WO2022233106A1 - Preparation method for alkaline phosphatase-labelled thyroxine - Google Patents

Abstract

The present invention relates to the technical field of biochemical detection, and in particular relates to a preparation method for alkaline phosphatase-labelled thyroxine. In the preparation method provided in the invention, after being activated, alkaline phosphatase is directly linked to thyroxine without the need to activate T4 and without the need to couple macromolecules such as BSA. The prepared ALP-T4 complex has good activity and does not contain by-products. The preparation method has simple processes and is low cost. It has been verified that the ALP-T4 prepared using the present method detects free thyroxine or total thyroxine with good sensitivity, accuracy, precision, and specificity.

Description

Preparation method of alkaline phosphatase-labeled thyroxine

This application claims the priority of the Chinese patent application filed on May 6, 2021 with the application number 202110490666.X and the invention titled "Method for the Preparation of Alkaline Phosphatase-labeled Thyroxine", the entire contents of which are approved by Reference is incorporated in this application.

technical field

The invention relates to the technical field of biochemical detection, in particular to a preparation method of alkaline phosphatase-labeled thyroxine.

Background technique

The detection of thyroxine (T4) includes the detection of free thyroxine (FT4) and total thyroxine (TT4), the results of which can directly reflect thyroid function. Because the radioimmunoassay method pollutes the environment and there are many factors that affect the detection results, chemiluminescence method is often used to detect thyroxine.

The components to be included in the free thyroxine (FT4) and total thyroxine (TT4) chemiluminescence kits include alkaline phosphatase (ALP)-labeled thyroxine (ALP-T4). At present, most of the methods for labeling T4 are to conjugate macromolecules (such as BSA) through T4 and then label ALP enzyme or coat magnetic beads; or to conjugate T4 to NHS and then label ALP enzyme; there are also some methods The coupling is carried out by activating ALPase and T4 respectively.

Among these technologies, some are complicated and expensive, while others are complicated and time-consuming due to the coupling process, and some are due to the low activation efficiency of the cross-linking agent glutaraldehyde, which may cause T4 molecules during the activation process. Self-connection, the by-products in the final product also affect the accuracy of detection.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide a preparation method of thyroxine labeled with alkaline phosphatase, and the thyroxine-labeled alkaline phosphatase can be used for more accurate detection of thyroxine.

The preparation method of alkaline phosphatase-labeled thyroxine provided by the present invention comprises:

After the alkaline phosphatase is activated in an acidic solution, the free activator is removed, and then it is coupled with thyroxine in an alkaline solution, and the alkaline phosphatase-labeled thyroxine is obtained after the reaction is terminated.

The acidic solution in the present invention is MES buffer with pH<7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer.

In the present invention, the activator is selected from EDC/NHS, glutaraldehyde or Sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide at least one of ester sodium salt).

In the present invention, the method for removing the activator includes dialysis or ultrafiltration.

In the present invention, the alkaline solution is MES buffer with pH>7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer.

In the present invention, in the coupling step, the carboxyl group of alkaline phosphatase is coupled with the amino group of thyroxine.

In the present invention, an amino group-containing buffer is used for the termination reaction.

After the termination reaction of the present invention, the method further includes: mixing the product with the preservation solution and removing by-products.

In some embodiments, the preservation solution is MES buffer, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer with a pH of 7.0-7.5;

The by-products include free thyroxine;

Methods of such removal include dialysis or ultrafiltration.

The present invention also provides a thyroxine detection reagent, which includes R1 reagent, R2 reagent and R3 reagent;

The R1 reagent is a magnetic bead suspension coated with T4 antibody;

The R2 reagent is an alkaline phosphatase-labeled thyroxine solution;

The R3 reagent is a magnetic bead buffer;

The R2 reagent includes alkaline phosphatase-labeled thyroxine, enzyme-labeled buffer C and glycerol prepared by the preparation method of the present invention.

The preparation method provided by the present invention directly connects the alkaline phosphatase with thyroxine after activation, and does not need to activate T4, and does not need to couple macromolecules such as BSA. The prepared ALP-T4 complex has good activity and does not contain by-products. And the preparation method has simple process and low cost. It has been verified that the ALP-T4 prepared by this method can detect free thyroxine or total thyroxine with good sensitivity, accuracy, precision and specificity.

Detailed ways

The present invention provides a method for preparing alkaline phosphatase-labeled thyroxine, which can be achieved by those skilled in the art by appropriately improving the process parameters for reference. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

In the preparation method of alkaline phosphatase-labeled thyroxine provided by the present invention, firstly, under acidic conditions, an activating group is connected to the carboxyl group of ALP enzyme to form a stable intermediate product, and then the free activating agent is removed. Then in alkaline conditions, without further addition of activating agent, the activating group on ALP enzyme is removed, and the carboxyl group of ALP enzyme is coupled with the amino group of T4. After that, the uncoupled carboxyl groups on ALP were blocked with buffer containing amino groups, and then free small molecules such as T4 were removed to obtain ALP-T4. In the obtained ALP-T4, the amino group of T4 is directly coupled with the carboxyl group of ALP without connecting other groups or molecules in the middle.

In the present invention, the acidic solution is MES buffer with pH<7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer. In the embodiment of the present invention, the acidic solution is denoted as enzyme labeling buffer A. In some embodiments, the enzyme labeling buffer A includes water and: MES and NaCl. The pH of the enzyme labeling buffer A was 4.5. In some specific embodiments, the enzyme labeling buffer A is made of water and 5.33g/L MES, 7.305g/L NaCl. Adjust pH=4.5 with 1mol/L NaOH or 1mol/L HCl solution.

The activator of the present invention is used to activate the carboxyl group of ALP. After activation, the carboxyl group of ALP is connected with an activating group. The selection of the activator is based on its ability to activate ALP, and the activator with better activation performance is preferred. Commonly used activators include EDC/NHS, or glutaraldehyde, or Sulfo-SMCC. In the embodiment of the present invention, the activator is EDC/NHS, and EDC and NHS are used as the activator of ALP. Specifically, the NHS is sulfo-NHS. The mass ratio of the EDC to sulfo-NHS was 500:50. The mass ratio of alkaline phosphatase, EDC and sulfo-NHS was 250:500:50. The concentration of alkaline phosphatase in the activated system was 1 μg/μL, the concentration of EDC was 2 μg/μL, and the concentration of sulfo-NHS was 0.1 μg/μL; the activation conditions included 25° C., 1100 rpm shaking for 1 h.

In the present invention, the free activator is removed before coupling, and other possible small molecule impurities are also removed, so as to ensure the accurate coupling of ALP-T4. The method of removal may include dialysis or ultrafiltration. In the embodiment of the present invention, the method of ultrafiltration is adopted. In the ultrafiltration step, the molecular weight cut-off of the filter membrane of the ultrafiltration tube is 30 kDa. The conditions of the ultrafiltration were centrifugation at 9000 rpm/min for 25 min. In order to ensure the effect of ultrafiltration, ultrafiltration is carried out under low temperature conditions. The low temperature condition is 0-4°C.

In the present invention, the alkaline solution is MES buffer with pH>7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer. In the embodiment of the present invention, the acidic solution is denoted as enzyme labeling buffer B. _In some embodiments, the enzyme labeling buffer B includes water and _{: Na2HPO4.12H2O , NaH2PO4.2H2O} , and _NaCl . The pH of the enzyme labeling buffer B was 9.0. The enzyme labeling buffer B was made of water and 25.786 g/L Na ₂ HPO ₄ ·12H ₂ O, 4.36 g/L NaH ₂ PO ₄ ·2H ₂ O and 8.78 g/L NaCl. Adjust pH=9.0 with 1 mol/L NaOH or 1 mol/L HCl solution.

In the coupling step, no activator or other components such as coupling agent are added, and only the activated ALP and T4 are subjected to a coupling reaction in an alkaline solution. Through continuous exploration, the reaction conditions described in the present invention are determined to be the optimum conditions. Coupling under this condition not only avoids the self-linking of T4 molecules, but also avoids the generation of other by-products, thereby improving the accuracy of coupling, which has positive significance for improving the accuracy of detection. The coupling step specifically includes: mixing the activated ALP solution with thyroxine, and incubating with enzyme labeling buffer B to a constant volume. The volume ratio of the activated ALP solution to the enzyme labeling buffer B was 250:150. The mass ratio of the alkaline phosphatase to thyroxine was 250:20. The enzyme labeling buffer B was used to make the volume to a concentration of 0.2 μg/μL of thyroxine. The incubation conditions were 25°C for 3h.

Following the coupling step, treatment with an amino group-containing buffer terminates the coupling reaction and blocks uncoupled activated carboxyl groups to avoid by-product formation. The buffer solution containing amino group is recorded as blocking solution in the present invention. The blocking solution is Tris buffer. In some embodiments, the blocking solution is Tris buffer with a pH value of 8.0. The blocking solution was made of water and 0.3 mol/L Tris, and adjusted to pH=8.0 with 1 mol/L NaOH or 1 mol/L HCl solution. The volume ratio of the blocking solution and the enzyme labeling buffer B after constant volume was 50:100. In the present invention, the blocking condition is room temperature blocking for 0.5 h. The room temperature is preferably 18-30°C.

In the present invention, after the termination of the reaction, the step further includes: mixing the product with the preservation solution, and then removing by-products. The by-products include free T4 unconjugated ALP and possibly other small molecule impurities. The method of removing by-products includes dialysis or ultrafiltration. In the ultrafiltration step, the molecular weight cut-off of the filter membrane of the ultrafiltration tube is 30 kDa. The conditions of the ultrafiltration were centrifugation at 9000 rpm/min for 25 min. In order to ensure the effect of ultrafiltration, ultrafiltration is carried out under low temperature conditions. The low temperature condition is 0-4°C. After ultrafiltration, collect the liquid in the ultrafiltration tube after ultrafiltration.

In the present invention, the preservation solution of the product is denoted as enzyme labeling buffer C. The enzyme labeling buffer C included water Na ₂ HPO ₄ ·12H ₂ O, NaH ₂ PO ₄ ·2H ₂ O and NaCl. In the present invention, the pH value of the enzyme labeling buffer C is 7.4. In some embodiments, the enzyme labeling buffer C is made of water and 25.786 g/L Na ₂ HPO ₄ ·12H ₂ O, 4.36 g/L NaH ₂ PO ₄ ·2H ₂ O and 8.78 g/L NaCl. Adjust pH=7.4 with 1 mol/L NaOH or 1 mol/L HCl solution. After the reaction was terminated, the volume ratio of the enzyme labeling buffer C and the blocking solution was 50:350.

After ultrafiltration, the liquid in the ultrafiltration tube can be directly used for the detection of T4. But it is usually diluted and made up to volume before use. In the present invention, the enzyme labeling buffer C is used to make constant volume, and glycerol is added as a cryoprotectant.

In some embodiments, the preparation method of alkaline phosphatase-labeled thyroxine provided by the present invention comprises:

Step 1: Mix alkaline phosphatase, EDC, sulfo-NHS and enzyme labeling buffer A, after activation, mix with enzyme labeling buffer B, and perform ultrafiltration to obtain an activated alkaline phosphatase solution;

Step 2: Mix the activated alkaline phosphatase solution with thyroxine, make up the volume with enzyme labeling buffer B, block after incubation, mix with enzyme labeling buffer C, and obtain alkaline phosphatase after ultrafiltration labeled thyroxine;

The enzyme labeling buffer A is made of water, 5.33g/L MES, 7.305g/L NaCl, and is adjusted to pH=4.5 with 1mol/L NaOH or 1mol/L HCl solution.

The enzyme labeling buffer B is made of water and 25.786g/L Na ₂ HPO ₄ .12H ₂ O, 4.36g/L NaH ₂ PO ₄ .2H ₂ O and 8.78g/L NaCl, with 1mol/L NaOH or Adjust pH=9.0 with 1 mol/L HCl solution.

The enzyme labeling buffer C was made of water and 25.786g/L Na ₂ HPO ₄ ·12H ₂ O, 4.36g/L NaH ₂ PO ₄ ·2H ₂ O and 8.78g/L NaCl, with 1mol/L NaOH or Adjust pH=7.4 with 1 mol/L HCl solution.

Described blocking solution is made of water and 0.3mol/L Tris, adjust pH=8.0 with 1mol/L NaOH or 1mol/L HCl solution.

The present invention also provides a thyroxine detection reagent, which includes R1 reagent, R2 reagent and R3 reagent;

The R1 reagent is a magnetic bead suspension coated with T4 antibody;

The R2 reagent is an alkaline phosphatase-labeled thyroxine solution;

The R3 reagent is a magnetic bead buffer.

The R2 reagent includes alkaline phosphatase-labeled thyroxine, enzyme-labeled buffer C and glycerol prepared by the preparation method of the present invention.

For the reagent for detecting total thyroxine, a dissociating agent is also included, and the dissociating agent is 0.5 mg/mL ANS solution.

The present invention also provides a method for detecting thyroxine, which is detected by using the detecting reagent of the present invention. The thyroxine is free thyroxine or total thyroxine.

The preparation method provided by the present invention directly connects the alkaline phosphatase with thyroxine after activation, and does not need to activate T4, and does not need to couple macromolecules such as BSA. The prepared ALP-T4 complex has good activity and does not contain by-products. Preliminary experiments show that, compared with the scheme using other activators, the technical scheme provided by the present invention has reasonable selection of process parameters, which not only greatly shortens the preparation process, but also improves the coupling effect and reduces the content of by-products, so that the prepared Reagents allow for more accurate detection of thyroxine.

The test materials used in the present invention are all common commercial products and can be purchased in the market. Below in conjunction with embodiment, the present invention is further elaborated:

Example 1:

1. Reagent preparation:

Enzyme Labeling Buffer A (pH=4.5)

materials	unit	Dosage
MES	g	10.66
NaCl	g	14.61

Configuration method: Weigh the above reagents in a clean beaker, add 400mL of double distilled water, stir well to dissolve completely, adjust pH=4.5 with 1M NaOH or 1M HCl solution; dilute to 500mL, filter with 0.45μm filter for use.

Enzyme labeling buffer B (pH=9.0)

materials	unit	Dosage
Na 2 HPO 4 ·12H 2 O	g	25.786
NaH 2 PO 4 ·2H 2 O	g	4.36
NaCl	g	8.78

Configuration method: Weigh the above reagents into a clean beaker, add 800mL of double distilled water, stir to dissolve completely, adjust pH=9.0 with 1M NaOH or 1M HCl solution; make up to 1000mL, filter with a 0.45μm filter for use.

Blocking solution (0.3M Tris, pH=8.0)

materials	unit	Dosage
Tris	g	36.342

Configuration method: Weigh the above reagents in a clean beaker, add 800mL of double distilled water, stir well to dissolve completely, adjust pH=8.0 with 1M NaOH or 1M HCl solution; make up to 1000mL, filter with 0.45μm filter for use.

Enzyme labeling buffer C (pH=7.4)

materials	unit	Dosage
Na 2 HPO 4 ·12H 2 O	g	25.786
NaH 2 PO 4 ·2H 2 O	g	4.36
NaCl	g	8.78

Configuration method: Weigh the above reagents in a clean beaker, add 800mL of double distilled water, stir thoroughly to dissolve completely, adjust pH=7.4 with 1M NaOH or 1M HCl solution; dilute to 1000mL, filter with 0.45μm filter head for use.

2. Preparation of ALP-T4 mother liquor

1) Take ALP enzyme 250ug, EDC 10μL (50mg/mL stock solution, enzyme labeling buffer A configuration), sulfo-NHS 1μL (50mg/mL stock solution, enzyme labeling buffer A configuration), adjust the volume of enzyme labeling buffer A to 250μL , 25 ℃, 1100rpm shaking activation for 1h.

2) Add enzyme labeling buffer B to 400 μL, perform ultrafiltration (30k ultrafiltration tube), 9000rpm/min, 25min.

3) Aspirate the activated ALP solution after centrifugation, add 20ug of T4, adjust the volume of enzyme labeling buffer B to 100μL, and react at 25°C for 3h.

4) Block with 50 μL blocking solution for 0.5 h.

5) After blocking, add enzyme labeling buffer C to 400 μL in an ultrafiltration tube, 9000 rpm/min, 25 min.

6) Aspirate the centrifuged ALP-T4 solution, add enzyme labeling buffer C to supplement the volume of ALP-T4 to 125 μL, then add 125 μL of glycerol, and store at -20°C for later use to prepare ALP-T4 stock solution.

3. Preparation and detection of free thyroxine reagent

3.1 Reagent preparation

R1: Dilute the magnetic bead-coated T4 antibody to 0.2 mg/mL with magnetic bead buffer to prepare

R1.

R2: Dilute the ALP-T4 stock solution 16,000 times with enzyme labeling buffer to prepare R2.

R3: Magnetic Bead Buffer.

3.2 Test method

Linear range: in the range of [2.00～100.00]pmol/L, with four-parameter fitting, the linear correlation coefficient (r) of the dose-response curve is ≥0.9900. The fitted curve is:

y=(4538520.61943-81.4275)/(1+(x/6.1623) ^1.29662 )+81.4275

R ² =0.9994

Minimum detection limit: test 20 zero-concentration calibrators, and calculate the average of the luminescence values

and the standard deviation SD, will

Substitute into the dose-response curve equation, the obtained concentration value is the minimum detection limit, which should not be higher than 2.00pmol/L. The test results are shown in Table 1

Table 1 Minimum detection limit test of free thyroxine reagent

Accuracy: To test the enterprise reference products with traceability, and calculate the test deviation, it should not exceed ±10.0%. The results are shown in Table 2:

Table 2 Accuracy test of free thyroxine reagent

Precision: The high-concentration and low-concentration quality control materials were tested 10 times respectively, and the coefficient of variation (CV) was calculated, which should not be greater than 8.0%. The results are shown in Table 3:

Table 3 Free thyroxine reagent intra-analytical precision test

Specificity: T3 with a concentration of not less than 200ng/mL, rT3 with 100ng/mL, 3,3'-diiodothyronine with a concentration of 200ng/mL, and the test result should not be higher than 2.00pmol/L. The results are shown in Table 4:

Table 4 Free thyroxine reagent specificity test

crossover	Concentration (ng/mL)	Calculated concentration (pmol/L)
T3	200	0.42
rT3	100	0.21
3,3'-Diiodothyronine	200	0.18

4. Preparation and detection of free thyroxine reagent

4.1 Reagent preparation

R1: Dilute the magnetic bead-coated T4 antibody to 0.2 mg/mL with magnetic bead buffer to prepare R1.

R2: Dilute ALP-T4 stock solution 8000 times with enzyme labeling buffer to prepare R2.

R3: dissociating agent ANS, 0.5 mg/mL.

4.2 Test method

Linear range: in the range of [10.0～300.0]nmol/L, with four-parameter fitting, the linear correlation coefficient (r) of the dose-response curve is ≥0.9900.

The fitted curve is:

y=(2984616.30526-4925.28)/(1+(x/10.3169) ^0.90479 )+4925.28

R ² =0.9981

Minimum detection limit: test 20 zero-concentration calibrators, and calculate the average of the luminescence values

and the standard deviation SD, will

Substitute into the equation of dose-response curve, the obtained concentration value is the minimum detection limit, which should not be higher than 10.0nmol/L. The results are shown in Table 5

Table 5 Minimum detection limit test of TT4 reagent

Accuracy: To test the enterprise reference products with traceability, and calculate the test deviation, it should not exceed ±10.0%. The results are shown in Table 6:

Table 6 Accuracy test of TT4 reagent

Precision: The high-concentration and low-concentration quality control materials were tested 10 times respectively, and the coefficient of variation (CV) was calculated, which should not be greater than 8.0%. The results are shown in Table 7:

Table 7 Intra-analytical precision test of TT4 reagent

Specificity: T3 with a concentration of not less than 500ng/mL, rT3 with a concentration of 50ng/mL, and the test result should not be higher than 10.0nmol/L. The results are shown in Table 8:

Table 8 TT4 reagent specificity test

crossover	Concentration (ng/mL)	Calculated concentration (nmol/L)
T3	500	0.3
rT3	50	0.1

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims (10)

1. A method for preparing alkaline phosphatase-labeled thyroxine, comprising:

   After the alkaline phosphatase is activated in an acidic solution, the free activator is removed, and then it is coupled with thyroxine in an alkaline solution, and the alkaline phosphatase-labeled thyroxine is obtained after the reaction is terminated.
2. The preparation method according to claim 1, wherein the acidic solution is MES buffer with pH<7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer.
3. The preparation method according to claim 1, wherein the activator is selected from at least one of EDC/NHS, glutaraldehyde, and Sulfo-SMCC.
4. The preparation method according to claim 1, wherein the method for removing the activator comprises dialysis or ultrafiltration.
5. The preparation method according to claim 1, wherein the alkaline solution is MES buffer with pH>7, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer .
6. The preparation method according to claim 1, wherein in the coupling step, the carboxyl group of alkaline phosphatase is coupled with the amino group of thyroxine.
7. The preparation method according to claim 6, wherein the termination reaction adopts an amino-containing buffer.
8. The preparation method according to claim 1, characterized in that, after the termination of the reaction, the method further comprises: mixing the product with the preservation solution to remove by-products.
9. preparation method according to claim 8, is characterized in that,

   The preservation solution is MES buffer, PBS buffer, Tris-HCl buffer, phosphate buffer, citrate buffer or glycine buffer with pH 7.0-7.5;

   The by-products include free thyroxine and/or alkaline phosphatase;

   Methods of such removal include dialysis or ultrafiltration.
10. A thyroxine detection reagent, characterized in that it comprises R1 reagent, R2 reagent and R3 reagent;

    The R1 reagent is a magnetic bead suspension coated with T4 antibody;

    The R2 reagent is an alkaline phosphatase-labeled thyroxine solution;

    The R3 reagent is a magnetic bead buffer;

    The R2 reagent includes alkaline phosphatase-labeled thyroxine, enzyme-labeled buffer C and glycerol prepared by the preparation method according to any one of claims 1 to 9.