Method for quantitatively detecting specific activity of HI antigen in recombinant staphylococcus aureus vaccine or stock solution

Abstract

The invention discloses a method for quantitatively detecting the specific activity of an HI antigen in a recombinant staphylococcus aureus vaccine or stock solution. The method comprises the following steps: (1) coating an elisa plate by using IsdB-318 as a capture antibody; (2) adding a sample to be detected into the coated ELISA plate; (3) carrying out a chromogenic reaction by using an enzyme-labeled Hla-26 monoclonal antibody, and determining an OD450 / 630 nm value; and (4) preparing a standard curve by using the HI reference substance, and calculating the specific activity of the HI antigen in the detected sample according to the standard curve. The specific activity of the HI protein in the recombinant staphylococcus aureus vaccine or stock solution can be quantified, and good stability and repeatability are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for quantitatively detecting the specific activity of HI antigen in a recombinant Staphylococcus aureus vaccine or a stock solution. Background Art

[0002] Staphylococcus aureus is a typical Gram-positive bacterium, which is associated with purulent skin infections, pneumonia, endocarditis and severe sepsis in humans. The selection pressure caused by excessive use of antibiotics has led to the emergence and widespread spread of methicillin-resistant Staphylococcus aureus (MRSA), a drug-resistant strain. Currently, most of the Staphylococcus aureus strains detected in clinical practice are drug-resistant strains, among which MRSA strains with broad-spectrum drug resistance are the main ones. MRSA exerts its pathogenicity by invading the host, immune evasion, invading tissues and maintaining 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, which induces interleukin production and cell apoptosis by activating the nucleotide binding domain and leucine-rich repeat gene family and inflammasome. Antibodies targeting α-hemolysin can reduce the virulence of MRSA by inhibiting its activity.

[0004] Staphylococcus aureus needs to continuously acquire nutrients during the process of infecting the host. Trace element iron is one of the important nutrients to be acquired. Iron is also an electron transfer carrier in metabolic activities. There is an iron uptake system composed of multiple proteins on the surface of Staphylococcus aureus. Iron-regulated surface determinant B (lsdB) is one of the members of this system. IsdB is a protein with a cell wall anchoring motif LPXTG. At the site of Staphylococcus aureus infection, as the redox potential changes and the pH changes, iron ions will become oxidized, which is beneficial to the conversion and utilization of iron and provides nutrients for the invading Staphylococcus aureus.

[0005] At present, there is no corresponding detection method developed for the determination of specific activity in recombinant Staphylococcus aureus vaccines and stock solutions. In the development of recombinant Staphylococcus aureus vaccines, Hlα and IsdB genes are recombined to obtain a fusion protein (HI). In order to ensure the quality and effect of the recombinant Staphylococcus aureus vaccine, it is necessary to perform specific activity detection on the component proteins in the vaccine and stock solution, which is used as the detection and release standard. Summary of the invention

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

[0007] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for quantitatively detecting the specific activity of HI antigen in a recombinant Staphylococcus aureus vaccine or a stock solution, comprising the following steps: (1) Coating the ELISA plate with IsdB-318 as the capture antibody; (2) Add the sample to be tested to the coated ELISA plate; (3) Use enzyme-labeled Hla-26 monoclonal antibody for color development and measure OD 450 / 630 nm value; (4) Prepare a standard curve using the HI reference material and calculate the specific activity of HI antigen in the sample under test based on the standard curve.

[0008] Furthermore, the OD 450 / 630 nm The values ​​were measured as OD 450 nm and OD 630 nm value.

[0009] Furthermore, the specific activity of the HI antigen is not less than 0.5×10 6 YU / mg is qualified.

[0010] Furthermore, after the ELISA plate in (1) was coated, it was refrigerated overnight, washed three times with PBST, and blocked with 3% BSA for 1 hour.

[0011] Furthermore, in (2), the plate needs to be washed three times with 1×PBST before the sample is added to the ELISA plate, and the plate needs to be incubated at 37° C. for 60 min after the sample is added to the ELISA plate.

[0012] Furthermore, the enzyme-labeled Hla-26 monoclonal antibody in (3) was added to the ELISA plate and incubated at 37°C for 60 min. After washing the plate three times with PBST, TMB colorimetric solution was added for color development, and 2 M sulfuric acid stop solution was added after color development.

[0013] Furthermore, the standard curve in (4) is based on the concentration of the reference product as the horizontal axis and the OD 450 / 630 nm The double logarithmic function constructed with the values ​​as the ordinate.

[0014] Furthermore, when the concentration of the standard curve reference substance in (4) is 3.125-100 YU / mL, R 2 =0.9931.

[0015] Furthermore, the IsdB-318 antibody includes a heavy chain and a light chain, and the heavy chain and light chain respectively contain three CDRs, wherein the heavy chain CDR1 is GGSLNKYY, the heavy chain CDR2 is IQDSGRT, and the heavy chain CDR3 is ARDRVDSPYSNSWSRFDY; the light chain CDR1 is NLGSKS, the light chain CDR2 is DDD, and the light chain CDR3 is QVWDSTSDVVV.

[0016] Further, the IsdB-318 antibody heavy chain and light chain also include FR regions, and the heavy chain and light chain respectively contain four FR regions, wherein heavy chain FR1 QVQLQESGPGLVKPSETLSLTCTVS, heavy chain FR2 WTWIRQSPNNGLEWIGY, heavy chain FR3 DSNPSLKSRVSISLDTSTNQISLKLTSVAAADTAIYYC, heavy chain FR4 WGQGKLVTVSS; light chain FR1QSVLTQPPSVPVAPGRSAVITCGGH, light chain FR2 VHWYQQKPGQAPVLVVY, light chain FR3 DDRPSGIPARFSGSTSERTATLTISRVEAGDEADYYC, light chain FR4 FGGGTKLAVLGQP.

[0017] Furthermore, the Hla-26 antibody comprises a heavy chain and a light chain, and the heavy chain and the light chain respectively comprise three CDRs, wherein the heavy chain CDR1 is GFSVGDNY, the heavy chain CDR2 is IWSDGSK, and the heavy chain CDR3 is AREWRNYFDY; the light chain CDR1 is ALPKKY, the light chain CDR2 is EDN, and the light chain CDR3 is YSTDISGNLGV.

[0018] Further, the Hla-26 antibody heavy chain and light chain also include FR regions, and the heavy chain and light chain respectively include four FR regions, wherein the heavy chain FR1 EVQLVQSGGGLVQPGGSLRLSCAAS, the heavy chain FR2 MTWVRQAPGKGLEWVSI, the heavy chain FR3 YYADSVQDRFTISRDNSENSLYLHLSSLRSEDTAVYYC, and the heavy chain FR4 WGQGTLVTVSS; the light chain FR1ELELTQPPSVSVSPGQTARITCSGD, the light chain FR2 AYWYQQKSGQAPVLVIY, the light chain FR3 NKRPSGIPERFSGSSTGTMATLTISGAQVEDEADYYC, and the light chain FR4 FGGGTQLTVLRTG.

[0019] Furthermore, the PBST is Na2HPO48 mM, NaCl 0.136 M, KH2PO42 mM, KCl 2.6 mM, and Tween-20 0.05% v / v.

[0020] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects: The specific activity of HI protein in Staphylococcus aureus vaccine or stock solution can be quantified with good stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0022] Figure 1 4 is a linear verification result diagram of an embodiment of the present invention. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The IsdB-318 antibody includes a heavy chain and a light chain, each of which contains three CDRs, wherein the heavy chain CDR1GGSLNKYY, the heavy chain CDR2 IQDSGRT, and the heavy chain CDR3 ARDRVDSPYSNSWSRFDY; the light chain CDR1 NLGSKS, the light chain CDR2 DDD, and the light chain CDR3 QVWDSTSDVVV.

[0029] The heavy chain and light chain of IsdB-318 antibody also include FR regions, and the heavy chain and light chain respectively include four FR regions, wherein heavy chain FR1 QVQLQESGPGLVKPSETLSLTCTVS, heavy chain FR2 WTWIRQSPNNGLEWIGY, heavy chain FR3 DSNPSLKSRVSISLDTSTNQISLKLTSVAAADTAIYYC, heavy chain FR4 WGQGKLVTVSS; light chain FR1 QSVLTQPPSVPVAPGRSAVITCGGH, light chain FR2 VHWYQQKPGQAPVLVVY, light chain FR3 DDRPSGIPARFSGSTSERTATLTISRVEAGDEADYYC, light chain FR4 FGGGTKLAVLGQP.

[0030] The Hla-26 antibody includes a heavy chain and a light chain, each of which comprises three CDRs, wherein heavy chain CDR1GFSVGDNY, heavy chain CDR2 IWSDGSK, heavy chain CDR3 AREWRNYFDY; light chain CDR1 ALPKKY, light chain CDR2 EDN, light chain CDR3 YSTDISGNLGV.

[0031] The Hla-26 antibody heavy chain and light chain also include FR regions, and the heavy chain and light chain respectively include four FR regions, wherein the heavy chain FR1 EVQLVQSGGGLVQPGGSLRLSCAAS, heavy chain FR2 MTWVRQAPGKGLEWVSI, heavy chain FR3 YYADSVQDRFTISRDNSENSLYLHLSSLRSEDTAVYYC, heavy chain FR4 WGQGTLVTVSS; light chain FR1ELELTQPPSVSVSPGQTARITCSGD, light chain FR2 AYWYQQKSGQAPVLVIY, light chain FR3 NKRPSGIPERFSGSSTGTMATLTISGAQVEDEADYYC, light chain FR4 FGGGTQLTVLRTG.

[0032] PBST is Na2HPO48mM, NaCl 0.136M, KH2PO42mM, KCl 2.6mM, Tween-20 0.05%v / v.

[0033] Example 1 Optimization of detection conditions for capture and detection antibodies 1. Experimental methods The capture antibody IsdB-318 was coated at different concentrations at 4°C overnight, washed three times with PBST, and blocked with 3% BSA at 200 μL / well at 37°C for 60 min. Washed three times with PBST, incubated with HI antigen protein at 37°C for 60 min. Washed three times with PBST, the enzyme-labeled Hla-26 monoclonal antibody was diluted at different concentrations, incubated at 37°C for 60 min, and TMB substrate colorimetric solution was added after washing, and color was developed at 37°C for 10 min. 50 μL of 2 M sulfuric acid stop solution was added to each well, and the plate was read at OD 600 at 4 °C. 450 / 630nm reading.

[0034] 2. Experimental results Criteria: (1) Calculate the OD ratio of each experimental well to the control well; (2) The OD ratio of the experimental well (P) to the control well (N) is greater than 2.1. The maximum P / N value is selected as the preferred combination.

[0035] As shown in Table 1, the preferred concentration of capture antibody IsdB-318 is 10 μg / mL, and the enzyme-labeled detection antibody Hla-26 is diluted 1:1000 as the combined working concentration.

[0036] Table 1 P / N values ​​of capture antibody and detection antibody Example 2: Establishment of standard curve 1. Experimental methods IsdB-318 (10 μg / mL) was used as the capture antibody to coat the ELISA plate, 100 μL per well, coated overnight at 4°C, washed three times with PBST, blocked with 3% BSA, 200 μL per well, blocked at 37°C for 1 h. The plate was washed three times with PBST, and the HI reference was diluted to 300, 200, 150, 100, 75, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 YU / mL, 100 μL per well; the negative control was PBST diluent, 2 replicates, incubated at 37°C for 60 min. After washing the plate three times with a plate washer, the enzyme-labeled Hla-26 monoclonal antibody was diluted with diluent at 1:1000, 100 μL per well was added to the ELISA plate, and incubated in a 37°C incubator for 60 min. Wash the plate three times with PBST, add 100 μL of TMB colorimetric solution to each well, place at 37°C in the dark for 10 min, and add 50 μL of 2 M sulfuric acid stop solution to each well. Detect OD with an enzyme reader 450 / 630nm value.

[0037] 2. Experimental results 2.1 Data processing Export detection OD 450 / 630nm Use EXCEL to calculate the OD of different concentrations of reference products 450 / 630nm The logarithm of the reference concentration is used as the horizontal axis, and the OD 450 / 630nm The logarithmic value constructs a double logarithmic function for the ordinate.

[0038] 2.2 Data Analysis When the reference sample concentration is 3.125-100 YU / mL, R 2 =0.9931, which has good linearity and is established.

[0039] Example 3: Methodological Validation 1. Exclusiveness verification (1) Experimental methods Since the recombinant Staphylococcus aureus vaccine is composed of four proteins (HI, MntC, SpA5, and mSEB), in order to evaluate the specific response of the established method to the HI protein and verify the specificity of the method.

[0040] The finished vaccine, other protein mixtures (MntC, SpA5, mSEB), and PBST buffer were used as samples to be tested, respectively, and the concentration range of the reference product 3.125-100 YU / mL was used as the standard curve, and double antibody sandwich ELISA detection was performed according to the steps listed in Example 2. The data analysis was completed with reference to the analysis method shown in Example 2.

[0041] (2) Experimental results As shown in Table 2: OD values ​​of other protein mixtures and PBST buffer 450 / 630 There was no difference in the values, and the detected values ​​were all less than the lowest value of the standard curve. The HI specific activity of the desorbed Staphylococcus aureus vaccine product was 1.1*10 6 YU / mg, and the recovery rate was 102%, indicating that this method is specific for HI antigen protein recognition and has good specificity.

[0042] Table 2 Specificity verification results 2. Repeatability Verification (1) Experimental methods According to the established calibration curve and operation steps, the same person tests the same sample 6 times. Calculate the relative standard deviation (RSD) of the 6 test results of the same sample.

[0043] (2) Experimental results The relative standard deviation is within 10% (RSD≤15%), and the values ​​measured by the method of the present invention have good repeatability, as shown in Table 3.

[0044] Table 3 Repeatability verification results 3. Accuracy Verification (1) Experimental methods According to the established standard curve and operation steps, low concentration 6.25 YU / mL, medium concentration 50 YU / mL, and high concentration 80 YU / mL HI reference products were selected for accuracy verification. The determination was repeated three times, and the recovery rate of three tests at the same concentration was calculated.

[0045] (2) Experimental results As shown in Table 4, the accuracy of low, medium and high HI reference samples detected by the established double antibody sandwich ELISA method was between 85% and 115% for three tests at the same concentration, indicating that the method had good accuracy.

[0046] Table 4 Accuracy verification results 4. Linear Verification (1) Experimental methods The experiment was repeated six times according to the calibration curve and operation steps established in Example 2, with the logarithm of the reference product concentration as the horizontal axis and the OD as the horizontal axis. 450 / 630nm Construct a double logarithmic function with the logarithmic value as the ordinate and calculate R 2 .

[0047] (2) Experimental results like Figure 1 As shown, the R 2They are 0.9832, 0.9915, 0.9909, 0.9867, 0.9825, 0.9918, R 2 All of them are greater than 0.98, and the method has good linearity.

[0048] Example 4: Detection of HI specific activity of multiple batches of finished vaccine products and stock solutions (1) Experimental methods According to the standard curve and operating steps established in Example 2, three batches of vaccine products and three batches of HI stock solutions were tested for specific activity.

[0049] (2) Experimental results If the specific activity of the sample is less than 0.5*10 6 YU / mg, the sample is judged to be unqualified. The HI antigen activity test of the three batches of finished vaccine products was qualified, and the RSD was less than 10%, indicating that the process of different batches of finished products was stable, as shown in Table 4. The HI antigen activity test of the three batches of stock solutions was qualified, and the RSD was less than 10%, indicating that the process of different batches of stock solutions was stable, as shown in Table 5.

[0050] Table 4 Results of HI antigen activity test for finished vaccine products Table 5 HI stock solution antigen specific activity test results Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 stock solution, characterized in that: The following steps are involved: (1) Coating the ELISA plate with IsdB-318 as the capture antibody; (2) Add the sample to be tested to the coated ELISA plate; (3) Use enzyme-labeled Hla-26 monoclonal antibody for color development and measure OD 450 / 630 nm value; (4) Prepare a standard curve using the HI reference material and calculate the specific activity of HI antigen in the sample under test based on the standard curve.

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

3. The method for quantitatively detecting the specific activity of HI antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: After the ELISA plate in (1) was coated, it was refrigerated overnight, washed three times with PBST, and blocked with 3% BSA for 1 hour.

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

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

6. The method for quantitatively detecting the specific activity of HI antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The standard curve in (4) is based on the logarithm of the reference sample concentration as the horizontal axis and the OD 450 / 630 nm The logarithmic function is constructed with the logarithmic value as the ordinate.

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

8. The method for quantitatively detecting the specific activity of HI antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The IsdB-318 antibody includes a heavy chain and a light chain, and the heavy chain and light chain respectively contain three CDRs, wherein the heavy chain CDR1 is GGSLNKYY, the heavy chain CDR2 is IQDSGRT, and the heavy chain CDR3 is ARDRVDSPYSNSWSRFDY; the light chain CDR1 is NLGSKS, the light chain CDR2 is DDD, and the light chain CDR3 is QVWDSTSDVVV.

9. The method for quantitatively detecting the specific activity of HI antigen in a recombinant Staphylococcus aureus vaccine or a stock solution according to claim 1, characterized in that: The Hla-26 antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise three CDRs, wherein the heavy chain CDR1 is GFSVGDNY, the heavy chain CDR2 is IWSDGSK, and the heavy chain CDR3 is AREWRNYFDY; the light chain CDR1 is ALPKKY, the light chain CDR2 is EDN, and the light chain CDR3 is YSTDISGNLGV.

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

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