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

Abstract

The application discloses a method for detecting specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution, and comprises the following steps: (1) using OprI-B052 as a capture antibody to coat an enzyme-labeled plate; (2) adding a sample to be detected into the coated enzyme-labeled plate; (3) diluting an enzyme-labeled antibody PcrV-A039 and then performing a color reaction to measure an OD 450 / 630 nm value; and (4) preparing a standard curve by using a rPO reference product, and calculating the specific activity of the rPO antigen in the sample according to the standard curve. In the application, OprI-B052 is used as a coating antibody, and 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 application has good stability and repeatability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution. Background Technology

[0002] Pseudomonas aeruginosa (PA), commonly known as Pseudomonas aeruginosa, is one of the most common nosocomial pathogens in clinical practice. It can cause a variety of acute and chronic infections, occurring in multiple sites and tissues of the body, commonly in the respiratory tract, especially the lungs; burn or wound sites; the middle ear; the cornea; and the urethra. It can also cause necrotic skin lesions, 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 classified as "critical," and despite aggressive antibiotic treatment, the morbidity and mortality rates are as high as 40%, highlighting the urgent need for research and development of new prevention and control methods.

[0003] Safe and effective Pseudomonas aeruginosa (PA) vaccines are the most practical and economical means of preventing Pseudomonas aeruginosa infection and its drug resistance. The most important core component of a 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 a key protein in the PA type III secretion system, located in the needle tip region of the type III secretion system. It is a soluble protein present in the bacterial cytoplasm and is a core component for bacterial secretion and toxin transport. Conformational changes in PcrV can regulate the T3SS, altering its structure and thus controlling the secretion of effector proteins. Antibodies against PcrV can protect cultured cells and animal models from Pseudomonas aeruginosa infection, making PcrV a key component of T3SS and a potential therapeutic target.

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

[0005] In the development of recombinant protein vaccines, the specific activity of the protein stock solution is a crucial quality control indicator, reflecting the biological activity of a unit mass of protein. For vaccines, the specific activity of the protein stock solution is closely related to vaccine efficacy. First, by detecting specific activity, the activity level of the active ingredient in the vaccine protein stock solution can be more accurately assessed, thereby inferring the vaccine's ability to elicit an immune response. Second, batch consistency control: Specific activity testing of vaccine protein stock solutions helps ensure the quality consistency between different batches of vaccines. During large-scale production, slight fluctuations in raw materials, production processes, and other factors may lead to differences between different batches of vaccine protein stock solutions. Rigorous testing and monitoring of specific activity can promptly identify these differences and implement appropriate adjustments, ensuring that each batch of vaccine has stable and reliable quality.

[0006] Based on the above issues, the specific activity test of vaccine protein stock solution is of vital importance in vaccine research and development, production, quality control and safety assessment. A detection method has been developed that can accurately reflect the specific activity of protein stock solution, ensuring the stability and repeatability of vaccine protein stock solution production. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution, so as to ensure the stability and repeatability of vaccine protein stock solution production.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution, comprising the following steps:

[0010] (1) The enzyme-labeled plate was coated with OprI-B052 as the capture antibody;

[0011] (2) Add the sample to be tested into the coated ELISA plate;

[0012] (3) The enzyme-labeled antibody PcrV-A039 was diluted and subjected to a colorimetric reaction to determine the OD. 450 / 630 nm value;

[0013] (4) Prepare a standard curve using rPO reference, and calculate the specific activity of rPO antigen in the test sample based on the standard curve.

[0014] Furthermore, the OD 450 / 630 nm The values ​​were determined by measuring OD. 450 nm and OD 630 nm value.

[0015] Furthermore, the specific activity of the rPO antigen is not less than 0.5 × 10⁻⁶. 6 The YU / mg level is within acceptable limits.

[0016] Furthermore, the capture antibody OprI-B052 in (1) was coated at a concentration of 6 μg / mL overnight at 4°C, washed four times with PBST, blocked with 3% BSA, incubated at 37°C for 1 hour, and washed four times with PBST.

[0017] Furthermore, in step (2), the sample to be tested needs to be incubated at 37°C for 60 min after being added to the ELISA plate.

[0018] Furthermore, in step (3), the enzyme-labeled antibody PcrV-A039 was diluted 1000, 2000, 3000, 4000, 5000, and 6000 times, incubated at 37°C for 1 hour, washed, and then developed at 37°C for 10 minutes. Finally, 50 μL of 2M sulfuric acid was added to terminate the reaction.

[0019] Furthermore, the standard curve in (4) is constructed with the logarithm of antigen concentration as the abscissa and the logarithm of OD value as the ordinate.

[0020] Furthermore, when the standard curve reference concentration in (4) is between 78.125 and 2500 YU / mL, R 2 =0.9938.

[0021] Furthermore, the Opri-B052 includes a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1 GFTLSSYS, heavy chain CDR2 ISSTSSYI, and heavy chain CDR3 VRGVDFDY; and light chain CDR1 QSLLHRNGQKY, light chain CDR2 LG, and light chain CDR3 MQALQRPVT.

[0022] Furthermore, the OprI-B052 antibody heavy and light chains also include FR regions, with each heavy and light chain containing four FR regions: heavy chain FR1 EVQLLESGGGLVKPGGSLRLSCAAS, heavy chain FR2 MNWVRQAPGKGLEWVSS, heavy chain FR3 SYADSVRGRFTISRDNAKKSLFLQMNSLRAEDTAVYYC, and heavy chain FR4 WGQGTLVTVSS; light chain FR1 DVVMTQSPLSLPVTPGEPASISCRSS, light chain FR2 LDWYLQKPGQSPQLLIY, light chain FR3 SNRASGVPERFSGSGSGTDFTLKISRVEAEDVGVYYC, and light chain FR4 FGPGTTVDIKRTV.

[0023] Furthermore, the PcrV-A039 includes a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1 GDTLNNFA, heavy chain CDR2 IIPLLGIA, and heavy chain CDR3 ATSPVRGIDYGMDV; light chain CDR1 QSVSTD, light chain CDR2 DA, and light chain CDR3 QQRTTWPPMYT.

[0024] Furthermore, the PcrV-A039 antibody heavy and light chains also include FR regions, with each heavy and light chain containing four FR regions: heavy chain FR1 QVQLVQSGPEVKNPGSSVKVSCKAS, heavy chain FR2 ISWVRQAPGPGLQWVGG, heavy chain FR3 HYSQDFQGRVTITADKSTSTVSMELRSLRSQDTAVYYC, and heavy chain FR4 WGQGTTVTVSS; light chain FR1 EIVLTQSPATLSLSPGERATLSCRAS, light chain FR2 LAWYQQKPGQAPRLLIY, light chain FR3 SNRATGIPARFSGSGSGTDFTLTISGLEPEDFAVYFC, and light chain FR4 FGQGTKVETKRTV.

[0025] 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.

[0026] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0027] OprI-B052 was used as the coating antibody, and PcrV-A039 was used as the detection antibody. This method can detect the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution, exhibiting good stability and reproducibility. Attached Figure Description

[0028] 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.

[0029] Figure 1 This is a graph showing the linear verification results of an embodiment of the present invention. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] OprI-B052 includes a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1GFTLSSYS, heavy chain CDR2 ISSTSSYI, and heavy chain CDR3 VRGVDFDY; and light chain CDR1 QSLLHRNGQKY, light chain CDR2LG, and light chain CDR3 MQALQRPVT.

[0036] The OprI-B052 antibody heavy and light chains also include FR regions, each containing four FR regions: heavy chain FR1 EVQLLESGGGLVKPGGSLRLSCAAS, heavy chain FR2 MNWVRQAPGKGLEWVSS, heavy chain FR3 SYADSVRGRFTISRDNAKKSLFLQMNSLRAEDTAVYYC, and heavy chain FR4 WGQGTLVTVSS; light chain FR1 DVVMTQSPLSLPVTPGEPASISCRSS, light chain FR2 LDWYLQKPGQSPQLLIY, light chain FR3 SNRASGVPERFSGSGSGTDFTLKISRVEAEDVGVYYC, and light chain FR4 FGPGTTVDIKRTV.

[0037] PcrV-A039 includes a heavy chain and a light chain, each containing three CDRs: heavy chain CDR1GDTLNNFA, heavy chain CDR2IIPLLGIA, and heavy chain CDR3ATSPRVRGIDYGMDV; and light chain CDR1QSVSTD, light chain CDR2DA, and light chain CDR3QQRTTWPPMYT.

[0038] The PcrV-A039 antibody heavy and light chains also include FR regions. Each heavy and light chain contains four FR regions: FR1 QVQLVQSGPEVKNPGSSVKVSCKAS, FR2 ISWVRQAPGPGLQWVGG, FR3 HYSQDFQGRVTITADKSTSTVSMELRSLRSQDTAVYYC, and FR4 WGQGTTVTVSS; FR1 EIVLTQSPATLSLSPGERATLSCRAS, FR2 LAWYQQKPGQAPRLLIY, FR3 SNRATGIPARFSGSGSGTDFTLTISGLEPEDFAVYFC, and FR4 FGQGTKVETKRTV. PBST is composed of Na2HPO4 8 mM, NaCl 0.136 M, KH2PO4 2 mM, KCl 2.6 mM, and Tween-20 0.05% v / v.

[0039] The CDR is named using IMGT.

[0040] Example 1

[0041] Establishment of a double-antibody sandwich ELISA methodology

[0042] 1. The working concentrations of the coating antibody and enzyme-labeled antibody were determined using the checkerboard method.

[0043] (1) Experimental methods

[0044] Dilute the coating antibody OprI-B052 to 1, 4, 6, and 8 μg / mL, and add 100 μL to each well for overnight coating at 4°C. Wash four times with PBST, block with 100 μL of 3% BSA per well, and incubate at 37°C for 1 hour. Wash four times with PBST, add 1 μg / mL of rPO antigen, and incubate at 37°C for 1 hour. Dilute the enzyme-labeled antibody PcrV-A039 1000, 2000, 3000, 4000, 5000, and 6000 times, and incubate at 37°C for 1 hour. After washing, develop the color at 37°C for 10 minutes, add 50 μL of 2M sulfuric acid to stop the reaction, and record the results on an OD500 microplate reader. 450 / 630nm Read the readings. Calculate the P / N (test well / control well) ratio.

[0045] (2) Experimental results

[0046] Judgment criteria:

[0047] (1) Calculate the OD ratio of each experimental well to the control well;

[0048] (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.

[0049] As shown in Table 1, the preferred combination is a capture antibody OprI-B052 at a concentration of 6 μg / mL and an enzyme-labeled detection antibody PcrV-A039 diluted 1:1000.

[0050] Table 1. P / N values ​​of capture antibody and detection antibody

[0051]

[0052] 2. Establishment of the standard curve

[0053] (1) Experimental methods

[0054] The capture antibody OprI-B052 was coated at a concentration of 6 μg / mL and incubated overnight at 4°C. After washing four times with PBST, the mixture was blocked with 3% BSA and incubated at 37°C for 1 hour. After washing four times with PBST, rPO reference was diluted to 10000 YU / mL as the starting concentration and serially diluted 2-fold. The mixture was incubated at 37°C for 1 hour and washed four times with PBST. The enzyme-labeled PcrV-A039 antibody was diluted 1:1000 and incubated at 37°C for 1 hour. After washing four times with PBST, TMB was added for color development for 10 minutes. The reaction was terminated by adding 50 μL of 2M sulfuric acid. The solution was then transferred to an OD500 microplate reader. 450 / 630nm Readings. Construct a curve with the logarithm of antigen concentration on the x-axis and the logarithm of OD value on the y-axis, and obtain the curve equation and correlation coefficient.

[0055] (2) Experimental results

[0056] When the reference concentration is between 78.125-2500 YU / mL, R 2 =0.9938, which has good linearity, and the linearity holds.

[0057] 3. Optimization of the standard curve

[0058] (1) Experimental methods

[0059] The capture antibody OprI-B052 was coated at a concentration of 6 μg / mL and incubated overnight at 4°C. After washing three times with PBST, the mixture was blocked with 3% BSA and incubated at 37°C for 1 h. The rPO antigen was then diluted to 1000, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, and 0 YU / mL and incubated at 37°C for 1 h. After washing three times with PBST, the enzyme-labeled antibody PcrV-A039 was diluted 1:1000 and incubated at 37°C for 1 h. After washing three times with PBST, TMB was added for color development for 10 minutes, and the color development was stopped. The sample was then transferred to an OD300 microplate reader. 450 / 630nm Readings. Construct a curve with the logarithm of antigen concentration on the x-axis and the logarithm of OD value on the y-axis, and obtain the curve equation and correlation coefficient.

[0060] (2) Experimental results

[0061] When the reference concentration is between 25-1000 YU / mL, R 2 =0.9979, which has good linearity, and the linearity holds.

[0062] Example 2

[0063] Methodological Validation

[0064] 1. Specificity verification

[0065] (1) Experimental methods

[0066] To verify whether the rPO protein stock solution buffer system affects the specific activity assay results. The rPO protein stock solution buffer system consisted of 1 mol / L sodium chloride and 10 mmol / L L-histidine.

[0067] rPO protein stock solution buffer, PBST buffer and rPO stock solution were used as test samples, and specificity verification was performed according to the steps listed in Example 1, using the reference concentration range of 25-1000 YU / mL as the standard curve.

[0068] (2) Experimental results

[0069] Table 2 shows the OD values ​​of rPO protein stock solution buffer and PBST buffer. 450 / 630nmThe values ​​were not different and none were detected. The specific activity of the rPO stock solution was 0.96*10. 6 The recovery rate was 102.5% (YU / mg), indicating that the method is specific for rPO antigen recognition and has good specificity.

[0070] Table 2 Specificity Validation Results

[0071]

[0072] 2. Repeatability verification

[0073] (1) Experimental methods

[0074] Following the standard curve and operating procedures established in Example 1, the same person took the same sample to be tested 6 times.

[0075] (2) Experimental results

[0076] The relative standard deviation is within 5% (RSD≤15%), indicating that the method measured in this invention has high repeatability, as shown in Table 3.

[0077] Table 3 Intermediate precision verification results

[0078]

[0079] 3. Linearity verification

[0080] (1) Experimental methods

[0081] Following the standard curve and operating procedures established in Example 1, two personnel conducted tests on the same sample at different 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 function with the logarithmic value as the ordinate and calculate R. 2 .

[0082] (2) Experimental results

[0083] like Figure 1 As shown, R in six experiments 2 The values ​​are 0.9917, 0.9899, ​​0.9912, 0.9923, 0.9979, and 0.9901, respectively, R0 2 All values ​​are greater than 0.98, indicating that the method exhibits good linearity.

[0084] 4. Accuracy Verification

[0085] (1) Experimental methods

[0086] Following the standard curve and operating procedures established in Example 1, the specific activity of the same rPO stock solution was tested six times to verify its accuracy. The recovery rates of the six tests were calculated.

[0087] (2) Experimental results

[0088] As shown in Table 4, the specific activity of the same batch of rPO protein stock solution was detected by the established double-antibody sandwich ELISA method. The recovery rate was between 90% and 120% in six tests, indicating that the method has high accuracy.

[0089] Table 4 Accuracy Verification Results

[0090]

[0091] Example 3

[0092] Specific activity testing of multiple batches of rPO stock solution

[0093] (1) Experimental methods

[0094] The specific activity of six batches of rPO protein stock solution was tested according to the method and operation steps established in Example 1.

[0095] (2) Experimental results

[0096] 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 six batches of rPO protein stock solution passed the specific activity test, with an RSD of less than 10%, indicating that the production process of different batches of stock solution was stable, as shown in Table 5.

[0097] Table 5. Specific activity test results of different batches of rPO protein stock solution

[0098]

[0099] Finally, it should be noted that:

[0100] 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 detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution, characterized in that, Includes the following steps: (1) The enzyme-labeled plate was coated with OprI-B052 as the capture antibody; (2) Add the sample to be tested into the coated ELISA plate; (3) The enzyme-labeled antibody PcrV-A039 was diluted and subjected to a colorimetric reaction to determine the OD. 450 / 630 nm value; (4) Prepare a standard curve using rPO reference, and calculate the specific activity of rPO stock solution in the test sample based on the standard curve; Specifically, OprI-B052 comprises a heavy chain and a light chain, each containing three CDRs: the heavy chain CDR1 amino acid sequence is GFTLSSYS, the heavy chain CDR2 amino acid sequence is ISSTSSYI, and the heavy chain CDR3 amino acid sequence is VRGVDFDY; the light chain CDR1 amino acid sequence is QSLLHRNGQKY, the light chain CDR2 amino acid sequence is LG, and the light chain CDR3 amino acid sequence is MQALQRPVT. Similarly, PcrV-A039 comprises a heavy chain and a light chain, each containing three CDRs: the heavy chain CDR1 amino acid sequence is GDTLNNFA, the heavy chain CDR2 amino acid sequence is IIPLLGIA, and the heavy chain CDR3 amino acid sequence is ATSPVRGIDYGMDV; the light chain CDR1 amino acid sequence is QSVSTD, the light chain CDR2 amino acid sequence is DA, and the light chain CDR3 amino acid sequence is QQRTTWPPMYT.

2. The method for detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution according to claim 1, characterized in that, The specific activity of the rPO stock solution is not less than 0.5 × 10⁻⁶. 6 The YU / mg level is within acceptable limits.

3. The method for detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution according to claim 1, characterized in that, In step (1), the capture antibody OprI-B052 was coated at a concentration of 6 μg / mL overnight at 4°C, 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 recombinant Pseudomonas aeruginosa vaccine rPO stock solution according to claim 1, characterized in that, In step (2), 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 detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution according to claim 1, characterized in that, In step (3), the enzyme-labeled antibody PcrV-A039 was diluted 1000, 2000, 3000, 4000, 5000, and 6000 times, incubated at 37°C for 1 hour, washed, and then developed at 37°C for 10 minutes. Finally, 50 μL of 2M sulfuric acid was added to terminate the reaction.

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

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

8. The method for detecting the specific activity of recombinant Pseudomonas aeruginosa vaccine rPO stock solution according to claim 3, characterized in that, The PBST was prepared as follows: 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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