CN116359146A - Method for preparing conjugate - Google Patents

Abstract

The present application relates to a method of preparing conjugates. 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

Method for preparing conjugate

The application is a patent application 6-phosphoglucose dehydrogenase mutant and application number of the mutant in preparation of theophylline detection reagent, which are filed on the 12 th month 26 of 2019, and is as follows: 2019113654393.

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 theophylline 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.

Theophylline (Theo) belongs to the class of xanthine alkaloids. Theophylline is usually formulated as a relatively water-soluble salt (e.g., aminophylline) for pharmaceutical use, but is still dissociated in the body to function.

Theophylline is a medicine for relaxing bronchial smooth muscle, and is an effective medicine for treating respiratory diseases such as bronchial asthma, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, etc. It is recorded in both of "diagnosis and treatment guidelines for chronic obstructive pulmonary disease" and "guidelines for preventing and treating bronchial asthma". In recent years, the research shows that the theophylline also has the effects of tonifying heart, promoting urination, resisting inflammation, regulating immunity, dilating coronary artery, exciting central nervous system and the like.

Because of the wide application of theophylline drugs, adverse reaction cases in patients are correspondingly increased, and more common adverse reactions include nausea, vomiting, stomach cold and tachycardia, and some patients have nervous system symptoms. According to the research, the adverse reaction occurrence condition of the patient after the theophylline medicament is used and the blood concentration of the patient are directly related. For most patients, theophylline blood levels between 10 and 20 μg/ml are effective in alleviating chronic asthma and other bronchospasm symptoms. The blood concentration of theophylline is between 5 and 10 mug/ml, which can control neonatal apnoea and has no obvious side effect.

However, when the blood concentration exceeds 20. Mu.g/ml, the following adverse reactions occur: nausea, headache, diarrhea. At higher blood levels, vomiting, gastrointestinal bleeding, seizures and arrhythmias may occur.

Because theophylline has a narrow therapeutic index, and the individual differences of metabolic rate and clearance rate are large, adverse reactions or poor therapeutic effects are caused. Therefore, monitoring the blood concentration of the patient and adjusting the dosage at any time are very important for reducing the occurrence rate of adverse reactions of the patient and improving the treatment effect.

The currently known theophylline detection methods mainly comprise: it has high performance liquid chromatography, emulsion enhanced immunoturbidimetry, chemiluminescence microparticle immunization, enzyme-linked immunosorbent assay, 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-theophylline) and non-labeled antigen (theophylline) compete for being combined with quantitative antibody (theophylline antibody), when the more the antibody is combined with the non-labeled 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 the theophylline in the 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 a theophylline 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 1:1 is coupled.

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., theophylline) 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, and amino acids.

Hapten such as, but not limited to: theophylline, phenytoin, 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 theophylline or a derivative thereof.

In a specific embodiment, the hapten is a theophylline derivative bearing a sulfhydryl reactive group such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine. In a specific embodiment, the hapten is a theophylline 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 theophylline detection reagent.

According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a theophylline 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 theophylline detection kit comprising:

-a first reagent comprising a substrate and a theophylline 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 theophylline; and

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

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

a first reagent comprising:

10mM to 500mM buffer,

0.5g/L to 20g/L of substrate,

0.1mg/L to 10mg/L of theophylline 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,

Conjugates according to the present application,

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 7 to 8, preferably 7.2 or 7.0. In some specific embodiments, the concentration of the buffer is 100mM.

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 specific embodiments, the concentration of the stabilizer is 1.0g/L.

In some embodiments, the surfactant is selected from one or a combination of the following: brij35, tritionX-100, tritionX-405, tween20, tween30, tween80, coconut fatty acid diethanolamide, AEO7, preferably Tween20. In some specific embodiments, the concentration of surfactant is 1.0g/L.

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 specific embodiments, the concentration of preservative is 1.0g/L.

In some embodiments, the substrate comprises: glucose-6-phosphate, beta-nicotinamide adenine dinucleotide. In some specific embodiments, the substrate concentration of the G6PDH enzyme catalyzed reaction is 15G/L.

In some specific embodiments, the concentration of theophylline antibody is 5.7mg/L.

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

providing a glucose 6-phosphate dehydrogenase mutant;

providing theophylline or a derivative thereof;

the mol ratio of the glucose 6-phosphate dehydrogenase mutant to the theophylline or the derivative thereof is 1:1 coupling.

In some specific embodiments, the method of preparing the conjugate comprises the steps of:

1) Providing a theophylline derivative in an aprotic solvent;

2) Providing a glucose 6-phosphate dehydrogenase mutant in a buffer;

3) Contacting said theophylline derivative and said mutant glucose 6-phosphate dehydrogenase at 18 ℃ to 28 ℃ for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that said theophylline derivative and said mutant glucose 6-phosphate dehydrogenase are coupled to obtain said conjugate;

4) Optionally, the conjugate is purified, preferably desalted.

Wherein steps 1) and 2) are interchangeable.

In some specific embodiments, the aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, dimethyl sulfoxide.

In some specific embodiments, prior to step 3), the glucose 6-phosphate dehydrogenase mutant comprises one or more (preferably one) free sulfhydryl group, in particular a free sulfhydryl group is present at position 306, 375 or 426.

Drawings

Figure 1. Theophylline structure diagram.

FIG. 2 shows the structure of theophylline derivatives.

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 theophylline derivatives

1. Synthesis of Compound 3

Theophylline (1.0 g,5.55 mmol) and K ₂ CO ₃ (1.53 g,11.10 mmol) was dissolved in 50mL of DMF and compound 2 (0.52 g,5.55 mmol) was added thereto at room temperature (20-25 ℃ C.) and stirred for 16h. The solvent was removed under reduced pressure, the mixture was dissolved in 50mL of water, extracted three times with ethyl acetate (40 mL), washed three times with 50mL of saturated brine, and concentrated in anhydrous Na ₂ SO ₄ And (5) drying. The solvent was removed under reduced pressure, and purified by column chromatography to give compound 3 (1.0 g, yield 76%).

2. Synthesis of theophylline derivatives

Compound 3 (100 mg,0.72 mmol) and compound 4 (126 mg,0.72 mmol) were dissolved in 5mL of DCM, triethylamine (218 mg,2.16 mmol) was added dropwise thereto, HATU (328 mg,0.86 mmol) was added and stirred at room temperature for 5h. Water (30 mL) was added to the reaction system, the mixture was extracted with DCM, and the organic phase was washed with saturated brine and dried Na ₂ SO ₄ Drying and removal of the solvent under reduced pressure gave the theophylline derivative as a colourless oil (125 mg, 48% yield).

The product structure was verified by conventional methods. This example provides theophylline with a group that can bind to enzymes.

EXAMPLE 2 coupling of theophylline derivatives with G6PDH molecules

The G6 PDH-theophylline conjugate according to the present application was coupled as follows: thiol-reactive groups (such as but not limited to maleimide groups) on theophylline derivative molecules are covalently bound to thiol groups on G6PDH molecules.

1. Preparing a solution:

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

g6pdh solution: g6pdh (mutant or prior art mutant of the present application) was dissolved in 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: 0.6ml of G6PDH solution, 4.18ml of coupling solution and 0.22ml of theophylline 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-theophylline conjugate.

EXAMPLE 3 preparation of the kit

The following kit for detecting theophylline was prepared, which comprises:

reagent R1 comprising:

100mM PB buffer, pH 7.2

15mM glucose 6-phosphate

15mM beta-nicotinamide adenine dinucleotide

5.7mg/L theophylline antibody (commercially available antibody, without particular limitation)

150mM NaCl

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

reagent R2, comprising:

100mM PB buffer, pH 7.2

0.1mg/L G6 PDH-theophylline conjugate

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

calibration material: 100mM PB buffer, pH 7.2, 0, 2.4, 5.0, 10, 20, 40mg/L theophylline (or added as needed);

quality control product: 100mM PB buffer, pH 7.2, and 5.0, 15.0, 25mg/L theophylline (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 theophylline content in the sample through a calibration curve:

theophylline = sample tube absorbance × calibrator concentration/calibrator absorbance.

The theophylline detection kit prepared in example 3 was subjected to performance detection, and the main detection performances are total inaccuracy, repeatability, recovery, linearity and 37 ℃ accelerated stability.

TABLE 1 full automatic Biochemical instrument parameters

Detecting machine type	Yaban C16000
		Analysis/time/point	Rate/10 min/25-33 points
R1/R2/S	150:50:2
		Wavelength (auxiliary/main)	405/340
Reaction type	Incremental increases
		Calibration type	Spine type
Calibration point	6
		Concentration of calibrator	0/2.4/5.00/10.00/20.00/40.00

Detection example 1 theophylline detection kit calibration absorbance

TABLE 2 calibration absorbance for theophylline detection kit

Note that: the mutant with the code of A45C in the prior art has a mutation site corresponding to the 46 th position of the FIG. 3A.

Detection example 2 Total inaccuracy of theophylline detection kit

TABLE 3 Total imprecision

Detection example 3 theophylline detection kit reproducibility

TABLE 4 repeatability

Detection example 4 recovery of theophylline detection kit

TABLE 5 recovery

Detection example 5 theophylline detection kit linearity

TABLE 6 linearity

Test example 6.37℃acceleration stability

TABLE 7 accelerated stability at 37℃

The calibrated absorbance drops by about 15% after 7 days of acceleration at 37℃for the reagent of the present application, and by about 95% after 7 days of acceleration at 37℃for the control reagent.

Detection example 7 antibody inhibition Rate

1. Principle of detection of antibody inhibition

When the antibody is combined with the G6 PDH-theophylline 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	Yaban C16000
		Analysis/time/point	Rate/10 min/25-33
R1/S	120:20
		Wavelength (auxiliary/main)	405/340
Reaction type	Incremental increases

3. Results:

and comparing the absorbance measurement value of the G6 PDH-theophylline 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 = [1- (absorbance change value of G6 PDH-theophylline with antibody/absorbance change value of G6 PDH-theophylline without antibody) ] ×100%.

Compared with published mutation sites, the mutant has obviously improved antibody inhibition rate, and can reach more than 30% and up to 55%. Whereas the inhibition rate of the mutation sites (e.g.A45C, K C) which have been used before is only about 40% at most, 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.

The mutant of the enzyme has obvious improvement on the inhibition rate of the antibody. After the conjugate of the enzyme mutant and theophylline is prepared into a kit, the reagent has obvious performance improvement in the aspects of repeatability, total inaccuracy, linearity, stability and the like.

Claims (2)

1. A method of preparing a conjugate comprising the steps of:

providing a glucose 6-phosphate dehydrogenase mutant;

providing a theophylline derivative;

the glucose 6-phosphate dehydrogenase mutant is coupled with the theophylline derivative;

the glucose 6-phosphate dehydrogenase mutant comprises any one of the mutations selected from the group consisting of: d306C, D375C, G426C; the glucose 6-phosphate dehydrogenase mutant is represented by any one of the following sequences: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4;

the theophylline derivative is shown in a formula I:

2. the method of preparing a conjugate according to claim 1, comprising the steps of:

1) Providing a theophylline derivative, preferably in an aprotic solvent;

2) Providing a glucose 6-phosphate dehydrogenase mutant, preferably providing said glucose 6-phosphate dehydrogenase mutant in a buffer;

3) Contacting said theophylline derivative and said mutant glucose 6-phosphate dehydrogenase at 18 ℃ to 28 ℃ for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that said theophylline derivative and said mutant glucose 6-phosphate dehydrogenase are coupled to obtain said conjugate;

4) Optionally, purifying, preferably desalting, the conjugate;

steps 1) and 2) are interchangeable;

the buffer is selected from the group consisting of: PBS, tris, TAPS, TAPSO, the buffer pH is from 6.0 to 8.0;

the aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, dimethyl sulfoxide;

preferably, prior to step 3), the glucose 6-phosphate dehydrogenase mutant has a free thiol group at position 306, 375 or 426.

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