazole buffer. Elution was accomplished using a linear sodium chloride gradient (0 to 1.0 n/l) at a flow rate of 6 ml/h. Fractions comprising the mono[ “‘I] iodogastrin peak were pooled, dispensed into five vials, and stored at -70” C. Gastrin antibody production and gastrin assay conditions were those of Walsh [ 21. Our assay format was defined by a computer program [ 3 1 and separation was with second antibody. Following iodination, at weekly intervals, a new vial of crude labeled gastrin from the G-10 column and purified monoiodinated gastrin were compared in an assay. The dilution of each was adjusted to 10 000 * 250 cpm per tube. Gastrin antibody was diluted at the beginning of the study and used throughout. Three assay parameters that reflect tracer integrity were examined: (1) nonspecific binding, determined by separation in the absence of antibody and expressed as the percent of total counts/assay tube; (2) total binding (measured by the percent of tracer that is bound to antibody in the absence of added unlabeled gastrin) minus non-specific binding; (3) the 50% intercept, or the point on the dose response curve where 50% of the tracer bound to antibody is displaced. Results and discussion The elution pattern in Fig. 1 shows good separation of mono[ 12’1] iodogastrin from di[ 1251]iodogastrin and other early eluting labeled material. The effect of storage on total binding, non-specific binding, and 50% intercept values of crude labeled gastrin compared to monoiodinated gastrin is shown in Table I. Compared to the crude G-10 preparation, total binding to antibody of the monoiodinated gastrin was consistently higher and decreased at a lower rate
“0 3 p 4
30.
25.
-
1251
3 w
zo-
.-.
NaCl
5 5
l5-
z i B
- I 00
-75
E
-
50
g z
-
25
IO -
5-
I
IO
20
x)
40
50
60
70
80
90
Kxz
110
120
I30
140
I50
FRACTION NUMBER
Fig. 1. Elution profile of crude 1 2 5 I-labeled gastrin on DEAE-Sephadex demonstrating separation of mono[12sI]iodogastrin from di[12!j Iliodogastrin and other labeled gastrin fragments. A linear, O-l.0 M sodium chloride gradient in 0.05 M imidazole buffer, pH 7.5 was used at a flow rate of 6 ml/h. The column (0.9 cm X 15 cm) and collector were kept at 4’C. The fractions (1.3 ml) were analyzed for radioactivity and conductance. Identity of mono and di[ 1 2 5 I] iodogastrin was confirmed by comparing the specific activity of the peaks with a fixed antiserum titer. Fractions 98 through 106 were pooled and used in the comparative gastrin radioimmunoassay studies.
323
TABLE
I
EFFECT TOTAL
OF
STORAGE
BINDING,
AND
CRUDE
Days
after
ON
THE
PROPERTIES
NOW-SPECIFIC
G-10
BINDING,
12SI-LABELED
Total
Binding
GASTRIN
(%)
OF AND
TWO
LABELED
SENSITIVITY
IN GASTRIN
GASTRIN OF
PREPARATIONS:
MON0[1251]IODOGASTRIN
RADIOIMMUNOASSAYS
Non-Specific
Binding G-10
50%
(“0)
intercept
(gastrin
pg/ml)
iodination MOllO
G-10
Mono
Mono
G-10
7
50.7
31.1
5.0
5.8
114
166
323
14
50.2
29.8
5.3
5.9
126
195
359
*
21
47.9
27.8
5.9
6.7
140
228
420
*
28
46.5
25.9
6.3
8.5
158
278
542
*
35
45.1
20.1
7.0
11.2
177
353
768
*
* Values
with
antibody
titer
adjusted
to a total
binding
*
of 50%.
over time. This indicates that the pure monoiodinated gastrin contains a significantly greater proportion of labeled immunoreactive material which is less susceptible to degradation than the unpurified preparation. Non-specific binding increased at essentially the same rate in the two preparations until about the fourth week after iodination, when the crude G-10 preparation began to deteriorate more rapidly. Mono-substituted gastrin also gave a more sensitive assay as the 50% intercept values indicate. For example, the 50% intercept value of four week old monoiodinated gastrin was attained with 158 pg unlabeled gastrin while the 85% intercept occured with 32 pg gastrin. Therefore, normal fasting serum levels of gastrin would fall on a portion of the standard curve where the coefficient of variation is quite acceptable. In contrast, the unpurified labeled gastrin was not satisfactory in this respect. For example, when the antiserum titer was readjusted to a total binding of 50%, we were unable to discern fasting gastrin values from buffer controls even within the first week following iodination. In conclusion, we have described a new method of isolating monoiodinated gastrin, using a DEAE-Sephadex/imidazole chromatography system which is readily available to investigators who are not able to obtain the Whatman AE41 resin described in earlier reports. Our data establishes that the monoiodinated gastrin thus obtained is superior to crude preparations of iodinated gastrin both in the stability of the reagent and in the sensitivity of the assay. Finally, the Sephadex/imidazole chromatography system may offer greater reproducibility since the pH changes that usually occur in an AE-4l/ammonium bicarbonate system are obviated. Acknowledgement The authors assistance.
wish to thank
Ms. Karen
Sulikowski
for her skillful technical
References 1
Stadil,
F. and
2
Walsh.
J.H.
3
Duddleson,
Rehfeld,
(1974) W.G.,
J.F.
(1972)
in Nuclear Midgley.
Stand.
Medicine
A.R.
and
J. CIin.
in Vitro
Niswender.
Lab.
Invest.
(Rothfeld, G.D.
(1972)
R..
30, ed.), Camp.
361 p. 231, Biomed.
Lippincott, Res.
Philadelphia.
5, 205
Pa.
Clinica Chimica Acta, 67 (1976) 321-323 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CCA 7665
ISOLATION OF MONOIODINATED GASTRIN USING DEAE-SEPHADEX AND ITS CHARACTERISTICS IN THE GASTRIN RADIOIMMUNOASSAY
T.R. BROWN, N. BAGCHI,
R.E. MACK, E. BOOTH and D.P. JONES
Departments of Pathology and Medicine, Hutzel Hospital Medical Unit and Wayne State University, Detroit, Mich. 48201 (U.S.A.) (Received
September
15, 1975)
Introduction The radioimmunoassay of gastrin is considerably improved when monoiodinated gastrin is used instead of a crude mixture of labeled gatrin which contains mono and diiodinated gastrin as well as other labeled gastrin fragments produced during iodination procedures. Following iodination, monoiodinated gastrin has been isolated satisfactorily by ion exchange chromatography on aminoethyl cellulose (Whatman AE-41) [ 1,2] , but recently the production of this resin has been discontinued. We report here a new method to isolate monoiodinated gastrin using DEAE-Sephadex, and compare the purified product to the unpurified mixture of iodination products in gastrin radioimmtmoassays. Materials Synthetic human gastrin I was obtained from Imperial Chemical Industries, Cheshire, England. ‘*‘I was obtained from New England Nuclear. DEAESephadex A-25 and a 0.9 cm X 15 cm column was purchased from Pharmacia Fine Chemicals. Second antibody was produced in our laboratory from goats immunized with rabbit gamma globulins (Miles Laboratories). Pooled antisera were fractionated three times with 45% saturated ammonium sulfate and stored at -70” C. Methods Iodination was carried out according to Walsh [2] excepting that the G-10 column was equilibrated with 0.05 M imidazole buffer, pH 7.5. An aliquot of the pooled tubes from the labeled gastrin peak was dispensed into five vials and
32%
stored at -70°C. The remaining portion was applied to a DEAE-Sephadex column (0.9 cm X 15 cm) equilibrated with imidazole buffer. Elution was accomplished using a linear sodium chloride gradient (0 to 1.0 n/l) at a flow rate of 6 ml/h. Fractions comprising the mono[ “‘I] iodogastrin peak were pooled, dispensed into five vials, and stored at -70” C. Gastrin antibody production and gastrin assay conditions were those of Walsh [ 21. Our assay format was defined by a computer program [ 3 1 and separation was with second antibody. Following iodination, at weekly intervals, a new vial of crude labeled gastrin from the G-10 column and purified monoiodinated gastrin were compared in an assay. The dilution of each was adjusted to 10 000 * 250 cpm per tube. Gastrin antibody was diluted at the beginning of the study and used throughout. Three assay parameters that reflect tracer integrity were examined: (1) nonspecific binding, determined by separation in the absence of antibody and expressed as the percent of total counts/assay tube; (2) total binding (measured by the percent of tracer that is bound to antibody in the absence of added unlabeled gastrin) minus non-specific binding; (3) the 50% intercept, or the point on the dose response curve where 50% of the tracer bound to antibody is displaced. Results and discussion The elution pattern in Fig. 1 shows good separation of mono[ 12’1] iodogastrin from di[ 1251]iodogastrin and other early eluting labeled material. The effect of storage on total binding, non-specific binding, and 50% intercept values of crude labeled gastrin compared to monoiodinated gastrin is shown in Table I. Compared to the crude G-10 preparation, total binding to antibody of the monoiodinated gastrin was consistently higher and decreased at a lower rate
“0 3 p 4
30.
25.
-
1251
3 w
zo-
.-.
NaCl
5 5
l5-
z i B
- I 00
-75
E
-
50
g z
-
25
IO -
5-
I
IO
20
x)
40
50
60
70
80
90
Kxz
110
120
I30
140
I50
FRACTION NUMBER
Fig. 1. Elution profile of crude 1 2 5 I-labeled gastrin on DEAE-Sephadex demonstrating separation of mono[12sI]iodogastrin from di[12!j Iliodogastrin and other labeled gastrin fragments. A linear, O-l.0 M sodium chloride gradient in 0.05 M imidazole buffer, pH 7.5 was used at a flow rate of 6 ml/h. The column (0.9 cm X 15 cm) and collector were kept at 4’C. The fractions (1.3 ml) were analyzed for radioactivity and conductance. Identity of mono and di[ 1 2 5 I] iodogastrin was confirmed by comparing the specific activity of the peaks with a fixed antiserum titer. Fractions 98 through 106 were pooled and used in the comparative gastrin radioimmunoassay studies.
323
TABLE
I
EFFECT TOTAL
OF
STORAGE
BINDING,
AND
CRUDE
Days
after
ON
THE
PROPERTIES
NOW-SPECIFIC
G-10
BINDING,
12SI-LABELED
Total
Binding
GASTRIN
(%)
OF AND
TWO
LABELED
SENSITIVITY
IN GASTRIN
GASTRIN OF
PREPARATIONS:
MON0[1251]IODOGASTRIN
RADIOIMMUNOASSAYS
Non-Specific
Binding G-10
50%
(“0)
intercept
(gastrin
pg/ml)
iodination MOllO
G-10
Mono
Mono
G-10
7
50.7
31.1
5.0
5.8
114
166
323
14
50.2
29.8
5.3
5.9
126
195
359
*
21
47.9
27.8
5.9
6.7
140
228
420
*
28
46.5
25.9
6.3
8.5
158
278
542
*
35
45.1
20.1
7.0
11.2
177
353
768
*
* Values
with
antibody
titer
adjusted
to a total
binding
*
of 50%.
over time. This indicates that the pure monoiodinated gastrin contains a significantly greater proportion of labeled immunoreactive material which is less susceptible to degradation than the unpurified preparation. Non-specific binding increased at essentially the same rate in the two preparations until about the fourth week after iodination, when the crude G-10 preparation began to deteriorate more rapidly. Mono-substituted gastrin also gave a more sensitive assay as the 50% intercept values indicate. For example, the 50% intercept value of four week old monoiodinated gastrin was attained with 158 pg unlabeled gastrin while the 85% intercept occured with 32 pg gastrin. Therefore, normal fasting serum levels of gastrin would fall on a portion of the standard curve where the coefficient of variation is quite acceptable. In contrast, the unpurified labeled gastrin was not satisfactory in this respect. For example, when the antiserum titer was readjusted to a total binding of 50%, we were unable to discern fasting gastrin values from buffer controls even within the first week following iodination. In conclusion, we have described a new method of isolating monoiodinated gastrin, using a DEAE-Sephadex/imidazole chromatography system which is readily available to investigators who are not able to obtain the Whatman AE41 resin described in earlier reports. Our data establishes that the monoiodinated gastrin thus obtained is superior to crude preparations of iodinated gastrin both in the stability of the reagent and in the sensitivity of the assay. Finally, the Sephadex/imidazole chromatography system may offer greater reproducibility since the pH changes that usually occur in an AE-4l/ammonium bicarbonate system are obviated. Acknowledgement The authors assistance.
wish to thank
Ms. Karen
Sulikowski
for her skillful technical
References 1
Stadil,
F. and
2
Walsh.
J.H.
3
Duddleson,
Rehfeld,
(1974) W.G.,
J.F.
(1972)
in Nuclear Midgley.
Stand.
Medicine
A.R.
and
J. CIin.
in Vitro
Niswender.
Lab.
Invest.
(Rothfeld, G.D.
(1972)
R..
30, ed.), Camp.
361 p. 231, Biomed.
Lippincott, Res.
Philadelphia.
5, 205
Pa.