A method for quantitatively detecting bxb1 integrase, a kit and application thereof

Abstract

The present disclosure relates to the field of biotechnology, and particularly relates to detection of BXB1 integrase. The present disclosure establishes a method for detecting BXB1 integrase based on double-antibody sandwich ELISA technology, which has good sensitivity and specificity, and the method is simple to operate and can quickly realize accurate quantification of BXB1 integrase; in addition, the present disclosure also provides an ELISA kit for detecting BXB1 integrase. By using the BXB1 integrase detection method or kit provided by the present disclosure, accurate quantification of the residual amount of BXB1 integrase in various intermediate products, semi-finished products or finished products in the process of preparing recombinant biological products by the BXB1 integrase system can be realized, which is beneficial to the quality control of products by biopharmaceutical enterprises.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, specifically to a double-antibody sandwich ELISA detection method, kit, and application of BXB1 integrase. Background Technology

[0002] Site-specific integration (SSI) is a technique that inserts target genes (genes of interest, GOIs) into specific genomic sites to achieve continuous and stable expression of the target genes. Using site-specific recombinase-mediated SSI, multiple host cells carrying marker genes are screened, and the marker genes at specific sites are replaced with different types of target protein genes.

[0003] BXB1 integrase is a commonly used site-specific recombinase, often used for targeted gene integration. This BXB1 integrase may be introduced into the final biopharmaceutical. As an exogenous protein, BXB1 integrase can trigger an immune response, increasing the risk of adverse drug reactions in the target protein biopharmaceutical. Therefore, monitoring the content of BXB1 integrase is crucial. Consequently, a method for detecting BXB1 integrase residues in biopharmaceuticals needs to be developed. Summary of the Invention

[0004] In a first aspect, this disclosure provides a method for detecting BXB1 integrase based on a double-antibody sandwich ELISA, comprising the following steps:

[0005] S1. Preparation of solid-phase antibodies: preparing solid-phase antibodies by coating enzyme-linked immunosorbent assay (ELISA) plates with coated antibodies;

[0006] S2. Blocking treatment: Add blocking solution to block the microplate;

[0007] S3. Sample detection: Add the sample to be tested, incubate and wash, add the diluted detection antibody, incubate and wash.

[0008] S4. Color development and termination: Add TMB substrate color development solution, incubate, then add stop solution to terminate the reaction, and measure the absorbance (OD value) at a wavelength of 450 nm.

[0009] In some embodiments, the solid-phase antibody preparation step S1 includes: adding the coated antibody diluted with coating buffer (pH 9.6, 50mM carbonate buffer) to the ELISA plate and incubating overnight at 4°C; wherein the coated antibody is a BXB1 rabbit polyclonal antibody. In some embodiments, the coating amount of the coated antibody is further selected from 100–1000 ng / well, preferably 100–500 ng / well; more preferably 300–500 ng / well. In some embodiments, the coating amount of the coated antibody is selected from 100 ng / well, 200 ng / well, 300 ng / well, 400 ng / well, or 500 ng / well.

[0010] In some embodiments, the blocking process in step S2 includes adding the blocking solution to the ELISA plate and incubating at 37°C for 2 hours. In some embodiments, the blocking solution is a skim milk powder solution, preferably a 3wt%-10wt% skim milk powder solution; more preferably a 5wt% skim milk powder solution.

[0011] In some embodiments, the detection antibody in step S3 is an HRP-labeled BXB1 rabbit polyclonal antibody. Further in some embodiments, the dilution ratio of the detection antibody can be selected from 1:1000 to 1:10000; preferably 1:2500 to 1:5000; more preferably 1:2500; wherein the diluent is a 1 wt% skim milk powder solution.

[0012] In some implementations, the BXB1 integrase detection method further includes step S5, the preparation of a standard curve.

[0013] In some embodiments, the preparation of the standard curve includes: preparing BXB1 integrase standard solutions with concentrations of 200, 100, 50, 25, 10, 5, 2.5, and 1 ng / mL; measuring the OD values ​​of the standard solutions at different concentrations; and constructing a standard curve with the corresponding concentrations of the standard solutions on the x-axis and the measured OD values ​​of the standard solutions on the y-axis. In some embodiments, the liquid used to prepare the BXB1 integrase standard solutions at different concentrations is a 1 wt% skim milk powder solution.

[0014] Furthermore, in some embodiments, the BXB1 integrase detection method further includes step S6, calculating the concentration of BXB1 integrase in the sample to be tested based on a standard curve.

[0015] In some embodiments, the BXB1 rabbit polyclonal antibody is prepared using BXB1 integrase with an amino acid sequence as shown in SEQ ID NO: 1 as the antigen.

[0016] In some embodiments, the amino acid sequence of the BXB1 integrase standard is shown in SEQ ID NO: 1.

[0017] In some embodiments, the nucleotide sequence of the BXB1 integrase is further shown in SEQ ID NO: 2.

[0018] In some embodiments, the limit of detection for BXB1 integrase using the BXB1 integrase detection method described in this disclosure is 0.5 ng / mL.

[0019] Secondly, this disclosure provides the application of the BXB1 integrase detection method based on double-antibody sandwich ELISA described above in the preparation of recombinant biological products, including the detection of residual BXB1 integrase in recombinant biological products, wherein the recombinant biological products are prepared by BXB1 integrase.

[0020] In some implementations, the recombinant biological products include, but are not limited to, recombinant proteins, recombinant peptides, recombinant nucleic acids, recombinant plasmids, vaccines, cell lines, strains, drugs, and intermediate products of the above-mentioned biological products.

[0021] In some embodiments, when the recombinant biological product is a recombinant cell line prepared from BXB1 integrase, the test sample is the fermentation supernatant of the recombinant cell line or a diluted solution of the fermentation supernatant. In some embodiments, the test sample is a diluted solution of the fermentation supernatant of the recombinant cell line, and the dilution factor can be selected from 2 to 100 times, preferably 10 to 100 times, and more preferably 50 to 100 times. In some embodiments, further, the test sample is a diluted solution of the dilute fermentation supernatant of the recombinant cell line, and the dilution factor is preferably 50 times or 100 times.

[0022] In some embodiments, when the recombinant biological product is a recombinant protein prepared and purified from BXB1 integrase, the test sample is a solution of the recombinant protein or a dilution of the recombinant protein solution. In some embodiments, further, the test sample is a dilution of the recombinant protein solution, with a dilution factor selectable from 2 to 100 times, preferably 10 to 100 times, and more preferably 20 to 100 times. In some embodiments, the test sample is a dilution of the recombinant protein solution, with a dilution factor preferably 20 times, 50 times, or 100 times.

[0023] In some implementations, the solution used to prepare the diluent for the test sample is a 1 wt% solution of skim milk powder.

[0024] Thirdly, this disclosure provides an ELISA kit for quantitative detection of BXB1 integrase, the kit comprising: an enzyme-labeled plate coated with BXB1 rabbit polyclonal antibody, HRP-labeled BXB1 rabbit polyclonal antibody, BXB1 integrase standard, diluent, washing buffer, chromogenic solution, and stop solution.

[0025] In some embodiments, the BXB1 rabbit polyclonal antibody is prepared using BXB1 integrase with an amino acid sequence as shown in SEQ ID NO: 1 as the antigen.

[0026] In some embodiments, the amino acid sequence of the BXB1 integrase standard is shown in SEQ ID NO: 1.

[0027] In some embodiments, the nucleotide sequence of the BXB1 integrase is further shown in SEQ ID NO: 2.

[0028] In some embodiments, the preparation of the ELISA plate coated with BXB1 rabbit polyclonal antibody includes: adding the BXB1 rabbit polyclonal antibody diluted with coating solution (pH 9.6, 50 mM carbonate buffer) to the ELISA plate and incubating it overnight at 4°C. In some embodiments, the preparation of the ELISA plate further includes: blocking with a 5 wt% skim milk powder solution.

[0029] In some embodiments, the amount of antibody coated on the ELISA plate can be selected from 100 to 1000 ng / well, preferably 100 to 800 ng / well; more preferably 100 to 500 ng / well. In some embodiments, the amount of antibody coated on the ELISA plate can be selected from 100 ng / well, 300 ng / well, or 500 ng / well.

[0030] In some embodiments, the diluent is a 1 wt% skim milk powder solution.

[0031] In some embodiments, the washing solution is a PBST solution at pH 7.4 (a 10 mM PBS solution containing 0.05 v / v% Tween 20).

[0032] In some implementations, the limit of detection for the ELISA kit described in this disclosure is 0.5 ng / mL.

[0033] Fourthly, this disclosure also provides the application of the above-mentioned ELISA kit for quantitative detection of BXB1 integrase in the preparation of recombinant biological products, including: using the kit to detect BXB1 integrase residues in recombinant biological products; wherein the recombinant biological products are prepared by BXB1 integrase.

[0034] In some implementations, the recombinant biological products include, but are not limited to, recombinant proteins, recombinant peptides, recombinant nucleic acids, recombinant plasmids, vaccines, cell lines, strains, drugs, and intermediate products of the above-mentioned biological products.

[0035] In some implementations, detecting BXB1 integrase residues in recombinant biological products using the kit includes the following steps:

[0036] S1. Obtain the sample to be tested;

[0037] S2. Add the test sample and BXB1 integrase standard solution to the ELISA plate coated with BXB1 rabbit polyclonal antibody, incubate at 37°C for 1 hour, wash, add the detection antibody, incubate at 37°C for 1 hour, and wash.

[0038] S3. Add diluted HRP-labeled BXB1 rabbit polyclonal antibody, incubate at 37°C for 1 hour, and then wash.

[0039] S4. Add TMB substrate colorimetric solution, let stand at room temperature in the dark for color development, then add stop solution to terminate the reaction, and measure the absorbance (OD value) at a wavelength of 450 nm.

[0040] S5. Construction of standard curves;

[0041] S6. Calculate the concentration of BXB1 integrase in the sample to be tested based on the standard curve.

[0042] In some implementations, the sample to be tested in step S1 may be a diluted or undiluted recombinant biological product.

[0043] In some embodiments, in step S1, when the recombinant biological product is a recombinant cell line prepared from BXB1 integrase, the sample to be tested is the fermentation supernatant of the recombinant cell line or a diluted solution of the fermentation supernatant. In some embodiments, the sample to be tested is a diluted solution of the fermentation supernatant of the recombinant cell line, and the dilution factor can be selected from 2 to 100 times, preferably 10 to 100 times, and more preferably 50 to 100 times. In some embodiments, further, the sample to be tested is a diluted solution of the dilute fermentation supernatant of the recombinant cell line, and the dilution factor is preferably 50 times or 100 times.

[0044] In some embodiments, in step S1, when the recombinant biological product is a recombinant protein prepared and purified from BXB1 integrase, the test sample is a solution of the recombinant protein or a dilution of the recombinant protein solution. In some embodiments, further, the test sample is a dilution of the recombinant protein solution, with a dilution factor selectable from 2 to 100 times, preferably 10 to 100 times, and more preferably 20 to 100 times. In some embodiments, the test sample is a dilution of the recombinant protein solution, with a dilution factor preferably 20 times, 50 times, or 100 times.

[0045] In some embodiments, in step S3, the dilution ratio of the HRP-labeled BXB1 rabbit polyclonal antibody can be selected from 1:1000 to 1:10000, preferably 1:2500 to 1:5000, and more preferably 1:2500.

[0046] In some implementations, the liquid used for diluting the antibody, BXB1 integrase standard, and test sample is the diluent in the kit, i.e., a 1 wt% skim milk powder solution.

[0047] Beneficial effects:

[0048] This disclosure establishes a method for detecting BXB1 integrase in samples using a double-antibody sandwich ELISA technique. This method has a detection limit as low as 0.5 ng / mL, exhibiting good sensitivity and specificity, and enables accurate quantification of BXB1 integrase. Furthermore, this disclosure provides an ELISA kit for detecting BXB1 integrase. When the above method or kit is used to detect residual BXB1 integrase in intermediates, semi-finished products, or finished products prepared from the BXB1 integrase system, the intra-batch variation of the detection results is <10%, and the inter-batch variation is <15%. Moreover, this method is simple to operate, has good reproducibility, and high stability, making it of significant value for product quality control by manufacturing enterprises. Attached Figure Description

[0049] Figure 1 The procedure for double antibody sandwich ELISA;

[0050] Figure 2 The standard curve established in Example 1;

[0051] Figure 3 This is the standard curve established in the matrix interference effect detection experiment of Example 2;

[0052] Figure 4 This is the standard curve established in the intra-batch precision verification test of Example 2;

[0053] Figure 5 This is the standard curve established in the inter-batch precision verification test of Example 2;

[0054] Figure 6 The standard curve established in the linear range confirmation experiment of Example 2;

[0055] Figure 7 The linear detection range of BXB1 in the diluted fermentation supernatant sample is given.

[0056] Figure 8 To determine the linear detection range of BXB1 in the purified sample dilution;

[0057] Figure 9 It is a plasmid containing BXB1 integrase nucleic acid. Detailed Implementation

[0058] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this disclosure.

[0059] The main reagents and consumables used in the following examples are as follows:

[0060] Antibody: BXB1 rabbit polyclonal antibody, prepared from BXB1 integrase (amino acid sequence as shown in SEQ ID NO: 1) as antigen (prepared by Wuhan Jinkairui Biotechnology Co., Ltd.)

[0061] Standard: BXB1 protein, the amino acid sequence of which is shown in SEQ ID NO: 1; the nucleotide sequence was codon-optimized to E. coli-preferred codons, as shown in SEQ ID NO: 2 (prepared by Wuhan Jinkairui Biotechnology Co., Ltd.)

[0062] Coating solution: pH 9.6, 50 mM carbonate buffer

[0063] Blocking solutions: 5 wt% skim milk powder solution (PBST solution containing 5 wt% skim milk powder), 1 wt% BSA solution (PBST solution containing 1 wt% BSA).

[0064] Diluents: 1 wt% skim milk powder solution (PBST solution containing 1 wt% skim milk powder), 1 wt% BSA solution (PBST solution containing 1 wt% BSA).

[0065] Color developer: 1-Step TM UltraTMB-ELISA substrate solution (Thermo Fisher, catalog number: 34028)

[0066] Washing solution: PBST solution at pH 7.4 (10mM PBS solution containing 0.05 v / v% Tween 20)

[0067] Termination solution: 2M sulfuric acid solution

[0068] 96-well microplate

[0069] Example 1: Establishment of a double-antibody sandwich ELISA

[0070] (1) Procedure for double-antibody sandwich ELISA (see...) Figure 1 ):

[0071] ① Antibody coating: Dilute BXB1 rabbit polyclonal antibody with coating buffer, add 100 μL / well to a 96-well microplate, and incubate overnight at 4°C;

[0072] ② Blocking: Discard the liquid in the 96-well microplate that has been coated overnight, add 300 μL / well of PBST washing solution and wash the plate 4 times, pat dry on absorbent paper, then add 200 μL / well of blocking solution and incubate at 37°C for 2 hours.

[0073] ③ Sample addition: Discard the blocking solution in the 96-well microplate, add PBST washing solution at 300 μL / well and wash the plate 4 times. Pat the plate dry on absorbent paper. Take 100 μL each of BXB1 standard solution, sample solution to be tested, and blank solution (diluent) and add them to the 96-well microplate, repeating 2 times in parallel. Incubate at 37°C for 1 hour.

[0074] ④ Add detection antibody: Discard the sample solution in the 96-well plate, add PBST washing buffer at 300 μL / well and wash the plate 4 times. Pat dry on absorbent paper. Take HRP-BXB1 rabbit polyclonal antibody, dilute it with diluent, add 100 μL / well to the 96-well microplate, and incubate at 37°C for 1 hour.

[0075] ⑤ Color development: Discard the antibody solution in the 96-well microplate, add 300 μL / well of PBST washing buffer and wash the plate 4 times, pat dry on absorbent paper, add 100 μL / well of TMB substrate color development solution, and let stand at room temperature in the dark for 15 min for color development.

[0076] ⑥ Termination: Add 50 μL of stop solution to each well of the 96-well plate to terminate the colorimetric reaction;

[0077] ⑦ Read the plate: Read the OD450 value using an ELISA reader.

[0078] (2) Preparation of HRP enzyme-labeled antibody (HRP-BXB1 rabbit polyclonal antibody):

[0079] According to the HRP conjugation kit - (abcam, catalog number: ab102890) labeled the BXB1 rabbit polyclonal antibody and verified the conjugation effect using direct ELISA.

[0080] (3) Determination of coating antibody amount and detection antibody amount:

[0081] ① Blocking solution 1, diluent 1: 1wt% BSA solution

[0082] The checkerboard dilution method was used to examine the coating antibody (BXB1 rabbit polyclonal antibody) at concentrations of 1000 ng / well and 500 ng / well, and the detection antibody (HRP-BXB1 rabbit polyclonal antibody) at dilution ratios of 1:5000 and 1:1000.

[0083] BXB1 standard was diluted to 2000 ng / mL, 200 ng / mL, and 20 ng / mL, with 0 ng / mL (blank, i.e., dilution 1) used as the test sample; the layout of the test samples in the ELISA plate is shown in Table 1.

[0084] Table 1. Layout of samples to be tested in the ELISA plate

[0085]

[0086] The test results are shown in Table 2. The standards paired with rabbit polyclonal antibody BXB1 and HRP-BXB1 enzyme-labeled antibody both showed high responses, suggesting that the antibodies can be used in the double antibody sandwich method. However, the blank OD response was also high, which may indicate detection interference.

[0087] Table 2. OD values ​​of the samples to be tested

[0088] serial number 1 2 3 4 A Overflow Overflow Overflow Overflow B Overflow Overflow Overflow Overflow C 3.5946 3.5654 3.0079 2.8785 D 2.3128 2.287 1.8129 1.7947 E 3.3728 3.398 2.901 2.9419 F 2.3899 2.2773 1.8461 1.7757 G 1.031 0.9805 0.8062 0.7842 H 0.5769 0.5447 0.4559 0.4506

[0089] "Overflow" indicates that the concentration of the sample being tested exceeds the upper limit of the microplate reader.

[0090] ②Blocking solution 2 (5wt% skim milk powder solution), diluent 2 (1wt% skim milk powder solution)

[0091] The checkerboard dilution method was used, with coating amounts of 100 ng / well, 300 ng / well, and 500 ng / well for the coating antibody (BXB1 rabbit polyclonal antibody), and dilution ratios of 1:5000 and 1:2500 for the detection antibody (HRP-BXB1 rabbit polyclonal antibody).

[0092] BXB1 standard was diluted to 800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, and 0 ng / mL (blank, i.e., dilution 2) as test samples; the layout of test samples in the ELISA plate is shown in Table 3.

[0093] Table 3. Layout of samples to be tested in the ELISA plate

[0094]

[0095]

[0096] The test results are shown in Table 4. Under the same coating amount, the higher the amount of detection antibody, the higher the OD value. Under the same amount of detection antibody, the OD value gradually increases with the increase of the coating amount of the antibody. The test results of 300 ng / well and 500 ng / well of antibody are not significantly different.

[0097] In summary, the optimal conditions were selected: 300 ng / well for coating antibody and 1:2500 dilution for detection antibody.

[0098] Table 4. OD values ​​of the samples to be tested

[0099] / 1 2 3 4 5 6 7 8 9 10 11 12 A 1.040 1.081 2.096 2.089 2.554 2.598 1.699 1.827 3.514 3.524 3.787 3.956 B 0.827 0.839 1.722 1.764 1.978 1.973 1.463 1.437 2.997 2.928 3.149 3.235 C 0.681 0.669 1.345 1.341 1.561 1.566 1.092 1.174 2.301 2.460 2.576 2.753 D 0.494 0.482 1.047 1.003 1.126 1.168 0.809 0.849 1.777 1.791 2.100 2.216 E 0.348 0.337 0.706 0.700 0.805 0.809 0.570 0.614 1.208 1.266 1.388 1.508 F 0.235 0.236 0.475 0.468 0.551 0.466 0.381 0.388 0.786 0.838 0.988 0.925 G 0.154 0.130 0.236 0.226 0.266 0.243 0.204 0.212 0.388 0.433 0.425 0.465 H 0.111 0.051 0.048 0.055 0.059 0.056 0.058 0.065 0.069 0.062 0.061 0.075

[0100] (4) Determining the range of the standard curve:

[0101] Refer to the operation in step (1), where,

[0102] The coating antibody was BXB1 rabbit polyclonal antibody (3 μg / mL), 100 μL / well;

[0103] The detection antibody was HRP-BXB1 rabbit polyclonal antibody, diluted 1:2500.

[0104] The diluent was a 1 wt% skim milk powder solution, and the blocking solution was a 5 wt% skim milk powder solution.

[0105] BXB1 integrase standard: concentrations set at 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1 ng / mL, and 0 ng / mL (blank, i.e., dilution), 100 μL / well;

[0106] Plotting the concentration of BXB1 standard on the x-axis and the corresponding OD values ​​on the y-axis, a standard curve was fitted using four parameters. The results are shown in [Figure number missing]. Figure 2 The BXB1 standard concentration showed good fit in the range of 1–200 ng / mL, with the fitting equation being y = 1.90262 - 1.90461 / (1 + (x / 76.02066)). 0.98075 The correlation coefficient R of the curve fitting 2 =0.99895, but the OD value of 1 ng / mL is closer to the blank value, so it is better to choose a range of 2.5 to 200 ng / mL.

[0107] (5) Limit of detection:

[0108] Select low-concentration BXB1 standard solutions (1.0 ng / mL, 0.5 ng / mL), repeat the test twice, and the test results are shown in Table 5. Substitute the measured OD values ​​into the standard curve equation in (4) to calculate the actual measured concentration.

[0109] Table 5. Detection results of low concentration samples

[0110] Test sample OD value 1 OD value 2 average value Concentration ng / mL Recovery rate % 1 0.0978 0.086 0.0919 1.0834 108.3 0.5 0.0702 0.0706 0.0704 0.4492 89.8

[0111] When the standard was further diluted to a concentration as low as 0.5 ng / mL, the concentration obtained by substituting the OD value into the equation was 0.4492 ng / mL, which was still detectable with relatively high accuracy. Therefore, the concentration of 0.5 ng / mL can be used as the detection limit of this method.

[0112] Example 2: Validation of Double Antibody Sandwich ELISA

[0113] Sample 1: Fermentation supernatant sample after site-specific integration screening (hereinafter referred to as: fermentation supernatant sample)

[0114] ① Site-directed integration experiment: Plasmids containing nucleic acids encoding the target protein (rhEPO-Fc, whose amino acid sequence is referenced in SEQ ID NO: 6 in CN100436482C) (refer to the product plasmid in CN118726475A) and plasmids containing BXB1 integrase nucleic acids (see...) were used to integrate the protein into the target protein (rhEPO-Fc, whose amino acid sequence is referenced in CN100436482C). Figure 9Add 100 μL of AlfaCell cell suspension (refer to Targeted Integrative Cells in CN118726475A) and gently mix the cell and plasmid mixture thoroughly. Perform electrotransfection using an Invitrogen MPK5000 instrument. After culturing under pressure for 2 days in culture medium (Sigma, catalog number: 24366C-10L) containing 250 μg / mL Hygromycin, centrifuge the cells and resuspend them in fresh culture medium (Sigma, catalog number: 24366C-10L). Continue culturing for 3 days. Use FACS (BD Biosciences, Melody) to sort single clones and select non-fluorescent single clones. After culturing statically in an incubator for 14 days, seed the selected single clones into culture medium (Sigma, catalog number: 24366C-10L) and culture at 37°C, 5% CO2, and 120 rpm. Cells were cultured for 3 days, and thereafter, cell counts and supernatant were collected every other day. Sugar content and protein expression levels were measured using a biochemical analyzer. On days 3 and 5, 3% Cell Boost 7a (HyClone, catalog number: SH31026.07), 0.3% Cell Boost 7b (HyClone, catalog number: SH31027.01), and glucose (Sigma, catalog number: G8769) were added until the sugar concentration reached 6 g / L. On days 7, 9, 11, and 13, 5% Cell Boost 7a and 0.5% Cell Boost 7b were added, and the sugar concentration was replenished to 6 g / L according to the cells' sugar consumption. When cell viability fell below 70%, the cell culture medium was collected, centrifuged at 1000 rpm for 10 min, and the supernatant was retained. The supernatant was then centrifuged again at 9000 rpm for 20 min, and the collected supernatant was the fermentation supernatant sample.

[0115] Sample 2: Purified sample

[0116] ②Purification steps: Take the supernatant from ①, filter it through a 0.22μm sterile syringe filter, and then purify it through a Protein A column to obtain a relatively pure target protein (rhEPO-Fc), which is the purified sample.

[0117] (1) Exclusivity:

[0118] Take the recombinant protein (recombinant human erythropoietin (Fc) fusion protein, rhEPO-Fc, reference CN100436482C) that was not prepared using BXB1 integrase, dilute it to 1 mg / mL with diluent, prepare two parallel aliquots, and perform detection according to the ELISA method in Example 1.

[0119] The results are shown in Table 6. The samples prepared without BXB1 integrase did not show a significant response to this method and their OD values ​​were close to those of the blank sample, thus meeting the specificity requirements.

[0120] Table 6. Results of Specificity Examination

[0121]

[0122] (2) Interference from blocking solution and diluent

[0123] The blocking and dilution effects of BSA and skim milk powder solutions were compared at a coating antibody concentration of 100 ng / well and a detection antibody concentration of 1:5000:

[0124] Experiment 1 used 1 wt% BSA as the blocking solution and diluent for samples, standards, and detection antibodies;

[0125] Experiment 2 used 5 wt% skim milk powder as the blocking solution and 1 wt% skim milk powder as the diluent for samples, standards, and detection antibodies.

[0126] Preparation of standard curve: BXB1 integrase standard was diluted to 800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, and 0 ng / mL (blank, i.e., dilution).

[0127] The test results are shown in Tables 7 and 8. When BSA solution was used for blocking and dilution, the blank OD value was much higher than that when skim milk powder was used for dilution and blocking, indicating that there may be non-specific adsorption between BSA and antibody, and the spiked recovery rate was low. The samples blocked and diluted with skim milk powder also had better detection accuracy. Therefore, skim milk powder was selected as the blocking and dilution for subsequent ELISA experiments.

[0128] Table 7. Detection results of BSA solution as blocking and diluent solutions

[0129]

[0130]

[0131] S+0 is the addition of 1 wt% BSA solution to a purified sample diluted 20 times.

[0132] S+25 (or 100, 200) is a spiked sample containing 25 ng / mL (or 100, 200 ng / mL) of BXB1 integrase standard, prepared by adding BXB1 integrase standard to a purified sample diluted 20 times.

[0133] Table 8. Detection results of skim milk powder solution as blocking and diluent solutions

[0134] Test sample OD value 1 OD value 2 average value Concentration ng / mL Recovery rate % S+0 0.0475 0.0479 0.0477 ND N / A S+25 0.205 0.2044 0.2047 22.80914 91.24 S+25 0.2128 0.1953 0.20405 22.69608 90.78 S+100 0.5031 0.5079 0.5055 95.80078 95.80 S+100 0.5254 0.484 0.5047 95.54138 95.54 S+200 0.7155 0.7028 0.70915 180.02930 90.01 S+200 0.6922 0.6979 0.69505 172.80884 86.40

[0135] S+0 is a purified sample diluted 20 times with 1 wt% skim milk powder solution added;

[0136] S+25 (or 100, 200) is a spiked sample containing 25 ng / mL (or 100, 200 ng / mL) of BXB1 integrase standard, prepared by adding BXB1 integrase standard to a purified sample diluted 20 times.

[0137] (3) Matrix interference effect:

[0138] ① Preparation of the test sample (spiked sample):

[0139] Sample 1 to be tested: Take sample 1 and dilute it 2, 5, 10, 20, 50 and 100 times with 1% skim milk powder diluent. Add BXB1 integrase standard to each sample to prepare a sample containing 50 ng / mL BXB1 integrase.

[0140] Sample 2 to be tested: Take sample 2 and dilute it 2, 5, 10 and 20 times with 1% skim milk powder diluent. Add BXB1 integrase standard to each sample to prepare test samples containing 50 ng / mL BXB1 integrase.

[0141] ② Double antibody sandwich ELISA detection

[0142] A. Antibody coating: Dilute rabbit polyclonal antibody BXB1 to 3 μg / mL with coating buffer, add 100 μL / well to a 96-well microplate, and incubate overnight at 4°C.

[0143] B. Blocking: Discard the liquid in the 96-well microplate that has been coated overnight, add 300 μL / well of PBST washing buffer and wash the plate 4 times, pat dry on absorbent paper, then add 200 μL / well of 5 wt% skim milk powder solution and incubate at 37°C for 2 hours.

[0144] C. Sample addition: Discard the blocking solution in the 96-well microplate, add PBST washing solution at 300 μL / well and wash the plate 4 times. Pat dry on absorbent paper. Take 100 μL each of BXB1 integrase standard solution (200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL), test sample solution, and blank solution (1 wt% skim milk powder solution) and add them to the 96-well microplate, repeating 2 times. Incubate at 37°C for 1 h.

[0145] D. Add detection antibody: Discard the sample solution in the 96-well plate, add 300 μL / well of PBST washing buffer and wash the plate 4 times. Pat dry on absorbent paper. Take HRP-BXB1 rabbit polyclonal antibody, dilute it with 1 wt% skim milk powder solution at a ratio of 1:2500, add 100 μL / well to the 96-well microplate, and incubate at 37°C for 1 h.

[0146] E. Color development: Discard the antibody solution in the 96-well microplate, add 300 μL / well of PBST washing buffer and wash the plate 4 times, pat dry on absorbent paper, add 100 μL / well of TMB substrate color development solution, and let stand at room temperature in the dark for 15 min for color development.

[0147] F. Termination: Add 50 μL of stop solution to each well of the 96-well plate to terminate the colorimetric reaction;

[0148] G. Plate reading: Read the OD450 value using an ELISA reader;

[0149] H. Calculation: Plot a standard curve with the concentration of the BXB1 integrase standard solution on the x-axis and the corresponding OD value on the y-axis. Substitute the OD value of the test sample into the standard curve to calculate the relative BXB1 integrase content.

[0150] ③ Spike recovery rate:

[0151] Recovery rate (%) = (Spiked sample value - Standard sample value) / Amount of standard added × 100%

[0152] The test results are shown in Tables 9 and 10. When the fermentation supernatant sample was diluted more than 50 times and the purified sample was diluted more than 20 times, the spike recovery rate of the test samples was between 80.0% and 120.0%, indicating that the interfering substances had little impact on the accuracy of this method in the fermentation supernatant sample diluted more than 50 times and the purified sample diluted more than 20 times (the corresponding standard curves are shown in Tables 9 and 10). Figure 3 ).

[0153] Table 9. Detection results of BXB1 integrase in fermentation supernatant samples.

[0154]

[0155] S1 is the fermentation supernatant sample;

[0156] S1-1, S1-2, S1-3, S1-4, S1-5, and S1-6 represent S1 samples diluted 2, 5, 10, 20, 50, and 100 times, respectively.

[0157] S1-1(S1-2, S1-3, S1-4, S1-5, S1-6)+50 indicates a spiked sample containing 50 ng / mL BXB1 integrase, prepared by adding BXB1 integrase standard to S1 sample diluted 2 times (5 times, 10 times, 20 times, 50 times, 100 times).

[0158] Table 10 Detection of BXB1 integrase in purified samples

[0159]

[0160] S2 is the fermentation supernatant sample;

[0161] S2-1, S2-2, S2-3, and S2-4 represent S2 samples diluted 2, 5, 10, and 20 times, respectively.

[0162] S2-1(S2-2, S2-3, S2-4)+50 indicates a spiked sample containing 50 ng / mL BXB1 integrase, prepared by adding BXB1 integrase standard to S1 sample diluted 2 times (5 times, 10 times, 20 times).

[0163] (4) Precision verification

[0164] ① Repeatability (intra-batch precision)

[0165] Prepare BXB1 integrase standard solutions using diluents: 5 ng / mL and 200 ng / mL. The diluent is a 1 wt% skim milk powder solution.

[0166] Prepare test samples by mixing fermentation supernatant diluted 50 times or purified sample diluted 10 times with different concentrations of BXB1 integrase dilution buffer or dilution buffer 1:1 (volume ratio):

[0167] G0 is a 50-fold diluted fermentation supernatant sample with the same volume of diluent (1 wt% skim milk powder solution);

[0168] G1 (or G2) is a 50-fold diluted fermentation supernatant sample with the same volume of 5 ng / mL (or 200 ng / mL) BXB1 integrase standard solution added.

[0169] S0 is a purified sample diluted 10-fold, with the same volume of diluent (1 wt% skim milk powder solution) added.

[0170] S1 (or S2) is a 10-fold diluted fermentation supernatant sample with the same volume of 5 ng / mL (or 200 ng / mL) BXB1 integrase standard solution added;

[0171] Each sample to be tested is configured in parallel in two copies (e.g., G1-1, G1-2), and the RSD value is calculated.

[0172] The test results are shown in Table 11 (the standard curves are shown in Table 11). Figure 4 The accuracy and reproducibility of the solutions for both fermented and purified samples were between 80.0% and 120.0%, and the RSD values ​​of parallel samples of the same sample were all less than 10.0%, that is, the intra-batch difference was less than 10.0%, indicating good accuracy and repeatability.

[0173] Table 11. Results of Repeatability Testing

[0174]

[0175] ② Intermediate precision (batch-to-batch precision)

[0176] Prepare two parallel samples for the repeatability experiment on different days, and perform the tests to calculate the RSD value.

[0177] The test results are shown in Table 12 (the standard curve is shown in Table 12). Figure 5 The RSD values ​​of the concentrations detected in the same sample on different days were all less than 15.0%, that is, the inter-batch difference was less than 15.0%, the intermediate precision was good, and the repeatability was good.

[0178] Table 12 Results of intermediate precision testing

[0179]

[0180] (5) Linear range

[0181] Prepare BXB1 integrase standard solutions using diluents: 200 ng / ml, 160 ng / ml, 120 ng / ml, 100 ng / ml, 60 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, and 5 ng / ml. The diluent is a 1 wt% skim milk powder solution.

[0182] Take a fermentation supernatant sample diluted 50 times (S1) or a purified sample diluted 10 times (S2), and mix it with different concentrations of BXB1 integrase dilution buffer or a 1:1 (volume ratio) dilution buffer to prepare the test sample:

[0183] S1+0 is a sample of fermentation supernatant diluted 50 times, to which an equal volume of diluent (1% skim milk powder solution) is added.

[0184] S1+5 (10, 20, 40, 60, 100, 120, 160, 200) refers to adding an equal volume of 5 ng / mL (10, 20, 40, 60, 100, 120, 160, 200 ng / mL) BXB1 integrase standard solution to fermentation supernatant samples diluted 50 times.

[0185] S2+0 is the addition of an equal volume of diluent (1% skim milk powder solution) to a purified sample diluted 10 times.

[0186] S2+5 (10, 20, 40, 60, 100, 120, 160, 200) refers to adding an equal volume of 5 ng / mL (10, 20, 40, 60, 100, 120, 160, 200 ng / mL) BXB1 integrase standard solution to purified samples diluted 10 times.

[0187] Each sample to be tested has two duplicate wells set in parallel.

[0188] The test results are shown in Table 13 (the corresponding standard curves are shown in Table 13). Figure 6 The recoveries of all tested samples ranged from 80.0% to 120.0%. A linear fitting curve was constructed with the spiked concentration on the x-axis and the corresponding OD value on the y-axis (see [link to curve]). Figure 7 and Figure 8 In fermentation supernatant samples diluted 100-fold or purified samples diluted 20-fold, when the BXB1 integrase concentration was between 2.5 and 30 ng / mL, the linear equation showed a good fit, with a correlation coefficient R0. 2 All are greater than 0.990.

[0189] Table 13. Detection results of samples with different concentrations

[0190] sample OD value 1 OD value 2 average value Concentration ng / mL Recovery rate % S1+0 0.0654 0.0532 0.0593 ND N / A S1+5 0.1269 0.1301 0.1285 2.1840 87.4 S1+10 0.2023 0.2048 0.20355 4.51683 90.3 S1+20 0.3281 0.3251 0.3266 8.5626 85.6 S1+40 0.59 0.5906 0.5903 18.2498 91.2 S1+60 0.8554 0.8449 0.85015 29.4075 98.0 S1+100 1.1528 1.2971 1.22495 49.20654 98.4 S1+120 1.4028 1.4238 1.4133 61.3236 102.2 S1+160 1.4861 1.5349 1.5105 68.2938 85.4 S1+200 1.6982 1.7113 1.70475 83.9874 84.0 S2+0 0.0558 0.0554 0.0556 ND N / A S2+5 0.1327 0.1366 0.13465 2.3714 94.9 S2+10 0.2135 0.2274 0.22045 5.0560 101.1 S2+20 0.3402 0.4031 0.37165 10.1154 101.2 S2+40 0.5799 0.6524 0.61615 19.2821 96.4 S2+60 0.8104 0.9081 0.85925 29.8325 99.4 S2+100 1.162 1.2429 1.20245 47.8668 95.7 S2+120 1.2637 1.3276 1.29565 53.5568 89.3 S2+160 1.5633 1.5432 1.55325 71.5332 89.4 S2+200 1.7633 1.7793 1.7713 89.9872 90.0

Claims

1. A double-antibody sandwich ELISA method for detecting BXB1 integrase, comprising: S1. Coating antibody: Coating an enzyme-linked immunosorbent assay (ELISA) plate to prepare solid-phase antibody; S2. Add blocking solution to block the ELISA plate; S3. Add the sample to be tested, incubate and wash, add the diluted detection antibody, incubate and wash. S4. Add TMB substrate colorimetric solution, incubate, then add stop solution to terminate the reaction, and measure the absorbance (OD value) at a wavelength of 450 nm. in, The coating antibody is a BXB1 rabbit polyclonal antibody; The detection antibody is an HRP-labeled BXB1 rabbit polyclonal antibody.

2. The detection method according to claim 1, wherein the BXB1 rabbit polyclonal antibody is prepared using BXB1 integrase with an amino acid sequence as shown in SEQ ID NO: 1 as the antigen; and the nucleotide sequence of the BXB1 integrase is shown in SEQ ID NO:

2.

3. The detection method according to any one of claims 2, wherein the coating amount of the coated antibody in step S1 is selected from 100 to 1000 ng / well, preferably 100 to 500 ng / well, and more preferably 300 to 500 ng / well; In step S3, the dilution ratio of the detection antibody can be selected from 1:1000 to 1:10000, preferably 1:2500 to 1:5000, and more preferably 1:2500.

4. The detection method according to any one of claims 1-3, further comprising: S5. Construction of standard curves; and S6. Calculate the concentration of BXB1 integrase in the sample to be tested based on the standard curve.

5. The detection method as described in claim 4, wherein the limit of detection for BXB1 integrase is 0.5 ng / mL.

6. The application of the double-antibody sandwich ELISA detection method for BXB1 integrase as described in any one of claims 1-5 in the preparation of recombinant biological products, including the detection of residual BXB1 integrase in the recombinant biological products; wherein, The recombinant biological product was prepared using BXB1 integrase.

7. The application as described in claim 6, in step S3: (1) When the recombinant biological product is a recombinant cell line prepared by BXB1 integrase, the sample to be tested is the fermentation supernatant of the recombinant cell line or the diluted solution of the fermentation supernatant; the dilution factor of the fermentation supernatant can be selected from 2 to 100 times, preferably 10 to 100 times, and more preferably 50 to 100 times. Or (2) When the recombinant biological product is a recombinant protein prepared and purified from BXB1 integrase, the sample to be tested is a solution of the recombinant protein or a diluent of the recombinant protein solution; the diluent of the recombinant protein solution may be selected from 2 to 100 times, preferably 10 to 100 times, and more preferably 20 to 100 times.

8. An ELISA kit for quantitative detection of BXB1 integrase, the kit comprising: ELISA plates coated with BXB1 rabbit polyclonal antibody, HRP-labeled BXB1 rabbit polyclonal antibody, BXB1 integrase standard, diluent, washing buffer, chromogenic solution, and stop solution.

9. The kit according to claim 8, wherein the BXB1 rabbit polyclonal antibody is prepared using BXB1 integrase with an amino acid sequence as shown in SEQ ID NO: 1 as the antigen; The amino acid sequence of the BXB1 integrase standard is shown in SEQ ID NO: 1; The nucleotide sequence of the BXB1 integrase is shown in SEQ ID NO:

2.

10. The application of the ELISA kit for quantitative detection of BXB1 integrase as described in claim 8 or 9 in the preparation of recombinant biological products, comprising: The kit was used to detect residual BXB1 integrase in recombinant biological products; wherein, The recombinant biological product was prepared using BXB1 integrase.

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