CA1093962A - Bile acid competitive assay using protein binding inhibitor - Google Patents
Bile acid competitive assay using protein binding inhibitorInfo
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- CA1093962A CA1093962A CA324,498A CA324498A CA1093962A CA 1093962 A CA1093962 A CA 1093962A CA 324498 A CA324498 A CA 324498A CA 1093962 A CA1093962 A CA 1093962A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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Abstract
BILE ACID ASSAY
Inventor:
Phillip C. Miller 357 Behm Drive Grayslake, Illinois 60030 ABSTRACT OF THE DISCLOSURE
This invention relates to a method for facilitating the measurement of individual bile acids in biological fluids.
The invention has particular application to assays performed on unextracted biological fluids in which bile acids will ordinarily bind to endogenous proteins such as serum albumin.
The method disclosed and claimed herein obviates pre-extraction of the bile acid from the serum binding proteins by employing buffered binding inhibitory agents to displace bile acids from interfering binding proteins.
Inventor:
Phillip C. Miller 357 Behm Drive Grayslake, Illinois 60030 ABSTRACT OF THE DISCLOSURE
This invention relates to a method for facilitating the measurement of individual bile acids in biological fluids.
The invention has particular application to assays performed on unextracted biological fluids in which bile acids will ordinarily bind to endogenous proteins such as serum albumin.
The method disclosed and claimed herein obviates pre-extraction of the bile acid from the serum binding proteins by employing buffered binding inhibitory agents to displace bile acids from interfering binding proteins.
Description
3~iZ
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 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 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;
(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 .
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 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 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;
(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 .
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
.
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.
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
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.
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.
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.
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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.
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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 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
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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.
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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 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
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-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.
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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.
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-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.
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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.
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-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.
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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.
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%
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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.
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%
Claims (9)
1. In an assay for the detection and determination of a bile acid or conjugate thereof in an unextracted serum sample, said method comprising the steps of:
(a) providing an antiserum specific to the particular bile acid being assayed;
(b) mixing said 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 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;
(f) the improvement comprising conducting said assay in the presence of a 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.
(a) providing an antiserum specific to the particular bile acid being assayed;
(b) mixing said 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 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;
(f) the improvement comprising conducting said assay in the presence of a 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.
2. An assay according to Claim 1 wherein the bind-ing inhibitory agent is selected from the group consisting of 8-anilino-1-napthalene-sulfonic acid, sodium barbital or a salicylate.
3. An assay according to Claim 2 wherein the binding inhibitory agent is sodium salicylate.
4. An assay according to Claim 1 wherein the bile acid to be detected is cholic acid.
5. An assay according to Claim 1 wherein the bile acid to be detected is sulfolithocholic acid.
6. In an assay for the detection and determination of cholic acid in an unextracted serum sample, said method comprising the steps of:
(a) providing an antiserum specific to the particular bile acid being assayed;
(b) mixing said 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 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;
(f) the improvement comprising conducting said assay in the presence of barbital buffered 8-anilino-1-napthalene-sulfonic acid, the concentration being sufficient to inhibit the retention of cholic acid to binding proteins present in the unextracted serum sample.
(a) providing an antiserum specific to the particular bile acid being assayed;
(b) mixing said 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 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;
(f) the improvement comprising conducting said assay in the presence of barbital buffered 8-anilino-1-napthalene-sulfonic acid, the concentration being sufficient to inhibit the retention of cholic acid to binding proteins present in the unextracted serum sample.
7. An assay according to Claim 6, wherein the cholic acid is present as the glycine conjugate.
8. In an assay for the detection and determination of a sulfolithocholic acid in an unextracted serum sample, said method comprising the steps of:
(a) providing an antiserum specific to the particular bile acid being assayed;
(b) mixing said 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 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;
(f) the improvement comprising conducting said assay in the presence of sodium salicylate, the concen-tration being sufficient to inhibit the retention of sulfolithocholic acid to binding proteins present in the unextracted serum sample.
(a) providing an antiserum specific to the particular bile acid being assayed;
(b) mixing said 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 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;
(f) the improvement comprising conducting said assay in the presence of sodium salicylate, the concen-tration being sufficient to inhibit the retention of sulfolithocholic acid to binding proteins present in the unextracted serum sample.
9. An assay according to Claim 8 wherein the sulfolithocholic acid is present as the glycine conjugate.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US89991878A | 1978-04-26 | 1978-04-26 | |
| US899,918 | 1978-04-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1093962A true CA1093962A (en) | 1981-01-20 |
Family
ID=25411734
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA324,498A Expired CA1093962A (en) | 1978-04-26 | 1979-03-30 | Bile acid competitive assay using protein binding inhibitor |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS54149700A (en) |
| AU (1) | AU527381B2 (en) |
| BE (1) | BE875854A (en) |
| CA (1) | CA1093962A (en) |
| DE (1) | DE2916783C3 (en) |
| ES (1) | ES479985A1 (en) |
| FR (1) | FR2424536A1 (en) |
| GB (1) | GB2020014B (en) |
| IT (1) | IT1112580B (en) |
| NL (1) | NL7902396A (en) |
| SE (1) | SE7903645L (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5474907A (en) * | 1994-03-25 | 1995-12-12 | Eastman Kodak Company | Multilayer analytical element for salicylate assay |
| JP2638521B2 (en) * | 1994-11-24 | 1997-08-06 | 有限会社ビー・エス・アール | Determination of liver disease by measurement of serum bile acids by ELISA |
| CN108713141A (en) | 2016-02-29 | 2018-10-26 | 富士胶片株式会社 | Kit for the measured substance in quantitative Biosample |
| JP6602952B2 (en) | 2016-02-29 | 2019-11-06 | 富士フイルム株式会社 | Kit for quantifying bile acids in biological samples and method for quantifying bile acids in biological samples |
| CN106501535B (en) * | 2016-11-30 | 2018-11-09 | 迪瑞医疗科技股份有限公司 | A kind of kit of the compound releasing agent of ANS salt and detection Blood cortisol |
| CN109444399A (en) * | 2018-10-26 | 2019-03-08 | 重庆中元汇吉生物技术有限公司 | A kind of glycocholic acid detection kit |
| CN113721012B (en) * | 2021-08-27 | 2022-08-09 | 深圳上泰生物工程有限公司 | Composition and application of kit thereof in detection of glycocholic acid |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4111656A (en) * | 1975-06-26 | 1978-09-05 | Mallinckrodt, Inc. | Radioimmunoassay methods for the determination of l-triiodothyronine and thyroxine |
| JPS5851000B2 (en) * | 1976-04-16 | 1983-11-14 | アボツト ラボラトリ−ズ | Production method of sulfolitocholylglycyltyramine |
-
1979
- 1979-03-27 NL NL7902396A patent/NL7902396A/en not_active Application Discontinuation
- 1979-03-30 AU AU45634/79A patent/AU527381B2/en not_active Ceased
- 1979-03-30 CA CA324,498A patent/CA1093962A/en not_active Expired
- 1979-04-05 GB GB7911887A patent/GB2020014B/en not_active Expired
- 1979-04-24 FR FR7910391A patent/FR2424536A1/en active Pending
- 1979-04-24 JP JP4984979A patent/JPS54149700A/en active Pending
- 1979-04-24 IT IT22140/79A patent/IT1112580B/en active
- 1979-04-25 DE DE2916783A patent/DE2916783C3/en not_active Expired
- 1979-04-25 BE BE0/194838A patent/BE875854A/en not_active IP Right Cessation
- 1979-04-25 SE SE7903645A patent/SE7903645L/en not_active Application Discontinuation
- 1979-04-26 ES ES479985A patent/ES479985A1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| AU4563479A (en) | 1979-11-01 |
| GB2020014A (en) | 1979-11-07 |
| DE2916783A1 (en) | 1979-10-31 |
| GB2020014B (en) | 1982-10-20 |
| JPS54149700A (en) | 1979-11-24 |
| DE2916783B2 (en) | 1981-07-16 |
| AU527381B2 (en) | 1983-03-03 |
| IT1112580B (en) | 1986-01-20 |
| SE7903645L (en) | 1979-10-27 |
| DE2916783C3 (en) | 1982-04-01 |
| ES479985A1 (en) | 1980-08-16 |
| NL7902396A (en) | 1979-10-30 |
| FR2424536A1 (en) | 1979-11-23 |
| IT7922140A0 (en) | 1979-04-24 |
| BE875854A (en) | 1979-10-25 |
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| Date | Code | Title | Description |
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| MKEX | Expiry |