Hepatitis b pre s1 antigen, its preparation method and application

Abstract

The present application relates to the technical field of immunological detection, in particular to hepatitis B PreS1 antigen and its preparation method and application. The present application first selects an active PreS1 polypeptide sequence, which is coupled to a hepatitis B surface antigen HBsAg molecule through a crosslinking agent to obtain a hepatitis B PreS1 antigen having both hepatitis B surface antigen activity and PreS1 antigen activity, which can be used as a calibrator and positive control in a hepatitis B PreS1 antigen detection kit. The kit uses anti-PreS1 antibody as coating and anti-HBsAg antibody as enzyme label. The artificial coupled hepatitis B PreS1 antigen prepared by the present application can effectively replace the currently used PreS1 real positive sample on the market, has no biological safety risk, stable source, and various indexes such as reactivity, vehicle stability and thermal stability, which are all better than those of the real positive sample, and has high innovation and applicability.

Description

Technical Field

[0001] This invention relates to the field of immunological detection technology, and in particular to the hepatitis B PreS1 antigen, its preparation method, and its application. Background Technology

[0002] Hepatitis B virus serological markers (HBVM) are no longer sufficient for routine hepatitis B virus diagnosis in clinical practice. HBV-DNA, hepatitis B surface antigen (HBsAg), and hepatitis B virus pre-S1 (PreS1) antigen all serve as evidence of HBV infection and replication in the human body. In recent years, preS1 antigen testing has proven to be more reflective of HBV replication than HBeAg. Therefore, preS1 antigen is more sensitive than HBeAg as an indicator of HBV infection and replication, has independent detection value, and plays an important supplementary role to the detection of the "five markers (two and a half pairs)" of HBV markers.

[0003] The outer membrane protein of hepatitis B virus (HBV) comprises three components: S, preS2, and preS1. PreS1 antigen, encoded by the preS1 region of the HBV genome, is a preprotein with strong immunogenicity and is an important component of hepatitis B surface antigen (HBsAg). PreS1 antigen plays a crucial role in viral invasion of hepatocytes. The most important mediating site for viral attachment to hepatocytes is the amino acid (AA) 21-47 segment of the preS1 protein; mutated viruses are infectious as long as this segment remains intact. Proteins containing preS1 are mainly found on Dane particles and tubular particles. PreS1 protein plays a vital role in viral infection, assembly, replication, and stimulating the body's immune response. The HBV preS1 antigen (Pres1-Ag) is located on the outer membrane protein of HBV; it exists on the surface of intact HBV particles and is closely related to HBV infection and replication. Therefore, developing a method for detecting HBV preS1 antigen is of great significance.

[0004] The hepatitis B virus pre-S1 antigen test is a research achievement completed by the Shanghai Institute of Biochemistry, Chinese Academy of Sciences. It passed national certification in 2000 and was approved for clinical use by the State Drug Administration in December of the same year. Tertiary hospitals in the vast majority of regions across the country have already implemented S1 antigen testing. The official clinical rollout of the hepatitis B virus pre-S1 antigen assay kit, and its combined application with the hepatitis B "two-and-a-half pairs" test in nearly a hundred hospitals in Beijing, Sichuan, Zhejiang, Henan, Shenzhen, Hainan, and other provinces in recent years, fully demonstrates the important value of this reagent in the clinical diagnosis and treatment guidance of viral hepatitis B. The pre-S1 antigen (Pre-S1Ag) test is an important supplement and enhancement to the hepatitis B "two-and-a-half pairs" test, especially the e antigen and HBV-DNA tests.

[0005] However, the calibrators and positive controls currently used in hepatitis B virus pre-S1 antigen detection reagents are all added from real high-positive values ​​of PreS1, which has many problems, such as limited sources, difficulty in screening, storage risks and significant biosafety risks. In addition, they have low potency and poor stability during use, which seriously affects the promotion and use of hepatitis B virus pre-S1 antigen detection methods. Summary of the Invention

[0006] In view of this, the present invention provides a hepatitis B PreS1 antigen, its preparation method, and its applications. The present invention first selects an active PreS1 polypeptide sequence and conjugates it to the hepatitis B surface antigen (HBsAg) molecule using a cross-linking agent, obtaining a hepatitis B PreS1 antigen that simultaneously possesses HBsAg and PreS1 antigen activity. This antigen can be used as a calibrator and positive control in a hepatitis B PreS1 antigen detection kit, which uses anti-PreS1 antibody as coating and anti-HBsAg antibody as an enzyme label. The artificially conjugated hepatitis B PreS1 antigen prepared by the present invention can effectively replace currently used commercially available real PreS1 positive samples, posing no biosafety risks, having a stable source, and exhibiting superior reactivity, vehicle-borne stability, thermal stability, and other performance indicators compared to real positive samples. It possesses high innovation and applicability.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] The present invention provides a hepatitis B PreS1 antigen, comprising hepatitis B surface antigen HBsAg, a coupling agent, and a PreS1 polypeptide; wherein the hepatitis B surface antigen HBsAg is linked to the PreS1 polypeptide via the coupling agent; the amino acid sequence of the PreS1 polypeptide is shown in SEQ ID NO:1.

[0009] The present invention also provides a method for preparing the hepatitis B PreS1 antigen, comprising: taking the coupling agent to activate the amino group in the hepatitis B surface antigen HBsAg, dialyzing, and obtaining the activated hepatitis B surface antigen HBsAg; taking the PreS1 polypeptide and coupling it with the activated hepatitis B surface antigen HBsAg, dialyzing, and obtaining the hepatitis B PreS1 antigen.

[0010] In some specific embodiments of the present invention, the coupling agent includes SMCC or EDC.

[0011] In some specific embodiments of the present invention, the SMCC is used in the form of sulfo-SMCC.

[0012] In some specific embodiments of the present invention, the molar ratio of the coupling agent to the hepatitis B surface antigen HBsAg includes (20~40):1.

[0013] In some specific embodiments of the present invention, the molar ratio of the PreS1 polypeptide to the hepatitis B surface antigen HBsAg includes (10~20):1.

[0014] In some specific embodiments of the present invention, the initial concentration of the hepatitis B surface antigen HBsAg includes 5 mg / mL.

[0015] In some specific embodiments of the present invention, the concentration of the Sulfo-SMCC includes 5 mg / mL.

[0016] In some specific embodiments of the present invention, the concentration of the activated hepatitis B surface antigen HBsAg includes 4 mg / mL.

[0017] In some specific embodiments of the present invention, the concentration of the PreS1 peptide includes 1 mg / mL.

[0018] In some specific embodiments of the present invention, the activation conditions include a reaction at 25°C in the dark.

[0019] The coupling temperature includes 25°C.

[0020] In some specific embodiments of the present invention, the activation time includes 5 hours.

[0021] In some specific embodiments of the present invention, the coupling time includes 14 hours.

[0022] In some specific embodiments of the present invention, the dialysis membrane has a pore size of 50 kDa.

[0023] In some specific embodiments of the present invention, the volume ratio of dialysate to sample during dialysis is ≥50:1; the number of dialysis cycles includes 5.

[0024] In some specific embodiments of the present invention, the dialysate includes 0.01M PBS pH7.2 buffer or 0.067M PBS pH6.7 buffer.

[0025] In some specific embodiments of the present invention, the hepatitis B PreS1 antigen and an equal volume of glycerol are mixed and stored at low temperature in the dark.

[0026] This invention also provides the following applications as calibrators or positive controls:

[0027] (I) the hepatitis B PreS1 antigen; and / or

[0028] (II) Hepatitis B PreS1 antigen prepared by the above preparation method.

[0029] This invention also provides the application of any of the following in the preparation of a hepatitis B PreS1 antigen detection kit:

[0030] (I) the aforementioned hepatitis B PreS1 antigen; and / or

[0031] (II) Hepatitis B PreS1 antigen prepared by the above preparation method.

[0032] The present invention also provides a product comprising any of the following:

[0033] (I) the hepatitis B PreS1 antigen; and / or

[0034] (II) Hepatitis B PreS1 antigen prepared by the above preparation method.

[0035] This invention includes, but is not limited to, the following beneficial effects:

[0036] This invention first selects an active PreS1 polypeptide sequence and conjugates it to the hepatitis B surface antigen (HBsAg) molecule using a cross-linking agent, obtaining a HBsAg PreS1 antigen that simultaneously possesses HBsAg and PreS1 antigen activity. This antigen can be used as a calibrator and positive control in a HBsAg PreS1 antigen detection kit, which uses anti-PreS1 antibody as coating and anti-HBsAg antibody as an enzyme label. The artificially conjugated HBsAg PreS1 antigen prepared by this invention can effectively replace currently used commercially available real PreS1 positive samples, posing no biosafety risks, having a stable source, and exhibiting superior reactivity, vehicle-to-everything (V2X) stability, thermal stability, and other performance indicators compared to real positive samples. It demonstrates high innovation and applicability. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0038] Figure 1 This demonstrates the synthetic process route for the hepatitis B PreS1 antigen. Detailed Implementation

[0039] This invention discloses the hepatitis B PreS1 antigen, its preparation method, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0040] The purpose of this invention is to provide a method for preparing artificially conjugated hepatitis B PreS1 antigen to replace currently used commercially available PreS1 real positive samples. This method poses no biosafety risks, has a stable source, and effectively promotes the application of hepatitis B PreS1 detection kits. An active PreS1 polypeptide sequence is selected, and based on the amino acid composition of the polypeptide sequence, different cross-linking agents are chosen to conjugate it to the hepatitis B surface antigen (HBsAg) molecule, resulting in a hepatitis B PreS1 antigen that simultaneously possesses hepatitis B surface antigen activity and PreS1 antigen activity. This antigen can be used as a calibrator and positive control in hepatitis B PreS1 antigen detection kits. The artificial hepatitis B PreS1 antigen prepared by this invention can effectively replace currently used commercially available PreS1 real positive samples, posing no biosafety risks, having a stable source, low cost, and superior performance compared to currently used high-value PreS1 real positive samples.

[0041] First, highly active PreS1 peptides were screened using ELISA. Based on the amino acid composition of the peptide sequence, either EDC or SMCC coupling agents were selected to link them to the HBsAg molecule. In the method described in this invention, SMCC was selected as the cross-linking agent to activate the amino groups in the HBsAg molecule, introduce maleimide groups, and couple them with cysteine ​​residues in the peptide molecule.

[0042] The PreS1 polypeptide sequence selected in this invention is: CAFGANSNNPDWDFNP (SEQ ID NO:1). Because its cysteine ​​C contains a thiol group, it can be coupled using SMCC.

[0043] After selecting the polypeptide sequence, the method for preparing the artificial hepatitis B PreS1 antigen according to the present invention is as follows:

[0044] First, HBsAg antigen is dialyzed into PBS to determine its actual concentration for later use. Sulfo-SMCC (purchased from Thermo Fisher Scientific, catalog number: 22322) is weighed and dissolved in PBS to a concentration of 1 mg / mL. This 1 mg / mL solution is then added to the HBsAg antigen at a specific molar ratio. The mixture is incubated at 25°C in the dark for 5 hours to activate the amino group in the HBsAg antigen molecule, converting it to maleimide. This active functional group can selectively couple with the thiol group in the peptide molecule. After the reaction, excess sulfo-SMCC is removed by dialyzing into PBS at a ratio of at least 1 / 50, for a total of five dialyzes. The dialysis bag is then opened, and the SMCC-activated HBsAg is transferred to a glass vial, the volume is recorded, and the concentration is determined for later use.

[0045] Take 1 mg / vial of peptide (synthesized by bioengineering), dissolve it in PBS to a concentration of 1 mg / mL, and add it to the HBsAg antigen solution activated by SMCC at a molar ratio of 10 / 1 to 20 / 1. Incubate overnight at 25°C. The peptide molecules are used to achieve site-specific coupling with SMCC via cysteine ​​sulfhydryl groups. After the reaction, dialyze the peptide into PBS using a 50 kDa dialysis bag to remove excess peptide. The dialysis ratio should be no less than 1 / 50. Dialyze five times to completely remove the peptide. Remove the dialysis bag, transfer the PreS1 peptide-HBsAg conjugate to a glass vial and record the volume. Detect the concentration, add an equal volume of glycerol, and store at -20°C.

[0046] This invention designs a highly active PreS1 peptide and, for the first time, artificially conjugates the peptide to the hepatitis B surface antigen (HBsAg) molecule, resulting in a HBsAg PreS1 antigen possessing both HBsAg and PreS1 antigen activity. This antigen can be used as a calibrator and positive control in HBsAg PreS1 antigen detection kits. The process flow diagram is shown below. Figure 1 As shown.

[0047] Specifically, its advantages are reflected in the following aspects:

[0048] 1. Raw materials are readily available, low in cost, and there is no risk of stockouts; PreS1 peptides are synthesized through bioengineering and are reasonably priced; HBsAg antigens can be expressed through recombinant expression, a relatively mature technology.

[0049] 2. The coupling process is simple and mild. The designed peptide contains cysteine ​​and has thiol groups that can be used for site-specific coupling. After SMCC activation of HBsAg antigen, the finished product can be obtained by one-step coupling. The coupling process is all in neutral and has almost no effect on protein conformation.

[0050] 3. There are no biological risks during preparation and use; the calibrators and positive controls used in the current PreS1 antigen detection kits are all added based on the true high positive values ​​of PreS1, which poses a significant biosafety risk and a high risk during use and storage.

[0051] 4. Good stability of the finished product: Adding high-value PreS1 real positive results as calibrators and positive controls for the PreS1 antigen detection kit itself has serious stability problems. After 3 days of vehicle loading and 7 days at 37 degrees Celsius, the stability drop is as high as 27-60%, which seriously affects the use and promotion of the PreS1 antigen detection kit. However, this invention uses artificial coupling to combine two stable components, PreS1 peptide and HBsAg, together with chemical bonds. The resulting hepatitis B PreS1 antigen has good stability. Experimental tests show that after 3 days of vehicle loading and 7 days at 37 degrees Celsius, the stability drop is only less than 5%.

[0052] Unless otherwise specified, the hepatitis B PreS1 antigen, its preparation method, and the raw materials and reagents used in its application provided by this invention can all be purchased from the market.

[0053] The present invention will be further illustrated below with reference to the embodiments:

[0054] Preparation Example 1: Preparation of Artificially Conjugated Hepatitis B PreS1 Antigen

[0055] The first step is the activation of HBsAg.

[0056] Dialyze 5 mg / mL HBsAg to 0.01 M PBS pH 7.2, with a dialysis ratio of at least 1 / 50, for four dialysis cycles. Remove the dialysis bag and place it in a clear glass bottle. Determine the concentration using the A280 1Abs method. Dilute to 4 mg / mL and record the mass and volume. Remove Sulfo-SMCC (purchased from Thermofisher, A39268), allow it to reach room temperature, and dissolve it in 0.01 M PBS pH 7.2 to a concentration of 1 mg / mL. Add Sulfo-SMCC to the HBsAg solution at a Sulfo-SMCC / HBsAg molar ratio of 40 / 1. Incubate at 25°C in the dark for 5 hours. Dialyze with 0.01 M PBS pH 7.2 buffer at 4°C for 16 hours, changing the buffer four times during dialysis to completely remove residual Sulfo-SMCC. After dialysis, accurately measure the HBsAg-SMCC concentration using the A280 1Abs method, and record the volume and concentration for future reference.

[0057] Step 2: Preparation of PreS1 peptide solution

[0058] Take 1 mg of PreS1 peptide (purchased from Sangon Biotech), and after returning to room temperature, dissolve it in 0.01 M PBS pH 7.2 to make a 1 mg / mL solution. Mix thoroughly until colorless and transparent with no flocculent matter present, and set aside for later use.

[0059] The third step is the cross-linking reaction between PreS1 peptide and HBsAg.

[0060] PreS1 peptide and HBsAg activated by SMCC (HBsAg-SMCC) were mixed in a transparent glass bottle at a molar ratio of 10 / 1 and coupled in a water bath at 25°C for 14 hours.

[0061] Step 4: Post-processing and saving

[0062] After the reaction, the sample was dialyzed into 0.067M PBS pH 6.7 buffer using a 50KD dialysis bag and dialyzed at 4°C for 16 hours, with the buffer changed 4 times during dialysis to completely remove residual PreS1 peptides. After dialysis, the HBsAg-SMCC-Peptide concentration was accurately measured using the A280 1Abs method. Three measurements were taken, the average value was recorded, and the volume was added. An equal volume of glycerol was added, mixed, and stored at -20°C in the dark to obtain the hepatitis B PreS1 antigen.

[0063] Preparation Example 2: Preparation of Artificially Conjugated Hepatitis B PreS1 Antigen

[0064] The first step is the activation of HBsAg.

[0065] Dialyze 5 mg / mL HBsAg to 0.01 M PBS pH 7.2 at a ratio of at least 1 / 50, repeating four times. Transfer the dialysis bag to a clear glass bottle and determine the concentration using the A280 1Abs method. Dilute to 4 mg / mL and record the mass and volume. Remove Sulfo-SMCC (purchased from Thermofisher, A39268), allow it to reach room temperature, and dissolve it in 0.01 M PBS pH 7.2 to a concentration of 1 mg / mL. Add Sulfo-SMCC to the HBsAg solution at a Sulfo-SMCC / HBsAg molar ratio of 20 / 1. Incubate at 25°C in the dark for 5 hours. Dialyze with 0.01 M PBS pH 7.2 buffer at 4°C for 16 hours, changing the buffer four times during dialysis to completely remove residual Sulfo-SMCC. After dialysis, accurately measure the HBsAg-SMCC concentration using the A280 1Abs method, recording the volume and concentration for later use.

[0066] Step 2: Preparation of PreS1 peptide solution

[0067] Take 1 mg of PreS1 peptide (purchased from Sangon Biotech), and after returning to room temperature, dissolve it in 0.01 M PBS pH 7.2 to make a 1 mg / mL solution. Mix thoroughly until colorless and transparent with no flocculent matter present, and set aside for later use.

[0068] The third step is the cross-linking reaction between PreS1 peptide and HBsAg.

[0069] PreS1 peptide and HBsAg activated by SMCC (HBsAg-SMCC) were mixed in a transparent glass bottle at a molar ratio of 20 / 1 and coupled in a 25°C water bath for 14 hours.

[0070] Step 4: Post-processing and saving

[0071] After the reaction, the solution was dialyzed to the final concentration of 0.067M PBS pH 6.7 using a 50KD dialysis bag and dialyzed at 4°C for 16 hours, with the buffer changed four times during dialysis to completely remove residual PreS1 peptides. After dialysis, the HBsAg-SMCC-Peptide concentration was accurately measured using the A280 1Abs method. The average value of three measurements was recorded, and an equal volume of glycerol was added. The mixture was then incubated at -20°C in the dark to obtain the hepatitis B PreS1 antigen.

[0072] Example

[0073] The performance of the hepatitis B virus preS1 antigen prepared in Examples 1 and 2 was tested using the AutoLumo A2000 Plus hepatitis B virus preS1 antigen detection kit (batch number: 20230929) from Antu Bio. The stability data were compared with those obtained using the PreS1-added true positive high value method. The hepatitis B PreS1 antigen and PreS1 true positive high value prepared in Examples 1 and 2 were prepared into working solutions, and their stability was compared after 3 days in a vehicle and 7 days at 37℃. The results are shown in Table 1.

[0074] The results showed that adding high-value PreS1 real positive results as calibrators and positive controls for the PreS1 antigen detection kit itself had serious stability issues. After 3 days in a vehicle and 7 days at 37°C, the stability decreased by 27-60%, which seriously affected the use and promotion of the PreS1 antigen detection kit. However, the present invention uses artificial coupling to combine two stable components, PreS1 peptide and HBsAg, together with chemical bonds. The resulting hepatitis B PreS1 antigen has better stability. Experimental results showed that after 3 days in a vehicle and 7 days at 37°C, the stability decreased by less than 5%.

[0075] Table 1. Comparison of stability data of high-value true positive results for hepatitis B PreS1 antigen prepared in this invention with those using PreS1-added antigen.

[0076]

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Hepatitis B PreS1 antigen, characterized in that, It is composed of hepatitis B surface antigen HBsAg, a coupling agent, and a PreS1 polypeptide; the hepatitis B surface antigen HBsAg is linked to the PreS1 polypeptide via the coupling agent; the amino acid sequence of the PreS1 polypeptide is shown in SEQ ID NO:1; the coupling agent is SMCC.

2. The method for preparing hepatitis B PreS1 antigen as described in claim 1, characterized in that, include: The coupling agent is used to activate the amino group in the hepatitis B surface antigen HBsAg, and then dialyzed to obtain the activated hepatitis B surface antigen HBsAg. The PreS1 polypeptide was conjugated with the activated hepatitis B surface antigen HBsAg, and then dialyzed to obtain the hepatitis B PreS1 antigen.

3. The preparation method according to claim 2, characterized in that, The SMCC is used in the form of sulfo-SMCC.

4. The preparation method according to claim 2 or 3, characterized in that, The molar ratio of the coupling agent to the hepatitis B surface antigen HBsAg is (20~40):

1.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the PreS1 polypeptide to the hepatitis B surface antigen HBsAg is (10~20):

1.

6. The preparation method according to claim 2, characterized in that, The activation conditions include a reaction at 25°C in the dark. The coupling temperature includes 25°C.

7. Any of the following may be used in the preparation of calibrators or positive controls for the hepatitis B PreS1 antigen detection kit: (I) The hepatitis B PreS1 antigen as described in claim 1; and / or (II) Hepatitis B PreS1 antigen prepared by the preparation method according to any one of claims 2 to 6.

8. Any of the following applications in the preparation of the hepatitis B PreS1 antigen detection kit: (I) The hepatitis B PreS1 antigen as described in claim 1; and / or (II) Hepatitis B PreS1 antigen prepared by the preparation method according to any one of claims 2 to 6.

9. A hepatitis B PreS1 antigen detection kit, characterized in that, Includes any of the following: (I) The hepatitis B PreS1 antigen as described in claim 1; and / or (II) Hepatitis B PreS1 antigen prepared by the preparation method according to any one of claims 2 to 6.

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