Journal of Immunological Methods, 130 (1990) 133-140
133
Elsevier JIM 05593
A quantitative dot immunobinding assay for coagulation factor XII in plasma Walter A. Wuillemin, Miha Furlan and Bernhard L~immle Central Hematology Laboratory, University of Bern. Inselspital, Bern, Switzerland
(Received 13 November 1989, revised received 12 February 1990, accepted 20 February 1990)
A dot immunobinding assay on nitrocellulose (NC) membranes has been developed for the quantification of human coagulation factor XII (F XII). Plasma samples were dotted on to N C filters and F XII was detected using a polyclonal antiserum followed by a radiolabelled antigen overlay. Dilutions of either pooled normal human plasma ( N H P ) or purified F XII in F XII deficient plasma were used as standards. Quantification was performed by measuring the radioactivity of bound 125I-F XII. Precise measurements of F XII antigen (F X I I : A g ) were possible with a sensitivity down to 0.12 ng. Thus, dotting samples containing 0.5 ~1 of plasma permitted detection of a F XII concentration corresponding to 1% of the level in NHP. The intra-assay coefficient of variation (CV) was < 5% and the interassay CV was < 16%. F XII : Ag in plasma samples of 50 healthy adults ranged from 12 ~ g / m l to 47 p g / m l . A good correlation (r = 0.93) existed between F XII : Ag and F XII clot promoting activity (F XII : C) in these samples. N H P contained 24.1 ~ g / m l F X I I : A g confirming earlier results obtained by other methods. In 16 pregnant women levels of F X I I : A g as well as of F X I I : C were elevated, but F X I I : A g was disproportionately higher compared with F XII : C. The immunobinding assay has the following advantages: (1) rapid quantification of large numbers of samples is possible, (2) the sensitivity down to 1% of N H P is better than that of several other methods, (3) only very small amounts of both test material and reagents are needed. Key words: Dot immunobinding assay; Nitrocellulose filter; Human blood coagulation factor XII
Introduction H u m a n blood coagulation F XII, also known as Hageman factor, is a glycoprotein with a
Correspondence to: B. IAtmmle, Central Hematology Laboratory, Inselspital, CH-3010 Bern, Switzerland. Abbreviations: F XII:Ag, factor XII antigen; F XII:C, factor XII clotting activity; NC, nitrocellulose; TBS, 0.01 M Tris, 0.14 M NaCI, 0.02% NaN3, pH 7.4; BSA, bovine serum albumin; NHP, pooled normal human plasma; SDS-PAGE, sodium dodecyl sulphate-polyacrylamidegel electrophoresis.
molecular weight of about 80,000 that circulates in plasma as an inactive zymogen (Fujikawa and Davie, 1981). When plasma comes into contact with certain negatively charged surfaces, such as glass or kaolin, reciprocal proteolytic activation of F XII and plasma prekallikrein results in the formation of the serine proteases plasma kallikrein and activated F XII (F XIIa), respectively (Cochrane et al., 1973). This so called plasma contact activation initiates intrinsic blood coagulation, fibrinolysis and kinin generation (Griffin and Cochrane, 1979; Colman, 1984; Tans and Rosing, 1987).
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
134
F XII measurements are important in several clinical situations, such as: disseminated intravascular coagulation, bacteremic shock, thrombophilia and unexplained prolongation of the activated partial thromboplastin time (Mannhalter et al., 1987; Saito, 1987). The titer of F XII clotting activity (F XII:C) is usually assayed by measuring the ability of a test sample to shorten the prolonged activated partial thromboplastin time of a F XII deficient plasma. F XII can also be measured by various immunological assays (F XII:Ag). The techniques most commonly used are radial immunodiffusion (Revak et al., 1974), electroimmunoassay (Girolami et al., 1982), radioimmunoassay (Saito et al., 1976) and quantitative immunoblotting (Limmle et al., 1986a). In this paper we describe the quantification of F XII:Ag by a dot immunobinding assay. The dot immunobinding assay or antigen spot test was introduced in 1982 by Hawkes (Hawkes et al., 1982) and Herbrink (Herbrink et al., 1982). This method, based on protein immobilization on nitrocellulose has been widely used as a qualitative method for rapid screening of large numbers of samples or as a quantitative technique for measuring either antibody titer or antigen concentration in serum, culture media, tissue extracts or cell lysates (Renner, 1988; Stott, 1989). Dot immunobinding represents an effective tool for the diagnosis of various diseases (Derer et al., 1984; Gordon and Rosenthal, 1985). Using an adapted dot immunobinding assay, F XII:Ag levels were determined in healthy volunteers and pregnant women and the results compared with the F XII:C.
0.106 M trisodium citrate. Plasma samples, obtained by twice centrifuging at 1500 × g for 10 min at room temperature, were handled using plastic dispenses and containers in order to avoid any glass contact and were kept at - 7 0 ° C until use.
A normal human plasma pool (NHP) from 31 healthy male volunteers was stored in small aliquots at - 7 0 ° C . N H P was used as the standard for measurement of both F XII:C and F XII:Ag and was defined to contain 1 U / m l of both F XII:C and F XII:Ag. F XII deficient plasma was prepared by mixing equal plasma volumes of 17 individuals congenitally deficient in F XII (L~immle et al., 1989). All 17 subjects had F XII:C < 0.01 U / m l and no detectable F XII:Ag in immunoblot assays ( 0 or"
0
I 0
0.25
t
I
0
6
D 0.5 NHP (pl) i
12 F Xll (ng)
Fig. 2. Standard curve of the dot immunobinding assay. N H P diluted 1/40 with TBS was mixed at various ratios with TBS-BSA 2 mg/ml. F XII deficient plasma reconstituted with purified F XII was diluted 1/40 in TBS and then mixed with 1/40 diluted F XII deficient plasma. Samples contained various amounts of N H P (0-0.5 ~1) or purified F XII (0-12 ng). a: autoradiogram of N H P standards containing from 0 to 0.5 ~tl NHP. b: NC pieces were cut out and their radioactivity (cpm) was plotted versus the amount of N H P (O O) or purified F XII (A. . . . . . A).
137 TABLE I INTRA-ASSAY CV A N D I N T E R A S S A Y CV OF T H E F XII DOT I M M U N O B I N D I N G ASSAY Samples
S1
$2
$3
$4
$5
F XII ( t t g / m l ) a Intra-assay CV b (n = 10) lnterassay CV ~ (n = 10)
7.2 3.3%
11.4 3.7%
14.4 3.8%
14.5 5.3%
19.2 3.1%
16%
12%
9%
9%
9%
a Mean values of F X I i : A g in five plasma samples S1-$5. b Ten-fold determination of F XII:Ag in the same assay. c F X I I : A g determination on ten different occasions.
dilutions was parallel to that obtained with dilutions of purified F XII (Fig. 2b).
Intra-assay and interassay coefficient of variation (CV). The intra-assay CV, determined by ten-fold determinations in one assay of five samples (S1$5) containing various amounts of F XII, was between 3 and 5.5%. The interassay CV, determined by assaying aliquots of the same plasma samples S1-$5 on ten different occasions, was 9-16% (Table I). Dilution study. Plasma samples from four normal subjects were each diluted 1 / 4 0 with TBS and further dilutions (1/2, 1/4, 1 / 1 0 and 1/20) were made with TBS-BSA 2 m g / m l . The F XII content in these dilutions was then measured and calculated for the undiluted samples. The CV obtained was 3.5-5.5%, which was in the range of the intra-assay CV. No additional variation was obtained by diluting the samples. Detection limit. Samples with a F XII concentration corresponding to 1% of N H P gave a signal distinct from the background. The radioactivity measured in ten 1% samples was 1630 + 78 cpm compared with 1105 + 57 cpm found in ten 0% samples, corresponding to 20 /~1 TBS-BSA 2 mg/mi.
F XII:Ag values in various plasma samples NHP. We determined F X I I : A g in our normal human plasma pool ( N H P ) using dilutions of F XII-deficient plasma reconstituted with purified F XII for calibration. At various dilutions of N H P we found a F X I I : A g level of 24.1 :t: 1.4 /~g/ml (n = 6). This was defined to be 100% or 1 U / m l . Healthy volunteers. F XII:Ag was determined in plasma samples of 50 healthy individuals (25
men and 25 women). Each sample was assayed on three separate occasions and the mean value was calculated. F X I I : A g concentrations were expressed as t t g / m l or as a percentage of N H P (Table II). F X I I : A g levels were between 12 ~ g / m l and 4 7 / t g / m l with a median of 28 ~ g / m l . The normal range of F XII in citrated plasma representing the 95% interval was 17-35 /~g/ml (71144% of NHP). The observed differences between men and women on the one hand and between women in the follicular and luteal phase of the menstrual cycle, on the other, were not significant (Table II). T A B L E II F X I I : A G ( p , g / m l or U / m l ) A N D F X II: C ( U / m l ) IN DIFFERENT GROUPS The values are expressed as medians and ranges. F XII:Ag
Healthy volunteers (n = 50) Normal men (n = 25) Normal ~,omen (n = 25) Women foil. a (n = 11) Women lut. a (n = 14) Pregnant women (n = 16)
F X II: C
txg/ml
U/ml
U/ml
28 12-47 27 12-35 28 17-47 27 21-31 29 17-47 43 24-72
1.16 0.5-1.94 1.14 0.5-1.44 1.18 ~' 0.71-1.94 1.13 0.87-1.27 1.22 0.71-1.94 1.78 b.d 1.01-3.00
1.02 0.46-1.46 1.01 0.46-1.39 1.02 ~ 0.61-1.46 0.99 0.82-1.13 1.02 0.61-1.46 1.29 ~.d 0.86-1.88
a Women in the follicular or luteal phase, respectively, are indicated as: women foll. and women lut. b P < 0.001. P < 0.001. d P < 0.05.
138
Discussion
Ei
""
i1
o 0
~
2
3
F XlI:C (U/ml)
Fig. 3. F Xll:Ag values compared with F XII:C levels in 50 healthy volunteers (zx) and 16 pregnant women (O). The correlation coefficient for the 50 healthy volunteers was 0.93.
Pregnant women. F XII:Ag levels in the plasma samples of 16 pregnant women were significantly ( P < 0.001) higher than those of samples from non-pregnant women (Table II). The level of F XII:Ag increased with the duration of pregnancy (r = 0.82). F X I I : C values In addition to the dot immunobinding assay, F XII clotting assays were performed on plasma samples from the healthy adults and the pregnant women (Table II). Each plasma sample was tested on four separate occasions and the mean values of F XII:C were calculated. Again, there was no difference between men and women but pregnant women had significantly ( P
133
Elsevier JIM 05593
A quantitative dot immunobinding assay for coagulation factor XII in plasma Walter A. Wuillemin, Miha Furlan and Bernhard L~immle Central Hematology Laboratory, University of Bern. Inselspital, Bern, Switzerland
(Received 13 November 1989, revised received 12 February 1990, accepted 20 February 1990)
A dot immunobinding assay on nitrocellulose (NC) membranes has been developed for the quantification of human coagulation factor XII (F XII). Plasma samples were dotted on to N C filters and F XII was detected using a polyclonal antiserum followed by a radiolabelled antigen overlay. Dilutions of either pooled normal human plasma ( N H P ) or purified F XII in F XII deficient plasma were used as standards. Quantification was performed by measuring the radioactivity of bound 125I-F XII. Precise measurements of F XII antigen (F X I I : A g ) were possible with a sensitivity down to 0.12 ng. Thus, dotting samples containing 0.5 ~1 of plasma permitted detection of a F XII concentration corresponding to 1% of the level in NHP. The intra-assay coefficient of variation (CV) was < 5% and the interassay CV was < 16%. F XII : Ag in plasma samples of 50 healthy adults ranged from 12 ~ g / m l to 47 p g / m l . A good correlation (r = 0.93) existed between F XII : Ag and F XII clot promoting activity (F XII : C) in these samples. N H P contained 24.1 ~ g / m l F X I I : A g confirming earlier results obtained by other methods. In 16 pregnant women levels of F X I I : A g as well as of F X I I : C were elevated, but F X I I : A g was disproportionately higher compared with F XII : C. The immunobinding assay has the following advantages: (1) rapid quantification of large numbers of samples is possible, (2) the sensitivity down to 1% of N H P is better than that of several other methods, (3) only very small amounts of both test material and reagents are needed. Key words: Dot immunobinding assay; Nitrocellulose filter; Human blood coagulation factor XII
Introduction H u m a n blood coagulation F XII, also known as Hageman factor, is a glycoprotein with a
Correspondence to: B. IAtmmle, Central Hematology Laboratory, Inselspital, CH-3010 Bern, Switzerland. Abbreviations: F XII:Ag, factor XII antigen; F XII:C, factor XII clotting activity; NC, nitrocellulose; TBS, 0.01 M Tris, 0.14 M NaCI, 0.02% NaN3, pH 7.4; BSA, bovine serum albumin; NHP, pooled normal human plasma; SDS-PAGE, sodium dodecyl sulphate-polyacrylamidegel electrophoresis.
molecular weight of about 80,000 that circulates in plasma as an inactive zymogen (Fujikawa and Davie, 1981). When plasma comes into contact with certain negatively charged surfaces, such as glass or kaolin, reciprocal proteolytic activation of F XII and plasma prekallikrein results in the formation of the serine proteases plasma kallikrein and activated F XII (F XIIa), respectively (Cochrane et al., 1973). This so called plasma contact activation initiates intrinsic blood coagulation, fibrinolysis and kinin generation (Griffin and Cochrane, 1979; Colman, 1984; Tans and Rosing, 1987).
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
134
F XII measurements are important in several clinical situations, such as: disseminated intravascular coagulation, bacteremic shock, thrombophilia and unexplained prolongation of the activated partial thromboplastin time (Mannhalter et al., 1987; Saito, 1987). The titer of F XII clotting activity (F XII:C) is usually assayed by measuring the ability of a test sample to shorten the prolonged activated partial thromboplastin time of a F XII deficient plasma. F XII can also be measured by various immunological assays (F XII:Ag). The techniques most commonly used are radial immunodiffusion (Revak et al., 1974), electroimmunoassay (Girolami et al., 1982), radioimmunoassay (Saito et al., 1976) and quantitative immunoblotting (Limmle et al., 1986a). In this paper we describe the quantification of F XII:Ag by a dot immunobinding assay. The dot immunobinding assay or antigen spot test was introduced in 1982 by Hawkes (Hawkes et al., 1982) and Herbrink (Herbrink et al., 1982). This method, based on protein immobilization on nitrocellulose has been widely used as a qualitative method for rapid screening of large numbers of samples or as a quantitative technique for measuring either antibody titer or antigen concentration in serum, culture media, tissue extracts or cell lysates (Renner, 1988; Stott, 1989). Dot immunobinding represents an effective tool for the diagnosis of various diseases (Derer et al., 1984; Gordon and Rosenthal, 1985). Using an adapted dot immunobinding assay, F XII:Ag levels were determined in healthy volunteers and pregnant women and the results compared with the F XII:C.
0.106 M trisodium citrate. Plasma samples, obtained by twice centrifuging at 1500 × g for 10 min at room temperature, were handled using plastic dispenses and containers in order to avoid any glass contact and were kept at - 7 0 ° C until use.
A normal human plasma pool (NHP) from 31 healthy male volunteers was stored in small aliquots at - 7 0 ° C . N H P was used as the standard for measurement of both F XII:C and F XII:Ag and was defined to contain 1 U / m l of both F XII:C and F XII:Ag. F XII deficient plasma was prepared by mixing equal plasma volumes of 17 individuals congenitally deficient in F XII (L~immle et al., 1989). All 17 subjects had F XII:C < 0.01 U / m l and no detectable F XII:Ag in immunoblot assays ( 0 or"
0
I 0
0.25
t
I
0
6
D 0.5 NHP (pl) i
12 F Xll (ng)
Fig. 2. Standard curve of the dot immunobinding assay. N H P diluted 1/40 with TBS was mixed at various ratios with TBS-BSA 2 mg/ml. F XII deficient plasma reconstituted with purified F XII was diluted 1/40 in TBS and then mixed with 1/40 diluted F XII deficient plasma. Samples contained various amounts of N H P (0-0.5 ~1) or purified F XII (0-12 ng). a: autoradiogram of N H P standards containing from 0 to 0.5 ~tl NHP. b: NC pieces were cut out and their radioactivity (cpm) was plotted versus the amount of N H P (O O) or purified F XII (A. . . . . . A).
137 TABLE I INTRA-ASSAY CV A N D I N T E R A S S A Y CV OF T H E F XII DOT I M M U N O B I N D I N G ASSAY Samples
S1
$2
$3
$4
$5
F XII ( t t g / m l ) a Intra-assay CV b (n = 10) lnterassay CV ~ (n = 10)
7.2 3.3%
11.4 3.7%
14.4 3.8%
14.5 5.3%
19.2 3.1%
16%
12%
9%
9%
9%
a Mean values of F X I i : A g in five plasma samples S1-$5. b Ten-fold determination of F XII:Ag in the same assay. c F X I I : A g determination on ten different occasions.
dilutions was parallel to that obtained with dilutions of purified F XII (Fig. 2b).
Intra-assay and interassay coefficient of variation (CV). The intra-assay CV, determined by ten-fold determinations in one assay of five samples (S1$5) containing various amounts of F XII, was between 3 and 5.5%. The interassay CV, determined by assaying aliquots of the same plasma samples S1-$5 on ten different occasions, was 9-16% (Table I). Dilution study. Plasma samples from four normal subjects were each diluted 1 / 4 0 with TBS and further dilutions (1/2, 1/4, 1 / 1 0 and 1/20) were made with TBS-BSA 2 m g / m l . The F XII content in these dilutions was then measured and calculated for the undiluted samples. The CV obtained was 3.5-5.5%, which was in the range of the intra-assay CV. No additional variation was obtained by diluting the samples. Detection limit. Samples with a F XII concentration corresponding to 1% of N H P gave a signal distinct from the background. The radioactivity measured in ten 1% samples was 1630 + 78 cpm compared with 1105 + 57 cpm found in ten 0% samples, corresponding to 20 /~1 TBS-BSA 2 mg/mi.
F XII:Ag values in various plasma samples NHP. We determined F X I I : A g in our normal human plasma pool ( N H P ) using dilutions of F XII-deficient plasma reconstituted with purified F XII for calibration. At various dilutions of N H P we found a F X I I : A g level of 24.1 :t: 1.4 /~g/ml (n = 6). This was defined to be 100% or 1 U / m l . Healthy volunteers. F XII:Ag was determined in plasma samples of 50 healthy individuals (25
men and 25 women). Each sample was assayed on three separate occasions and the mean value was calculated. F X I I : A g concentrations were expressed as t t g / m l or as a percentage of N H P (Table II). F X I I : A g levels were between 12 ~ g / m l and 4 7 / t g / m l with a median of 28 ~ g / m l . The normal range of F XII in citrated plasma representing the 95% interval was 17-35 /~g/ml (71144% of NHP). The observed differences between men and women on the one hand and between women in the follicular and luteal phase of the menstrual cycle, on the other, were not significant (Table II). T A B L E II F X I I : A G ( p , g / m l or U / m l ) A N D F X II: C ( U / m l ) IN DIFFERENT GROUPS The values are expressed as medians and ranges. F XII:Ag
Healthy volunteers (n = 50) Normal men (n = 25) Normal ~,omen (n = 25) Women foil. a (n = 11) Women lut. a (n = 14) Pregnant women (n = 16)
F X II: C
txg/ml
U/ml
U/ml
28 12-47 27 12-35 28 17-47 27 21-31 29 17-47 43 24-72
1.16 0.5-1.94 1.14 0.5-1.44 1.18 ~' 0.71-1.94 1.13 0.87-1.27 1.22 0.71-1.94 1.78 b.d 1.01-3.00
1.02 0.46-1.46 1.01 0.46-1.39 1.02 ~ 0.61-1.46 0.99 0.82-1.13 1.02 0.61-1.46 1.29 ~.d 0.86-1.88
a Women in the follicular or luteal phase, respectively, are indicated as: women foll. and women lut. b P < 0.001. P < 0.001. d P < 0.05.
138
Discussion
Ei
""
i1
o 0
~
2
3
F XlI:C (U/ml)
Fig. 3. F Xll:Ag values compared with F XII:C levels in 50 healthy volunteers (zx) and 16 pregnant women (O). The correlation coefficient for the 50 healthy volunteers was 0.93.
Pregnant women. F XII:Ag levels in the plasma samples of 16 pregnant women were significantly ( P < 0.001) higher than those of samples from non-pregnant women (Table II). The level of F XII:Ag increased with the duration of pregnancy (r = 0.82). F X I I : C values In addition to the dot immunobinding assay, F XII clotting assays were performed on plasma samples from the healthy adults and the pregnant women (Table II). Each plasma sample was tested on four separate occasions and the mean values of F XII:C were calculated. Again, there was no difference between men and women but pregnant women had significantly ( P
