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Homogeneous enzyme immunoassay of chenodeoxycholate conjugates in serum
ANALYTICAL BIOCHEMISTRY 93, 361--365 (1979)
Homogeneous Enzyme Immunoassay of Chenodeoxycholate Conjugates in Serum Y. A.
BAQIR,
P. E. Ross, 1 AND IAN A. D. BOUCHIER
Department of Medicine, Ninewells Hospital and Medical School, Dundee, DDI 9SY, Scotland Received June 6, 1978 Enzyme immunoassay has been applied to the determination of conjugates of chenodeoxycholate in serum. The procedure does not require extraction of serum nor separation of antibody-bound from free antigen. These two factors reduce the analysis time to less than 5 h, with sensitivity comparable to that of radioimmunoassay. Results correlate well with values determined by radioimmunoassay (r = 0.99, N = 14) and precision (_+ 6%, N =8) compares favourably with reported values for radioimmunoassay.
The potential use of individual serum bile Liverpool, England) while antiserum to consalt concentrations as a sensitive index of jugated chenodeoxycholate was a gift from liver function was restricted by the lengthy Mr. J. Murison of this department. procedures required by gas-liquid chromatography (glc) methods with sufficient sensiMETHODS tivity to assay normal fasting sera (1,2). When Simmonds et al. (3) reported the first Enzyme-Hapten Complex radioimmunoassay of a serum bile salt this Horseradish peroxidase 2 (HRPO) was pointed the way and now radioimmunassays treated by the procedure of Nakane and Kafor the major salts in serum have been waoi (8). The enzyme (5 mg) was dissolved reported (4-6). in 1.0 ml of freshly prepared sodium bicarRecently many established radioimmuno- bonate solution (0.3 M, pH 8.1) and mixed assays have been changed to enzyme im- with 0.1 ml of fluorodinitrobenzene (1% w/v munoassays to benefit by the increased in ethanol) at room temperature. After 1 h speed of analysis and reduced costs afforded 1.0 ml of sodium m-periodate (0.06 M) was by this technique (7). This report describes added and the solution mixed for a further application of enzyme immunoassay to con- 1 h. During this time the solution became jugated chenodeoxycholate, using the ac- yellow-green. The reaction was terminated tivity of horseradish peroxidase (EC 1.11.1.7) by addition of 1.0 ml ethylene glycol (0.16 for quantitation. M), left for 1 h and the solution dialysed at 4°C against 0.01 M sodium carbonate (pH MATERIALS 9.5) for 12-16 h, using an Amicon 8MC ulHorseradish peroxidase (EC 1.11.1.7) trafiltration unit. The dialysed solution was was obtained from Miles Laboratories added to 1 mmol of glycochenodeoxycho(Slough, England) and had an activity of late containing 100,000 dpm of [3H]glyco61.6 purpuragallin units/rag. Other chemi- chenodeoxycholate (9 pmol) dissolved in 0.5 cals and solvents were AR grade (BDH Ltd., ml methanol. The resultant solution was I TO whom reprint requests should be addressed,
z Abbreviation used: HRPO, horseradish peroxidase. 361
0003-2697/79/040361-05502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
362
BAQIR, ROSS, AND BOUCHIER
stirred at room temperature for 2-3 h before addition of 5 mg of sodium borohydride. This solution was left for a further 3 h, overnight, at 4°C before a 1% aliquot was removed for liquid scintillation counting. The remainder was dialysed at 4°C against 0.1 M phosphate buffered saline (pH 7.4) for 6 h and a second 1% aliquot removed to determine the extent of enzyme-hapten complex formation by liquid scintillation counting.
Measurement of Enzyme-Hapten Activity
The enzyme activity of this complex was determined at increasing dilutions to establish an enzyme concentration suitable for further investigation. Under the conditions described a dilution of 1:10 gave an absorbance of 0.2-0.4 for three different preparations of enzyme-hapten. This dilution was used for all subsequent studies.
Effect of Glycochenodeoxycholate Antibodies on Enzyme-Hapten Activity
The enzyme-hapten complex was mixed The enzyme activity of this complex was with 0.1 ml of anti-glycochenodeoxycholate assayed by a modification of the procedure serum diluted in the range 1:100 to 1:1000 of Polis and Shmukler (9). Pyrogallol solu- and the enzyme activity determined (Table tion (1 ml of 5% w/v), hydrogen peroxide l). When it was established that the antisera (0.5 ml of 0.5% v/v), 1 ml phosphate buf- inhibited enzyme activity standards (50-500 fer (0.1 M, pH 6.3) and 5 ml distilled water pmol/incubation mixture) were included in were mixed with enzyme-hapten (0.1 ml) the enzyme incubation mixture. and incubated at 20°C for 15 min. Where antiserum (0.1 ml) and standards or serum Assay of Chenodeoxycholate Conjugates (0.1 ml) were included the volume of water was reduced to maintain an incubation volStandard glycochenodeoxycholate in 0.1 ume of 7.6 ml. After incubation sulphuric ml phosphate buffer (0.1 M, pH 7.4) was acid (1 ml, 2 M) was added and, after thor- mixed with antiserum (0.1 ml of 1:1000 diough mixing, the oxidised pyrogallol lution) and enzyme-hapten complex (0.1 (purpuragallin) was extracted by gently mix- ml). The activity of the enzyme under these ing three times with 5 ml of diethyl ether. conditions was determined as previously deBlanks were prepared where the enzyme scribed. Sera were assayed in the same way solution was replaced by phosphate buffer and the concentrations calculated from the and the optical density of samples and blanks calibration curve determined simultaneously. Other common bile salts were assayed was determined at 420 nm. TABLE 1 ANTISERUM CROSS REACTIVITY DETERMINED BY ENZYME IMMUNOASSAYAND RADIOIMMUNOASSAYa
Bile acid
Radioimmunoassay percentage cross reaction
Enzyme immunoassay percentage cross reaction
Chenodeoxycholic acid Glycocholic acid Glycolithocholic acid Glycoursodeoxycholic acid Glycodeoxycholic acid Cholic acid Lithocholic acid Deoxycholic acid
10 1.0 1.3 1.0 < 1.0 < 1.0 < 1.0 < 1.0
7 2.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
a Cross-reaction was identical for glycine and taurine conjugates of each bile acid studied.
363
BILE SALT ENZYME IMMUNOASSAY 0.2
TABLE2 INHIBITION OF E N Z Y M E - - H A P T E N ACTIVITY BY INCREASING D I L U T I O N S OF ANTI-GLYCOCHENO-
Y
DEOXYCHOLATE S E R U M
Antiserum dilution
Absorbance
Inhibition (%)
1:100 1:300 1:500 1:700 1:1000 No antiserum
0.003 0.031 0.032 0.032 0.046 0.203
98.5 84.7 84.2 84.2 77.3 0
to determine that concentration which restored 50% of enzyme-hapten activity inhibited by antiserum. The concentration of glycochenodeoxycholate which produced the same activity was expressed as a percentage of this concentration and this percentage termed the cross-reactivity. Accuracy of this assay was assessed by comparison with a radioimmunoassay similar to that reported by Schalm et al. (5). Both procedures used the same antiserum and cross-reactivities for each method are compared in Table 1. Radioimmunoassay accuracy, determined by addition of authentic standard (equivalent to 12.5/xmol litre -I) to sera, was ___8.5% and comparison with an established glc method (2) gave a correlation coefficient of 0.976 (N = 20). Precision of this radioimmunoassay was _ 8.6% for 10 replicate control samples.
Absorbance
0.1
420 nm
0. J p mol Chenodeoxycholate
FIG. 1. Correlation of serum chenodeoxycholate conjugates assayed by enzyme immunoassay and radioimrnunoassay.
ity, but 14/zg of enzyme/incubation proved satisfactory under the conditions described. Each reaction in the formation of this complex was necessary for optimal enzyme activity. Antisera diluted between 1:100 and 1:1000 inhibited the activity of the enzyme-bile salt complex (Table 2). A dilution of 1:1000 gave > 70% inhibition and was used for all subsequent studies. Standards analysed by this procedure gave absorbance values which were linear 25
/
/ /
20
/ 0
8, /
RESULTS
Different preparations of the bile salthorseradish peroxidase complex gave molar ratios of bile salt:HRPO ranging from approximately 1:1 to 8:1 measured both isotopically and by radioimmunoassay. The differences in this ratio were not important either in terms of enzyme activity or inhibition of enzyme activity by antisera. The procedure used to form the bile salt-HRPO complex caused loss of 55% of enzyme activ-
-
/
I0-
e/ / co/ o/ O-
/
Rodioimmunoassay p mol/litre
FIG. 2. Enzyme immunoassay standard curve for chenodeoxycholate conjugates.
364
BAQIR, ROSS, AND BOUCHIER
over the range 50-500 pmol (Fig. 1). Other common bile salts were assayed to assess their effect of this procedure and the crossreactivities reported in Table 1. For each bile acid taurine and glycine conjugates cross-reacted identically. Sera from controls and patients with mildly disturbed livers [as determined by Pennington et al. (10)] were assayed by radioimmunoassay and enzyme immunoassay. The results (Fig. 2) gave a correlation coefficient of 0.99 while precision, assessed by analysis of eight replicate samples, was ___6%.
Cross-reactivities of 7% for unconjugated chenodeoxycholate and 2% for conjugated cholate were similar to, but not identical with, those quoted for radioimmunoassay and are most likely a function of the antisera rather than the method of detecting the equilibrium point of the antigen-antibody reaction. The concentrations of serum bile salts assayed by radioimmunoassay and enzyme immunoassay correlated well (r = 0.990, N = 15) over the range 1-15 /zmol/litre. This range was selected as it includes normal patients and those with mild hepatic disorders (10), the clinical area where disDISCUSSION crimination is most important. Precision determined on eight replicate samples was The procedure of Nakane and Kawaoi (8) -+6%, a figure which compares well with for preparation of enzyme-hapten uses a glc and radioimmunoassay procedures used Schiff condensation between an aldehyde in this department. group and a primary amino group. The aldeThus enzyme immunoassay for serum bile hyde group is formed by periodate oxidation acids compares favourably in terms of assay of the enzyme but bile salts do not contain performance and, with an assay time of apa primary amino group and consequently no proximately 0.5 a working day, is quicker Schiff condensation can occur. However, it than either radioimmunoassay (1 working was found that glycochenodeoxycholate day) or glc (2 working days). Selection of bound to oxidised enzyme and could not be a different substrate, such as homovanillic removed by extensive untrafiltration. This acid (11), could improve sensitivity even furcomplex was investigated as the basis for ther and reduce assay time. However, senan enzyme immunoassay. sitivity using this cheap, unsophisticated enAlthough the bile salt-HRPO complex zyme assay is comparable to radioimmunoformation was accompanied by a 50% loss assay and consequently there is no need to of enzyme activity the remaining enzyme improve sensitivity for serum analysis alactivity was sufficient for useful assay at though such development may prove useful a concentration of 14/.tg enzyme/incubation. for the investigation of amniotic fluid or tisThe activity of this enzyme-bile salt comsue biopsies. plex was inhibited by antisera diluted 1:1000 and this inhibition could be prevented by ACKNOWLEDGMENT the addition of standard glycochenodeoxyThanks are due to Dr. G. Wilson for the helpful cholate. This inhibition and prevention of discussion which preceded this work. inhibition forms the basis for a successful enzyme immunoassay and this proved to be REFERENCES the case, for enzyme activity was directly 1. Van Berge Henegouwen, G. P., Ruben, A., and proportional to the concentration of conBrandt, K. H. (1974) Clin. Chim. Acta 54, 249jugated chenodeoxycholate added and the 261. relationship was linear between 50 and 500 2. Ross, P. E., Pennington, C. R., and Bouchier, I. A. D. (1977)Anal. Biochem 80, 458-465. pmol/incubation.
BILE SALT ENZYME IMMUNOASSAY 3. Simmonds, W. J., Korman, M. G., Go, V. L. W., and Hofmann, A. F. (1973) Gastroenterology 65, 705-711. 4. Demers, L. M., and Hepner, G. (1976) Clin. Chem. 22, 602-606. 5. Schalm, S. W., Van Berge Henegouwen, G. P., Hofmann, A. F., Cowen, A. E. i and Turcotte, J. (1977) Gastronenterology 73, 285-290. 6. Janne, D. A., and Maentausta, O. K. (1978) in Proceedings of International Symposium on Ra-
7. 8. 9. 10. 11.
365
di0immunoassay and Related Procedures in Medicine (International Atomic Energy Agency). Wisdom, G. B. (1976)Clin. Chem. 22, 1243-1255. Nakane, P. K., and Kawaoi, A. (1974) J. Histochem. Cytochem. 22, 1084-1091. Polis, B. D., and Shmukler, H. W. (1953) J. Biol. Chem. 201, 475-500. Pennington, C. R., Ross, P. E., and Bouchier, I. A. D. (1977) Gut 18, 903-908. Guilbault, G. G., Brignac, P., and Zimrner, M. (1966) Anal. Chem. 40, 190-196.
Homogeneous Enzyme Immunoassay of Chenodeoxycholate Conjugates in Serum Y. A.
BAQIR,
P. E. Ross, 1 AND IAN A. D. BOUCHIER
Department of Medicine, Ninewells Hospital and Medical School, Dundee, DDI 9SY, Scotland Received June 6, 1978 Enzyme immunoassay has been applied to the determination of conjugates of chenodeoxycholate in serum. The procedure does not require extraction of serum nor separation of antibody-bound from free antigen. These two factors reduce the analysis time to less than 5 h, with sensitivity comparable to that of radioimmunoassay. Results correlate well with values determined by radioimmunoassay (r = 0.99, N = 14) and precision (_+ 6%, N =8) compares favourably with reported values for radioimmunoassay.
The potential use of individual serum bile Liverpool, England) while antiserum to consalt concentrations as a sensitive index of jugated chenodeoxycholate was a gift from liver function was restricted by the lengthy Mr. J. Murison of this department. procedures required by gas-liquid chromatography (glc) methods with sufficient sensiMETHODS tivity to assay normal fasting sera (1,2). When Simmonds et al. (3) reported the first Enzyme-Hapten Complex radioimmunoassay of a serum bile salt this Horseradish peroxidase 2 (HRPO) was pointed the way and now radioimmunassays treated by the procedure of Nakane and Kafor the major salts in serum have been waoi (8). The enzyme (5 mg) was dissolved reported (4-6). in 1.0 ml of freshly prepared sodium bicarRecently many established radioimmuno- bonate solution (0.3 M, pH 8.1) and mixed assays have been changed to enzyme im- with 0.1 ml of fluorodinitrobenzene (1% w/v munoassays to benefit by the increased in ethanol) at room temperature. After 1 h speed of analysis and reduced costs afforded 1.0 ml of sodium m-periodate (0.06 M) was by this technique (7). This report describes added and the solution mixed for a further application of enzyme immunoassay to con- 1 h. During this time the solution became jugated chenodeoxycholate, using the ac- yellow-green. The reaction was terminated tivity of horseradish peroxidase (EC 1.11.1.7) by addition of 1.0 ml ethylene glycol (0.16 for quantitation. M), left for 1 h and the solution dialysed at 4°C against 0.01 M sodium carbonate (pH MATERIALS 9.5) for 12-16 h, using an Amicon 8MC ulHorseradish peroxidase (EC 1.11.1.7) trafiltration unit. The dialysed solution was was obtained from Miles Laboratories added to 1 mmol of glycochenodeoxycho(Slough, England) and had an activity of late containing 100,000 dpm of [3H]glyco61.6 purpuragallin units/rag. Other chemi- chenodeoxycholate (9 pmol) dissolved in 0.5 cals and solvents were AR grade (BDH Ltd., ml methanol. The resultant solution was I TO whom reprint requests should be addressed,
z Abbreviation used: HRPO, horseradish peroxidase. 361
0003-2697/79/040361-05502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
362
BAQIR, ROSS, AND BOUCHIER
stirred at room temperature for 2-3 h before addition of 5 mg of sodium borohydride. This solution was left for a further 3 h, overnight, at 4°C before a 1% aliquot was removed for liquid scintillation counting. The remainder was dialysed at 4°C against 0.1 M phosphate buffered saline (pH 7.4) for 6 h and a second 1% aliquot removed to determine the extent of enzyme-hapten complex formation by liquid scintillation counting.
Measurement of Enzyme-Hapten Activity
The enzyme activity of this complex was determined at increasing dilutions to establish an enzyme concentration suitable for further investigation. Under the conditions described a dilution of 1:10 gave an absorbance of 0.2-0.4 for three different preparations of enzyme-hapten. This dilution was used for all subsequent studies.
Effect of Glycochenodeoxycholate Antibodies on Enzyme-Hapten Activity
The enzyme-hapten complex was mixed The enzyme activity of this complex was with 0.1 ml of anti-glycochenodeoxycholate assayed by a modification of the procedure serum diluted in the range 1:100 to 1:1000 of Polis and Shmukler (9). Pyrogallol solu- and the enzyme activity determined (Table tion (1 ml of 5% w/v), hydrogen peroxide l). When it was established that the antisera (0.5 ml of 0.5% v/v), 1 ml phosphate buf- inhibited enzyme activity standards (50-500 fer (0.1 M, pH 6.3) and 5 ml distilled water pmol/incubation mixture) were included in were mixed with enzyme-hapten (0.1 ml) the enzyme incubation mixture. and incubated at 20°C for 15 min. Where antiserum (0.1 ml) and standards or serum Assay of Chenodeoxycholate Conjugates (0.1 ml) were included the volume of water was reduced to maintain an incubation volStandard glycochenodeoxycholate in 0.1 ume of 7.6 ml. After incubation sulphuric ml phosphate buffer (0.1 M, pH 7.4) was acid (1 ml, 2 M) was added and, after thor- mixed with antiserum (0.1 ml of 1:1000 diough mixing, the oxidised pyrogallol lution) and enzyme-hapten complex (0.1 (purpuragallin) was extracted by gently mix- ml). The activity of the enzyme under these ing three times with 5 ml of diethyl ether. conditions was determined as previously deBlanks were prepared where the enzyme scribed. Sera were assayed in the same way solution was replaced by phosphate buffer and the concentrations calculated from the and the optical density of samples and blanks calibration curve determined simultaneously. Other common bile salts were assayed was determined at 420 nm. TABLE 1 ANTISERUM CROSS REACTIVITY DETERMINED BY ENZYME IMMUNOASSAYAND RADIOIMMUNOASSAYa
Bile acid
Radioimmunoassay percentage cross reaction
Enzyme immunoassay percentage cross reaction
Chenodeoxycholic acid Glycocholic acid Glycolithocholic acid Glycoursodeoxycholic acid Glycodeoxycholic acid Cholic acid Lithocholic acid Deoxycholic acid
10 1.0 1.3 1.0 < 1.0 < 1.0 < 1.0 < 1.0
7 2.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
a Cross-reaction was identical for glycine and taurine conjugates of each bile acid studied.
363
BILE SALT ENZYME IMMUNOASSAY 0.2
TABLE2 INHIBITION OF E N Z Y M E - - H A P T E N ACTIVITY BY INCREASING D I L U T I O N S OF ANTI-GLYCOCHENO-
Y
DEOXYCHOLATE S E R U M
Antiserum dilution
Absorbance
Inhibition (%)
1:100 1:300 1:500 1:700 1:1000 No antiserum
0.003 0.031 0.032 0.032 0.046 0.203
98.5 84.7 84.2 84.2 77.3 0
to determine that concentration which restored 50% of enzyme-hapten activity inhibited by antiserum. The concentration of glycochenodeoxycholate which produced the same activity was expressed as a percentage of this concentration and this percentage termed the cross-reactivity. Accuracy of this assay was assessed by comparison with a radioimmunoassay similar to that reported by Schalm et al. (5). Both procedures used the same antiserum and cross-reactivities for each method are compared in Table 1. Radioimmunoassay accuracy, determined by addition of authentic standard (equivalent to 12.5/xmol litre -I) to sera, was ___8.5% and comparison with an established glc method (2) gave a correlation coefficient of 0.976 (N = 20). Precision of this radioimmunoassay was _ 8.6% for 10 replicate control samples.
Absorbance
0.1
420 nm
0. J p mol Chenodeoxycholate
FIG. 1. Correlation of serum chenodeoxycholate conjugates assayed by enzyme immunoassay and radioimrnunoassay.
ity, but 14/zg of enzyme/incubation proved satisfactory under the conditions described. Each reaction in the formation of this complex was necessary for optimal enzyme activity. Antisera diluted between 1:100 and 1:1000 inhibited the activity of the enzyme-bile salt complex (Table 2). A dilution of 1:1000 gave > 70% inhibition and was used for all subsequent studies. Standards analysed by this procedure gave absorbance values which were linear 25
/
/ /
20
/ 0
8, /
RESULTS
Different preparations of the bile salthorseradish peroxidase complex gave molar ratios of bile salt:HRPO ranging from approximately 1:1 to 8:1 measured both isotopically and by radioimmunoassay. The differences in this ratio were not important either in terms of enzyme activity or inhibition of enzyme activity by antisera. The procedure used to form the bile salt-HRPO complex caused loss of 55% of enzyme activ-
-
/
I0-
e/ / co/ o/ O-
/
Rodioimmunoassay p mol/litre
FIG. 2. Enzyme immunoassay standard curve for chenodeoxycholate conjugates.
364
BAQIR, ROSS, AND BOUCHIER
over the range 50-500 pmol (Fig. 1). Other common bile salts were assayed to assess their effect of this procedure and the crossreactivities reported in Table 1. For each bile acid taurine and glycine conjugates cross-reacted identically. Sera from controls and patients with mildly disturbed livers [as determined by Pennington et al. (10)] were assayed by radioimmunoassay and enzyme immunoassay. The results (Fig. 2) gave a correlation coefficient of 0.99 while precision, assessed by analysis of eight replicate samples, was ___6%.
Cross-reactivities of 7% for unconjugated chenodeoxycholate and 2% for conjugated cholate were similar to, but not identical with, those quoted for radioimmunoassay and are most likely a function of the antisera rather than the method of detecting the equilibrium point of the antigen-antibody reaction. The concentrations of serum bile salts assayed by radioimmunoassay and enzyme immunoassay correlated well (r = 0.990, N = 15) over the range 1-15 /zmol/litre. This range was selected as it includes normal patients and those with mild hepatic disorders (10), the clinical area where disDISCUSSION crimination is most important. Precision determined on eight replicate samples was The procedure of Nakane and Kawaoi (8) -+6%, a figure which compares well with for preparation of enzyme-hapten uses a glc and radioimmunoassay procedures used Schiff condensation between an aldehyde in this department. group and a primary amino group. The aldeThus enzyme immunoassay for serum bile hyde group is formed by periodate oxidation acids compares favourably in terms of assay of the enzyme but bile salts do not contain performance and, with an assay time of apa primary amino group and consequently no proximately 0.5 a working day, is quicker Schiff condensation can occur. However, it than either radioimmunoassay (1 working was found that glycochenodeoxycholate day) or glc (2 working days). Selection of bound to oxidised enzyme and could not be a different substrate, such as homovanillic removed by extensive untrafiltration. This acid (11), could improve sensitivity even furcomplex was investigated as the basis for ther and reduce assay time. However, senan enzyme immunoassay. sitivity using this cheap, unsophisticated enAlthough the bile salt-HRPO complex zyme assay is comparable to radioimmunoformation was accompanied by a 50% loss assay and consequently there is no need to of enzyme activity the remaining enzyme improve sensitivity for serum analysis alactivity was sufficient for useful assay at though such development may prove useful a concentration of 14/.tg enzyme/incubation. for the investigation of amniotic fluid or tisThe activity of this enzyme-bile salt comsue biopsies. plex was inhibited by antisera diluted 1:1000 and this inhibition could be prevented by ACKNOWLEDGMENT the addition of standard glycochenodeoxyThanks are due to Dr. G. Wilson for the helpful cholate. This inhibition and prevention of discussion which preceded this work. inhibition forms the basis for a successful enzyme immunoassay and this proved to be REFERENCES the case, for enzyme activity was directly 1. Van Berge Henegouwen, G. P., Ruben, A., and proportional to the concentration of conBrandt, K. H. (1974) Clin. Chim. Acta 54, 249jugated chenodeoxycholate added and the 261. relationship was linear between 50 and 500 2. Ross, P. E., Pennington, C. R., and Bouchier, I. A. D. (1977)Anal. Biochem 80, 458-465. pmol/incubation.
BILE SALT ENZYME IMMUNOASSAY 3. Simmonds, W. J., Korman, M. G., Go, V. L. W., and Hofmann, A. F. (1973) Gastroenterology 65, 705-711. 4. Demers, L. M., and Hepner, G. (1976) Clin. Chem. 22, 602-606. 5. Schalm, S. W., Van Berge Henegouwen, G. P., Hofmann, A. F., Cowen, A. E. i and Turcotte, J. (1977) Gastronenterology 73, 285-290. 6. Janne, D. A., and Maentausta, O. K. (1978) in Proceedings of International Symposium on Ra-
7. 8. 9. 10. 11.
365
di0immunoassay and Related Procedures in Medicine (International Atomic Energy Agency). Wisdom, G. B. (1976)Clin. Chem. 22, 1243-1255. Nakane, P. K., and Kawaoi, A. (1974) J. Histochem. Cytochem. 22, 1084-1091. Polis, B. D., and Shmukler, H. W. (1953) J. Biol. Chem. 201, 475-500. Pennington, C. R., Ross, P. E., and Bouchier, I. A. D. (1977) Gut 18, 903-908. Guilbault, G. G., Brignac, P., and Zimrner, M. (1966) Anal. Chem. 40, 190-196.