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 chenodeoxy- cholate in serum. The procedure does not require extraction of serum nor separation of anti- body-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 salt concentrations as a sensitive index of liver function was restricted by the lengthy procedures required by gas-liquid chroma- tography (glc) methods with sufficient sensi- tivity to assay normal fasting sera (1,2). When Simmonds et al. (3) reported the first radioimmunoassay of a serum bile salt this pointed the way and now radioimmunassays for the major salts in serum have been reported (4-6).

Recently many established radioimmuno- assays have been changed to enzyme im- munoassays to benefit by the increased speed of analysis and reduced costs afforded by this technique (7). This report describes application of enzyme immunoassay to con- jugated chenodeoxycholate, using the ac- tivity of horseradish peroxidase (EC 1.11.1.7) for quantitation.

MATERIALS

Horseradish peroxidase (EC 1.11.1.7) was obtained from Miles Laboratories (Slough, England) and had an activity of 61.6 purpuragallin units/rag. Other chemi- cals and solvents were AR grade (BDH Ltd.,

Liverpool, England) while antiserum to con- jugated chenodeoxycholate was a gift from Mr. J. Murison of this department.

METHODS

Enzyme-Hapten Complex

Horseradish peroxidase 2 (HRPO) was treated by the procedure of Nakane and Ka- waoi (8). The enzyme (5 mg) was dissolved in 1.0 ml of freshly prepared sodium bicar- bonate solution (0.3 M, pH 8.1) and mixed with 0.1 ml of fluorodinitrobenzene (1% w/v in ethanol) at room temperature. After 1 h 1.0 ml of sodium m-periodate (0.06 M) was added and the solution mixed for a further 1 h. During this time the solution became yellow-green. The reaction was terminated by addition of 1.0 ml ethylene glycol (0.16 M), left for 1 h and the solution dialysed at 4°C against 0.01 M sodium carbonate (pH 9.5) for 12-16 h, using an Amicon 8MC ul- trafiltration unit. The dialysed solution was added to 1 mmol of glycochenodeoxycho- late containing 100,000 dpm of [3H]glyco- chenodeoxycholate (9 pmol) dissolved in 0.5 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 © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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 remain- der was dialysed at 4°C against 0.1 M phos- phate buffered saline (pH 7.4) for 6 h and a second 1% aliquot removed to determine the extent of enzyme-hapten complex for- mation by liquid scintillation counting.

Measurement of Enzyme-Hapten Activity

The enzyme activity of this complex was assayed by a modification of the procedure of Polis and Shmukler (9). Pyrogallol solu- tion (1 ml of 5% w/v), hydrogen peroxide (0.5 ml of 0.5% v/v), 1 ml phosphate buf- fer (0.1 M, pH 6.3) and 5 ml distilled water were mixed with enzyme-hapten (0.1 ml) and incubated at 20°C for 15 min. Where antiserum (0.1 ml) and standards or serum (0.1 ml) were included the volume of water was reduced to maintain an incubation vol- ume of 7.6 ml. After incubation sulphuric acid (1 ml, 2 M) was added and, after thor- ough mixing, the oxidised pyrogallol (purpuragallin) was extracted by gently mix- ing three times with 5 ml of diethyl ether. Blanks were prepared where the enzyme solution was replaced by phosphate buffer and the optical density of samples and blanks was determined at 420 nm.

The enzyme activity of this complex was determined at increasing dilutions to estab- lish an enzyme concentration suitable for further investigation. Under the conditions described a dilution of 1:10 gave an absor- bance of 0.2-0.4 for three different prepara- tions 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 with 0.1 ml of anti-glycochenodeoxycholate serum diluted in the range 1:100 to 1:1000 and the enzyme activity determined (Table l). When it was established that the antisera inhibited enzyme activity standards (50-500 pmol/incubation mixture) were included in the enzyme incubation mixture.

Assay of Chenodeoxycholate Conjugates

Standard glycochenodeoxycholate in 0.1 ml phosphate buffer (0.1 M, pH 7.4) was mixed with antiserum (0.1 ml of 1:1000 di- lution) and enzyme-hapten complex (0.1 ml). The activity of the enzyme under these conditions was determined as previously de- scribed. Sera were assayed in the same way and the concentrations calculated from the calibration curve determined simultane- ously. Other common bile salts were assayed

TABLE 1

ANTISERUM CROSS REACTIVITY DETERMINED BY ENZYME IMMUNOASSAY AND RADIOIMMUNOASSAY a

Radioimmunoassay Enzyme immunoassay Bile acid percentage cross reaction percentage cross reaction

Chenodeoxycholic acid 10 7 Glycocholic acid 1.0 2.0 Glycolithocholic acid 1.3 < 1.0 Glycoursodeoxycholic acid 1.0 < 1.0 Glycodeoxycholic acid < 1.0 < 1.0 Cholic acid < 1.0 < 1.0 Lithocholic acid < 1.0 < 1.0 Deoxycholic acid < 1.0 < 1.0

a Cross-reaction was identical for glycine and taurine conjugates of each bile acid studied.

BILE SALT ENZYME IMMUNOASSAY 363

T A B L E 2

INHIBITION OF ENZYME--HAPTEN ACTIVITY BY

INCREASING DILUTIONS OF ANTI-GLYCOCHENO-

DEOXYCHOLATE SERUM

Antiserum Inhibition dilution Absorbance (%)

1:100 0.003 98.5 1:300 0.031 84.7 1:500 0.032 84.2 1:700 0.032 84.2 1:1000 0.046 77.3 No antiserum 0.203 0

to determine that concentration which re- stored 50% of enzyme-hapten activity in- hibited by antiserum. The concentration of glycochenodeoxycholate which produced the same activity was expressed as a per- centage of this concentration and this per- centage termed the cross-reactivity.

Accuracy of this assay was assessed by comparison with a radioimmunoassay simi- lar 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 (equiva- lent 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 radioim- munoassay was _ 8.6% for 10 replicate con- trol samples.

RESULTS

Different preparations of the bile salt- horseradish peroxidase complex gave molar ratios of bile salt:HRPO ranging from ap- proximately 1:1 to 8:1 measured both iso- topically and by radioimmunoassay. The differences in this ratio were not important either in terms of enzyme activity or inhi- bition of enzyme activity by antisera. The procedure used to form the bile sa l t -HRPO complex caused loss of 55% of enzyme activ-

0.2

Absorbance 420 nm Y 0.1

0. J

p mol Chenodeoxycholate

FIG. 1. Correlation of serum chenodeoxycholate con- jugates assayed by enzyme immunoassay and radio- imrnunoassay.

ity, but 14/zg of enzyme/incubation proved satisfactory under the conditions described. Each reaction in the formation of this com- plex 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 sub- sequent studies.

Standards analysed by this procedure gave absorbance values which were linear

25

20

0

8,

- I0-

c o / o /

O- /

e/ /

/

/

/

/ /

/

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 cross- reactivities 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 ra- dioimmunoassay and enzyme immunoassay. The results (Fig. 2) gave a correlation co- efficient of 0.99 while precision, assessed by analysis of eight replicate samples, was ___ 6%.

DISCUSSION

The procedure of Nakane and Kawaoi (8) for preparation of enzyme-hapten uses a Schiff condensation between an aldehyde group and a primary amino group. The alde- hyde group is formed by periodate oxidation of the enzyme but bile salts do not contain a primary amino group and consequently no Schiff condensation can occur. However, it was found that glycochenodeoxycholate bound to oxidised enzyme and could not be removed by extensive untrafiltration. This complex was investigated as the basis for an enzyme immunoassay.

Although the bile salt-HRPO complex formation was accompanied by a 50% loss of enzyme activity the remaining enzyme activity was sufficient for useful assay at a concentration of 14/.tg enzyme/incubation. The activity of this enzyme-bile salt com- plex was inhibited by antisera diluted 1:1000 and this inhibition could be prevented by the addition of standard glycochenodeoxy- cholate. This inhibition and prevention of inhibition forms the basis for a successful enzyme immunoassay and this proved to be the case, for enzyme activity was directly proportional to the concentration of con- jugated chenodeoxycholate added and the relationship was linear between 50 and 500 pmol/incubation.

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 equi- librium point of the antigen-antibody reaction.

The concentrations of serum bile salts as- sayed 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 dis- crimination is most important. Precision de- termined on eight replicate samples was -+6%, a figure which compares well with glc and radioimmunoassay procedures used in this department.

Thus enzyme immunoassay for serum bile acids compares favourably in terms of assay performance and, with an assay time of ap- proximately 0.5 a working day, is quicker than either radioimmunoassay (1 working day) or glc (2 working days). Selection of a different substrate, such as homovanillic acid (11), could improve sensitivity even fur- ther and reduce assay time. However, sen- sitivity using this cheap, unsophisticated en- zyme assay is comparable to radioimmuno- assay and consequently there is no need to improve sensitivity for serum analysis al- though such development may prove useful for the investigation of amniotic fluid or tis- sue biopsies.

ACKNOWLEDGMENT

Thanks are due to Dr. G. Wilson for the helpful discussion which preceded this work.

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BILE SALT ENZYME IMMUNOASSAY 365

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