Method for detecting rPO antigen content of recombinant pseudomonas aeruginosa vaccine

Abstract

The invention discloses a method for detecting the rPO antigen content of a recombinant pseudomonas aeruginosa vaccine. 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 rPO antigen content 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 rPO protein content in the pseudomonas aeruginosa vaccine can be rapidly and accurately quantified, and the method has good stability and repeatability.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for detecting the content of rPO antigen in a recombinant Pseudomonas aeruginosa vaccine. Background Art

[0002] Pseudomonas aeruginosa is one of the most virulent opportunistic Gram-negative bacterial pathogens. It causes many acute and chronic infections, including pneumonia and infections involving the urinary tract, wounds, burns, and blood. Especially in the care of critically ill patients, its infection is associated with high morbidity and mortality. In 2017, WHO released a list of pathogens for priority development of new antibiotics. Pseudomonas aeruginosa was listed as the "most critical" level, and its emergence and spread has become a serious public health problem. Data show that in some regions such as Europe and the United States, multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa account for 15% to 30%. In November 2022, The Lancet reported that the number of people who died from Pseudomonas aeruginosa infection in 2019 was as high as 559,000. PA infection and its drug resistance have become a thorny medical problem.

[0003] Vaccines are an important means of preventing and controlling Pseudomonas aeruginosa infections. In the past 50 years, scientists have been looking for an effective Pseudomonas aeruginosa vaccine, studying a variety of antigens and strategies. Many protective antigens have been tested, such as PcrV, OprF, OprI, FlgE, etc. However, the development of an effective Pseudomonas aeruginosa vaccine is difficult and there are many obstacles, such as the complexity of PA pathogenesis, the diversity of virulence factors, its high adhesion in the lungs, and the high diversity of serotypes. These are all problems that need to be faced, so there is currently no approved PA vaccine. To date, more than 60 candidate PA vaccines have been tested in animals or clinical human trials.

[0004] Pseudomonas aeruginosa uses its own complex type III secretion apparatus, which is a needle-like protein complex that can inject effector proteins into host cells to cause infection. Pseudomonas aeruginosa V antigen (also known as PcrV protein) is a pinhole structure in the type III secretion apparatus, which is necessary for the formation of pore channels during the infection of the host. Antibodies against PcrV can protect cultured cells and model animals from Pseudomonas aeruginosa infection, making PcrV a potential therapeutic target.

[0005] 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. Studies have shown that OprI has an adjuvant effect, because the OprI lipid tail binds to the Toll-like receptors of antigen presenting cells (APCs), thereby stimulating APCs activation and promoting IL-12 production, thereby initiating innate immunity and inducing specific T cell responses. OprI has also become a candidate antigen for a variety of vaccines.

[0006] In the research and development of recombinant protein vaccines, the antigen content in the finished vaccine product after the protein antigen and adjuvant are adsorbed is used as one of the stability detection indicators. During the vaccine production process, measuring the concentration of the antigen after desorption can determine whether the content of effective antigen components in the vaccine meets the standards. This is a key link in ensuring the stability and reliability of vaccine quality, and helps to avoid the effects of insufficient or excessive antigen content on the efficacy and safety of the vaccine. Summary of the invention

[0007] The object of the present invention is to provide a method for detecting the rPO antigen content of a recombinant Pseudomonas aeruginosa vaccine, which can quickly and accurately quantify the rPO protein content in the Pseudomonas aeruginosa vaccine.

[0008] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for detecting the content of rPO antigen in a recombinant Pseudomonas aeruginosa vaccine, comprising the following steps: (1) Coating the 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 color development reaction, and the OD450 / 630 nm value was measured; (4) Use rPO reference material to prepare a standard curve and calculate the rPO antigen content in the sample according to the standard curve.

[0009] Furthermore, the OD450 / 630 nm value is the OD450 nm and OD630 nm values ​​measured respectively.

[0010] Furthermore, the capture antibody OprI-B052 in (1) was coated at a concentration of 6 μg / mL at 4°C overnight, washed four times with PBST, blocked with 3% BSA, incubated at 37°C for 1 hour, and washed four times with PBST.

[0011] Furthermore, the sample to be tested in (2) needs to be incubated at 37°C for 60 min after being added to the ELISA plate.

[0012] Furthermore, the enzyme-labeled antibody PcrV-A039 in (3) 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, and 50 μL of 2M sulfuric acid was added to terminate the reaction.

[0013] Furthermore, the standard curve in (4) is constructed with the logarithm of the antigen concentration as the horizontal axis and the logarithm of the OD value as the vertical axis.

[0014] Furthermore, when the concentration of the standard curve reference substance in (4) is 78.125-2500 ng / mL, R2=0.9938.

[0015] Furthermore, the OprI-B052 includes a heavy chain and a light chain, and the heavy chain and the light chain respectively contain three CDRs, wherein the heavy chain CDR1 GFTLSSYS, the heavy chain CDR2 ISSTSSYI, and the heavy chain CDR3 VRGVDFDY; the light chain CDR1 QSLLHRNGQKY, the light chain CDR2 LG, and the light chain CDR3 MQALQRPVT.

[0016] Further, the OprI-B052 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 EVQLLESGGGLVKPGGSLRLSCAAS, heavy chain FR2 MNWVRQAPGKGLEWVSS, heavy chain FR3 SYADSVRGRFTISRDNAKKSLFLQMNSLRAEDTAVYYC, heavy chain FR4 WGQGTLVTVSS; light chain FR1 DVVMTQSPLSLPVTPGEPASISCRSS, light chain FR2 LDWYLQKPGQSPQLLIY, light chain FR3 SNRASGVPERFSGSGSGTDFTLKISRVEAEDVGVYYC, light chain FR4 FGPGTTVDIKRTV.

[0017] Further, the PcrV-A039 includes a heavy chain and a light chain, and the heavy chain and light chain respectively contain three CDRs, wherein the heavy chain CDR1 GDTLNNFA, the heavy chain CDR2 IIPLLGIA, the heavy chain CDR3 ATSPVRGIDYGMDV; the light chain CDR1QSVSTD, the light chain CDR2 DA, the light chain CDR3 QQRTTWPPMYT.

[0018] Further, the PcrV-A039 antibody heavy chain and light chain also include FR regions, and the heavy chain and light chain respectively contain four FR regions, wherein the heavy chain FR1 QVQLVQSGPEVKNPGSSVKVSCKAS, heavy chain FR2 ISWVRQAPGPGLQWVGG, heavy chain FR3 HYSQDFQGRVTITADKSTSTVSMELRSLRSQDTAVYYC, heavy chain FR4 WGQGTTVTVSS; light chain FR1EIVLTQSPATLSLSPGERATLSCRAS, light chain FR2 LAWYQQKPGQAPRLLIY, light chain FR3 SNRATGIPARFSGSGSGTDFTLTISGLEPEDFAVYFC, light chain FR4 FGQGTKVETKRTV.

[0019] Furthermore, the PBST is Na2HPO4 8 mM, NaCl 0.136M, KH2PO4 2mM, 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: OprI-B052 was used as the coating antibody and paired with PcrV-A039 as the detection antibody. The rPO protein content in the Pseudomonas aeruginosa vaccine can be quantified quickly and accurately with good stability and repeatability. Accurate antigen concentration determination results are important evidence of vaccine safety and effectiveness, which helps to enhance the public's trust in vaccines and willingness to be vaccinated, and is of great significance for public health and epidemic prevention work. 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 This is the SDS-PAGE test result after desorption of the vaccine of the embodiment of the present invention; Figure 2 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] OprI-B052 includes a heavy chain and a light chain, each of which comprises three CDRs, wherein heavy chain CDR1GFTLSSYS, heavy chain CDR2 ISSTSSYI, heavy chain CDR3 VRGVDFDY; light chain CDR1 QSLLHRNGQKY, light chain CDR2LG, light chain CDR3 MQALQRPVT.

[0029] The heavy chain and light chain of OprI-B052 antibody also include FR regions, and the heavy chain and light chain respectively include four FR regions, wherein heavy chain FR1 EVQLLESGGGLVKPGGSLRLSCAAS, heavy chain FR2 MNWVRQAPGKGLEWVSS, heavy chain FR3 SYADSVRGRFTISRDNAKKSLFLQMNSLRAEDTAVYYC, heavy chain FR4 WGQGTLVTVSS; light chain FR1 DVVMTQSPLSLPVTPGEPASISCRSS, light chain FR2 LDWYLQKPGQSPQLLIY, light chain FR3 SNRASGVPERFSGSGSGTDFTLKISRVEAEDVGVYYC, light chain FR4 FGPGTTVDIKRTV. PcrV-A039 includes a heavy chain and a light chain, each of which contains three CDRs, wherein heavy chain CDR1 GDTLNNFA, heavy chain CDR2 IIPLLGIA, heavy chain CDR3ATSPVRGIDYGMDV; light chain CDR1 QSVSTD, light chain CDR2 DA, light chain CDR3 QQRTTWPPMYT.

[0030] The heavy chain and light chain of the PcrV-A039 antibody also include FR regions, wherein the heavy chain and light chain respectively include four FR regions, wherein the heavy chain FR1 QVQLVQSGPEVKNPGSSVKVSCKAS, the heavy chain FR2 ISWVRQAPGPGLQWVGG, the heavy chain FR3 HYSQDFQGRVTITADKSTSTVSMELRSLRSQDTAVYYC, the heavy chain FR4 WGQGTTVTVSS; the light chain FR1 EIVLTQSPATLSLSPGERATLSCRAS, the light chain FR2 LAWYQQKPGQAPRLLIY, the light chain FR3 SNRATGIPARFSGSGSGTDFTLTISGLEPEDFAVYFC, the light chain FR4 FGQGTKVETKRTV. PBST is Na2HPO4 8 mM, NaCl 0.136M, KH2PO4 2mM, KCl 2.6 mM, Tween-20 0.05% v / v.

[0031] The CDR naming adopts the IMGT naming.

[0032] Example 1

[0033] Desorption of recombinant Pseudomonas aeruginosa vaccine (1) Experimental methods Prepare 20% diethanolamine solution and 40% sodium citrate solution. Take 5 mL of 40% sodium citrate solution and add 5 mL of 20% diethanolamine solution to make 20% sodium citrate (10.0% diethanolamine) dissociation solution. Take 500 μL of dissociation solution and add it to 500 μL of finished product, put it into a vertical suspension instrument and place it in a 4℃ refrigerator overnight for dissociation. Centrifuge and take the supernatant for use.

[0034] (2) Experimental results like Figure 1 As shown, the recombinant Pseudomonas aeruginosa vaccine has been successfully dissociated by SDS-PAGE detection of the desorbed sample supernatant.

[0035] Example 2

[0036] Establishment of double antibody sandwich ELISA methodology 1. Determination of the working concentration of coating antibody and enzyme-labeled antibody (1) Experimental methods The coated antibody OprI-B052 was coated at different concentrations at 4°C overnight, washed four times with PBST, blocked with 3% BSA, incubated at 37°C for 1 hour, washed four times with PBST, added with 1 μg / mL rPO antigen, incubated at 37°C for 1 hour, the enzyme-labeled antibody PcrV-A039 was diluted at different dilution multiples, incubated at 37°C for 1 hour, washed, developed at 37°C for 10 minutes, and 50 μL of 2M sulfuric acid was added to terminate the reaction, and the OD450 / 630nm reading was performed on the microplate reader. The P / N (test well / control well) ratio was calculated.

[0037] (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.

[0038] As shown in Table 1, the preferred combination is a capture antibody OprI-B052 concentration of 6 μg / mL and an enzyme-labeled detection antibody PcrV-A039 diluted 1:1000.

[0039] Table 1 P / N values ​​of capture antibody and detection antibody

[0040] 2. Establishment of standard curve (1) Experimental methods The capture antibody OprI-B052 was coated at 6 μg / mL overnight at 4°C, washed four times with PBST, blocked with 3% BSA, incubated at 37°C for 1 hour, washed four times with PBST, the rPO reference was diluted to 10 μg / mL as the starting concentration and diluted 2-fold, incubated at 37°C for 1 hour, washed four times with PBST, the enzyme-labeled PcrV-A039 antibody was diluted 1:1000, incubated at 37°C for 1 hour, washed four times with PBST, TMB was added for color development for 10 minutes, 50 μL of 2M sulfuric acid was added to terminate the reaction, and the OD450 / 630nm reading was performed on the microplate reader. The logarithm of the antigen concentration was used as the abscissa and the logarithm of the OD value was used as the ordinate to construct the curve, and the curve equation and correlation coefficient were obtained.

[0041] (2) Experimental results When the reference sample concentration was between 78.125-2500 ng / mL, R2=0.9938, which showed good linearity and was established.

[0042] 3. Optimization of the standard curve (1) Experimental methods The capture antibody OprI-B052 was coated at a concentration of 6 μg / mL at 4°C overnight, 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, and 0 ng / mL, incubated at 37°C for 1 h, washed three times with PBST, the enzyme-labeled PcrV-A039 antibody was diluted 1:1000, incubated at 37°C for 1 h, washed three times with PBST, TMB was added for color development for 10 min, the color development was terminated, and the plate was analyzed by OD spectroscopy. 450 / 630nm The logarithm of the antigen concentration was used as the horizontal axis, and the logarithm of the OD value was used as the vertical axis to construct a curve, and the curve equation and correlation coefficient were obtained.

[0043] (2) Experimental results When the reference sample concentration was between 25 and 1000 ng / mL, R2 was 0.9979, which showed good linearity and was established.

[0044] Example 3 Methodological validation 1. Exclusiveness verification (1) Experimental methods The recombinant Pseudomonas aeruginosa vaccine is composed of a mixture of two antigens, rPO and rTF. In order to evaluate the specific reaction of the established method, rTF protein, PBST buffer and desorbed Pseudomonas aeruginosa vaccine were used as test samples to verify the specificity of the method.

[0045] The rTF protein, PBST buffer and desorbed Pseudomonas aeruginosa vaccine were used as samples to be tested, respectively, and the concentration range of 25-1000 ng / mL of the reference product was used as the standard curve, and the specificity verification was performed according to the steps listed in Example 3.

[0046] (2) Experimental results As shown in Table 2: There was no difference in the OD450 / -630nm value between rTF protein and PBST buffer, and no detectable value was found. The content of desorbed Pseudomonas aeruginosa vaccine rPO antigen was 50.52 μg / mL, and the recovery rate was 102.5%, indicating that this method was specific for rPO antigen recognition and had good specificity.

[0047] Table 2 Specificity verification results

[0048] 2. Intermediate precision verification (1) Experimental methods According to the calibration curve and operation steps established in Example 2, two persons took the same sample for testing at different times.

[0049] (2) Experimental results The relative standard deviation is within 5% (RSD≤15%), indicating that the values ​​determined by the method of the present invention have high intermediate precision, as shown in Table 3.

[0050] Table 3 Intermediate precision verification results

[0051] 3. Linear verification (1) Experimental methods According to the calibration curve and operation steps established in Example 1, two personnel tested the same sample at different times, and constructed a function with the logarithm of the reference sample concentration as the abscissa and the logarithm of OD450 / -630nm as the ordinate, and calculated R2.

[0052] (2) Experimental results like Figure 2 As shown, the R2 of the six experiments are 0.9918, 0.9923, 0.9909, 0.9937, 0.9934, and 0.9954, respectively. R2 is greater than 0.99, and the method has good linearity.

[0053] 4. Accuracy Verification (1) Experimental methods According to the calibration curve and operation steps established in Example 2, the rPO antigen content of the same finished product was tested 6 times to verify its accuracy. The recovery rates of the six tests were calculated.

[0054] (2) Experimental results As shown in Table 4, the rPO antigen content of the same finished product was detected by the established double antibody sandwich ELISA method, and the recovery rates of six tests were between 90-115%, indicating that the method has high accuracy.

[0055] Table 4 Accuracy verification results

[0056] Example 4 Detection of rPO content in multiple batches of finished vaccine products (1) Experimental methods According to the method and operating procedures established in Example 2, three batches of finished vaccine products were tested for rPO content.

[0057] (2) Experimental results Compared with the theoretical value, the recovery rates of the rPO antigen content of the three batches of vaccine products were between 90% and 110%, as shown in Table 5. This indicates that the detection method has high accuracy and can be used to detect the rPO content of recombinant Pseudomonas aeruginosa vaccines.

[0058] Table 5 Test results of rPO antigen content in finished vaccine products

[0059] 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 detecting the content of rPO antigen in a recombinant Pseudomonas aeruginosa vaccine, characterized in that: The following steps are involved: (1) Coating the 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 color development reaction, and the OD450 / 630 nm value was measured; (4) Use rPO reference material to prepare a standard curve and calculate the rPO antigen content in the sample according to the standard curve.

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

3. The method for detecting the rPO antigen content of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that: After the sample to be tested in (2) is added to the ELISA plate, it needs to be incubated at 37°C for 60 minutes.

4. The method for detecting the rPO antigen content of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that: The enzyme-labeled antibody PcrV-A039 in (3) 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, and 50 μL of 2M sulfuric acid was added to terminate the reaction.

5. The method for detecting the rPO antigen content of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that: The standard curve in (4) is constructed with the logarithm of the antigen concentration as the horizontal axis and the logarithm of the OD value as the vertical axis.

6. The method for detecting the rPO antigen content of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that: When the concentration of the reference material of the standard curve in (4) is 78.125-2500 ng / mL, R2=0.9938.

7. The method for detecting the rPO antigen content of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, characterized in that: The OprI-B052 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 GFTLSSYS, the heavy chain CDR2 is ISSTSSYI, and the heavy chain CDR3 is VRGVDFDY; Light chain CDR1 QSLLHRNGQKY, light chain CDR2LG, light chain CDR3 MQALQRPVT.

8. The method for detecting the rPO antigen content 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 respectively contain three CDRs, wherein the heavy chain CDR1GDTLNNFA, the heavy chain CDR2 IIPLLGIA, and the heavy chain CDR3 ATSPVRGIDYGMDV; the light chain CDR1 QSVSTD, the light chain CDR2DA, and the light chain CDR3 QQRTTWPPMYT.

9. The method for detecting the rPO antigen content of the recombinant Pseudomonas aeruginosa vaccine according to claim 1, 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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