Note: Descriptions are shown in the official language in which they were submitted.
3~iZ
--1--
BACKGROUND OF THE INVENTION
The synthesis of bile acids in the liver from
cholesterol is one of the most important catabolic pathways
for cholesterol. It has been estimated that more than 80~ of
the biological cholesterol in man is transformed in the liver
into various bile acids and bile acid conjugates.
The primary bile acids, cholic and chenodeoxy-
cholic, are secreted by the liver usually as the glycine or
taurine conjugates and stored in the gall bladder. Although
10 some are absorbed from the gall bladder into the blood, most
are secreted with the bile through the common bile duct into
the lumen of the duodenum where they serve to facilitate the
absorption of cholesterol and the digestion and absorption
of fatty acids. The unused conjugated bile acids are ab-
15 sorbed into the blood vessels perfusing the duodenum andreturned to the liver through the hepatic-portal system.
Those bile acids not absorbed in the duodenum axe converted
to secondary bile acids, e.g., lithocholic and deoxycholic,
by the action of intestinal flora. These are partially
20 absorbed into the blood and returned to the liver via the
hepatic-portal system. ~here lithocholic is further metabo-
lized in the liver to sulfolithocholic. In normal individuals,
the bile acids are removed from the periteral circulation by the liver
and recycled. When the hepatocytes have been damaged by in-
25 fection, chemicals or mechanical obstructions, the entero-
hepatic circulation of the bile acids is disturbed. This
disturbance can be reflected by increased levels of bile
acids in the peripheral circulation.
Therefore, metabolic disturbances in hepatic
30 disease or disorders can be reflected by the level of bile
acids or bile acid conjugates present in sera. As early
as 1948, Sherlock and Walshe, Clin. Sci. 6:223, observed and
reported that total serum bile acids were increased in hepato-
biliary disease. This observation was confirmed in subsequent
35 years. Serum bile acids measured as the conjugates of cholic
acid proved to be the most sensitive indication of hepatic
disease in comparison to tests for serum bilirubin, proteins,
prothrombin time and bromsulphthalein retention. Korman, M.G.,
.
,
. ~a33s6z
--2--
et al., J. New Engl. F. Med. 290 (1974) 1399. Demers and
Hepner, Am. J. Clin. Path. 66 (1976) 831, have reported that
serum bile acid levels, particularly sulfolithocholyglycine
(SLCG), are very sensitive indexes of hepatic cell dysfunction.
Determining the presence of specific bile acids or their con-
jugates in sera can be a valuable diagnostic aid for the
study of liver function and a number of assays have been de-
vised to measure the quantity of these bile acids.
The most successful tests currently employed in-
10 clude gas-liquid chromatography (Sandberg, et al., Lipid Res.
6 (1965) 182), enzymatic assay using hydroxysteroid dehy-
drogenase (Murphy, G.M., Ann. Clin. Biochem. 9 (1972) 67),
and radioimmunoassay (RIA) techniques (Simmonds, et al.,
Gastroenterology 65 (1973) 705). The RIA techniques are
15 preferred because of sensitivity, specificity and convenience.
An immunoassay for the detection and determination
of an unknown immunoreactant can be conducted by providing a
limited number of specific binding sites (antibodies) for a
mixture of labeled and unknown reactants. The larger the
20 number of labeled reactants bound by the antibody, the smaller
the concentration of unknown present in the sample. For an
accurate determination, it is essential that both the labeled
and unknown reactants be free to react with the binding sites
provided. This freedom to enter into competition with the
25 labeled reactant is often hindered by other substances,
usually proteins such as serum albumin or specific binding
proteins, in the serum sample. These binding proteins prevent
the reactant being assayed from binding exclusively with the
provided antibody and thereby effect the accuracy of the
30 assay procedure.
Elimination of binding protein material in serum
bile acid immunoassays has been achieved by various extraction
procedures that employ suitable solvents to precipitate the
interfering protein. ~
Passage of serum through an Amberlite ~AD-2 column
has also been employed, Matern, et al., Clin. Chim. Acta 72
(1976) 39.
~ D ~ k
:,
lU~3962
--3--
In all the prior art methods, the serum
is not analyzed directly: that is, some sort of time consum-
ing extraction procedure has to be employed prior to per-
formance of the immunoassay.
It is the object of this invention to provide
a means for the direct measurement of bile acids in fluids
such as serum, urine or bile without requiring an extraction
procedure for proteineous or other interfering material. In
particular, it is the object of this invention to provide a
10 means for the immunologic assay of bile acids directly in
biological fluids, especially serum, without an extraction of
the fluid proteins to which the bile acids may bind.
SUMMARY OF THE INVENTION
This invention relates to a novel method for the de-
15 tection and determination of individual bile acids and con-
jugates thereof. This invention is particularly applicable
when the assay is to be conducted on an unextracted serum
sample. Specifically the disclosed method comprises the steps
of:
(a) providing an antiserum specific to the
particular bile acid to be assayed;
(b) mixing said unknown serum sample with the
antiserum and the same bile acid tagged with a labeling
agent;
(c) incubating the mixture of step (b) so as to
, allow any bile acid of the type being assayed and the labeled
j bile acid to bind with the antibodies of the antiserum;
' (d) separating the antibody-bound reactants from
the unbound;
(e) measuring the labeled-bound fraction from
step (d) to determine the relative quantity of the bile acid
in the sample by comparison with standards;
396Z
(f) the improvement comprising conducting said
assay in the presence of a buffered binding inhibitory agent,
the concentration of said agent being sufficient to inhibit
the retention of bile acids to binding proteins present in
the unextracted serum sample.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The disclosed improvement in bile immunoassay
technique may be employed to determine the presence and
quantity of any bile acid such as sulfolithocholic, cholic,
chenodeoxycholic, deoxycholic, lithocholic, ursodeoxycholic
10 and their amino acid conjugates, sulfate esters and
glucuronide conjugates. The most prevalent conjugates will
be those of glycine and taurine.
The disclosed improvement is applicable to an
immunoassay regardless of the labeling technique employed.
15 Subsequent examples will demonstrate particular applicability
in radioimmunoassays, but assays employing enzyme and
fluorescent labeled bile acids will also benefit by the
application of the disclosed improvement.
Example I
Radioimmunoassay for Sulfolit~ocholylglycine
This example demonstrates a procedure for the
determination of sulfolithocholylglycine (SLCG) in an un-
e~tracted biological fluid. The following reagents were
employed:
1. SLCG powder for use in standards was prepared
by the method of Palmer and Bolt, J. Lipid Res. 12,671
` (1971). This material was fully characterized for identity
and purity using physical chemical methods and was used to
;~ prepare standards, tracer analog and immunogen. Standards
30 were prepared by first formulating a stock solution of SLCG.
A known concentration of SLCG powder was dissolved in a
solution of 50ml water-ethanol with 0.08% ammonia. This
stock solution was diluted to the desired concentration in a
solution of human serum albumin and bovine gamma globulin
35 (BGG) in 0.9% saline.
:~ '
.
~0~ 62
.
--5--
2. 1 5I SLCG tracer was prepared by first coupling
SLCG to histamine to form sulfolithocholylglycylhistamine
(SLCG-H). The SLCG-H was then iodinated using iodogen
(Pierce Chemical Co., Rockford, Illinois) and purified using
sephadex LH-20 (Pharmacia Fine Chemicals, Piscataway, New
Jersey).
3. SLCG antisera was obtained by immunizing
rabbits with SLCG covalently coupled to bovine serum albumin
using water soluble ethylcarbodiimide.
4. Sodium salicylate reagent grade was obtained
from several vendors (J.T. Baker Chemical Co., Phillipburg,
New Jersey and Mallinckrodt Chemical Co., St. Louis,
Missouri).
5. The buffer employed in the SLCG RIA is 0.05
-15 molar phosphate, pH 7.5, containing 0.9~ sodium chloride,
0.02 molar sodium salicylate, 0.75~ bovine gamma globulin
and 0.01% thimerosal. The radioimmunoassay procedure is as
follow~:
SLCG RIA Test Procedure
A 5tandard Curve must be prepared each time
that a group of unknown samples is assayed.
1. Bring all test kit reagents to room
temperature.
2. Label tubes for performance of the test
as follows:
a. Tubes 1 and 2, labeled TCT (Total
Count Tubes), will contain aliquots of the 1 5I-
Sulfolithocholylglycylhistamine reagent solution.
These are to be used in determination of total
radioactivity.
b. Tubes 3 and 4 are to be used to
detect Nonspecific Binding (NSB) if this determi-
nation is to be made.
c. Tubes 5 through 16 are standards
from which the standard curve is prepared.
Tubes 5 and 6, O~g/lOOml SLCG
Tubes 7 and 8, lO~g/lOOml SLCG
Tubes 9 and 10, 25~g/lOOml SLCG
1~39fiZ
.
--6--
Tubes 11 and 12, 50~g/lOOml SLCG
Tubes 13 and 14, lOO~g/lOOml SLCG
Tubes 15 and 16, 250~g/lOOml SLCG
d. Tubes 17, etc. are for unknown samples
in duplicate.
3. a. Pipette 25~1 of SLCG Standards into
appropriately labeled Tubes 5 through 16.
b. Pipette 25~1 of unknown samples into
properly identified tubes beginning with 17.
c. Pipette 25~1 SLCG Standard, O~g/lOOml,
and 200~1 PBS-BGG Buffer into Tubes 3 and 4 (NSB)~
4. Pipette 200~1 125I-Sulfolithocholylglycyl~
histamine reagent solution into all tubes. Mix on
sample mixer 3 to 5 seconds, or shake the test tube rack
manually.
5. Carefully and without delay, pipette 200~1
SLCG antiserum (rabbit) into all tubes except 1 through
4. Mix on sample mixer 3 to 5 seconds, or shake the
test tube rack manually.
6. Cover all tubes with Parafilm~ or equiva-
lent and incubate at 37+ 2C for one hour.
7. After incubation, pipette 2ml polyethylene
glycol solution into all tubes except 1 and 2 (Total
Count Tubes). Total elapsed time for PEG addition
should not exceed 10 minutes. Mix vigorously on sample
mixer for 5 seconds.
8. Immediately (within 15 minutes after PEG
addition) centrifuge tubes containing PEG for 10 minutes
at room temperature at 1000 x g.
9. Remove tubes carefully from centrifuge
(not disturbing precipitate). Decant the supernatant
solution and blot the lip of the tube on absorbent paper.
10. Count the radioactivity remaining in each
tube including TCT (1 and 2) for a minimum of one
minu~e each. If necessary, subtract background and
record as net cpm.
11. Calculate results.
.
.
,
1(~9396Z
-7- ~
Typical results for the SLCG radioimmunoassay
are as follows: Figure 1 shows a standard curve covering
the range of SLCG.concentrations from O to 250~g/lOOml.
The reproducibility of the SLCG RIA is illus-
trated in Table I, which summarizes the inter and intra assay
precisionA The precision of the SLCG RIA was evaluated by
assaying a panel of four (4) serum pools in replicates of 10
on three consecutive occasions, using one lot of material.
.Three estimates of the variability were computed: Inter
10 assay (between), Intra assay (within~ and total variabilityn
X is defined as the grand mean over the three occasions of
testing.
39~Z
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Figure 2 shows the effect of serum proteins on the
SLCG RIA. Two standard curves are compared, one prepared in
buEfer (1), and a second (2) prepared in SLCG-free serum
prepared by the method of Simmonds et al., Gastroenterology,
19'73:65:705. The serum was checked by following the removal
of 3HSLCG tracer to'be sure the serum was free of SLCG. The
two curves are not superimposable, thus, demonstrating the
inhibitory effects of serum proteins on the SLCG RIA when no
effort is made to extract or free the SLCG from the serum
10 proteins.
Figure 3 shows the same comparison of two standard~
curves one in buffer and one in bile acid free seru~; however~
this comparison was done with the salicylate buffer pre-
viously described. The two curves are now superimposable,
15 thus, demonstrating that salicylate'eliminates the serum
protein binding effects (Rudman J. Clin. Invest. 36 (1957)
530). Thus, the salicylate buffer systems allow the SLCG
RIA to be performed directly on unextracted specimens.
In normal fasting volunteers, the mean value
20 obtained, using the SLCG RIA, is 0.7mM. This is in good
agreement with a mean for normals of 0.6mM obtained by
Campbell (Clinica Chimica Acta 631 (1975) 248-262) using a
Gas Liquid chromatographic method employing organic solvents
and ion exchange columns to extract the SLCG from serum.
Example 2
Radioimmunoassay for Determination of Choly-lglycine
This example demonstrates that cholylglycine can
be assayed in an unextracted biological fluid by e~ploying a
barbital buffered ANS as a binding inhibitory agent~ The
30 following reagents were formulated for use in the assay:
1. Cholylglycine (CG) (Sigman Chemical Co.,
St. Louis, Missouri) was used in standards, immunogen and
tracer. This powder was fully characterized for identity and
purity using physical chemical methods and found to be ~99%
35 pure CG. Standards were prepared by dissolving a known amount
of the CG powder in a 50~ water ethanol solution. This stock
--11--
solution was then diluted in either a protein buffer or bile
acid free serum. The bile acid free serum was prepared by the
method of Simmonds et al., Gastroenterology 1973:65:705. The
serum was checked by following the removal of 3HCG tracer to
be sure the serum was free of CG.
2. 125I CG tracer was prepared by covalently
coupling tyrosine to cholylglycine to form cholylglycltyrosine
(CGT). The CGT was then iodinated using chloramine-T and
purified using LH-20 ~ephadex (Pharmacia Fine Chemicals,
10 Piscataway, New Jersey).
30CG antisera was obtained by immunizing rabbits
with CG covalently coupled to bovine serum albumin using water
soluble ethylcarbodiimide.
4. 8~anilino-1-napthalene-sulfonic acid (ANS) was
15 obtained from Eastman Organic Chemicals (Rochester,.New York),
5. Buffer employed in the assay was Q.05M
barbital pH 8.6, with 0.4 milimolar ANS, 0.75g ~ bovine gamma
globulin and 0.01% thimerosal.
The radioimmunoassay procedure is as follows:
CGRIA Test Procedure
A standard curve is prepared each time that
a group of unknown samples is assayed.
1. Bring all test kit reagents to room
temperature.
2. Label tubes for perfor~ance of the test
as follows:
a. Tubes 1 and 2, labeled TCT (Total
Count Tubes) will contain aliquots of the 5I-
Cholylglycyltyrosine reagent solution. These are
to be used in determination of total radioactivityO
b. Tubes 3 and 4 are to be used to de-
tect Nonspecific Binding (NSB) if this determination
is to be made.
c. Tubes 5 through 16 are standards from
which the standard curve is prepared.
Tubes 5 and 6, O~g/lOOml CG
Tubes 7 and 8, 25~g/lOOml CG
Tubes 9 and 10, lOO~g/lOOml CG
J~ ~ ~D~ ~
., ~ -
~V~C,~l~9t;Z
-12-
Tubes 11 and 12, 250~g/lOOml CG
Tubes 13 and 14, lOOO~g/lOOml CG
Tubes 15 and 16, 4000~g/lOOml CG
d. Tubes 17, etc. are for unknown sar~ples
in duplicate.
3. a. Pipette 25~1 of CG Standards into
appropriately labeled Tubes 5 through 16.
b. Pipette 25~1 of unknown samples into
properly identified tubes beginning with 17.
; 10 c. Pipette 25~1 CG Standard, O~g/lOOml,
and 200~1 0.06M Barbital Buffer into Tubes 3 and
4 (NSB).
4. Pipet~e 200 1 125I-Cholylglycyltryosine
reagent solution into all tubes. Mix all tubes except
1 and 2 (TCT), on sample mixer for 3 to 5 seconds, or
shake the test tube rack manually.
5. Carefully and without delay, pipette
200~1 CG antiserum (Rabbit) into all tubes except 1
through 4. Mix on sample mixer 3 to 5 seconds, or
shake the test tube rack manually.
6. Cover all tubes with Parafilm~ or equiva~
lent and incubate at room temperature for one hour.
~l 7. After incubation, pipette 2ml Polyethylene
'! Glycol solution into all tubes except 1 and 2 (TCT).
/~ 25 Totaly elapsed time far PEG addition should not exceed
10 minutes. Mix vigorously on sample mixer for 5 seconds.
, 8. Immediately (within 15 minutes after
PEG addition~ centrifuge tubes containing PEG for 10
minutes at room temperature at 1000 x g.
9. Remove tubes carefully from centrifuge
(not disturbin~ the precipitate). Decant the superna
tant solution and blot the lips of the tubes on
absorbent paper.
10. Count the radioactivity remaining in each
tube including TCT (1 and 2) for a minimum of one
minute each. If necessary, subtract background and
record as net cpm.
11. Calculate results.
~ `
Z
-13-
Figure 4 shows the effect of serum proteins on the
CG RIA. Two standard curves are compared, one prepared in
buffer and a second prepared in CG free serum as described
above. The two curves are not superimposable, thus, demon-
strating the inhibitory effects of the serum proteins on theCG RIA when no effort is made to extract or free the CG from
serum proteins.
Figure 5 shows the same comparison of two standard
curves, one in buffer and one in CG free serum; however,
10 this comparison was done with the Barbital-ANS buffer pre-
viously described ~see reagent section). The two curves are
now superimposable, thus, demonstrating that salicylate
eliminates the serum protein binding of CG (Rudman JO ClinO
Invest. 36 (1957) 530). Thus, the Barbital-ANS buffer system
15 allows the CG RIA to be performed directly on unextracted
specimens.
In normal fasting volunteers, the mean value ob-
tained using this CG RIA was 0.43mM. This is in agreement
with previously published normal values for a CG RIA which
20 used extraction methodology (Maters, Clinicia Chimica Acta
725 (1976) 39-48).
The reproducibility of the CG RIA is illustrated
in ~able 3, which summarizes the inter and intra assay
precision of the CG RIA. The precision was evaluated by assay~
25 ing a panel of four serum pools in replicates of 10 on three
consecutive occasions using one lot of materialO The three
estimates of variability were computed: Interassay,(between)
Intra-assay (within) and total variability. X is defined as
the grand mean over the three occasions of testing~
The accuracy of the CG RIA is illustrated in
Table 4, which shows good recovery of CG added to normal
human sera. The good recovery is indicative of good
accuracy. As further validation, this recovery study was
repeated using sera with various abnormal protein levels
35 (hypoalbumin, hypergamma globulin and hypogamma globulin).
The CG RIA employing the Barbital-ANS buffer shows good
recovery with various levels of abnormal proteins thus
validating the assay for use in sera with protein abnormalities.
:
t~,Z
-14-
To further validate the assay for use with urine
and bile, parallelism studies were performed by diluting
several specimens of urine and bile. These diluted specimens
gave curves parallel to the standard curve.
~0~96Z
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-16-
ACCURACY
Table 4 shows the actual recovery of the powdered Cholyglycine
from human serum. Specimens A, B, and C are normal human sera.
Specimens 14, 20, 21, 22, 23, 28, 29 and 30 are human sera with
abnormal low concentrations of immunoglobulin G. Specimens 16 r
17, 18, 24, 27, 33, 34, 35, and 36 are human sera with abnormal
elevated concentrations of immunoglobulin G.
TABLE 4
CG RECOVERY STUDY
Specimen Ospike 9.25~gm/ml % Rec.
Blank 0.02 ~gm/ml 9.25 100%
Normal A 0.80 9.3 92%
B 2.37 11.2 95%
C 6.97 15.43 92%
14 0.16 9.27 98%
0.21 9.58 101%
Hypo Ig G 21 0.15 10.0 106%
22 1.33 10.03 94%
23 0.23 9.87 104%
28 0.16 9.39 100%
29 0.17 9.65 102%
0.19 9.30 98%
16 0.26 9.36 98%
Hyper Ig G17 0.11 9ol0 97
18 0.08 8.08 86% '
24 0.31 9.25 97%
27 0.13 8.61 92%
33 0.18 9.19 97% 1,
34 0.83 9.47 97%
0.02 9.20 99%
36 3.42 12.59 99%
X % Rec. Normal = 93%
Hyper Ig G = 96%
Hypo Ig G = 101%
