CN116008201A - Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of detection reagent - Google Patents

Abstract

The application relates to a glucose 6-phosphate dehydrogenase mutant and application thereof in preparing detection reagents. Specifically, the mutant glucose 6-phosphate dehydrogenase of the present application comprises one mutation or a combination thereof selected from the group consisting of: d306C, D375C, G426C. The detection kit prepared by using the glucose 6-phosphate dehydrogenase mutant has the advantages of strong specificity, high sensitivity, convenient operation, short detection time, accurate quantification and suitability for high-throughput detection.

Description

Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of detection reagent

The present application claims priority from patent application 201910017764.4 filed on 1 month 9 in 2019 and patent application 201910423122.4 filed on 21 month 5 in 2019. The application is a divisional application of patent application 2019113721472 of glucose 6-phosphate dehydrogenase mutant and application of mutant in preparation of detection reagent, which are filed on the 12 th month and 27 th 2019.

Technical Field

The application relates to the field of biological detection, in particular to a multi-site mutant enzyme glucose 6-phosphate dehydrogenase (G6 PDH for short) and application thereof in a detection kit.

Background

Hapten, some small molecule substances (molecular weight less than 4000 Da) alone are not able to induce an immune response, i.e. are not immunogenic, but are immunogenic when crosslinked or conjugated to a carrier such as a macromolecular protein or non-antigenic polylysine, inducing an immune response. These small molecule substances can bind to the response effect products, are antigenic, are only immunoreactive, are not immunogenic, and are also called incomplete antigens.

Hapten can bind to the corresponding antibody to generate antigen-antibody reaction, and antigen which can not independently excite human or animal body to generate antibody can not be generated. It is only immunoreactive, not immunogenic, also known as incomplete antigen. Most polysaccharides, lipids, hormones and small molecule drugs belong to the hapten group. If hapten is chemically bound to a protein molecule (carrier), new immunogenicity is obtained and the animal is stimulated to produce the corresponding antibody. Hapten, once bound to a protein, constitutes an antigenic cluster of the protein. Some chemically active substances (such as penicillin, sulfonamides, etc.) which have a smaller molecular weight than the general hapten but a specific structure are called simple haptens.

Small molecule antigens or haptens, which lack two or more sites available for sandwich methods, cannot be assayed by the double antibody sandwich method, and are often in competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete for binding with the solid phase antibody. The more the antigen content in the specimen, the less the enzyme-labeled antigen is bound on the solid phase, and the lighter the color development. ELISA assay for small molecule hormones, drugs and the like is commonly used.

Glycocholic acid (CG), which is a specific example of hapten, is a conjugated cholic acid formed by combining cholic acid with glycine, which is one of the main components of bile acid. Cholesterol undergoes a series of complex enzymatic reactions in hepatocytes to form primary bile acids, including Cholic Acid (CA) and chenodeoxycholic acid (CDCA), with three hydroxyl groups (C3, C7, C12) on the steroid nucleus of cholic acid, and the hydroxyl groups at the end of the side chain are combined with glycine as glycocholic acid by peptide bonds (fig. 1).

Glycocholic acid is synthesized by hepatocytes, is discharged into gallbladder via capillary bile duct and bile duct, and enters duodenum together with bile to help digestion and absorption of fat in food. 95% of bile acid is reabsorbed in ileum and colon, returns to liver through portal vein, is absorbed by liver cells and reused, and the reabsorbed glycocholic acid enters liver-intestine circulation, so that the organism can fully utilize the glycocholic acid.

Under normal conditions, the content of cholic acid in peripheral blood is very low, and the content of glycocholic acid in peripheral blood is very low in normal people, both on an empty stomach and after meal. When human liver cells are damaged or cholestasis occurs, metabolism and circulation disturbance of the glycocholic acid can be caused, the capacity of the liver cells for taking the glycocholic acid is reduced, the content of the glycocholic acid in blood is increased, and the value of the glycocholic acid is related to the severity of the damage of the liver cells and the disorder of bile acid metabolism.

The measurement of the content of glycocholate in serum is one of the sensitive indexes for evaluating the liver cell function and the liver and gall substance circulation function. Glycocholic acid is more sensitive than conventional liver function assays such as ALT, AST, total Bilirubin (TBIL), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), serum Albumin (ALB), etc. Therefore, in liver function detection of chronic hepatitis, acute hepatitis, liver cirrhosis, liver cancer, obstructive liver disease, liver and intestine circulatory disturbance, bile duct, gall bladder excretion dysfunction and the like, glycocholic acid can be used as a better detection index.

The currently known glycocholic acid detection methods mainly comprise: radioimmunoassay, enzyme-linked immunoassay, chemiluminescent immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography, mass spectrometry, etc. However, the detection methods have many defects, such as radioactive pollution, short effective period, inconvenient operation and the like of radioimmunoassay isotopes, and the ELISA method has the defects of complicated operation, long time consumption and inapplicability to clinical use. The chemiluminescence has better sensitivity, but needs matched special equipment, and has higher input use cost and is not beneficial to popularization. In the clinical detection and diagnosis process, the homogeneous enzyme immune method (EMIT) and the latex enhanced turbidimetric immunoassay are mainly adopted.

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6 PDH-CG) and unlabeled antigen (CG) compete for combining with quantitative antibody (CG antibody), when the more the antibody is combined with unlabeled antigen, the more the activity of the enzyme-labeled antigen is released, the more the enzyme-catalyzed substrate NAD+ generates NADH, and the absorbance change of NADH is detected at the wavelength of 340nm, so that the content of CG in liquid can be deduced.

Disclosure of Invention

In view of the needs in the art, the present application provides a novel glucose 6-phosphate dehydrogenase mutant and its use in preparing glycocholic acid detection kit.

According to some embodiments, a glucose 6-phosphate dehydrogenase mutant is provided. Unlike the mutants of glucose 6 phosphate dehydrogenase of the prior published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the glucose 6-phosphate dehydrogenase mutants of the present application comprise a mutation selected from the group consisting of: d306C, G426C, D375C.

According to some embodiments, there is provided a glucose 6-phosphate dehydrogenase mutant, the glucose 6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

According to some embodiments, a polynucleotide encoding a glucose 6-phosphate dehydrogenase mutant of the present application is provided.

According to some embodiments, there is provided an expression vector comprising a polynucleotide of the present application.

According to some embodiments, a host cell is provided comprising an expression vector of the present application. The host cell may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast).

According to some embodiments, there is provided a conjugate which is a glucose 6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1: and n is coupled.

In some embodiments, n is 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

In some specific embodiments, the molar ratio of the glucose 6-phosphate dehydrogenase mutant to hapten of the present application is preferably 1: 1.

In some specific embodiments, the hapten has a molecular weight of 100Da to 4000Da, for example: 100. 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

In light of the present application, the skilled artisan will appreciate that "hapten" also includes the form of its derivative. In order to facilitate coupling with glucose-6-phosphate dehydrogenase, haptens (e.g., CG) that do not themselves carry a coupling group (e.g., a group that reacts with a thiol group) may be engineered to carry a linker for covalent binding to the thiol group. Thus, in the present application, hapten derivatives refer to haptens engineered to bear a thiol-reactive group.

The hapten is selected from the group consisting of: small molecule drugs (e.g., antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids, short peptides (molecular weight less than 4000kDa, or amino acids no more than 50 residues in length).

Hapten such as, but not limited to: vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside, zymophenolic acid, lei Paming, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, uronikotin metabolite cotinine, uromorphine, uromonophenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactation, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, anti-triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urine 17-hydroxycortisterol urinary 17-ketosteroid, dehydroepiandrosterone, sulfate, aldosterone, urovanillyl mandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 alpha hydroxy progesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotrophin, insulin, proinsulin, C peptide, gastrin, plasma prostaglandin, plasma 6-keto prostaglandin f1α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, natriuretic acid, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

In a specific embodiment, the hapten is glycocholic acid or a derivative thereof. Although glycocholic acid is taken as a specific example, the skilled person will appreciate that the technical effect of the present application is independent of the particular type of hapten, which is applicable to any hapten that can be detected immunologically by means of competition methods.

In a specific embodiment, the hapten is a glycocholic acid derivative bearing a sulfhydryl reactive group, such as a maleimide, bromoacetyl, vinyl sulfone, or aziridine. In a specific embodiment, the hapten is a glycocholic acid derivative, as shown in formula I:

according to some embodiments, there is provided an agent comprising a conjugate of the present application.

According to some embodiments, there is provided the use of a glucose 6-phosphate dehydrogenase mutant of the present application in the preparation of a detection reagent.

According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a detection reagent.

In specific embodiments, the detection reagent is selected from the group consisting of: ELISA detection reagent, chemiluminescent detection reagent, homogeneous ELISA detection reagent and latex enhanced turbidimetry detection reagent.

In a specific embodiment, the detection reagent is preferably a reagent for competition-based detection.

According to some embodiments, there is provided a glycocholic acid detection kit comprising:

-a first reagent comprising a substrate and a glycocholic acid antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;

-a second agent comprising a conjugate of the present application;

-optionally, a calibrator comprising 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid; and

-optionally, a quality control comprising 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid.

According to one embodiment, there is provided a glycocholic acid detection kit comprising:

a first reagent comprising:

10mM to 500mM buffer,

5mM to 25mM substrate,

0.1mg/L to 1mg/L of glycocholic acid antibody,

10mM to 300mM NaCl,

0.1g/L to 5g/L of stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer,

The conjugate according to claim 5,

0.1g/L to 5g/L of stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative.

In some embodiments, the buffer is selected from one or a combination of the following: tromethamine buffer, phosphate buffer, tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, tris buffer; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 100mM; the pH of the buffer is from 6.5 to 7.5, preferably 7.2 or 7.0.

In some embodiments, the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; bovine serum albumin is preferred.

In some embodiments, the surfactant is selected from one or a combination of the following: brij35, triton X-100, triton X-405, tween20, tween30, tween80, coconut fatty acid diethanolamide, AEO7, preferably Tween20.

In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, PC-300, merthiolate; the azide is selected from: sodium azide and lithium azide.

In some embodiments, the substrate comprises: glucose-6-phosphate, beta-nicotinamide adenine dinucleotide.

In some embodiments, the concentration of the buffer is 100mM.

In some embodiments, the substrate concentration of the G6PDH enzyme catalyzed reaction is 5mM.

In some embodiments, the concentration of glycocholic acid antibody is 0.1mg/L.

In some embodiments, the concentration of NaCl is 300mM.

In some embodiments, the concentration of the stabilizer is 0.5g/L.

In some embodiments, the concentration of surfactant is 0.1g/L.

In some embodiments, the concentration of preservative is 1g/L.

According to some embodiments, there is provided a method of preparing a conjugate comprising the steps of:

1) Providing a hapten or derivative thereof according to the present application, particularly in an aprotic solvent (such as, but not limited to, acetonitrile, dimethylformamide, dimethylsulfoxide);

2) Providing a glucose 6-phosphate dehydrogenase mutant of the present application, preferably in a buffer (which provides a reaction environment such as, but not limited to PBS, tris, TAPS, TAPSO, said buffer having a pH of 6.0 to 8.0);

3) At 18 ℃ to 28 ℃, the glucose 6-phosphate dehydrogenase mutant and the hapten or derivative thereof are mixed according to a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow coupling of the hapten or derivative thereof and the glucose 6-phosphate dehydrogenase mutant to occur to give the seed conjugate;

4) The seed conjugate is optionally purified, e.g., desalted, etc., as desired.

In some embodiments, n is 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

In some specific embodiments, steps 1) and 2) may be interchanged or in parallel.

In some specific embodiments, the glucose 6-phosphate dehydrogenase comprises a free thiol group prior to coupling, thereby allowing for 1: 1.

Drawings

FIG. 1 is a glycocholic acid structure diagram.

FIG. 2 shows the structure of glycocholic acid derivative.

FIG. 3A. G6PDH (wild type) amino acid sequence (SEQ ID No. 1); is derived from Leuconostoc pseudomesenteroides Leuconostoc pseudomesenteroides.

FIG. 3B.G6PDH (D306C) amino acid sequence (SEQ ID No. 2).

FIG. 3C.G6PDH (D375C) amino acid sequence (SEQ ID No. 3).

FIG. 3D.G6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

EXAMPLE 1 Synthesis of Glycocholic acid derivatives

To a dry and clean 25mL two-necked flask were added glycocholic acid (1.0 eq), maleimide ethylamine (1.0 g,1.0 eq) and triethylamine (3.0 eq);

dimethylformamide (5 mL) was then added and stirred to complete dissolution, dichloroethane (1.25 eq) was added and stirred at 25℃for 2h;

HPLC monitoring until the reaction is completed;

the reaction mixture was added to water (25 mL), and extraction was performed by adding ethyl acetate 20 mL. Times.3;

the organic phases were combined, anhydrous Na ₂ SO ₄ Drying, concentrating under reduced pressure, and purifying the obtained oily substance by column chromatography to obtain 1.04g of milky powdery solid with a yield of 45%, M+:602.72.

the effect of this example is to make CG carry a group that can bind to the enzyme, the technical effect of this application is independent of the particular hapten derivative.

EXAMPLE 2 coupling of glycocholic acid derivative to G6PDH molecule

The G6 PDH-glycocholic acid conjugate according to the present application was coupled as follows: the thiol-reactive group (e.g., maleimide group) on the glycocholic acid derivative molecule is covalently bound to the thiol group on the G6PDH molecule.

1. Preparing a solution:

glycocholic acid derivative solution: 10mg/ml of the glycocholic acid derivative prepared in example 1 was dissolved in DMF;

g6pdh solution: 6.7mg/mL G6PDH (mutant of the present application or control mutant), PB 100mmol, naCl 100mmol, ph=8.0;

coupling solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH=7.2;

desalination solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH=7.2.

2. Coupling operation: 1.6ml of G6PDH solution, 6ml of coupling solution and 0.40ml of glycocholic acid derivative solution were reacted at room temperature (20 to 25 ℃) for 4 hours.

3. After the reaction system is subjected to oscillation reaction for 4 hours at room temperature, eluting by using a desalting column by using the desalting solution, and collecting protein peaks to obtain a product, namely the G6 PDH-glycocholic acid conjugate.

EXAMPLE 3 preparation of the kit

The following kit for detecting glycocholic acid was prepared, which comprises:

reagent R1 comprising:

100mM PB buffer, pH 7.2

15mM glucose 6-phosphate

15mM beta-nicotinamide adenine dinucleotide

0.1mg/L glycocholic acid antibody

200mM NaCl

0.5g/L bovine serum albumin

0.1g/L Tween20

1g/L sodium azide;

reagent R2, comprising:

100mM PB buffer, pH 7.2

0.1mg/L G6PDH-CG conjugate

0.5g/L bovine serum albumin

0.1g/L Tween 20

1g/L sodium azide;

calibration material: 100mM PB buffer, pH 7.2, 0, 2.5, 5.0, 10, 20, 40mg/L glycocholic acid (or added as needed);

quality control product: 100mM PB buffer, pH 7.2, and 1.5, 8.0, 25, 35mg/L glycocholic acid (or added as needed).

Test case

Reaction time: 10min, wherein the incubation time is 4.7min, after 1min incubation after addition of reagent R2, the read absorbance A1 is determined, after 1min incubation, the read absorbance A2 is determined, and Δa= (A2-A1)/min is calculated. Calculating the content of glycocholate in the sample through a calibration curve: CG = sample tube absorbance calibrator concentration/calibrator absorbance.

The glycocholic acid detection kit prepared in example 3 was subjected to performance detection, and the main detection performances are total inaccuracy, repeatability, recovery, linearity, specificity and the like.

TABLE 1 full automatic Biochemical instrument parameters

Detecting machine type	Hitachi 7180
		Analysis/time/point	2-point rate/10 min/20-24 points
R1/R2/S	120:40:9
		Wavelength (auxiliary/main)	405/340
Reaction type	Incremental increases
		Calibration type	Spine type
Calibration point	6
		Concentration of calibrator	0/2.50/5.00/10.00/20.00/40.00

Detection example 1 glycocholic acid detection kit calibration absorbance

TABLE 2 Glycocholic acid detection kit calibration absorbance

Detection example 2 Glycocholic acid detection kit Total inaccuracy

TABLE 3 Total imprecision

Detection example 3 Glycocholic acid detection kit repeatability

TABLE 4 repeatability

Detection example 4 recovery of glycocholic acid detection kit

TABLE 5 recovery

/>

Detection example 5 Glycocholic acid detection kit linearity

TABLE 6 linearity

/>

Detection example 6 glycocholic acid detection kit anti-specificity

TABLE 7 specificity

Interferents (40. Mu.g/ml)	Reagent of the application (D306C)	Control reagent (A45C mutant)
			Glycodeoxycholic acid	18.61％	30.20％
Glycine chenodeoxycholic acid	1.63％	37.91％
			Chenodeoxycholic acid	0.61％	16.28％
Ursodeoxycholic acid	-0.42％	6.55％
			Sodium cholate	61.21％	61.90％
Deoxycholate sodium	4.22％	11.74％

The reagent of the application has little or no cross reaction with the structural analogue of glycocholic acid.

Detection example 7 antibody inhibition Rate

1. Principle of detection of antibody inhibition

When the antibody is combined with the G6PDH-CG conjugate, the activity of the G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing NAD to be converted into NADH is reduced, and the difference between an experiment group to which the antibody is added and an experiment group to which the antibody is not added is compared by detecting the change of the NADH amount, and the difference is expressed as the inhibition capability of the antibody on the G6 PDH.

2. The reaction system:

TABLE 8 preparation of reagents for detection of antibody inhibition

TABLE 9 detection of on-machine parameters for antibody inhibition

Detecting machine type	Hitachi 7180
		Analysis/time/point	2-point rate/10 min/20-24 points
R1/S	120:20
		Wavelength (auxiliary/main)	405/340
Reaction type	Incremental increases

3. Results:

and comparing the absorbance measurement value of the G6PDH-CG conjugate when the antibody is added with the antibody is not added with the antibody, and obtaining the inhibition condition of the antibody on the G6 PDH.

Antibody inhibition ratio = absorbance change value for G6PDH-CG with antibody/absorbance change value for G6PDH-CG without antibody.

Compared with published mutation sites, the mutant has obviously improved antibody inhibition rate, and can reach more than 30% and up to 50%. Whereas the inhibition rate of the mutation sites (e.g.A45C, K C) which have been used before is only about 20% or even lower.

TABLE 10 antibody inhibition by different G6PDH mutants

While not being limited to a particular theory, it may be explained in part as: compared with the G6PDH mutant in the prior art, the mutation site (i.e. the site for introducing free sulfhydryl) in the enzyme mutant (D306C, D375C, G C) is the coupling site with hapten (such as hormone, small molecule drug and the like). When hapten is combined with hapten specific antibody at this position, the steric hindrance formed has the greatest effect on the activity of G6PDH enzyme, and after mutation is introduced, the steric folding of the molecule cannot be substantially influenced. Therefore, the position of this mutation site is very important, and it is necessary to combine the activity of the G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

Since mutants of the enzyme have a significant increase in antibody inhibition, there can be significant advantages in scaling absorbance. After the conjugate of the enzyme mutant and hapten is prepared into a kit, the reagent has obvious performance improvement in the aspects of repeatability, total inaccuracy, linearity, specificity and the like due to the improvement of a calibration curve.

Claims (7)

1. Use of a conjugate in the preparation of a detection reagent, wherein:

the conjugate is prepared from a glucose 6-phosphate dehydrogenase mutant and hapten according to a molar ratio of 1:1, coupling;

the glucose 6-phosphate dehydrogenase mutant comprises a mutation selected from any one of the following compared to the wild-type leuconostoc mesenteroides: d306C, D375C, G426C;

the hapten is glycocholic acid or glycocholic acid derivative.

2. The use according to claim 1, wherein the glucose 6-phosphate dehydrogenase mutant is represented by any one of the sequences selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

3. The use according to claim 1, wherein the detection reagent is an enzyme-linked immunosorbent assay detection reagent.

4. The use according to claim 1, wherein the detection reagent is a chemiluminescent immunoassay detection reagent.

5. The use according to claim 1, wherein the detection reagent is a homogeneous enzyme immunoassay detection reagent.

6. The use according to claim 1, wherein the detection reagent is a latex-enhanced immunoturbidimetry detection reagent.

7. The use according to claim 1, wherein the glycocholic acid derivative is of formula I:

/>

Images