Method for detecting specific activity of recombinant pseudomonas aeruginosa vaccine rPO stock solution

Abstract

The invention discloses a method for detecting the specific activity of a recombinant pseudomonas aeruginosa vaccine rPO stock solution. The method comprises the following steps: (1) coating an elisa plate by using OprI-B052 as a capture antibody; (2) adding a sample to be detected into the coated ELISA plate; (3) diluting the enzyme-labeled antibody PcrV-A039, then carrying out a chromogenic reaction, and measuring the OD450 / 630 nm value; and (4) preparing a standard curve by using the rPO reference, and calculating the specific activity of the rPO antigen in the detected sample according to the standard curve. According to the invention, OprI-B052 is used as a coating antibody, and paired PcrV-A039 is used as a detection antibody. The specific activity of the recombinant pseudomonas aeruginosa vaccine rPO stock solution can be quantitatively detected, and the method has good stability and repeatability.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a method for detecting the specific activity of the rPO stock solution of a recombinant Pseudomonas aeruginosa vaccine. Background Art

[0002] Pseudomonas aeruginosa (PA), commonly known as Pseudomonas aeruginosa, is one of the most common nosocomial infection pathogens clinically. It can cause many acute and chronic infections, and the infections can occur in multiple parts and tissues of the human body, commonly in the respiratory tract, especially the lungs, burn or trauma sites, middle ear, cornea and urethra; it can also cause necrotic skin damage, endocarditis, severe otitis externa and folliculitis, osteochondritis of the foot, gastroenteritis, empyema and even sepsis. In the World Health Organization's list of bacterial pathogens, it is listed in the "critical" category. Despite active antibiotic treatment, the morbidity and mortality rates are as high as 40%, and there is an urgent need to research and develop new prevention and treatment methods.

[0003] A safe and effective PA vaccine is the most practical and economical means for preventing Pseudomonas aeruginosa infection and its drug resistance. The most important core part of the vaccine is the antigen. Pseudomonas aeruginosa V antigen (PcrV) and outer membrane lipoprotein OprI are two widely studied targets in PA vaccine research. PcrV is one of the key proteins of the PA type III secretion system, located at the tip of the type III secretion system, and is a soluble protein. It exists in the cytoplasm of the bacteria and is the core component for the bacteria to secrete and transport toxins. The conformational change of PcrV can regulate the T3SS, causing its structure to change, thereby controlling the secretion of effector proteins. Antibodies against PcrV can protect cultured cells and model animals from Pseudomonas aeruginosa infection, making PcrV a key component of the T3SS and a potential therapeutic target.

[0004] Lipoprotein I (OprI) is conserved and highly immunogenic in PA strains of different serotypes and is also the most widely studied outer membrane protein in PA. In fact, the protein subunit vaccine IC43, which has undergone phase III clinical trials, uses OprF (190 - 342) and OprI (21 - 83) as the antigens of the recombinant protein. OprI plays an important role in the drug resistance of Pseudomonas aeruginosa and is of great significance in the research of Pseudomonas aeruginosa vaccines.

[0005] In the research and development of recombinant protein vaccines, the specific activity of the protein bulk solution is an important quality control index. Specific activity reflects the biological activity possessed by a unit mass of protein. For vaccines, the specific activity of their protein bulk solution is closely related to the efficacy of the vaccine. First, through specific activity detection, the activity level of the active ingredients in the vaccine protein bulk solution can be more accurately evaluated, and then the ability of the vaccine to stimulate the immune response can be inferred. Second, batch consistency control: The specific activity detection of the vaccine protein bulk solution helps to ensure the quality consistency between different batches of vaccines. During large-scale production, due to minor fluctuations in factors such as raw materials and production processes, there may be differences in the vaccine protein bulk solutions of different batches. By strictly detecting and monitoring the specific activity, these differences can be discovered in a timely manner, and corresponding measures can be taken for adjustment to ensure that each batch of vaccines has stable and reliable quality.

[0006] Based on the above problems, the specific activity detection of the vaccine protein bulk solution is of crucial significance in aspects such as the research and development, production, quality control, and safety assessment of vaccines. A detection method has been developed that can accurately reflect the specific activity of the protein bulk solution and ensure the stability and repeatability in the production of the vaccine protein bulk solution. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting the specific activity of the rPO bulk solution of the recombinant Pseudomonas aeruginosa vaccine to ensure the stability and repeatability in the production of the vaccine protein bulk solution.

[0008] To achieve the above purpose, the present invention provides the following technical solutions: A method for detecting the specific activity of the rPO bulk solution of the recombinant Pseudomonas aeruginosa vaccine provided by the present invention includes the following steps: (1) Coating an enzyme-linked immunosorbent assay (ELISA) plate with OprI-B052 as the capture antibody; (2) Adding the sample to be tested to the coated ELISA plate; (3) Diluting the enzyme-labeled antibody PcrV-A039 and performing a color reaction, and measuring the OD 450 / 630 nm value; (4) Preparing a standard curve using the rPO reference product, and calculating the specific activity of the rPO antigen in the sample to be tested according to the standard curve.

[0009] Further, the OD 450 / 630 nm value is the OD 450 nm and OD 630 nm values measured respectively.

[0010] Further, the specific activity of the rPO antigen is considered qualified if it is not less than 0.5×10 6 YU / mg.

[0011] Further, the capture antibody OprI-B052 in (1) was coated overnight at 4°C at a concentration of 6 μg / mL, washed four times with PBST, blocked with 3% BSA, incubated at 37°C for 1 hour, and washed four times with PBST.

[0012] Further, after the test sample in (2) was added to the ELISA plate, it needed to be incubated at 37°C for 60 min.

[0013] Further, the enzyme-labeled antibody PcrV-A039 in (3) was diluted 1000, 2000, 3000, 4000, 5000, 6000 times, incubated at 37°C for 1 hour, developed color at 37°C for 10 minutes after washing, and 50 μL of 2M sulfuric acid was added to terminate the reaction.

[0014] Further, the standard curve in (4) was a curve constructed with the logarithm of the antigen concentration as the abscissa and the logarithm of the OD value as the ordinate.

[0015] Further, when the concentration of the standard curve reference product in (4) was 78.125 - 2500 YU / mL, R 2 = 0.9938.

[0016] Further, the OprI-B052 comprises a heavy chain and a light chain, and the heavy chain and the light chain respectively contain three CDRs, wherein the heavy chain CDR1 is GFTLSSYS, the heavy chain CDR2 is ISSTSSYI, and the heavy chain CDR3 is VRGVDFDY; the light chain CDR1 is QSLLHRNGQKY, the light chain CDR2 is LG, and the light chain CDR3 is MQALQRPVT.

[0017] Further, the heavy chain and the light chain of the OprI-B052 antibody also include FR regions, and the heavy chain and the light chain respectively contain four FR regions, wherein the heavy chain FR1 is EVQLLESGGGLVKPGGSLRLSCAAS, the heavy chain FR2 is MNWVRQAPGKGLEWVSS, the heavy chain FR3 is SYADSVRGRFTISRDNAKKSLFLQMNSLRAEDTAVYYC, and the heavy chain FR4 is WGQGTLVTVSS; the light chain FR1 is DVVMTQSPLSLPVTPGEPASISCRSS, the light chain FR2 is LDWYLQKPGQSPQLLIY, the light chain FR3 is SNRASGVPERFSGSGSGTDFTLKISRVEAEDVGVYYC, and the light chain FR4 is FGPGTTVDIKRTV.

[0018] Further, the PcrV-A039 comprises a heavy chain and a light chain, and each of the heavy chain and the light chain contains three CDRs. Among them, the heavy chain CDR1 is GDTLNNFA, the heavy chain CDR2 is IIPLLGIA, and the heavy chain CDR3 is ATSPVRGIDYGMDV; the light chain CDR1 is QSVSTD, the light chain CDR2 is DA, and the light chain CDR3 is QQRTTWPPMYT.

[0019] Further, the heavy chain and the light chain of the PcrV-A039 antibody further comprise FR regions. Each of the heavy chain and the light chain contains four FR regions. Among them, the heavy chain FR1 is QVQLVQSGPEVKNPGSSVKVSCKAS, the heavy chain FR2 is ISWVRQAPGPGLQWVGG, the heavy chain FR3 is HYSQDFQGRVTITADKSTSTVSMELRSLRSQDTAVYYC, and the heavy chain FR4 is WGQGTTVTVSS; the light chain FR1 is EIVLTQSPATLSLSPGERATLSCRAS, the light chain FR2 is LAWYQQKPGQAPRLLIY, the light chain FR3 is SNRATGIPARFSGSGSGTDFTLTISGLEPEDFAVYFC, and the light chain FR4 is FGQGTKVETKRTV.

[0020] Further, the PBST is 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% v / v Tween-20.

[0021] Based on the above technical solutions, the embodiments of the present invention can at least produce the following technical effects: Using OprI-B052 as the coating antibody and pairing PcrV-A039 as the detection antibody, the specific activity of the recombinant Pseudomonas aeruginosa vaccine rPO stock solution can be detected, and it has good stability and repeatability. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0023] Figure 1 It is the linear verification result diagram of the embodiments of the present invention. Detailed Embodiments

[0024] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0028] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0029] OprI - B052 comprises a heavy chain and a light chain, and the heavy chain and the light chain each contain three CDRs, where the heavy chain CDR1 is GFTLSSYS, the heavy chain CDR2 is ISSTSSYI, and the heavy chain CDR3 is VRGVDFDY; the light chain CDR1 is QSLLHRNGQKY, the light chain CDR2 is LG, and the light chain CDR3 is MQALQRPVT.

[0030] The OprI-B052 antibody heavy and light chains also include FR regions. The heavy and light chains each contain four FR regions. Among them, the heavy chain FR1 is EVQLLESGGGLVKPGGSLRLSCAAS, the heavy chain FR2 is MNWVRQAPGKGLEWVSS, the heavy chain FR3 is SYADSVRGRFTISRDNAKKSLFLQMNSLRAEDTAVYYC, and the heavy chain FR4 is WGQGTLVTVSS; the light chain FR1 is DVVMTQSPLSLPVTPGEPASISCRSS, the light chain FR2 is LDWYLQKPGQSPQLLIY, the light chain FR3 is SNRASGVPERFSGSGSGTDFTLKISRVEAEDVGVYYC, and the light chain FR4 is FGPGTTVDIKRTV.

[0031] PcrV-A039 includes a heavy chain and a light chain. The heavy and light chains each contain three CDRs. Among them, the heavy chain CDR1 is GDTLNNFA, the heavy chain CDR2 is IIPLLGIA, and the heavy chain CDR3 is ATSPVRGIDYGMDV; the light chain CDR1 is QSVSTD, the light chain CDR2 is DA, and the light chain CDR3 is QQRTTWPPMYT.

[0032] The PcrV-A039 antibody heavy and light chains also include FR regions. The heavy and light chains each contain four FR regions. Among them, the heavy chain FR1 is QVQLVQSGPEVKNPGSSVKVSCKAS, the heavy chain FR2 is ISWVRQAPGPGLQWVGG, the heavy chain FR3 is HYSQDFQGRVTITADKSTSTVSMELRSLRSQDTAVYYC, and the heavy chain FR4 is WGQGTTVTVSS; the light chain FR1 is EIVLTQSPATLSLSPGERATLSCRAS, the light chain FR2 is LAWYQQKPGQAPRLLIY, the light chain FR3 is SNRATGIPARFSGSGSGTDFTLTISGLEPEDFAVYFC, and the light chain FR4 is FGQGTKVETKRTV. PBST is 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% v / v Tween-20.

[0033] The CDR naming uses the IMGT nomenclature.

[0034] Example 1 Establishment of the double antibody sandwich ELISA methodology 1. Use the checkerboard method to determine the working concentrations of the coating antibody and the enzyme-labeled antibody (1) Experimental method Dilute the coated antibody OprI-B052 to 1, 4, 6, 8 μg / mL, coat overnight at 4°C with 100 μL per well, wash four times with PBST, block with 100 μL per well of 3% BSA, incubate at 37°C for 1 hour, wash four times with PBST, add rPO antigen at a concentration of 1 μg / mL, incubate at 37°C for 1 hour, dilute the enzyme-labeled antibody PcrV-A039 1000, 2000, 3000, 4000, 5000, 6000 times, incubate at 37°C for 1 hour, after washing, develop color at 37°C for 10 minutes, add 50 μL of 2M sulfuric acid to terminate the reaction, and read the OD 450 / 630nm value. Calculate the P / N (test well / control well) ratio.

[0035] (2)Experimental results Judgment criteria: (1)Calculate the OD ratio of the experimental wells and control wells in each group; (2)If the OD ratio of the experimental well (P) to the control well (N) > 2.1, select the maximum P / N value as the preferred combination.

[0036] As shown in Table 1, the preferred concentration of the capture antibody OprI-B052 is 6 μg / mL, and the enzyme-labeled detection antibody PcrV-A039 is diluted 1:1000 as the combination.

[0037] Table 1 P / N values of capture antibody and detection antibody 2. Establishment of standard curve (1)Experimental method Coat the capture antibody OprI-B052 at a concentration of 6 μg / mL overnight at 4°C, wash four times with PBST, block with 3% BSA, incubate at 37°C for 1 hour, wash four times with PBST, dilute the rPO reference product to 10000 YU / mL as the starting concentration for two-fold serial dilution, incubate at 37°C for 1 hour, wash four times with PBST, dilute the enzyme-labeled PcrV-A039 antibody at 1:1000, incubate at 37°C for 1 hour, wash four times with PBST, add TMB to develop color for 10 minutes, add 50 μL of 2M sulfuric acid to terminate the reaction, and read the OD 450 / 630nm value. Construct a curve with the logarithm of the antigen concentration as the abscissa and the logarithm of the OD value as the ordinate, and obtain the curve equation and correlation coefficient.

[0038] (2)Experimental results When the concentration of the reference product is between 78.125 - 2500 YU / mL, R 2 = 0.9938, showing good linearity and the linearity holds.

[0039] 3. Optimization of standard curve (1) Experimental method The capture antibody OprI-B052 was coated overnight at 4 °C at a concentration of 6 μg / mL, washed three times with PBST, blocked with 3% BSA, incubated at 37 °C for 1 h, washed three times with PBST, the rPO antigen was diluted to 1000, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, 0 YU / mL, incubated at 37 °C for 1 hour, washed three times with PBST, the enzyme-labeled antibody PcrV-A039 was diluted 1:1000, incubated at 37 °C for 1 hour, washed three times with PBST, added TMB for color development for 10 minutes, terminated the color development, and read the OD value on an enzyme-linked immunosorbent assay (ELISA) reader. 450 / 630nm Using the logarithm of the antigen concentration as the abscissa and the logarithm of the OD value as the ordinate, a curve was constructed, and the curve equation and correlation coefficient were obtained.

[0040] (2) Experimental results When the concentration of the reference product was between 25 - 1000 YU / mL, R 2 = 0.9979, showing good linearity and the linearity was established.

[0041] Example 2 Methodology verification 1. Specificity verification (1) Experimental method Verify whether the buffer system of the rPO protein stock solution affects the specific activity detection result. The buffer system of the rPO protein stock solution is 1 mol / L sodium chloride and 10 mmol / L L-histidine.

[0042] Using the rPO protein stock solution buffer, PBST buffer, and rPO stock solution as the test samples respectively, with the concentration range of the reference product 25 - 1000 YU / mL as the standard curve, the specificity verification was carried out according to the steps listed in Example 1.

[0043] (2) Experimental results As shown in Table 2: There was no difference in the OD values between the rPO protein stock solution buffer and the PBST buffer, and no detectable values were found. The specific activity of the rPO stock solution was 0.96*10 450 / 630nm YU / mg, and the recovery rate was 102.5%, indicating that the method is specific for the recognition of rPO antigen and has good specificity. 6

[0044] Table 2 Specificity verification results 2. Repeatability verification (1) Experimental method According to the standard curve and operation steps established in Example 1, the same person took the same test sample for 6 detections.

[0045] (2) Experimental results The relative standard deviation is within 5% (RSD ≤ 15%), indicating that the measured values of the method in the present invention have high repeatability, as shown in Table 3 below.

[0046] Table 3 Intermediate precision verification results 3. Linear verification (1) Experimental method According to the standard curve and operation steps established in Example 1, two personnel respectively detected the same sample at different times. Taking the logarithm of the reference product concentration as the abscissa and the logarithm of the OD value as the ordinate, a function was constructed, and R was calculated. 450 / 630nm 2 .

[0047] (2) Experimental results As Figure 1 shown, the Rs of the six experiments were 0.9917, 0.9899, 0.9912, 0.9923, 0.9979, and 0.9901 respectively. All Rs were greater than 0.98, indicating that the method has good linearity. 2 2

[0048] 4. Accuracy verification (1) Experimental method According to the standard curve and operation steps established in Example 1, the specific activity of the same rPO stock solution was detected 6 times to verify its accuracy. The recovery rates of the six detections were calculated.

[0049] (2) Experimental results As shown in Table 4, when using the established double antibody sandwich ELISA method to detect the specific activity of the same batch of rPO protein stock solution, the recovery rates of the six detections were between 90 - 120%, indicating that the method has high accuracy.

[0050] Table 4 Accuracy verification results Example 3 Detection of specific activity of multiple batches of rPO stock solution (1) Experimental method According to the method and operation steps established in Example 1, the specific activities of six batches of rPO protein stock solutions were detected.

[0051] (2) Experimental results If the specific activity value of the test sample is less than 0.5×10 6 YU / mg, the sample is determined to be unqualified. The detection results of the specific activities of six batches of rPO protein stock solutions were all qualified, and the RSD was less than 10%, indicating that the production processes of the stock solutions of different batches were stable, as shown in Table 5 below. ​​​

[0052] Table 5 Specific activity detection results of rPO protein stock solution in different batches 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements 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 detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine, characterized in that, It includes the following steps: (1) Coat an ELISA plate with OprI-B052 as the capture antibody; (2) Add the sample to be tested to the coated ELISA plate; (3)The enzyme-labeled antibody PcrV-A039 was diluted and subjected to a color reaction to measure the OD 450 / 630 nm value; (4) Prepare a standard curve using the rPO reference product, and calculate the specific activity of rPO antigen in the sample to be tested according to the standard curve.

2. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, wherein The specific activity of the rPO is not less than 0.5×10 6 YU / mg to be qualified.

3. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, In step (1), the capture antibody OprI-B052 is coated overnight at 4 °C at a concentration of 6 μg / mL, washed four times with PBST, blocked with 3% BSA, incubated at 37 °C for 1 hour, and washed four times with PBST.

4. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, In step (2), after adding the sample to be tested to the ELISA plate, it needs to be incubated at 37 °C for 60 min.

5. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, In step (3), the enzyme-labeled antibody PcrV-A039 is diluted 1000, 2000, 3000, 4000, 5000, and 6000 times, incubated at 37 °C for 1 hour, washed, developed at 37 °C for 10 minutes, and 50 μL of 2M sulfuric acid is added to terminate the reaction.

6. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, wherein In step (4), the standard curve is a curve constructed with the logarithm of the antigen concentration as the abscissa and the logarithm of the OD value as the ordinate.

7. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, When the concentration of the standard curve reference product in (4) is between 78.125 - 2500 YU / mL, R 2 = 0.9938.

8. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, The OprI-B052 includes a heavy chain and a light chain, and the heavy chain and the light chain each contain three CDRs. Among them, the heavy chain CDR1 is GFTLSSYS, the heavy chain CDR2 is ISSTSSYI, and the heavy chain CDR3 is VRGVDFDY; The light chain CDR1 is QSLLHRNGQKY, the light chain CDR2 is LG, and the light chain CDR3 is MQALQRPVT.

9. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that, The PcrV-A039 includes a heavy chain and a light chain, and the heavy chain and the light chain each contain three CDRs. Among them, the heavy chain CDR1 is GDTLNNFA, the heavy chain CDR2 is IIPLLGIA, and the heavy chain CDR3 is ATSPVRGIDYGMDV; the light chain CDR1 is QSVSTD, the light chain CDR2 is DA, and the light chain CDR3 is QQRTTWPPMYT.

10. The method for detecting the specific activity of the rPO stock solution of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, wherein The PBST is 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% v / v Tween-20.

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