Kit for quantitatively detecting HBsAg-HBsAb immune complex and application thereof

Abstract

The invention discloses a kit for quantitatively detecting an HBsAg-HBsAb immune complex and application of the kit. The kit provided by the invention is a detection kit based on an ELISA sandwich method, a novel double-antibody sandwich ELISA detection platform provided by the invention adopts an HBsAg specific mSA1 antibody as a capture carrier, and efficient solid phase coating is realized through specific binding of the HBsAg specific mSA1 antibody with HBsAg; meanwhile, an anti-human IgG-HRP conjugate is introduced as a detection antibody, and high-sensitivity and high-specificity quantitative detection of the HBsAg-HBsAb immune complex is realized by utilizing special epitopes of different subtypes of human IgG and the catalytic amplification effect of HRP. The detection kit disclosed by the invention provides a reliable tool for quantitatively detecting the HBsAg-HBsAb immune complex for clinical use.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a kit for quantitative detection of HBsAg-HBsAb immune complexes and its application. Background Technology

[0002] Chronic hepatitis B (CHB) is a liver disease caused by persistent infection with the hepatitis B virus (HBV), and it remains a significant global public health issue. Although the widespread use of hepatitis B vaccines and antiviral drugs has significantly reduced the incidence of new infections and some related complications, a complete cure for CHB still faces enormous challenges.

[0003] Currently, the treatment of chronic hepatitis B mainly relies on nucleoside (acid) analogs (such as entecavir and tenofovir) and interferon. These drugs can effectively inhibit viral replication and slow disease progression, but they are difficult to completely eliminate the virus. Clinical cure (also known as functional cure) is defined as persistent negative hepatitis B surface antigen (HBsAg) with or without the appearance of hepatitis B surface antibody (HBsAb) after stopping treatment, and undetectable HBV DNA. This is the current treatment goal. However, the HBsAg clearance rate is still not ideal, and most patients need to take medication for a long time or even for life.

[0004] Against this backdrop, the dynamic changes of HBsAg and HBsAb have become a focus of attention. Especially during clinical recovery, a serological state where HBsAg and HBsAb coexist is common, at which point they may form immune complexes (ICs). Theoretically, these complexes may affect HBsAg test results (leading to false negatives or low values) and even participate in immune regulatory mechanisms, such as affecting viral clearance and the liver immune microenvironment by masking antigen epitopes or activating complement pathways. Recent studies suggest that HBsAg-HBsAb immune complexes may be associated with persistent chronic infection, poor treatment response, and clinical outcomes, but their specific mechanisms of action and clinical significance remain unclear.

[0005] Currently, there is a lack of standardized, commercially available detection platforms for HBsAg-HBsAb immune complexes. Conventional serological tests (such as ELISA or chemiluminescence assays) can only quantify HBsAg and HBsAb separately, but cannot identify and measure the complex form in which they bind. Therefore, clinicians cannot accurately assess the level of immune complexes and their association with disease progression. Secondly, during HBsAg seroconversion (the disappearance of HBsAg and the appearance of HBsAb), the formation of complexes may underestimate the true HBsAg concentration, misleading the assessment of treatment efficacy. Furthermore, basic research struggles to deeply explore the role of these complexes in immune regulation and viral persistence.

[0006] Developing a technological platform for the specific detection of HBsAg-HBsAb immune complexes is of great significance. On the one hand, it can provide clinicians with a more comprehensive serological assessment tool, aiding in personalized treatment decisions and optimizing cure criteria. On the other hand, it holds promise for elucidating the mechanisms of immune complexes in the natural history and treatment response of chronic hepatitis B, providing targets for the development of new therapies (such as combined immunomodulatory therapies). Ultimately, this breakthrough may drive a shift in chronic hepatitis B management from a strategy centered on viral suppression to one focused on immune reconstitution, accelerating the achievement of higher levels of clinical cure. Summary of the Invention

[0007] The purpose of this invention is to provide a kit for quantitative detection of HBsAg-HBsAb immune complexes and its application.

[0008] To achieve the above objectives, the present invention provides a hepatitis B virus antigen-antibody complex detection kit, the kit comprising a capture antibody and a detection antibody; The capture antibody is mSA1, whose heavy chain CDR1, CDR2 and CDR3 have amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively, and whose light chain CDR1, CDR2 and CDR3 have amino acid sequences shown in SEQ ID NO.4, QDT and SEQ ID NO.5 respectively.

[0009] The detection antibody is an anti-human IgG-HRP conjugate.

[0010] It is understandable that, for antibodies, the regions that affect their precise complementarity with antigenic determinants are complementarity-determining regions (CDRs), specifically CDR1, CDR2, and CDR3 of the heavy chain and CDR1, CDR2, and CDR3 of the light chain. Therefore, as long as the CDR1, CDR2, and CDR3 sequences of the heavy and light chains of the capture antibody of this invention remain unchanged, its function and role can be basically realized.

[0011] Preferably, the detection antibody is selected from at least one of anti-human IgG1-HRP conjugate, anti-human IgG3-HRP conjugate, and anti-human IgG4-HRP conjugate.

[0012] Preferably, the kit further includes: a microplate, a diluent, a blocking solution, a washing solution, a colorimetric solution, and a stop solution.

[0013] Preferably, the diluent is a PBS solution.

[0014] Preferably, the washing solution is a PBST solution.

[0015] Preferably, the sealing solution is prepared by mixing deionized water and skim milk powder in a ratio of 100 mL: 5 g.

[0016] Preferably, the colorimetric solution is prepared by mixing equal volumes of TMB and H2O2.

[0017] Preferably, the stop solution is an ELISA stop solution.

[0018] The present invention also provides a method for quantitative detection of HBsAg-HBsAb immune complexes using the above-mentioned detection kit, comprising the following steps: (1) Dilute the capture antibody to 0.1-10 g / L with diluent and add it to the microplate at 100 μL / well, and incubate at room temperature for 10 min-12 h; (2) Add blocking solution at 200 μL / well and incubate at room temperature for 10 min – 12 h; (3) Dilute the sample to be tested at the original volume ratio, or dilute it with the blocking solution at a volume ratio of 1:(1–1000) and add it to the microplate at 100 μL / well, and incubate at room temperature for 10 min - 4 h; (4) Add 100 μL of the detection antibody (originally 100 μL) or the blocking solution (1:(1–50000)) to the microplate and incubate at room temperature for 10 min–2 h. (5) Add 100 μL of colorimetric solution to each well and develop the color in the dark for 20 s – 30 min; (6) Add stop solution at 50 μL / well, and obtain the OD of the sample at 450 nm using a microplate reader or spectrometer. 450 value.

[0019] Preferably, after the incubation in steps (1)-(4), the process further includes washing with a washing solution.

[0020] Due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: This invention provides a detection kit based on an ELISA sandwich method. The novel double-antibody sandwich ELISA detection platform utilizes an HBsAg-specific mSA1 antibody as a capture carrier, achieving efficient solid-phase coating through its specific binding to HBsAg. Simultaneously, an anti-human IgG-HRP conjugate is introduced as the detection antibody. Leveraging the specific epitopes of different human IgG subtypes and the catalytic amplification effect of HRP, highly sensitive and specific quantitative detection of HBsAg-HBsAb immune complexes is achieved. This invention provides a reliable tool for the clinical quantitative detection of HBsAg-HBsAb immune complexes. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This refers to the gate pathway of HBsAg-DY650 / 550 double-positive B cells in PBMCs during antibody screening in Example 1 of this invention.

[0023] Figure 2 This shows the antibody identification results from the Western Blot experiment during antibody screening in Example 1 of this invention. Wherein, M represents a marker, + represents a positive control (anti-HBsAg-IgG1), - represents a negative control (anti-A29L-IgG1), and 1-270 are antibody serial numbers.

[0024] Figure 3 The results are SDS-PAGE analysis of the mSA1 antibody in Example 2 of this invention.

[0025] Figure 4 This is the experimental result from Example 3 of the present invention, verifying that the enzyme-labeled antibody does not specifically react with the blocking solution.

[0026] Figure 5 In Example 3 of this invention, the dilution of the enzyme-labeled antibody is used as the x-axis, and COI (OD) is... 450 The experimental results are plotted using Graphpad software, with the sample / threshold (CIO) on the ordinate. COI < 1.0 represents the OD of the sample. 450 If the value is below the negative threshold, it is judged as a negative signal.

[0027] Figure 6 The results are from Example 4 of this invention, which demonstrate that human plasma does not specifically react with the blocking solution. A, B, and C are the experimental results for plasma samples HC231129-(1-12), HC231129-(13-23), and HC231130-(1-11), respectively.

[0028] Figure 7 This is the result of quantitative detection of HBsAg-HBsAb immune complexes in different samples in Example 5 of the present invention. COI (OD) 450 The experimental results are plotted using Graphpad software, with Sample / Threshold as the ordinate. COI > 1.0 represents the OD of the sample. 450 A value higher than the negative threshold is considered a positive signal.

[0029] Figure 8 This is the result of detecting the HBsAg-HBsAb simulated complex in Example 6 of the present invention. Detailed Implementation

[0030] The embodiments of the present invention will be clearly and completely described below with reference to the examples. These described embodiments are merely some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the instruments and materials used in the following embodiments are all commonly used laboratory equipment, and the technical solutions described are all conventional technologies in the field.

[0032] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (Approval No.: Shenzhen Third People's Hospital Ethics Review and Research No. [2023-105-3]). Participants provided written informed consent for sample collection and subsequent analysis.

[0033] The experimental techniques and procedures involved in this invention are described below:

[0034] I. Fluorescently labeled proteins

[0035] HBsAg was labeled using DyLight 650 and DyLight 550 dual fluorescent markers to identify HBsAg-specific B cells. Taking HBsAg labeling as an example, the labeling method is as follows:

[0036] (1) Protein preparation

[0037] For best results, the optimal labeling buffer is 50 mM boric acid at pH 8.5; the optimal concentration of the labeled protein is approximately 2 mg / mL of 1 mg protein.

[0038] Protein dissolved in PBS: Add 40 μL of borate buffer (0.67 M) to 0.5 mL of HBsAg protein at a concentration of 2 mg / mL. If the protein concentration is greater than 2 mg / mL, adjust the concentration to 2 mg / mL using labeling buffer.

[0039] (2) Protein labeling

[0040] Briefly centrifuge the DyLight reagent for a few seconds to ensure the dye is at the bottom of the test tube. Add 0.5 mL of the prepared HBsAg protein to the test tube, gently vortex and repeatedly aspirate and blow to mix thoroughly. Briefly centrifuge the test tube to collect the sample at the bottom, and incubate the reaction mixture at room temperature in the dark for 60 minutes.

[0041] (3) Protein purification

[0042] Place the two centrifuge columns into two separate centrifuge collection tubes. Mix the purified resin several times by pipetting to ensure uniform suspension. Add 250 μL of the resin suspension to each centrifuge column. Centrifuge at approximately 1000 g for 1 minute to remove the preservative solution. Discard the collection tubes and place the centrifuge columns into new collection tubes.

[0043] Add 250–270 µL of labeled reaction solution to each column and briefly vortex to mix the sample with the resin. Centrifuge the column at approximately 1000 g for 1 minute to collect the purified protein. Combine the samples from both columns (approximately 0.5 mL in total) and discard the used columns.

[0044] Labeled HBsAg protein can be stored at 4°C protected from light for up to one month. Alternatively, aliquot the labeled protein into single-use portions and store at -20°C. Avoid repeated freeze-thaw cycles.

[0045] II. Flow cytometry staining

[0046] After PBMCs were resuscitated, they were washed once with flow cytometry staining buffer (FACS, PBS containing 2% FBS), centrifuged at approximately 350g for 5 minutes, and then diluted with 50μL / 10 6 The cell system was resuspended, and the HBsAg-specific B cell staining protocol is shown in Table 1.

[0047] Table 1. Staining Protocol fluorescent antibody Volume / 50µL brand Item number Cloned ID CD3 BV421 1µL BioLegend 300434 UCHT1 CD19 BUV396 1µL BD Bioscience 612938 SJ25C1 L / D BV510 0.5µL BD Bioscience 564406 / HBsAg-Dylight 650 0.5µL / / / HBsAg-Dylight 550 0.5µL / / /

[0048] After staining according to the above procedure, incubate on ice in the dark for 30 minutes, add an appropriate amount of FACS for washing, centrifuge at 350g for 5 minutes, discard the supernatant, repeat twice to reduce non-specific antibody binding, resuspend in 200µL FACS after centrifugation, and perform single cell sorting using the FACSymphony™ S6 cell sorting instrument.

[0049] III. Enzyme-linked immunosorbent assay (ELISA) for identifying HBsAg-specific antibodies

[0050] (1) Antigen coating: Dilute HBsAg with PBS to 7µg / mL, or prepare HBsAg (10µg) with 10mM DTT solution, add 100µL / well to a 96-well plate, seal the plate and incubate overnight at 4°C or for 1 hour at 37°C.

[0051] (2) Prepare PBST washing solution: Add 1×PBS powder and 500µL Tween to 1L of pure water.

[0052] (3) Preparation of sealing solution: Deionized water and skim milk powder are prepared at a ratio of 100mL:5g.

[0053] (4) Washing: Pour PBST washing solution into the Bio-tek plate washer, wash the plate once, and then spin dry with a microplate dehydrator.

[0054] (5) Sealing: Use 200µL / well sealing solution to seal at room temperature for 1 hour, and repeat step (4).

[0055] (6) Incubation with primary antibody: Dilute 270 antibody strains synthesized by Genscript Pharmaceuticals with PBS at a ratio of 1:10, add 100 µL / well to a 96-well plate, incubate at 37°C or room temperature for 1 hour, wash the plate 5 times and spin dry. Set up 3 positive control wells and 3 negative control wells in each plate. Positive control: Anti-HBsAg-IgG3 (Innosys, catalog number: A2217), negative control: YZ0708A2 (prepared by the applicant in the previous stage, which has been proven to be unable to bind HBsAg, its heavy chain variable region amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYL QMNSLKTEDTAVYYCTTALGGGTPLQRGYWGQGTLVTVSS, light chain variable region amino acid sequence: QLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAWHQQQPEKGPRYLMKLNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQTWGTGLWVFGGGTKLTVL, antibody customized expression and purification by Sinocare), 10µg / well.

[0056] (7) Incubation of secondary antibody: Dilute the secondary antibody Goat anti-Human IgG-Fc Fragment Antibody HRPConjugated (Southern Biotech, catalog number: 2048-05) with PBS at a ratio of 1:10,000, add 100µL / well to a 96-well plate, incubate at 37°C or room temperature for 30 minutes, wash the plate 5 times and spin dry.

[0057] (8) Color Development: The ELISA color development solution (BBI, catalog number: E661007-0100) should be removed from the 4℃ refrigerator half an hour in advance and allowed to return to room temperature. Mix color development solution A and color development solution B in a 1:1 ratio and use immediately. Add 100µL of the prepared color development solution to each well of a 96-well plate and incubate at room temperature in the dark for 7-8 minutes.

[0058] (9) Stop color development: Add 50µL of ELISA stop solution (Solepro, catalog number: C1058) to each well.

[0059] (10) Reading the plate: Read the absorbance value at a wavelength of 450nm on a multi-functional microplate reader within 10 minutes after the color development is terminated, otherwise the accuracy of the results will be affected.

[0060] (11) Data processing standard: Take the average OD of negative wells 450 The value plus 1.2 standard deviations is used as the cutoff, COI = OD 450 The value / cutoff indicates that a COI greater than 1.2 in four replicate experiments is considered positive.

[0061] IV. Western Blot Identification of HBsAg-Specific Antibodies

[0062] (1) Protein denaturation: Prepare 0.13 mg / mL of HBsAg and 2×loading buffer, mix them at a ratio of 1:1, denature at 95°C for 10 minutes in a constant temperature metal bath, and centrifuge at 300g for 2 minutes before use.

[0063] (2) SDS-PAGE electrophoresis: Dissolve Tris-MOPS-SDS Running Buffer Powder in 1L ddH2O, and add 500-700mL of electrophoresis solution to each electrophoresis tank; Take out the 12% precast gel, carefully remove the comb, and place it in the electrophoresis tank. Add 10 μL of HBsAg protein sample to each well. The electrophoresis conditions are as follows: first, electrophoresis at 80V for 30 minutes, then increase the voltage to 120V and run for 40 minutes. The voltage should not be too high, otherwise it will affect the stability of the protein. Stop electrophoresis when the bromophenol blue indicator is close to the bottom of the gel.

[0064] (3) Transfer: After electrophoresis, remove the SDS-PAGE gel and carefully remove the gel by peeling off the plastic cover. Thoroughly wet the NC membrane with transfer buffer beforehand. Place the sponge, filter paper, SDS-PAGE gel, NC membrane, filter paper, and sponge onto the transfer clamp using a "sandwich" method. During this process, remove air bubbles after each layer is added to avoid affecting the protein transfer. (The transfer buffer consists of 700 mL ddH2O, 200 mL methanol, and 100 mL 10× transfer buffer. Add approximately 700 mL of transfer buffer to each transfer tank. Transfer according to the molecular weight of the target protein. In this experiment, transfer is typically performed at 200 mA on ice for 40 minutes.)

[0065] (4) Blocking: Prepare blocking solution by mixing deionized water and skim milk powder at a ratio of 100mL:5g. After the transfer, rinse the NC membrane with TBST, add an appropriate amount of blocking solution and block on a shaker at room temperature for 1-2 hours.

[0066] (5) Primary antibody incubation: After blocking, use tweezers to pick up the NC membrane, rinse it twice in TBST, and place it on a disposable PE glove, avoiding contact with the membrane surface as much as possible. Cut a whole NC membrane into small strips about 5 mm wide, and record the front and back sides of the membrane. Each strip needs to incubate one antibody. Add 1 mL of blocking buffer to each 2 mL EP tube, and dilute all antibodies with blocking buffer at a ratio of 1:50, for a total of 270 antibodies. Incubate overnight at 4°C on a rotary mixer. Positive control: Anti-HBsAg-IgG1 (Innosys, catalog number: A2245), negative control: Anti-A29L-IgG1 (Innosys, catalog number: A2526), ​​1 μg / mL.

[0067] (6) Rinse: Wash 3 times with 1×TBST, 10 minutes each time.

[0068] (7) Secondary antibody incubation: Dilute the secondary antibody Goat anti-Human IgG-Fc Fragment Antibody HRP (Southern Biotech, catalog number: 2048-05) at a ratio of 1:5,000 with PBS, add 1 mL to each EP tube, and incubate at room temperature for 1 hour on a mixer.

[0069] (8) Rinse: Wash 3 times with 1×TBST, 10 minutes each time.

[0070] (9) Color development and exposure: Mix the ultrasensitive ECL luminescent substrate A solution and B solution (Merck, catalog number: WBKLS0100) in a 1:1 ratio, immerse the membrane completely in the color development solution for 1-2 seconds, and expose it on a WB chemiluminescence imaging instrument. The positive target band can be seen to be around 25kD after exposure.

[0071] (10) Data processing standards: The intensity of the immunoblot bands was measured by ImageJ software, and the gray value of each band was calculated. COI was expressed as the ratio of the target protein to the control protein. A ratio greater than 1 was considered a positive antibody.

[0072] Example 1: Antibody Screening

[0073] Peripheral blood was collected from children with hepatitis B. Mononuclear cells were isolated and HBsAg was labeled with Dylight 650 and Dylight 550. HBsAg-DY650 / 550 double-positive B cells were screened. Flow cytometry sorting was performed, and the HBsAg-DY650 / 550 double-positive B cells were preliminarily identified as HBsAg-specific B cells. The flow cytometry phenotypic analysis pathway for PBMCs is as follows: Figure 1 As shown, HBsAg-DY650 / 550 double-positive B cells were circled from total CD3-CD19+ B cells.

[0074] A total of 188 specific HBsAbs were identified by ELISA and Western Blot. All antibodies were engineered to the IgG1 subtype. Through repeated ELISA experiments, 38 of the synthesized antibodies showed strong specificity for HBsAg (Table 1), and 14 antibodies showed strong binding affinity to DTT-treated HBsAg (Table 2). SA1 was originally the IgG3 subtype, and SA2 was the IgG1 subtype; the average COI value of the two modified antibodies reached 47.

[0075] Table 1. 38 antibodies exhibiting strong specificity against HBsAg

[0076] Table 2. 14 antibodies with strong binding affinity to DTT-treated HBsAg

[0077] The binding of antibodies to linear HBsAg was detected by Western blotting. Under denaturing SDS-PAGE electrophoresis, the native conformation of HBsAg was fully opened. Each antibody was then incubated with the linear HBsAg protein, and antibody specificity was identified based on the WB band exposure results. Figure 2 As shown, a total of 182 specific antibodies and 53 non-specific antibodies were identified.

[0078] Example 2: Preparation of murine antibody mSA1

[0079] The target gene of the SA1 antibody obtained through PCR amplification and screening was used to construct a plasmid, which was then transfected into HEK293 cells. After culturing for 6 days, the cell culture medium was centrifuged, and the supernatant was used to extract and purify the protein. The murine antibody mSA1 was then recombined. The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain of the mSA1 antibody are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, while the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are shown in SEQ ID NO.4, QDT, and SEQ ID NO.5, respectively.

[0080] The purified protein was analyzed by SDS-PAGE. Figure 3 The results show the SDS-PAGE analysis of the mSA1 antibody. The results indicate that the mSA1 antibody has an SDS-PAGE purity of 98.7%, a concentration of 1.1 mg / mL, and an extinction coefficient of 1.46 L·mol⁻¹. -1 ·cm -1 .

[0081] Example 3: Verification that enzyme-labeled antibody does not specifically react with blocking solution

[0082] A blocking buffer (5% NFM) prepared with deionized water and skim milk powder at a ratio of 100 mL: 5 g was used as the coating solution for coating. Subsequently, enzyme-labeled antibodies of different subtypes were added for detection to investigate whether the blocking buffer interfered with the enzyme-labeled antibodies. The detection results are as follows: Figure 4 As shown in the figure, the detected OD values ​​were all less than 0.1 (COI value < 1.0), indicating a negative result. This shows that the blocking solution did not specifically react with the enzyme-labeled antibody, and the background values ​​presented by the detection were clean and uncontaminated.

[0083] Figure 5 The dilution of enzyme-labeled antibody is used as the x-axis, and COI (OD) is... 450 The experimental results are plotted using Graphpad software with Sample / Threshold as the ordinate.

[0084] Based on the experimental results, this invention selects 5% NFM blocking solution as the blocking solution for the kit to ensure the stability of the experimental environment and the accuracy of the experimental data.

[0085] Example 4: Verification that human plasma does not specifically react with the blocking solution

[0086] The 5% NFM blocking buffer prepared in Example 3 was used as the coating solution for coating treatment. Then, human plasma at 1 / 100 volume of the blocking buffer was added. Finally, enzyme-labeled antibodies of different subtypes were added for detection to investigate whether the blocking solution affected the plasma. The detection results are plotted with plasma sample as the x-axis, and COI (OD) as the y-axis.450 Using Sample / Threshold as the ordinate, plot the graph using Graphpad software, as shown below. Figure 6 As shown, the results indicate that the detected OD values ​​were all less than 0.1 and the COI values ​​were all less than 1, meaning that no specific reaction occurred between the plasma diluent diluted at a ratio of 1:100 and the blocking solution.

[0087] Based on the experimental results, this invention selects 5% NFM blocking solution as the blocking solution for the kit to ensure the purity of the experimental environment and the accuracy of the experimental data.

[0088] Example 5: Quantitative detection of HBsAg-HBsAb immune complexes using a kit

[0089] Thirty-nine plasma samples were collected from CHB patients before and after treatment. The kit of this invention was applied to the analysis of plasma samples from CHB patients before and after treatment to quantitatively detect HBsAg-HBsAb immune complexes.

[0090] The kit for quantitative detection of HBsAg-HBsAb immune complexes includes a capture antibody mSA1 and a detection antibody anti-human IgG-HRP conjugate; microplate, diluent, blocking buffer, washing buffer, chromogenic solution, and stop solution.

[0091] The microplate is an ELISA 96-well plate.

[0092] The diluent is a PBS solution.

[0093] The washing solution is PBST solution.

[0094] The sealing solution is prepared by mixing deionized water and skim milk powder in a ratio of 100mL:5g.

[0095] The colorimetric solution is prepared by mixing equal volumes of TMB and H2O2.

[0096] The stop solution is an ELISA stop solution.

[0097] The quantitative detection of HBsAg-HBsAb immune complexes using a kit includes the following steps: (1) Coating: Dilute mSA1 to 1 g / L with diluent and add 100 μL / well to the microplate. Incubate at room temperature for 1 h and wash once with washing solution. (2) Blocking: Add blocking solution at 200 μL / well, incubate at room temperature for 2 h, and wash once with washing solution; (3) Add the sample to be tested: Dilute the plasma sample and blocking solution at a volume ratio of 1:100 and add 100 μL / well to the microplate. Incubate at room temperature for 1 h and wash five times with washing solution. (4) Add secondary antibody: Dilute anti-human-IgG1-HRP (Southern Biotech, catalog number: 9052-05), or anti-human-IgG3-HRP (Southern Biotech, catalog number: 9210-05) or anti-human-IgG4-HRP (Southern Biotech, catalog number: 9200-05) with blocking buffer at a volume ratio of 1:1000 and add 100 μL / well to the microplate. Incubate at room temperature for 30 min and wash five times with washing buffer. (5) Color development: Add 100 μL of color development solution to each well and develop color for 3 min in the dark; (6) Termination: Add 50 μL of stop solution per well, and obtain the OD of the sample at 450 nm using a microplate reader or spectrometer. 450 value.

[0098] Figure 7 This embodiment presents the results of quantitative detection of HBsAg-HBsAb immune complexes in different samples. The results show that different patients exhibit unique antigen-antibody complex composition patterns before and after treatment. Among them, antigen-antibody complexes were detectable in patients A, C, E, F, G, and H before and after treatment, and HBsAg decreased or even cleared after treatment. In patient B, IC-IgG4 data was only detected late after treatment, and HBsAg remained at a high level in the early stages. In contrast, patient D had a low HBsAg level, so no complexes were detected.

[0099] Example 6: Detection of HBsAg-HBsAb simulated complex

[0100] This embodiment is based on the HBsAg-HBsAb immune complex detection procedure of Example 5, but replaces the plasma sample with a pre-incubated HBsAg-HBsAb mimic complex for the experiment. Specifically, each well of the HBsAg-HBsAb mimic complex contains 1 μg of recombinant HBsAg (Innox, catalog number: P2303), the first well contains 20 μg of recombinant SA2 antibody, and the second to eighth wells contain 5 μg, 1.25 μg, 0.3125 μg, 0.078125 μg, 0.01953125 μg, 0.004882813 μg, and 0.001220703 μg of recombinant SA2 antibody, respectively.

[0101] The results are as follows Figure 8As shown, the simulated complex results indicate that the dynamic pattern of the antigen-antibody simulated complex conforms to theoretical predictions: when the antibody is supersaturated, HBsAg is recognized by SA2 and cannot be captured by mSA1, so the complex detection signal is low; as the SA2 antibody gradually decreases, the HBsAg epitopes are gradually exposed, and the complex signal gradually increases until the antibody continues to decrease to an extremely low concentration, which cannot be detected by anti-human IgG-HRP, so the complex signal decreases again.

[0102] In summary, the novel double-antibody sandwich ELISA detection platform provided by this invention uses an HBsAg-specific mSA1 antibody as a capture carrier, achieving efficient solid-phase coating through its specific binding with HBsAg. Simultaneously, an anti-human IgG-HRP conjugate is introduced as the detection antibody. Utilizing the specific epitopes of different human IgG subtypes and the catalytic amplification effect of HRP, it enables highly sensitive and specific quantitative detection of HBsAg-HBsAb immune complexes, providing a reliable tool for clinical quantitative detection of HBsAg-HBsAb immune complexes.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A kit for quantitative detection of HBsAg-HBsAb immune complex, characterized in that, The kit comprises a capture antibody and a detection antibody; The capture antibody is mSA1, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of mSA1 are SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 in sequence, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of mSA1 are SEQ ID NO. 4, QDT and SEQ ID NO. 5 in sequence. The detection antibody is an anti-human IgG-HRP conjugate.

2. The kit according to claim 1, characterized in that, The detection antibody is at least one selected from the group consisting of an anti-human IgG1-HRP conjugate, an anti-human IgG3-HRP conjugate and an anti-human IgG4-HRP conjugate.

3. The kit of claim 1, wherein The kit further comprises a micro-well reaction plate, a diluent, a blocking solution, a washing solution, a color developing solution and a termination solution.

4. The kit of claim 3, wherein The diluent is a PBS solution.

5. The kit of claim 3, wherein The washing solution is a PBST solution.

6. The kit of claim 3, wherein The blocking solution is prepared by mixing deionized water and skimmed milk powder at a ratio of 100 mL:5 g.

7. The kit of claim 3, wherein The color developing solution is prepared by mixing TMB and H2O2 in equal volumes.

8. The kit of claim 3, wherein The termination solution is an ELISA termination solution.

9. A method for quantitative detection of HBsAg-HBsAb immune complex using the kit according to any one of claims 1-8, characterized in that, The kit comprises the following steps: (1) The capture antibody is diluted to 0.1-10 g / L with the diluent, and then 100 μL / well is added to the micro-well reaction plate, and incubated at room temperature for 10 min-12 h; (2) 200 μL / well of the blocking solution is added, and incubated at room temperature for 10 min-12 h; (3) The sample to be tested is added to the micro-well reaction plate at 100 μL / well, or diluted with the blocking solution at a volume ratio of 1:(1-1000), and incubated at room temperature for 10 min-4 h; (4) The detection antibody is added to the micro-well reaction plate at 100 μL / well, or diluted with the blocking solution at a volume ratio of 1:(1-50000), and incubated at room temperature for 10 min-2 h; (5) 100 μL / well of the color developing solution is added, and color developed in the dark for 20 s-30 min; (6) Add 50 μL / well of stop solution, and use a microplate reader or a spectrometer to obtain the OD value of the sample at 450 nm wavelength. 450 value.

10. The method of claim 9, wherein, After the incubation of steps (1)-(4), the micro-well reaction plate is washed with the washing solution.

Images