- Email: [email protected]
A study of isoelectric focusing in polyacrylamide gels of serum proteins as a cystic fibrosis screening test
103
Clinicrr Chimicu Actu, I17 (1981) 103- 1 IO Elsevier/North-Holland Biomedical Press
CCA
1944
Short communication
A study of isoelectric focusing in po~yacryla~de gels of serum proteins as a cystic fibrosis screening test F.M. Hallinan *, D. Kenny and E. Tempany Children’s Research Centre, Our Lady’s Hospital for Sick Children, Crumliv, Dublin I? (Republic of Irelund) Received
April 6th, 198 1)
Introduction In view of the high incidence of the cystic fibrosis (CF) gene in Caucasian populations [l] there is clearly a great need for a screening test which would enable the ready detection of CF heterozygotes. The reports [2-41 that isoelectric focusing in polyacrylamide gels (IEF-PA) of serum proteins provides a reliable CF screening test, therefore, generated much interest, particularly as it was proposed [S] that this factor was related to the previously described ciliary dyskinesia factor [8]. However, attempts to confirm the original findings have given mixed results [9,10- 131. It has been suggested that the failure of some workers with this technique was due to their modifications of the original procedure [4,6]. We have, therefore, attempted to assess the utility of Wilson’s IEF-PA procedure while conforming to the details of his technique and we here report our results with this original method. In addition, the effects of a number of variations of the original procedure on the results obtained are described. Materials and methods Blood samples were collected from normal laboratory staff and from clinically confirmed CF patients and their parents attending the CF clinic at this hospital and serum prepared, as described [2]. The IgG concentration of each sample was determined by nephelometry 1151and the IgG fraction of some samples was isolated using Protein A-Sepharose 1141.The isoelectric focusing procedure used was demonstrated to one of us (DK) by Dr. G.B. Wilson and was essentially the same as the published procedure [3].
* To whom correspondence
0009~89R
I ,/81/~-~/$02.7~
should be addressed. Q 198 I Elsevier/North-Holland
Biomedical
Press
104
Experimental
results
A. Standard technique 84 different serum samples were initially examined by the standard technique [3]. The typical type of focusing pattern obtained is demonstrated in Fig. 1. Samples were considered positive for the CF factor of Wilson, if a band of Coomassie Blue staining material was consistently detectable on repeated analysis in the most cathodal centimeter of the gel (the pH region of 8.5-9.0) in duplicate lanes. It is evident from our results that the criteria of positivity used here included a number of different types of patterns ranging from a distinct single band (Fig. 2A) through a diffuse staining (Fig. 2B) to doublet or, even multiple bands (Fig. 2C). This heterogeneity appears to be characteristic of the individual patient and is not related in any obvious way to genetic type or procedural variations. If the criteria of positivity were made more stringent the percentage positivity was lowered. The overall result (Table I) was disappointing in that there was a substantial amount of uncertainty and the degree of positivity was low. Thus, the unmodified technique could not be used as a screening test for CF homozygotes or heterozygotes. In an attempt to improve the usefulness of the technique the effect of variations in a number of important experimental parameters was then examined. B. Variations in sample processing The failure of certain other workers to reproduce the original results has been attributed [3,4,6] to incorrect preparation of the samples. However, we have found (Table I) the sample score to be unaffected by (a) changes in the sample preparation protocol, (b) pre-incubation of sample prepared by a standardised technique. Therefore, the material in serum giving rise to the band which we observe is quite stable. On protein A Sepharose chromatography the putative CF factor band of positive samples was detectable in both the IgG fraction and the unbound fraction but in the absence of accurate quantitation the degree of fractionation achieved cannot be determined. However, isolation of the IgG fraction did not alter the score obtained with any sample previously classified as negative, irrespective of genotype. Therefore, the addition of this step to the procedure did not improve the diagnostic value of the test. C. Variations in focusing procedure The effect of substitution of different ampholytes, Pharmalyte (Pharmacia Ltd., London, UK) and Biolyte (Bio-Rad Labs, Watford, UK) for the LKB pH 3.5-10 ampholines was then examined. Although the overall protein banding pattern was quite similar, certain details were altered with each of these ampholyte preparations. Thus, with Pharmalyte the CF factor band could not be distinguished from background whereas with Biolyte a cathodal band analogous to the CF factor detectable with ampholines was apparent. There was, however, no improvement in diagnostic value on altering the source of ampholyte. It has been suggested [7] that the low pH near the anode may facilitate dissocia-
3
4
S6
7
8
'9
10
11
12
-13
14
15
16
Fig. I. IEF-PA of serum in a pH 2.5- 10 gradient. Serum samples were applied on 5 X IO mm or 8 X IO mm 3MM pads at the anode and focused for 95 min. at 30 W, as described in “Materials and methods”. All samples were run in duplicate except for tracks 15 and 16 where different ~01s. (150 1.18 and 300 ng, respectively) of the same serum samples were applied. The genetic type of the samples was as follows: I .2. CF hetcrozygote: 3-X. unaffected siblings; 9- 12, controls; 13- 16, CF homozygotes. Samples marked with a black dot at the right of the lane were scored positive.
12
'a-9
ma-2
4-55
5.1 Q
5.7 I
6-35
7-2
7995
8-7
I
27 23 22 12
CF homozygotes CF heterozygotes Unaffected sibs *** Normal control
8 12 8 7
M
Sex
BAND
19 II I4 5
F 2-19 30-54 I-21 20-35
Age range &ears)
IN SERA USING
9.6244 10.2r2.3 8.1 t2.6 9.oi: 1.2
(mg/mI)
_
.
7 (25.9%) 3 (13%) 0 (0%) 1(8.3%)
c
CF Factor
6 2 2 0
?
to unequivocally
14 (51.8%) 18 (78.3%) 20 (90.0%) 11(91.7%)
_
runs or inability
(22.2%) (8.7%) (9.1%) (0%)
score **
between electrofocusing
PROTOCOL
IgG cont. *
STANDARD
* Results are as meant- standard deviation, ** +. CF factor band present; ?, no score possible either because of score variation in an otherwise acceptable electrofocusing run. *** Unaffected siblings of parents with known CF children,
No. tested
OF CF FACTOR
Serum type
DISTRIBUTION
TABLE
Predicted
attribute
100 100 66 4
(%)
score
II
a b ’ d e ’ s h
None None None None None None
Glass Plastic Plastic Glass Glass Glass
56°C 56°C 37°C 37’C 37’C 37°C 4OC 4°C
Smin 15 min 1.5 min 15 min, 15 min, 15 min, 15 min, 15 min, HCI
BAND
2.5 mmol/l PMSF d 10 mmol/l EDTA 25 mmoI/l benzamidine 1 mmol/l HCl 10 mmol/l HCI
OF THE CF FACTOR
Blood samples were processed, either by the standard protocol or by the modified procedure indicated Serum samples, prepared by the standard protocol were preincubated at the indicated conditions immediately Samples were previously classified + or - by the standard focusing procedure. Phenyl methyl sulphonyl fluoride. Not determined. 0.38% (w/v) sodium citrate, final concentration. 20 units/ml sodium heparin, final concentration. 10 mmol/l EDTA final concentration.
tube, room temp. tube, room temp. tube, 4°C tube, titrated plasma ’ tube, heparin plasma g tube, EDTA plasma h
Preinc. Preinc. Preinc. Preinc. Preinc. Preinc. Preinc. Preinc.
Sample treatment
ON THE DETECTION
Standard Standard Standard Standard Standard Standard Standard Standard
a
TREATMENTS
protocol
OF VARIOUS
Sample preparation
EFFECT
TABLE
before
None None None None None None
focusing
None None None None None None
-sample None NDe ND ND ND ND ND ND
+ sample ’ None None None None None None None None
Effect on sample score of ’
A Fig. 2. Enlargement of cathodal represent (A) CF heterozygote; indicated by the arrow.
areas of IEF-PA gels showing heterogeneity of positives. (B) and (C) homozygotes. The position of the CF protein
Samples band is
tion of an IgG-CF factor complex and, thereby, demonstration of the CF factor band. However, we have found that the score of a strongly positive sample was unaffected by its site of application even when it was applied at the cathodal IgG region of the gel.
TABLE
III
DISTRIBUTION VOLUME
OF
CF FACTOR
IN SERA
USING
Serum type
No. tested
IgG cont.
CF homozygotes CF heterozygotes Normal
8 6 9
9.2 i 4.4 iO.li2.1 9.0* 1.4
* Results are as mean&standard deviation. ** Results are presented as the % of individual of serum.
* (g/l)
MODIFIED
PROTOCOL
R CF factor+
OF
VARYING
** at
5 pl
20pl
so PI
25 0 0
62.5 x3.3 22
I00
62.5 33
sera which were factor positive with the different
volume.\
109
We re-examined a number of samples but while using 5 ~1, 20 ~1 and 50 ~1 aliquots of serum on 8 X 10 pads and the standard focusing technique (Table III), to assess the effect of sample volume on score. Clearly the overall score is dependent on the volume applied and although the score of the CF gene carriers looks promisingly good in this series the degree of positivity in the normal group is higher than that predicted by the gene frequency. In addition the overall score at the 209~1 level (approximately 200 pg IgG) is higher than in the previous series (Table I), although the serum samples are a subset of those used earlier. In our view this is a reflection of the inherently subjective nature of the scoring procedure. Discussion The primary goal of this study was an evaluation of the utility of the IEF-PA method as a CF screening test. Our findings of an unusual and heterogeneous cathodal banding pattern in some CF homozygotes and heterozygotes is in agreement with the observations of Wilson and others [10,13]. However, such observations are not a sufficient basis for a reliable screening test, and, in our view, there are a number of difficulties with the technique which make any such claims premature. First, as the cathodal banding pattern is, in almost all cases, extremely faint a reliable estimate of positivity rests on repeated analysis and a verdict agreed by at least two experienced observers. Second, the heterogeneity among positive samples increases the subjective element of the scoring procedure. Third, the increasing percentage positivity among normals on increasing the serum volume and the type of positivity pattern seen with Biolyte in place of ampholines suggests that normal sera possess materials similar to the CF factor band. Thus, it may be that the difference between normal, heterozygote and homozygote represents a continuum of concentrations rather than discrete levels, in which case screening by an electrophoretic procedure is inappropriate. However, although this method cannot be recommended for CF screening the nature of the materials constituting the band and the development of more unequivocal methods e.g. immunochemical [16] for their determination, is desirable. The discrepancies between the results of different groups using the IEF-PA technique have been controversial [7,11,12] and have been attributed to a presumed lability of the material constituting the diagnostic band [7]. However, experimentally we found that this band is quite stable and that such discrepancies may therefore result from individual variations in the sample scoring process. In addition, the question of CF factor band IgG association cannot be satisfactorily resolved in the absence of an accurate quantitation of its distribution in different serum fractions but, in any case, isolation of the IgG fraction did not alter our estimation of the score of any sample. Acknowledgement We are indebted work.
to the Cystic Fibrosis
Association
of Ireland
for supporting
this
110
References I Nadler HL. Rao GJS, Taussig LM. Cystic fibrosis. in: Stanbury JB, Wyngaarden JB, Fredrickson DS. eds. The metabolic basis of inherited disease. 4th ed. New York: McGraw-Hill, 1978: 1683- 1710. 2 Wilson GB, Fudenberg HH, Jahn TL. Studies on cystic fibrosis using isoelectric focusing. I. An assay for detection of cystic fibrosis homozygotes and heterozygote carriers from serum. Pediatr Res 1975; 9: 635-640. 3 Wilson GB, Arnaud P, Fudenberg HH. Improved method for the detection of cystic fibrosis protein in serum using the LKB Multiphor electrofocusing apparatus. Pediatr Res 1977; I I : 986-989. 4 Wilson GB, Jahn TL. Fonesca JR. Demonstration of serum protein differences in cystic fibrosis by isoelectric focusing in thin layer polyacrylamide gels. Clin Chim Acta 1973; 49: 79-91. 5 Wilson GB. Monsher MT, Fudenberg HH. Studies on cystic fibrosis using isoelectric focusing. III. Correlation between cystic fibrosis protein and ciliary dyskinesia activity in serum shown by a modified rabbit tracheal bioassay. Pediatr Res 1977; I I : l43- 146. 6 Wilson GB, Fudenberg HH. Is cystic fibrosis protein a diagnostic marker for individuals who harbor the defective gene? Pediatr Res 1978: 12: 80 I -R04. 7 Wilson GB. Cystic fibrosis protein, a confirmed diagnostic marker for detecting heterozygote carriers: significance in relation to future screening and to a proposed primary defect in alpha*-macroglobulin. Pediatr Res 1979; 13: 1079-1080. 8 Bowman BH, Barnett D, Metalon R. Detection and characterisation of the cystic fibrosis ciliary inhibitor. In: Mangos JA, Talamo R, eds. Fundamental problems in cystic fibrosis and related diseases. Intercontinental Medical Book Corp., 1973: 29-46. 9 Altland K, Schmidt SR, Kaiser G, Knoche W. Demonstration of a factor in the serum of homozygotes and heterozygotes for cystic fibrosis by a non biological technique. Humangenetik 1975; 28: 207-2 16. IO Scholey J. Applegarth DA, Davidson GF, Wong LTK. Detection of cystic fibrosis protein by electrofocusing. Pediatr Res 1978; 12: 800. I I Smith QT. Hamilton MJ, Shapiro BL. Letter to the editor. Pediatr Res 1976; IO: 999-1000. 12 Thomas JM, Merritt AD, Hodes ME. Electrophoretic analysis of serum proteins in cystic fibrosis. Pediatr Res 1977; 115: 1148-l 154. I3 Tully GW, Nevin GB. Young IR, Nevin NC. Detection of cystic fibrosis protein by isoelectric focusing of serum. Pediatr Res 1979; 13: 107% 14 Protein A, Protein A-Scpharose CL-4B Manual. Pharmacia (GB) Ltd., 75 Uxbridge Road, London W5 5SS. England. I5 Walker WHC, Gauldic J. Automated determination of immunoglobulins. In: Ritchie RF, ed. Automated immunoanalysis, Part I. New York, Marcel Dekker, 197X: 203-226. I6 Manson JC, Brock DJH. Development of a quantitative immunoassay for the cystic fibrosis gene. Lancet 1980; I: 330-331.
Clinicrr Chimicu Actu, I17 (1981) 103- 1 IO Elsevier/North-Holland Biomedical Press
CCA
1944
Short communication
A study of isoelectric focusing in po~yacryla~de gels of serum proteins as a cystic fibrosis screening test F.M. Hallinan *, D. Kenny and E. Tempany Children’s Research Centre, Our Lady’s Hospital for Sick Children, Crumliv, Dublin I? (Republic of Irelund) Received
April 6th, 198 1)
Introduction In view of the high incidence of the cystic fibrosis (CF) gene in Caucasian populations [l] there is clearly a great need for a screening test which would enable the ready detection of CF heterozygotes. The reports [2-41 that isoelectric focusing in polyacrylamide gels (IEF-PA) of serum proteins provides a reliable CF screening test, therefore, generated much interest, particularly as it was proposed [S] that this factor was related to the previously described ciliary dyskinesia factor [8]. However, attempts to confirm the original findings have given mixed results [9,10- 131. It has been suggested that the failure of some workers with this technique was due to their modifications of the original procedure [4,6]. We have, therefore, attempted to assess the utility of Wilson’s IEF-PA procedure while conforming to the details of his technique and we here report our results with this original method. In addition, the effects of a number of variations of the original procedure on the results obtained are described. Materials and methods Blood samples were collected from normal laboratory staff and from clinically confirmed CF patients and their parents attending the CF clinic at this hospital and serum prepared, as described [2]. The IgG concentration of each sample was determined by nephelometry 1151and the IgG fraction of some samples was isolated using Protein A-Sepharose 1141.The isoelectric focusing procedure used was demonstrated to one of us (DK) by Dr. G.B. Wilson and was essentially the same as the published procedure [3].
* To whom correspondence
0009~89R
I ,/81/~-~/$02.7~
should be addressed. Q 198 I Elsevier/North-Holland
Biomedical
Press
104
Experimental
results
A. Standard technique 84 different serum samples were initially examined by the standard technique [3]. The typical type of focusing pattern obtained is demonstrated in Fig. 1. Samples were considered positive for the CF factor of Wilson, if a band of Coomassie Blue staining material was consistently detectable on repeated analysis in the most cathodal centimeter of the gel (the pH region of 8.5-9.0) in duplicate lanes. It is evident from our results that the criteria of positivity used here included a number of different types of patterns ranging from a distinct single band (Fig. 2A) through a diffuse staining (Fig. 2B) to doublet or, even multiple bands (Fig. 2C). This heterogeneity appears to be characteristic of the individual patient and is not related in any obvious way to genetic type or procedural variations. If the criteria of positivity were made more stringent the percentage positivity was lowered. The overall result (Table I) was disappointing in that there was a substantial amount of uncertainty and the degree of positivity was low. Thus, the unmodified technique could not be used as a screening test for CF homozygotes or heterozygotes. In an attempt to improve the usefulness of the technique the effect of variations in a number of important experimental parameters was then examined. B. Variations in sample processing The failure of certain other workers to reproduce the original results has been attributed [3,4,6] to incorrect preparation of the samples. However, we have found (Table I) the sample score to be unaffected by (a) changes in the sample preparation protocol, (b) pre-incubation of sample prepared by a standardised technique. Therefore, the material in serum giving rise to the band which we observe is quite stable. On protein A Sepharose chromatography the putative CF factor band of positive samples was detectable in both the IgG fraction and the unbound fraction but in the absence of accurate quantitation the degree of fractionation achieved cannot be determined. However, isolation of the IgG fraction did not alter the score obtained with any sample previously classified as negative, irrespective of genotype. Therefore, the addition of this step to the procedure did not improve the diagnostic value of the test. C. Variations in focusing procedure The effect of substitution of different ampholytes, Pharmalyte (Pharmacia Ltd., London, UK) and Biolyte (Bio-Rad Labs, Watford, UK) for the LKB pH 3.5-10 ampholines was then examined. Although the overall protein banding pattern was quite similar, certain details were altered with each of these ampholyte preparations. Thus, with Pharmalyte the CF factor band could not be distinguished from background whereas with Biolyte a cathodal band analogous to the CF factor detectable with ampholines was apparent. There was, however, no improvement in diagnostic value on altering the source of ampholyte. It has been suggested [7] that the low pH near the anode may facilitate dissocia-
3
4
S6
7
8
'9
10
11
12
-13
14
15
16
Fig. I. IEF-PA of serum in a pH 2.5- 10 gradient. Serum samples were applied on 5 X IO mm or 8 X IO mm 3MM pads at the anode and focused for 95 min. at 30 W, as described in “Materials and methods”. All samples were run in duplicate except for tracks 15 and 16 where different ~01s. (150 1.18 and 300 ng, respectively) of the same serum samples were applied. The genetic type of the samples was as follows: I .2. CF hetcrozygote: 3-X. unaffected siblings; 9- 12, controls; 13- 16, CF homozygotes. Samples marked with a black dot at the right of the lane were scored positive.
12
'a-9
ma-2
4-55
5.1 Q
5.7 I
6-35
7-2
7995
8-7
I
27 23 22 12
CF homozygotes CF heterozygotes Unaffected sibs *** Normal control
8 12 8 7
M
Sex
BAND
19 II I4 5
F 2-19 30-54 I-21 20-35
Age range &ears)
IN SERA USING
9.6244 10.2r2.3 8.1 t2.6 9.oi: 1.2
(mg/mI)
_
.
7 (25.9%) 3 (13%) 0 (0%) 1(8.3%)
c
CF Factor
6 2 2 0
?
to unequivocally
14 (51.8%) 18 (78.3%) 20 (90.0%) 11(91.7%)
_
runs or inability
(22.2%) (8.7%) (9.1%) (0%)
score **
between electrofocusing
PROTOCOL
IgG cont. *
STANDARD
* Results are as meant- standard deviation, ** +. CF factor band present; ?, no score possible either because of score variation in an otherwise acceptable electrofocusing run. *** Unaffected siblings of parents with known CF children,
No. tested
OF CF FACTOR
Serum type
DISTRIBUTION
TABLE
Predicted
attribute
100 100 66 4
(%)
score
II
a b ’ d e ’ s h
None None None None None None
Glass Plastic Plastic Glass Glass Glass
56°C 56°C 37°C 37’C 37’C 37°C 4OC 4°C
Smin 15 min 1.5 min 15 min, 15 min, 15 min, 15 min, 15 min, HCI
BAND
2.5 mmol/l PMSF d 10 mmol/l EDTA 25 mmoI/l benzamidine 1 mmol/l HCl 10 mmol/l HCI
OF THE CF FACTOR
Blood samples were processed, either by the standard protocol or by the modified procedure indicated Serum samples, prepared by the standard protocol were preincubated at the indicated conditions immediately Samples were previously classified + or - by the standard focusing procedure. Phenyl methyl sulphonyl fluoride. Not determined. 0.38% (w/v) sodium citrate, final concentration. 20 units/ml sodium heparin, final concentration. 10 mmol/l EDTA final concentration.
tube, room temp. tube, room temp. tube, 4°C tube, titrated plasma ’ tube, heparin plasma g tube, EDTA plasma h
Preinc. Preinc. Preinc. Preinc. Preinc. Preinc. Preinc. Preinc.
Sample treatment
ON THE DETECTION
Standard Standard Standard Standard Standard Standard Standard Standard
a
TREATMENTS
protocol
OF VARIOUS
Sample preparation
EFFECT
TABLE
before
None None None None None None
focusing
None None None None None None
-sample None NDe ND ND ND ND ND ND
+ sample ’ None None None None None None None None
Effect on sample score of ’
A Fig. 2. Enlargement of cathodal represent (A) CF heterozygote; indicated by the arrow.
areas of IEF-PA gels showing heterogeneity of positives. (B) and (C) homozygotes. The position of the CF protein
Samples band is
tion of an IgG-CF factor complex and, thereby, demonstration of the CF factor band. However, we have found that the score of a strongly positive sample was unaffected by its site of application even when it was applied at the cathodal IgG region of the gel.
TABLE
III
DISTRIBUTION VOLUME
OF
CF FACTOR
IN SERA
USING
Serum type
No. tested
IgG cont.
CF homozygotes CF heterozygotes Normal
8 6 9
9.2 i 4.4 iO.li2.1 9.0* 1.4
* Results are as mean&standard deviation. ** Results are presented as the % of individual of serum.
* (g/l)
MODIFIED
PROTOCOL
R CF factor+
OF
VARYING
** at
5 pl
20pl
so PI
25 0 0
62.5 x3.3 22
I00
62.5 33
sera which were factor positive with the different
volume.\
109
We re-examined a number of samples but while using 5 ~1, 20 ~1 and 50 ~1 aliquots of serum on 8 X 10 pads and the standard focusing technique (Table III), to assess the effect of sample volume on score. Clearly the overall score is dependent on the volume applied and although the score of the CF gene carriers looks promisingly good in this series the degree of positivity in the normal group is higher than that predicted by the gene frequency. In addition the overall score at the 209~1 level (approximately 200 pg IgG) is higher than in the previous series (Table I), although the serum samples are a subset of those used earlier. In our view this is a reflection of the inherently subjective nature of the scoring procedure. Discussion The primary goal of this study was an evaluation of the utility of the IEF-PA method as a CF screening test. Our findings of an unusual and heterogeneous cathodal banding pattern in some CF homozygotes and heterozygotes is in agreement with the observations of Wilson and others [10,13]. However, such observations are not a sufficient basis for a reliable screening test, and, in our view, there are a number of difficulties with the technique which make any such claims premature. First, as the cathodal banding pattern is, in almost all cases, extremely faint a reliable estimate of positivity rests on repeated analysis and a verdict agreed by at least two experienced observers. Second, the heterogeneity among positive samples increases the subjective element of the scoring procedure. Third, the increasing percentage positivity among normals on increasing the serum volume and the type of positivity pattern seen with Biolyte in place of ampholines suggests that normal sera possess materials similar to the CF factor band. Thus, it may be that the difference between normal, heterozygote and homozygote represents a continuum of concentrations rather than discrete levels, in which case screening by an electrophoretic procedure is inappropriate. However, although this method cannot be recommended for CF screening the nature of the materials constituting the band and the development of more unequivocal methods e.g. immunochemical [16] for their determination, is desirable. The discrepancies between the results of different groups using the IEF-PA technique have been controversial [7,11,12] and have been attributed to a presumed lability of the material constituting the diagnostic band [7]. However, experimentally we found that this band is quite stable and that such discrepancies may therefore result from individual variations in the sample scoring process. In addition, the question of CF factor band IgG association cannot be satisfactorily resolved in the absence of an accurate quantitation of its distribution in different serum fractions but, in any case, isolation of the IgG fraction did not alter our estimation of the score of any sample. Acknowledgement We are indebted work.
to the Cystic Fibrosis
Association
of Ireland
for supporting
this
110
References I Nadler HL. Rao GJS, Taussig LM. Cystic fibrosis. in: Stanbury JB, Wyngaarden JB, Fredrickson DS. eds. The metabolic basis of inherited disease. 4th ed. New York: McGraw-Hill, 1978: 1683- 1710. 2 Wilson GB, Fudenberg HH, Jahn TL. Studies on cystic fibrosis using isoelectric focusing. I. An assay for detection of cystic fibrosis homozygotes and heterozygote carriers from serum. Pediatr Res 1975; 9: 635-640. 3 Wilson GB, Arnaud P, Fudenberg HH. Improved method for the detection of cystic fibrosis protein in serum using the LKB Multiphor electrofocusing apparatus. Pediatr Res 1977; I I : 986-989. 4 Wilson GB, Jahn TL. Fonesca JR. Demonstration of serum protein differences in cystic fibrosis by isoelectric focusing in thin layer polyacrylamide gels. Clin Chim Acta 1973; 49: 79-91. 5 Wilson GB. Monsher MT, Fudenberg HH. Studies on cystic fibrosis using isoelectric focusing. III. Correlation between cystic fibrosis protein and ciliary dyskinesia activity in serum shown by a modified rabbit tracheal bioassay. Pediatr Res 1977; I I : l43- 146. 6 Wilson GB, Fudenberg HH. Is cystic fibrosis protein a diagnostic marker for individuals who harbor the defective gene? Pediatr Res 1978: 12: 80 I -R04. 7 Wilson GB. Cystic fibrosis protein, a confirmed diagnostic marker for detecting heterozygote carriers: significance in relation to future screening and to a proposed primary defect in alpha*-macroglobulin. Pediatr Res 1979; 13: 1079-1080. 8 Bowman BH, Barnett D, Metalon R. Detection and characterisation of the cystic fibrosis ciliary inhibitor. In: Mangos JA, Talamo R, eds. Fundamental problems in cystic fibrosis and related diseases. Intercontinental Medical Book Corp., 1973: 29-46. 9 Altland K, Schmidt SR, Kaiser G, Knoche W. Demonstration of a factor in the serum of homozygotes and heterozygotes for cystic fibrosis by a non biological technique. Humangenetik 1975; 28: 207-2 16. IO Scholey J. Applegarth DA, Davidson GF, Wong LTK. Detection of cystic fibrosis protein by electrofocusing. Pediatr Res 1978; 12: 800. I I Smith QT. Hamilton MJ, Shapiro BL. Letter to the editor. Pediatr Res 1976; IO: 999-1000. 12 Thomas JM, Merritt AD, Hodes ME. Electrophoretic analysis of serum proteins in cystic fibrosis. Pediatr Res 1977; 115: 1148-l 154. I3 Tully GW, Nevin GB. Young IR, Nevin NC. Detection of cystic fibrosis protein by isoelectric focusing of serum. Pediatr Res 1979; 13: 107% 14 Protein A, Protein A-Scpharose CL-4B Manual. Pharmacia (GB) Ltd., 75 Uxbridge Road, London W5 5SS. England. I5 Walker WHC, Gauldic J. Automated determination of immunoglobulins. In: Ritchie RF, ed. Automated immunoanalysis, Part I. New York, Marcel Dekker, 197X: 203-226. I6 Manson JC, Brock DJH. Development of a quantitative immunoassay for the cystic fibrosis gene. Lancet 1980; I: 330-331.