A method for quantitatively detecting the specific activity of HI antigen in a recombinant staphylococcus aureus vaccine or bulk

Abstract

The application discloses a method for quantitatively detecting specific activity of HI antigen in recombinant staphylococcus aureus vaccine or stock solution, comprising the following steps: (1) coating an enzyme-labeled plate with IsdB-318 as a capture antibody; (2) adding a sample to be measured into the coated enzyme-labeled plate; (3) performing color development reaction by using an enzyme-labeled Hla-26 monoclonal antibody, and measuring OD 450 / 630 nm value; and (4) preparing a standard curve by using an HI reference product, and calculating specific activity of the HI antigen in the sample according to the standard curve. The application can quantitatively detect specific activity of the HI protein in the recombinant staphylococcus aureus vaccine or stock solution, and has good stability and repeatability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for quantitatively detecting the specific activity of HI antigen in recombinant Staphylococcus aureus vaccine or stock solution. Background Technology

[0002] Staphylococcus aureus is a typical Gram-positive bacterium associated with purulent skin infections, pneumonia, endocarditis, and severe sepsis in humans. The selective pressure caused by antibiotic overuse has led to the emergence and widespread transmission of methicillin-resistant Staphylococcus aureus (MRSA). Currently, most Staphylococcus aureus strains detected clinically are drug-resistant, with broadly resistant MRSA strains being the most prevalent. MRSA exerts its pathogenicity through host invasion, immune evasion, tissue invasion, and maintenance of infection, and this process is closely related to the various virulence factors it produces.

[0003] α-Hemolysin (Hlα) is one of the important virulence factors of Staphylococcus aureus. It induces interleukin production and apoptosis by activating nucleotide-binding domains and gene families rich in leucine repeat sequences, as well as inflammasomes. Antibodies targeting α-hemolysin can reduce the virulence of MRSA by inhibiting its activity.

[0004] Staphylococcus aureus requires a constant supply of nutrients during its infection of the host, and iron is one of the essential trace elements it needs. Iron also acts as an electron transporter in metabolic activities. A multi-protein iron uptake system exists on the surface of Staphylococcus aureus, and iron-regulated surface determinant B (ISdB) is one member of this system. ISdB is a protein carrying the cell wall anchoring motif LPTTG. At the site of Staphylococcus aureus infection, changes in redox potential and pH cause iron ions to oxidize, thus facilitating iron conversion and utilization and providing nutrients for the invading Staphylococcus aureus.

[0005] Currently, there are no corresponding detection methods developed for determining the specific activity of recombinant Staphylococcus aureus vaccines and their original proteins. During the development of recombinant Staphylococcus aureus vaccines, the Hlα and IsdB genes are recombinated to obtain a fusion protein (HI). To ensure the quality and efficacy of the recombinant Staphylococcus aureus vaccine, it is necessary to detect the specific activity of the component proteins in the vaccine and its original solution, using this as a release standard. Summary of the Invention

[0006] The purpose of this invention is to provide a method for quantitatively detecting the specific activity of HI antigen in recombinant Staphylococcus aureus vaccine or stock solution, so as to solve the problem that there are few existing methods for detecting the specific activity of Staphylococcus aureus vaccine antigen.

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

[0008] This invention provides a method for quantitatively detecting the specific activity of HI antigen in recombinant Staphylococcus aureus vaccine or stock solution, comprising the following steps:

[0009] (1) The enzyme-labeled plate was coated with IsdB-318 as the capture antibody;

[0010] (2) Add the sample to be tested into the coated ELISA plate;

[0011] (3) Use enzyme-labeled Hla-26 antibody to perform a colorimetric reaction and measure OD. 450 / 630 nm value;

[0012] (4) Prepare a standard curve using HI reference standard, and calculate the specific activity of HI antigen in the test sample based on the standard curve.

[0013] Furthermore, the OD 450 / 630 nm The values ​​were determined by measuring OD. 450 nm and OD 630 nm value.

[0014] Furthermore, the specific activity of the HI antigen is not less than 0.5 × 10⁻⁶. 6 The YU / mg level is within acceptable limits.

[0015] Furthermore, in step (1), after the enzyme-labeled plate is coated, it is refrigerated overnight, washed three times with PBST, and blocked with 3% BSA for 1 hour.

[0016] Furthermore, in step (2), the sample to be tested needs to be washed three times with 1×PBST before being added to the ELISA plate, and the sample to be tested needs to be incubated at 37°C for 60 min after being added to the ELISA plate.

[0017] Furthermore, in step (3), the enzyme-labeled Hla-26 antibody is added to the ELISA plate and incubated at 37°C for 60 min. After washing the plate three times with PBST, TMB chromogenic solution is added for color development. After color development, 2 M sulfuric acid stop solution is added.

[0018] Furthermore, in (4), the standard curve uses the reference concentration as the abscissa and OD as the ordinate. 450 / 630 nm The value is a double logarithmic function constructed from the ordinate.

[0019] Furthermore, in (4), when the concentration of the standard curve reference is between 3.125 and 100 YU / mL, R 2 =0.9931.

[0020] Furthermore, the IsdB-318 antibody comprises a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1 GGSLNKYY, heavy chain CDR2 IQDSGRT, and heavy chain CDR3 ARDRVDSPYSNSWSRFDY; light chain CDR1 NLGSKS, light chain CDR2 DDD, and light chain CDR3 QVWDSTSDVVV.

[0021] Furthermore, the IsdB-318 antibody heavy and light chains also include FR regions, with each heavy and light chain containing four FR regions: heavy chain FR1 QVQLQESGPGLVKPSETLSLTCTVS, heavy chain FR2 WTWIRQSPNNGLEWIGY, heavy chain FR3 DSNPSLKSRVSISLDTSTNQISLKLTSVAAADTAIYYC, and heavy chain FR4 WGQGKLVTVSS; light chain FR1 QSVLTQPPSVPVAPGRSAVITCGGH, light chain FR2 VHWYQQKPGQAPVLVVY, light chain FR3 DDRPSGIPARFSGSTSERTATLTISRVEAGDEADYYC, and light chain FR4 FGGGTKLAVLGQP.

[0022] Furthermore, the Hla-26 antibody comprises a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1 GFSVGDNY, heavy chain CDR2 IWSDGSK, and heavy chain CDR3 AREWRNYFDY; and light chain CDR1 ALPKKY, light chain CDR2 EDN, and light chain CDR3 YSTDISGNLGV.

[0023] Furthermore, the Hla-26 antibody heavy and light chains also include FR regions, each containing four FR regions: heavy chain FR1 EVQLVQSGGGLVQPGGSLRLSCAAS, heavy chain FR2 MTWVRQAPGKGLEWVSI, heavy chain FR3 YYADSVQDRFTISRDNSENSLYLHLSSLRSEDTAVYYC, and heavy chain FR4 WGQGTLVTVSS; light chain FR1ELELTQPPSVSVSPGQTARITCSGD, light chain FR2 AYWYQQKSGQAPVLVIY, light chain FR3 NKRPSGIPERFSGSSTGTMATLTISGAQVEDEADYYC, and light chain FR4 FGGGTQLTVLRTG.

[0024] Furthermore, the PBST is composed of Na2HPO4 8 mM, NaCl 0.136M, KH2PO4 2 mM, KCl 2.6 mM, and Tween-20 0.05% v / v.

[0025] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0026] It can quantify the specific activity of HI protein in Staphylococcus aureus vaccine or stock solution, and has good stability and repeatability. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this 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 this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0028] Figure 1 This is a graph showing the linear verification results of an embodiment of the present invention. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] The IsdB-318 antibody comprises a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1 GGSLNKYY, heavy chain CDR2 IQDSGRT, and heavy chain CDR3 ARDRVDSPYSNSWSRFDY; and light chain CDR1 NLGSKS, light chain CDR2 DDD, and light chain CDR3 QVWDSTSDVVV.

[0035] The IsdB-318 antibody heavy and light chains also include FR regions, each containing four FR regions: heavy chain FR1 QVQLQESGPGLVKPSETLSLTCTVS, heavy chain FR2 WTWIRQSPNNGLEWIGY, heavy chain FR3 DSNPSLKSRVSISLDTSTNQISLKLTSVAAADTAIYYC, and heavy chain FR4 WGQGKLVTVSS; light chain FR1 QSVLTQPPSVPVAPGRSAVITCGGH, light chain FR2 VHWYQQKPGQAPVLVVY, light chain FR3 DDRPSGIPARFSGSTSERTATLTISRVEAGDEADYYC, and light chain FR4 FGGGTKLAVLGQP.

[0036] The Hla-26 antibody comprises a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1 GFSVGDNY, heavy chain CDR2 IWSDGSK, and heavy chain CDR3 AREWRNYFDY; and light chain CDR1 ALPKKY, light chain CDR2 EDN, and light chain CDR3 YSTDISGNLGV.

[0037] The Hla-26 antibody heavy and light chains also include FR regions, each containing four FR regions: heavy chain FR1 EVQLVQSGGGLVQPGGSLRLSCAAS, heavy chain FR2 MTWVRQAPGKGLEWVSI, heavy chain FR3 YYADSVQDRFTISRDNSENSLYLHLSSLRSEDTAVYYC, and heavy chain FR4 WGQGTLVTVSS; light chain FR1ELELTQPPSVSVSPGQTARITCSGD, light chain FR2 AYWYQQKSGQAPVLVIY, light chain FR3 NKRPSGIPERFSGSSTGTMATLTISGAQVEDEADYYC, and light chain FR4 FGGGTQLTVLRTG.

[0038] PBST is Na2HPO4 8 mM, NaCl 0.136M, KH2PO4 2mM, KCl 2.6 mM, Tween-20 0.05% v / v.

[0039] Example 1

[0040] Optimization of detection conditions for antibody capture and detection

[0041] 1. Experimental Methods

[0042] Capture antibody IsdB-318 was coated at different concentrations and incubated overnight at 4°C. After washing three times with PBST, 200 μL / well was blocked with 3% BSA at 37°C for 60 min. After washing three times with PBST, the HI antigen protein was incubated at 37°C for 60 min. After washing three times with PBST, enzyme-labeled Hla-26 antibody was diluted at different concentrations and incubated at 37°C for 60 min. After washing, TMB substrate chromogenic solution was added, and the mixture was incubated at 37°C for 10 min. 50 μL of 2 M sulfuric acid stop solution was added to each well, and the mixture was then transferred to an OD platen microplate reader. 450 / 630nm reading.

[0043] 2. Experimental Results

[0044] Judgment criteria:

[0045] (1) Calculate the OD ratio of each experimental well to the control well;

[0046] (2) The OD ratio of the experimental well (P) to the control well (N) is >2.1. The maximum value of P / N is selected as the preferred combination.

[0047] As shown in Table 1, the preferred working concentrations are 10 μg / mL for the capture antibody IsdB-318 and 1:1000 for the enzyme-labeled detection antibody Hla-26.

[0048] Table 1. P / N values ​​of capture antibody and detection antibody

[0049]

[0050] Example 2: Establishment of the Standard Curve

[0051] 1. Experimental Methods

[0052] ELISA plates were coated with IsdB-318 (10 μg / mL) as the capture antibody, 100 μL per well, and incubated overnight at 4°C. The plates were washed three times with PBST and blocked with 200 μL of 3% BSA per well at 37°C for 1 h. After washing three times with PBST, HI reference solutions were diluted to 300, 200, 150, 100, 75, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 YU / mL, 100 μL per well. The negative control was PBST diluted solution, with two replicates, and incubated at 37°C for 60 min. After washing three times with a plate washer, the enzyme-labeled Hla-26 antibody was diluted 1:1000 with diluent, 100 μL per well, and incubated at 37°C for 60 min. Wash the plate three times with PBST, add 100 μL of TMB chromogenic buffer to each well, incubate at 37°C in the dark for 10 min, and then add 50 μL of 2 M sulfuric acid stop solution to each well. OD values ​​are measured using a microplate reader. 450 / 630 nm value.

[0053] 2. Experimental Results

[0054] 2.1 Data Processing

[0055] Export OD detection 450 / 630nm Values ​​were calculated using Excel for the OD values ​​of reference standards at different concentrations. 450 / 630nm Values, plotted with the logarithm of the reference concentration on the x-axis and OD value on the y-axis. 450 / 630nm Construct a double logarithmic function with the logarithmic value as the ordinate.

[0056] 2.2 Data Analysis

[0057] When the reference concentration is between 3.125-100 YU / mL, R 2 =0.9931, which has good linearity, and the linearity holds.

[0058] Example 3: Methodological Validation

[0059] 1. Specificity verification

[0060] (1) Experimental methods

[0061] Since the recombinant Staphylococcus aureus vaccine is composed of four proteins (HI, MntC, SpA5, and mSEB), this study aims to evaluate the specific response of the established method to the HI protein and verify the specificity of the method.

[0062] The finished vaccine, other protein mixtures (MntC, SpA5, mSEB), and PBST buffer were used as test samples. A double-antibody sandwich ELISA was performed using a reference standard concentration range of 3.125-100 YU / mL, following the steps outlined in Example 2. Data analysis was completed using the analytical method described in Example 2.

[0063] (2) Experimental results

[0064] Table 2 shows the OD values ​​of other protein mixtures and PBST buffer. 450 / 630 The values ​​showed no difference, and all detected values ​​were lower than the lowest value of the standard curve. The HI specific activity of the desorbed Staphylococcus aureus vaccine product was 1.1*10. 6 The recovery rate was 102% (YU / mg), indicating that the method is specific for the recognition of HI antigen protein and has good specificity.

[0065] Table 2 Specificity Validation Results

[0066]

[0067] 2. Repeatability verification

[0068] (1) Experimental methods

[0069] Following the established standard curve and operating procedures, the same personnel performed six tests on the same sample. The relative standard deviation (RSD) of the six test results for the same sample was calculated.

[0070] (2) Experimental results

[0071] The relative standard deviation is within 10% (RSD≤15%), and the values ​​measured by the method of this invention have good repeatability, as shown in Table 3.

[0072] Table 3. Repeatability verification results

[0073]

[0074] 3. Accuracy Verification

[0075] (1) Experimental methods

[0076] Following the established standard curve and operating procedures, accuracy was validated using HI reference standards at low concentrations of 6.25 YU / mL, medium concentrations of 50 YU / mL, and high concentrations of 80 YU / mL. The measurements were repeated three times, and the recoveries for the same concentration were calculated.

[0077] (2) Experimental results

[0078] As shown in Table 4, the accuracy of the established double-antibody sandwich ELISA method for detecting low, medium, and high HI reference standards was good. The recovery rate of the same concentration in three tests was between 85% and 115%, indicating that the method has good accuracy.

[0079] Table 4. Accuracy Verification Results

[0080]

[0081] 4. Linearity Validation

[0082] (1) Experimental methods

[0083] Following the standard curve and operating procedures established in Example 2, the experiment was repeated six times. The logarithm of the reference concentration was plotted on the x-axis, and the OD value was plotted on the y-axis. 450 / 630nm Construct a double logarithmic function with the logarithmic value as the ordinate and calculate R. 2 .

[0084] (2) Experimental results

[0085] like Figure 1 As shown, R in six experiments 2 The values ​​are 0.9832, 0.9915, 0.9909, 0.9867, 0.9825, and 0.9918 respectively, R 2 All values ​​are greater than 0.98, indicating that the method exhibits good linearity.

[0086] Example 4: HI specific activity test of multiple batches of finished vaccine products and raw materials

[0087] (1) Experimental methods

[0088] Following the standard curve and operating procedures established in Example 2, specific activity tests were performed on three batches of finished vaccine products and three batches of HI stock solution.

[0089] (2) Experimental results

[0090] If the specific activity value of the sample is less than 0.5*10 6 If the concentration of YU / mg is less than 10%, the sample is deemed unqualified. All three batches of finished vaccine products passed the HI antigen specific activity test, with an RSD of less than 10%, indicating stable processing across different batches, as shown in Table 4. All three batches of raw material solutions also passed the HI antigen specific activity test, with an RSD of less than 10%, indicating stable processing across different batches, as shown in Table 5.

[0091] Table 4 Results of HI antigen specificity test in finished vaccine products

[0092]

[0093] Table 5 Results of HI stock antigen specific activity assay

[0094]

[0095] Finally, it should be noted that:

[0096] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitatively detecting the specific activity of HI antigen in a recombinant Staphylococcus aureus vaccine or bulk, characterized by, Includes the following steps: (1) The enzyme-labeled plate was coated with IsdB-318 antibody as the capture antibody; (2) Add the sample to be tested into the coated ELISA plate; (3) The color reaction was performed using enzyme-labeled Hla-26 antibody, and OD values were measured 450 / 630 nm values were measured (4) A standard curve was prepared using the HI reference standard, and the specific activity of the HI antigen in the test sample was calculated based on the standard curve. The HI antigen is a fusion protein formed by the recombination of IsdB and Hlα. The IsdB-318 capture antibody includes a heavy chain and a light chain, and the heavy chain and light chain each contain three CDRs. The amino acid sequence of heavy chain CDR1 is GGSLNKYY, the amino acid sequence of heavy chain CDR2 is IQDSGRT, and the amino acid sequence of heavy chain CDR3 is... The amino acid sequence is ARDRVDSPYSNSWSRFDY; the amino acid sequence of the light chain CDR1 is NLGSKS, the amino acid sequence of the light chain CDR2 is DDD, and the amino acid sequence of the light chain CDR3 is QVWDSTSDVVV; the Hla-26 antibody comprises a heavy chain and a light chain, each containing three CDRs, wherein the amino acid sequence of the heavy chain CDR1 is GFSVGDNY, the amino acid sequence of the heavy chain CDR2 is IWSDGSK, and the amino acid sequence of the heavy chain CDR3 is AREWRNYFDY; the amino acid sequence of the light chain CDR1 is ALPKKY, the amino acid sequence of the light chain CDR2 is EDN, and the amino acid sequence of the light chain CDR3 is YSTDISGNLGV.

2. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, The specific activity of the HI antigen is not less than 0.5 x 10 6 YU / mg is qualified.

3. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, After coating the enzyme-labeled plate in (1), it was refrigerated overnight, washed three times with PBST, and blocked with 3% BSA for 1 hour.

4. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, In step (2), the sample to be tested needs to be washed three times with 1×PBST before being added to the ELISA plate, and the sample to be tested needs to be incubated at 37°C for 60 min after being added to the ELISA plate.

5. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, In step (3), the enzyme-labeled Hla-26 antibody was added to the ELISA plate and incubated at 37°C for 60 min. After washing the plate three times with PBST, TMB chromogenic solution was added for color development. After color development, 2 M sulfuric acid stop solution was added.

6. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, The standard curve in the (4) is constructed with the logarithm value of the reference substance concentration as the horizontal coordinate and OD 450 / 630 nm The double logarithm function constructed with the logarithm value as the vertical coordinate.

7. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, The standard curve reference sample concentration in the (4) is 3.125-100 YU / mL, R 2 =0.9931.

8. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to any one of claims 3-5, characterized in that, The PBST contained 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% v / v Tween-20.

9. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, The IsdB-318 antibody heavy and light chains also include FR regions. Each heavy and light chain contains four FR regions: the heavy chain FR1 amino acid sequence is QVQLQESGPGLVKPSETLSLTCTVS, the heavy chain FR2 amino acid sequence is WTWIRQSPNNGLEWIGY, the heavy chain FR3 amino acid sequence is DSNPSLKSRVSISLDTSTNQISLKLTSVAAADTAIYYC, and the heavy chain FR4 amino acid sequence is WGQGKLVTVSS; the light chain FR1 amino acid sequence is QSVLTQPPSVPVAPGRSAVITCGGH, the light chain FR2 amino acid sequence is VHWYQQKPGQAPVLVVY, the light chain FR3 amino acid sequence is DDRPSGIPARFSGSTSERTATLTISRVEAGDEADYYC, and the light chain FR4 amino acid sequence is FGGGTKLAVLGQP.

10. The method for quantitative detection of HI antigen specific activity in recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, The Hla-26 antibody heavy and light chains also include FR regions. Each heavy and light chain contains four FR regions: the heavy chain FR1 amino acid sequence is EVQLVQSGGGLVQPGGSLRLSCAAS, the heavy chain FR2 amino acid sequence is MTWVRQAPGKGLEWVSI, the heavy chain FR3 amino acid sequence is YYADSVQDRFTISRDNSENSLYLHLSSLRSEDTAVYYC, and the heavy chain FR4 amino acid sequence is WGQGTLVTVSS; the light chain FR1 amino acid sequence is ELELTQPPSVSVSPGQTARITCSGD, the light chain FR2 amino acid sequence is AYWYQQKSGQAPVLVIY, the light chain FR3 amino acid sequence is NKRPSGIPERFSGSSTGTMATLTISGAQVEDEADYYC, and the light chain FR4 amino acid sequence is FGGGTQLTVLRTG.

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