Electrochemiluminescence detection method of PARP enzyme, kit and application thereof

Abstract

The invention discloses an electrochemiluminescence detection method of PARP enzyme, a kit and application thereof. The electrochemical luminescence detection method for detecting PARP enzyme comprises the following steps: (1) mixing human whole blood, a PARP inhibitor and a PARG inhibitor, incubating, and centrifuging to obtain human peripheral blood mononuclear cells; (2) collecting a human peripheral blood mononuclear cell precipitate for cracking, detecting the total protein concentration, adding a sample, adding a cell lysis solution into an electrochemical luminescent plate for incubation, and adding a PARP enzyme detection antibody for re-incubation; and (3) adding a detection secondary antibody for incubation to form a capture antibody-PARP enzyme-detection antibody-secondary antibody complex, and adding plate reading liquid for plate reading. The electrochemical luminescence detection method and the kit have the characteristics of high sensitivity, high specificity, high accuracy, high precision and simple operation method, and can quantitatively detect the content of PARP enzyme.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and relates to an electrochemiluminescence detection method for PARP enzyme, a kit and application thereof. BACKGROUND

[0002] Poly-ADP-ribosylation modification (PARylation) is a form of post-translational modification of proteins, which connects ADP ribose polymers (ADPr) to proteins through PARP enzyme catalysis to regulate cell metabolism and stress response. According to the degree of modification, it is divided into three categories: mono-PARylation, oligo-PARylation and poly-PARylation. The poly-ADP-ribosylation modification process is catalyzed by poly-ADP-ribose polymerase (PARP) and plays a key role in various biological processes, especially in the process of DNA damage response (DDR). When the cell occurs DDR, PAPR1 is activated and forms a PAR chain to help repair factors to gather at the damage site, promote chromatin rearrangement and repair process.

[0003] Through the detection of poly-ADP-ribosylation modification, it can directly reflect whether the function of PARP enzyme is inhibited, and understand the drug efficacy, so the accuracy and sensitivity of the detection of poly-ADP-ribosylation modification will directly affect the judgment of the drug efficacy, and the significant correlation between the detection of poly-ADP-ribosylation modification and the drug efficacy is a difficulty to be solved in the detection of poly-ADP-ribosylation modification.

[0004] “Synthetic lethality” is that any single mutation or inaction of two genes in the cell will not cause cell death, but the simultaneous mutation or inaction of the two genes will cause cell death, and the principle can be used to selectively kill tumor cells without affecting normal body cells. The anticancer drugs targeting DNA damage response use this pathway to treat tumors by using the gene or functional defects in this pathway. In solid tumors, human peripheral blood mononuclear cells are a normal tissue compared with cancer tissues, and the level of poly-ADP-ribosylation modification in normal peripheral blood mononuclear cells is extremely low, which is basically lower than the detection limit of many detection methods at present.

[0005] Therefore, how to accurately evaluate polyADP ribosylation modification in human peripheral blood mononuclear cells, and thus conveniently and promptly monitor the efficacy of PARP inhibitors and polyADP ribohydrolase (PARG) inhibitors without surgery or puncture to obtain tumor tissue, remains a problem that urgently needs to be solved by those skilled in the art. Detecting changes in polyADP ribosylation modification in human peripheral blood mononuclear cells using immunofluorescence activated cell sorting requires stimulation of the cells with stimulants. The methodology is of moderate difficulty to develop and carries a certain risk of development failure. The stability of whole blood collection and transportation needs to be carefully examined. Furthermore, the results are qualitative and cannot accurately quantify the content of polyADP ribosylation modification. This requires a high degree of instrumentation, fresh samples, and simultaneous detection of large samples, and demands a high level of expertise from operators, making it unsuitable for on-site monitoring and large-scale sample screening.

[0006] CN119044481A discloses a screening method for polyADP-ribose polymerase 1 inhibitors, an ELISA kit, and their applications. The screening method provided by this invention includes: coating a microplate with histone H3, then adding polyADP-ribose polymerase 1 and the drug to be tested for a first incubation, followed by adding NAD+ for a second incubation; after the second incubation, removing the reaction solution, adding a primary antibody that specifically recognizes polyADP-ribosylated modification for a third incubation; after the third incubation, removing the reaction solution, adding a secondary antibody that specifically recognizes the primary antibody and is labeled with horseradish peroxidase for a fourth incubation; after the fourth incubation, removing the reaction solution, adding TMB chromogenic solution for a colorimetric reaction, and calculating the inhibition rate of the drug against polyADP-ribose polymerase 1 based on the colorimetric results. However, this kit has low sensitivity and accuracy.

[0007] Therefore, there is an urgent need to provide a method and kit for detecting PARP enzymes that has high sensitivity, high specificity, high accuracy, high precision, and simple operation. Summary of the Invention

[0008] To address the shortcomings of existing technologies and practical needs, this invention provides an electrochemiluminescence detection method, kit, and application of PARP enzymes. The aim is to provide an electrochemiluminescence enzyme-linked immunosorbent assay kit with high sensitivity, high specificity, high accuracy, high precision, and simple operation method for quantitative detection of PARP enzyme content in human peripheral blood mononuclear cells, thereby indirectly evaluating the pharmacodynamics of synthetic lethal drugs such as PARP inhibitors and PARG inhibitors.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides an electrochemiluminescence detection method for detecting PARP enzyme, the electrochemiluminescence detection method for detecting PARP enzyme comprising the following steps:

[0011] (1) Human peripheral blood mononuclear cells were obtained by centrifugation after incubation with human whole blood, PARP inhibitor and PARG inhibitor.

[0012] (2) Collect human peripheral blood mononuclear cell pellets for lysis, detect total protein concentration, adjust the sample loading amount according to protein concentration and then add the sample. Add cell lysate to electrochemiluminescence plate for incubation, wash, add PARP enzyme detection antibody for incubation again, and wash.

[0013] (3) Add the detection secondary antibody and incubate to form a capture antibody-PARP enzyme-detection antibody-secondary antibody complex, then add the reading solution for plate reading.

[0014] The electrochemiluminescence detection method of the present invention has the characteristics of high sensitivity, high specificity, high accuracy, high precision, and simple operation. It can quantitatively detect the content of PARP enzyme, with a limit of quantification (LLOQ) of 82.30 pM and a linear range of 82.3 pM to 20000 pM.

[0015] Preferably, the centrifugation to obtain human peripheral blood mononuclear cells in step (1) further includes: mixing the obtained human peripheral blood mononuclear cells with a stimulant and incubating them, followed by centrifugation.

[0016] Preferably, the stimulant comprises hydrogen peroxide.

[0017] Preferably, the volume ratio of whole blood, PARP inhibitor, and PARG inhibitor in step (1) is 1:(1×10⁻⁶). -2 ~1×10 6 ):(1×10 -2 ~1×10 6 ).

[0018] The above 1×10 -2 ~1×10 6 The specific point value in the value can be selected as 1×10. -2 1×10 -1 1, 10, 10 2 10 3 10 4 10 5 Or 10 6 .

[0019] Preferably, the centrifugation speed in step (1) is 500×g to 1000×g (e.g., 500×g, 600×g, 800×g, 900×g or 1000×g), and the centrifugation time is 20 to 60 min (e.g., 20 min, 30 min, 40 min, 50 min or 60 min).

[0020] Preferably, the incubation time in step (2) is 1 to 3 hours (e.g., 1 hour, 2 hours or 3 hours), and the re-incubation time is 1 to 3 hours (e.g., 1 hour, 2 hours or 3 hours).

[0021] Preferably, the incubation time in step (3) is 0.5 to 1.5 hours (e.g., 0.5 hours, 1 hour or 1.5 hours).

[0022] Preferably, the electrochemiluminescent plate in step (2) is coated and sealed.

[0023] Preferably, the plate coating includes: sealing the unused wells of the electrochemiluminescence plate with a sealing film, adding a diluted capture antibody solution to each remaining well, sealing the plate, and then incubating it overnight at 500–1000 rpm (e.g., 500 rpm, 800 rpm, or 1000 rpm) for 20–60 s (e.g., 20 s, 40 s, or 60 s) at room temperature, followed by incubation at 2°C–8°C (e.g., 2°C, 4°C, 6°C, or 8°C) for 16–18 h (e.g., 16 h, 17 h, or 18 h).

[0024] Preferably, the plate sealing includes: adding 100-200 μL (e.g., 100 μL, 150 μL, or 200 μL) of sealing solution to each well, and then incubating at 23°C-27°C (e.g., 23°C, 25°C, or 27°C) and 500-1000 rpm (e.g., 500 rpm, 800 rpm, or 1000 rpm) for 0.5-2 h (e.g., 0.5 h, 1 h, or 2 h).

[0025] Preferably, the sample addition in step (2) includes adding 50-150 μL (e.g., 50 μL, 100 μL or 150 μL) of standard, 50-150 μL (e.g., 50 μL, 100 μL or 150 μL) of quality control and 50-150 μL (e.g., 50 μL, 100 μL or 150 μL) of test sample to each well, sealing the membrane, and incubating at 23℃-27℃ (e.g., 23℃, 25℃ or 27℃) and 500-1000 rpm (e.g., 500 rpm, 800 rpm or 1000 rpm) for 1-3 h (e.g., 1 h, 2 h or 3 h).

[0026] Preferably, the addition of PARP enzyme detection antibody in step (2) includes: adding 20-100 μL (e.g., 20 μL, 50 μL or 100 μL) of diluted detection antibody solution to each well, sealing the membrane, and incubating at 23°C-27°C (e.g., 23°C, 25°C or 27°C) and 500-1000 rpm (e.g., 500 rpm, 800 rpm or 1000 rpm) for 1-3 h (e.g., 1 h, 2 h or 3 h).

[0027] Preferably, the addition of the detection secondary antibody in step (3) includes: adding 20-100 μL (e.g., 20 μL, 50 μL or 100 μL) of diluted conjugated antibody solution to each well, sealing the membrane, and incubating at 23°C-27°C (e.g., 23°C, 25°C or 27°C) and 500-1000 rpm (e.g., 500 rpm, 800 rpm or 1000 rpm) for 0.5-2 h (e.g., 0.5 h, 1 h or 2 h).

[0028] Preferably, the plate reading in step (3) includes: adding 100-120 μL (e.g., 100 μL, 110 μL or 120 μL) of plate reading solution to each well and reading the plate immediately.

[0029] Secondly, the present invention provides an electrochemiluminescence detection kit for detecting PARP enzyme, the kit comprising: a high-affinity electrochemiluminescence plate, a capture antibody, a detection antibody, a standard, a quality control, a secondary antibody labeled with an electrochemiluminescence marker, and a reading solution.

[0030] Preferably, the PARP enzyme is derived from peripheral blood mononuclear cells.

[0031] Preferably, the capture antibody comprises a mouse monoclonal antibody.

[0032] Preferably, the detection antibody includes rabbit polyclonal antibodies.

[0033] Preferably, the standard comprises an ADP polymer.

[0034] Preferably, the secondary antibody comprises a goat anti-rabbit antibody.

[0035] Preferably, the electrochemiluminescence detection kit further includes an stimulant.

[0036] Preferably, the stimulant comprises hydrogen peroxide.

[0037] The preparation of quality control samples in this invention includes: using the standard stock solution as the highest concentration gradient dilution of the standard. Five quality control concentrations are prepared using sample blocking solution: upper limit of quantitation (ULOQ), high concentration quality control (HQC), medium concentration quality control (MQC), low concentration quality control (LQC), and lower limit of quantitation (LLOQ), as shown in Table 1.

[0038] Table 1

[0039]

[0040] Thirdly, the present invention provides the application of the electrochemiluminescence detection method for detecting PARP enzymes as described in the first aspect or the electrochemiluminescence detection kit for detecting PARP enzymes as described in the second aspect in the preparation of products for detecting PARP enzyme content or evaluating the efficacy of PARP inhibitors and / or PARG inhibitors.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) In this invention, mouse monoclonal antibodies were screened as capture antibodies, ADP polymers were used as standards, and polyclonal antibodies were screened as detection antibodies (the detection value of the highest point signal of the standard curve of antibody 89190S was increased from 10501 to 22034 compared with antibody 83732S, which is doubled), and an electrochemical enzyme-linked immunosorbent assay kit for detecting the content of PARP enzyme in human peripheral blood mononuclear cells was established.

[0043] (2) The detection kit of the present invention can accurately and sensitively detect the content of PARP enzyme in human peripheral blood mononuclear cell lysate, with a limit of quantitation (LLOQ) of 82.30 pM and a linear range of 82.3 pM-20000 pM. The sample pretreatment process is simple and time-saving, and a large number of samples can be detected simultaneously. The sample detection cost is much lower than that of traditional instrument detection methods. Moreover, the kit of the present invention has a long shelf life, a wide linear range, high sensitivity, and no radioactive contamination. The present invention has important practical significance for solving the problem of the inhibitory effect of PARP inhibitors on PARP enzyme and the effect of PARG inhibitors on the increase of PARP enzyme in large batches of samples. Attached Figure Description

[0044] Figure 1 The inhibition effect of adding different concentrations of PARG inhibitors to whole blood is shown in the graph. Detailed Implementation

[0045] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0046] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0047] Example 1

[0048] This embodiment provides an electrochemical detection enzyme-linked immunosorbent assay kit for detecting PARP enzyme content, specifically comprising the following components:

[0049] High-affinity electrochemiluminescence plate (96 wells) (QuickPlex 96-Well HighBindPlate Pack; Manufacturer: MSD; Catalog No. L55XB-3), coating antibody is mouse monoclonal antibody with a final concentration of 100 nM (Anti-PAR Mono Ab w / o; Manufacturer: R&DSYSTEMS; Catalog No. 4335-MC-100), detection antibody is rabbit polyclonal antibody with a final concentration of 100 nM (Poly / Mono-ADP Ribose (D9P7Z) RabbitmAb; Manufacturer: CST; Catalog No. 89190S), standard is ADP polymer with a final concentration of 100,000 pM, quality control is ADP polymer with final concentrations of 200 pM, 1500 pM, 15000 pM, and 20000 pM (Poly(ADP-ribose) Pol; Manufacturer: R&D). SYSTEMS; Catalog No. 4336-100-01), the electrochemiluminescent labeled secondary antibody was goat anti-rabbit antibody (SULFO-TAG Labeled Anti-Rabbit Antibody (Goat); Manufacturer: MSD; Catalog No. R32AB-1) with a final concentration of 5 μg / mL, and the reading buffer was 4× ReadbufferT.

[0050] By comparing different catalog numbers of detection antibodies, catalog number 89190S is more effective than catalog number 83732S. By comparing different catalog numbers of detection secondary antibodies, catalog number R32AB-1 is more effective than catalog number R32AD-5. By comparing different catalog numbers of reaction plates, catalog number L55XB-3 is more effective and has a lower background value than catalog number L55XA-3.

[0051] Example 2

[0052] This embodiment provides an electrochemical detection method for detecting PARP enzyme content.

[0053] Whole blood from healthy volunteers was divided into three groups (Group 1: PBMCs were isolated first, and cells were lysed after stimulation with PARG inhibitors; Group 2: PBMCs were isolated and cells were lysed after stimulation with PARG inhibitors; Group 3: PBMCs were obtained by erythropoiesis after stimulation with PARG inhibitors, and then cells were lysed). Different concentrations of PARG inhibitors were added to simulate drug entry into the human bloodstream. The effect of the PARG inhibitor was evaluated by detecting the PARP enzyme content according to the electrochemical detection method of this invention, as detailed below:

[0054] 1. PARG inhibitor whole blood incubation

[0055] Whole blood of 40 mL was collected from healthy volunteers using EDTAK2 anticoagulant blood collection tubes (containing DMSO solvent). The volunteers were then divided into 6 groups, with 3 mL of whole blood in each group. PARG inhibitors were added at concentrations of 0 μM, 0.03 μM, 0.10 μM, 0.30 μM, and 1.00 μM, respectively, and incubated at room temperature for 4 hours. A group containing whole blood supplemented with a PARP inhibitor (Olaparib) was kept as a negative control.

[0056] 2. PBMC separation, hydrogen peroxide stimulation, and pyrolysis

[0057] 2.1 PBMC Separation

[0058] After incubation with the drug, the whole blood and control group were transferred to human lymphocyte separation medium and placed in a horizontal rotor (open head) centrifuge. The centrifuge was balanced and set to 800g for 30 minutes at 25°C without brakes (i.e., slow acceleration and deceleration).

[0059] After centrifugation, mononuclear cells will be in the white layer, immediately adjacent to the upper plasma layer. Carefully aspirate 2 / 3 of the upper plasma layer using a Pasteur pipette, being careful not to disturb the underlying cell layer. The upper plasma layer can be disposed of according to biomedical waste regulations. Then, carefully aspirate the white layer of cells (lymphocytes and mononuclear cells) into a 15 mL conical centrifuge tube using a new Pasteur pipette, and label the tube with the corresponding sample information using a marker.

[0060] PBMC washing: Add at least 10 times the volume of PBMC in PBS solution each time, 300g, 10min, 25°C. Wash at least twice.

[0061] Transferring PBMCs: After centrifugation, carefully aspirate as much supernatant as possible, avoiding blowing up the cell pellet. Resuspend the cells in 200 μL of resuspension buffer (RPMI 1640 + 10% FBS, ensuring it has been brought to room temperature (10℃-30℃), count the cells, and transfer them to a 1.5 mL centrifuge tube.

[0062] 2.2 Hydrogen peroxide stimulation of PBMCs

[0063] Stimulating PBMCs: Add 200 μL of hydrogen peroxide (5 mM) to the PBMC cell pellet (cell density approximately 1.5 × 10⁻⁶). 7 Mix the contents of the container ( / mL) immediately by pipetting and incubate at room temperature (10℃-30℃) for 5 minutes (incubation time must be strictly controlled).

[0064] Immediately centrifuge the PBMC suspension in a 1.5 mL centrifuge tube at 500 g for 3 min at 18℃-25℃. After centrifugation, remove as much supernatant as possible (ensuring less than 20 μL of liquid remains on the cell pellet).

[0065] 2.3 Pyrolysis of PBMC

[0066] Immediately after centrifugation, add 200 μL of cell lysis buffer to the cell pellet, mix well by pipetting, and lyse on ice for at least 30 minutes.

[0067] After lysis, centrifuge the 1.5 mL centrifuge tube at 12000g for 10 min at 4℃, transfer the supernatant to a new tube, add 10% SDS according to the volume of the lysis buffer to make the final concentration of SDS 1%, and then place it in a metal bath at 100℃ for 5 min.

[0068] After boiling the sample, label the 1.5mL centrifuge tubes and centrifuge at 12000g for 10min at 4℃. Transfer the supernatant to a new tube and store it in a -90℃ refrigerator for later use.

[0069] 2.4 Protein Quantification Using the BCA Method

[0070] The cell lysis buffer was removed from the -90°C freezer, thawed on wet ice, and then analyzed using the BCA protein quantification kit (Pierce). TM The BCA Protein Assay Kit (manufacturer: ThermoFisher; catalog number: 23227) was used to determine the protein concentration in each group. The purpose was to adjust the sample loading amount in each group to be consistent.

[0071] 3. PARP enzyme content detection

[0072] Antibody dilution: Dilute all the protein lysate after protein quantification to a concentration of 0.5 mg / mL using cell lysis buffer.

[0073] Experimental plate preparation: Remove the electrochemiluminescence microplate (QuickPlex 96-Well High Bind Plate Pack; Manufacturer: MSD; Catalog No. L55XB-3), and seal any unused wells with sealing film. Add 50 μL of diluted capture antibody solution (Anti-PAR Mono Ab w / o; Manufacturer: R&D SYSTEMS; Catalog No. 4335-MC-100) to each well, seal, and incubate at room temperature with shaking at 700 rpm for 30 s. Then transfer to 2℃~8℃ and incubate overnight for 16~18 h.

[0074] Plate sealing: Add 150 μL of blocking solution to each well, seal the membrane, and incubate at room temperature and 700 rpm for 1 h.

[0075] Add 100 μL of standard (Poly(ADP-ribose)Pol; manufacturer: R&D SYSTEMS; catalog number 4336-100-01) / quality control / sample to each well, seal the membrane, and incubate at room temperature and 700 rpm for 2 h.

[0076] The quality control samples were prepared according to the method in Table 1.

[0077] Add detection antibody: Add 50 μL of diluted detection antibody solution (Poly / Mono-ADP Ribose (D9P7Z) Rabbit mAb; manufacturer: CST; catalog number 89190S) to each well, seal the membrane, and incubate at room temperature and 700 rpm for 2 h.

[0078] Add secondary antibody: Add 50 μL of diluted conjugated antibody solution (SULFO-TAG Labeled Anti-Rabbit Antibody (Goat); Manufacturer: MSD; Catalog No. R32AB-1) to each well. After sealing, incubate at room temperature and 700 rpm for 1 h.

[0079] Reading the plate: Add 150 μL of reading solution to each well and read the plate immediately using a MESO QuickPlex SQ 120MM plate reader.

[0080] The plate samples are shown in Table 2, the signal values ​​are shown in Table 3, and the inhibitory effects of adding different concentrations of PARG inhibitors to whole blood are shown in Table 4. The method of the present invention can accurately quantify the content of PARP enzyme and has a wide range of applications.

[0081] Table 2

[0082]

[0083] Table 3

[0084]

[0085]

[0086] Table 4

[0087]

[0088] 4. Interpretation and Explanation of Results

[0089] Under all three conditions, PARP inhibitors (Olaparib) could inhibit PARP enzyme levels; PARG inhibitors could increase PARP enzyme levels, up to 10 times.

[0090] The inhibitory effects of adding different concentrations of PARG inhibitors to whole blood are as follows: Figure 1 As shown, the concentration of PARP enzyme decreased significantly after the addition of PARP inhibitors. Then, after adding different concentrations of PARP inhibitors, all three treatment methods could effectively inhibit the hydrolysis of PARP enzymes and increase the concentration of PARP enzymes, indicating that the method of the present invention is feasible and universal.

[0091] Example 3

[0092] Standard curve and quantitative range detection.

[0093] To determine the standard curve range, repeatability, and reproducibility, six analytical batches of standards will be repeatedly tested (standards for each experiment must be freshly prepared and used immediately). At least two people must complete the experiments for all six analytical batches within at least two days. Each analytical batch includes a standard curve to evaluate the accuracy and precision of each plate. The standard curve range is tentatively set at 82.30 pM to 20000.00 pM.

[0094] Acceptable standards for each batch of experiments: The relative deviation (RE) of the backcalculated average concentration at each concentration level within the quantitation range compared to the labeled value is within ±20% (RE within ±25% for the upper limit of quantitation (ULOQ) and lower limit of quantitation (LLOQ), and the coefficient of variation (CV) between replicates is ≤20% (CV ≤25% for ULOQ and LLOQ). Excluding anchor points, at least 75% of the calibration standards (including LLOQ and ULOQ) at at least six concentration levels should meet the above standards. Anchor points, being outside the quantitation range of the calibration curve, are not required to meet the above standards. Blank standards will be used to monitor the batch background values ​​but will not participate in curve regression analysis. Non-zero calibration standards will participate in curve fitting.

[0095] The goodness of fit of the intra-batch / inter-batch standard curves for six independent batches, as well as the coefficient of variation (CV) and relative deviation (RE) at each concentration point, were investigated.

[0096] Table 5 shows the inter-batch precision and accuracy data of the standard curves for the six analytical batches of standards.

[0097] Table 5

[0098]

[0099] Intra-batch results for PARPase in the concentration range of 82.30 pM to 20000.00 pM for six batches: CV for Standard 1: 0.4%–6.8%, RE: -3.4%–2.3%; CV for Standard 2: 1.6%–14.8%, RE: -6.5%–15.3%; CV for Standard 3: 0.2%–3.2%, RE: -13.1%–13.2%; CV for Standard 4: 0.6%–5.5%, RE: -7.3%–9.7%; CV for Standard 5: 3.3%–9.3%, RE: -7.5%–12.9%; CV for Standard 6: 0.2%–11.0%, RE: -6.2%–7.7%; CV for Standard 7: 0.5%–13.8%, RE: -3.5%–16.5%. Inter-batch results for 6 batches: CV: 3.0%–13.1%, RE: -3.2%–4.1%. All met the acceptance criteria, and the accuracy and precision of the standard curve passed. The quantitative range of the standard curve for PARP enzyme detection is 82.30 pM–20000.00 pM.

[0100] Example 4

[0101] Intra-batch and inter-batch precision and accuracy testing.

[0102] Intra-batch / inter-batch precision and accuracy testing is performed by at least two analysts over at least two days, analyzing six batches of data. Three sets of quality control samples are prepared for intra-batch / inter-batch precision and accuracy, provided the standard curve meets acceptance criteria: Upper limit of quantitation (ULOQ) (20000.00 pM), high concentration quality control (HQC) (15000.00 pM), medium concentration quality control (MQC) (1500.00 pM), low concentration quality control (LQC) (200.00 pM), and lower limit of quantitation (LLOQ) (82.30 pM). The back-calculated concentration of each QC sample is used to evaluate accuracy and precision. Accuracy and precision are evaluated in the following order: 1) replicate precision; 2) intra-batch accuracy and precision; 3) inter-batch accuracy and precision. The precision and accuracy of the next batch are evaluated only after the previous batch's precision and accuracy meet acceptance criteria.

[0103] Acceptable standards: For intra-batch and inter-batch accuracy, the average concentration (RE) of each quality control sample is within ±20% of the labeled value (RE for ULOQ and LLOQ is within ±25%). For intra-batch and inter-batch precision, the detection concentration CV of each quality control sample is ≤20% (CV for ULOQ and LLOQ is ≤25%), and the total intra-batch and inter-batch error (TE) is ≤30% (ULOQ / LLOQ ≤40%).

[0104] The inter-batch accuracy, precision, and minimum quantitation limits of the six analytical batches of standards are shown in Table 6.

[0105] Table 6

[0106]

[0107]

[0108] The results showed that, within the batch: ULOQ (20000.00pM) had a CV of 3.7%–12.3%, RE of -1.5%–5.3%, and TE of 4.9%–13.8%; HQC (15000.00pM) had a CV of 2.4%–8.5%, RE of -12.1%–1.2%, and TE of 2.5%–15.8%; MQC (1500.00pM) had a CV of 0.9%– 8.6%, RE: -6.5% to 16.4%, TE: 14.0% to 18.0%; CV of LQC (150.00pM): ​​3.7% to 9.7%, RE: -5.4% to 12. 8%, TE: 8.0% to 22.5%; LLOQ (82.30pM): ​​CV: 5.5% to 13.8%, RE: -4.7% to 11.1%, TE: 9.7% to 19.3%.

[0109] Inter-batch: CV: 6.1%–8.8%; RE: -5.0%–10.3%; TE: 7.1%–18.1%.

[0110] All quality control measures met acceptance criteria, and intra-batch and inter-batch accuracy and precision were passed.

[0111] Example 5

[0112] Linear dilution detection.

[0113] PBMCs were isolated from whole blood of 6 healthy individuals, lysed, and BCA protein concentration was determined. Serial dilutions were then performed to obtain linearly diluted samples. Linearity was assessed by performing 2-fold serial dilutions (at least 3 dilutions within the standard curve range).

[0114] Acceptable criteria: Within the detection range of the standard curve, the cross-sectional area (CV) between replicates of different dilution factors is ≤30%, and the linear dilution range is defined as dilution factors with a reactivity (RE) within ±30%. At least two-thirds of the samples should meet the acceptance criteria. Recommended loading concentrations for clinical samples are based on the linear dilution results.

[0115] The linear dilution data results for Sample 1 are shown in Table 7.

[0116] Table 7

[0117]

[0118]

[0119] Note: Sample 1 represents the PBMC sample of healthy volunteer 1. Samples 1-6, 1-7, and 1-8 have signal values ​​below the detection limit and are therefore not included in statistical analysis.

[0120] The linear dilution and hook effect data of samples 1-3 (S1-S3) are shown in Table 8.

[0121] Table 8

[0122]

[0123]

[0124]

[0125] Note: S1 to S3 represent PBMC samples from healthy volunteers 1 to 3. 50,000.00 pM of standard was added to the PBMC lysis buffer, followed by serial dilutions. The 50,000.00 pM and 25,000.00 pM samples were used as HOOK effect samples. NaN in S1-1 indicates values ​​greater than the upper limit of quantitation; no value is displayed. NA indicates not applicable.

[0126] The results showed that after diluting the lysed samples to 1000.00 μg / mL, the blocking buffer was serially diluted. The CV for samples 1-1 (1000.00 μg / mL) to 1-5 (62.50 μg / mL) ranged from 0.1% to 13.7%, and the RE ranged from -10.9% to 4.0%. The CV for samples 2-1 (1000.00 μg / mL) to 2-6 (31.25 μg / mL) ranged from 0.4% to 2.9%, and the RE ranged from -14.4% to 12.9%. The CV for samples 3-1 (1000.00 μg / mL) to 3-6 (31.25 μg / mL) ranged from 0.3% to 6%. The CV for samples 4-1 (1000.00 μg / mL) to 4-5 (62.50 μg / mL) ranged from 0.3% to 9.9%, with RE ranging from -8.8% to 13.5%. The CV for samples 5-1 (1000.00 μg / mL) to 5-5 (62.50 μg / mL) ranged from 3.2% to 4.9%, with RE ranging from 4.0% to 18.3%. The CV for samples 6-1 (700.00 μg / mL) to 6-5 (43.75 μg / mL) ranged from 0.5% to 5.2%, with RE ranging from -2.6% to 18.1%.

[0127] For PBMC samples, a total protein loading concentration of 1000.00 μg / mL to 62.50 μg / mL (not lower than the limit of detection) yields a CV of 0.1% to 13.7% and an RE of -14.4% to 18.3%, satisfying the established standards for 2 / 3 of the samples. The recommended total protein loading concentration is 1000.00 μg / mL to 200.00 μg / mL.

[0128] After adding an excess of ADP polymer standard stock solution to the PBMC lysis buffer of 6 healthy volunteers, the buffer was serially diluted 2-fold to 8 gradients. 50000.00 pM and 25000.00 pM were used as hook effect samples. The PAR content in PBMCs was low compared to the bid concentration and could be ignored; the bid concentration was used as the theoretical value. No signal suppression was observed at 50000.00 pM and 25000.00 pM, indicating no hook effect. The CV for sample S1 at dilutions of 16-256 times ranged from 0.1% to 5.5%, and the RE ranged from -28.7% to 19.3%. For sample S2, the CV at dilutions of 16-256 times ranged from 1.4% to 13.2%, and the RE ranged from -27.8% to 28.3%. For sample S3, the CV at dilutions of 16-128 times ranged from 1.7% to 5.7%, and the RE ranged from -26.5% to 7.5%. For sample S4, the CV at dilutions of 16-128 times ranged from 0.8% to 13.6%, and the RE ranged from -9.9% to 27.9%. For sample S5, the CV at dilutions of 8-128 times ranged from 2.3% to 29.5%, and the RE ranged from -14.5% to 29.2%. For sample S6, the CV at dilutions of 16-128 times ranged from 1.7% to 13.2%, and the RE ranged from -26.1% to -9.5%. At 16-128x, all samples met the acceptance criteria, and the exogenous standard ADP polymer did not interfere with the detection of PARP enzyme content in PBMC lysate.

[0129] Example 6

[0130] High-fat effect detection.

[0131] Three PBMC samples from healthy individuals were used for hemolysis validation. Triglyceride solution was added to the PBMC lysis buffer to prepare cell samples with a triglyceride concentration of 600 mg / dL and those without triglycerides (baseline). Finally, the protein in the cell lysis supernatant was quantified to the same concentration before sample loading and detection. The concentration of PARP enzyme in the high-lipid sample was calculated back based on the standard curve.

[0132] For high-fat studies, at least two-thirds of the samples must have a RE within ±20%, and the precision of the final concentration back-calculated must not exceed 30%. In such cases, the method is considered unaffected by high-fat conditions.

[0133] The summary results of the high-fat sample test data are shown in Table 9.

[0134] Table 9

[0135]

[0136] Note: The concentration detected in non-high-fat normal samples is used as the baseline, and the RE is calculated back from the concentration detected in high-fat samples. TRI-NC represents the blank control (non-high-fat sample) of the high-fat sample, and TRI-N represents the corresponding high-fat sample. NA indicates not applicable.

[0137] Based on the above results, the protein concentration in PBMC samples from healthy volunteers was low after lysis. Therefore, all samples were diluted to a total protein concentration of 300 μg / mL before loading for detection. After adding triglycerides, a high-fat sample with a concentration of 600 mg / dL was prepared. Compared with baseline, the CV of the high-fat sample was 0.9%–1.4%, and the RE was -2.7%–1.6%, meeting the acceptance criteria. High-fat did not affect the detection of PARP enzyme content.

[0138] Example 7

[0139] Hemolysis test.

[0140] Three PBMC samples from healthy individuals were used for hemolysis validation. Hemoglobin solution was added to the PBMC lysis buffer to prepare cell samples (baseline) with hemoglobin concentrations of 2.5 mg / mL and 0 mg / mL (i.e., no hemoglobin). Finally, the protein concentration in the cell lysis supernatant was quantified to the same concentration before sample loading and detection. The hemolyzed sample concentration was calculated back based on the standard curve.

[0141] For hemolysis testing, at least two-thirds of the samples must have a RE within ±30%, and the precision of the final concentration back calculation must not exceed 30%. In such cases, the method is considered unaffected by hemolysis.

[0142] The summary results of the hemolytic sample test data are shown in Table 10.

[0143] Table 10

[0144]

[0145]

[0146] Note: The concentration detected in the non-hemolyzed normal sample is used as the baseline, and the RE is calculated back from the concentration detected in the hemolyzed sample. HEM-NC represents the blank control (non-hemolyzed sample) of the hemolyzed sample, and HEM-N represents the corresponding hemolyzed sample. NA indicates not applicable.

[0147] Based on the above results, the protein concentration was low in PBMC lysis from healthy volunteers. Therefore, all samples were diluted to a concentration of 300 μg / mL for detection. After adding hemoglobin solution, samples with a hemoglobin concentration of 2.5 mg / mL were prepared. Compared with samples without hemoglobin, the CV was 1.7%–4.6%, and the RE was -2.3%–2.1%, which met the acceptance criteria. Hemolysis did not affect the detection of PARP enzyme content.

[0148] Example 8

[0149] Stability test.

[0150] Stability was assessed using PBMCs isolated from whole blood of six healthy individuals. All stability samples (except baseline samples) were frozen in liquid nitrogen for at least 24 hours before processing.

[0151] Freeze-thaw stability: 6 PBMCs were tested. Each sample underwent 3 repeated freeze-thaw cycles (the protein lysate was stored for freeze-thaw testing after PBMC lysis. The first freeze-thaw test was performed for at least 24 hours until the sample was completely frozen. The second and subsequent freeze tests were performed for at least 12 hours of complete freezing, followed by natural thawing at room temperature). The test results on the day of sample collection were used as the baseline.

[0152] Transportation stability: Six whole blood samples were transported under simulated conditions (2℃-8℃) for 24h, 48h, and 72h, respectively, and PBMCs were isolated. The test results on the day of sample collection were used as the baseline.

[0153] Storage stability: The 6 isolated PBMC samples were aliquoted and stored under different conditions and tested at different time points. The test results on the day of sample preparation were used as the baseline. The detection concentration of the samples at different conditions and time points was compared with the baseline. The sample stability time point was based on the actual test time point.

[0154] The baseline data results are shown in Table 11.

[0155] Table 11

[0156]

[0157] Based on the above results, after being placed at 2℃ to 8℃ for 3 days, the CV of the samples ranged from 0.0% to 5.8%, and the RE ranged from -16.0% to -5.2%. The short-term stability after three freeze-thaw cycles showed a CV of 0.8% to 2.6% and an RE of -11.5% to -4.7%. After 24 hours of transport, the CV of the samples ranged from 0.4% to 18.9%, and the RE ranged from -26.9% to 18.8%, with sample 5 showing an RE of -55.4%. After 48 hours of transport, the CV of the samples ranged from 0.7% to 15.3%, and the RE ranged from -25.1% to 20.7%, with sample 1 showing an RE of -46.8% and sample 5 showing an RE of -81.3%. After 72 hours of transport, the CV of the samples ranged from 0.3% to 67.2%, with sample 1 showing an RE of -45.1%, sample 4 showing an RE of -50.9%, sample 5 showing an RE of -80.3%, and sample 6 showing an RE of -61.0%, failing to meet the acceptance criteria. The CV of the reconstituted PBMC samples stored at -60℃ to -90℃ for one month was 0.6% to 7.5%, and the RE was -18.7% to 26.1%. The CV of the reconstituted PBMC samples stored in liquid nitrogen for one month was 0.7% to 6.1%, and the RE was -29.1% to 18.9%. Except for the 72-hour transport period (transport stability is tentatively set at 48 hours), all validation items in the other experiments met the requirement that at least 2 / 3 of the samples passed, and the stability test was passed.

[0158] In summary, the electrochemiluminescence detection method and kit of the present invention have the characteristics of high sensitivity, high specificity, high accuracy, high precision, and simple operation. It can quantitatively detect the content of PARP enzyme, with a limit of quantitation (LLOQ) of 82.30 pM and a linear range of 82.3 pM-20000 pM.

[0159] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An electrochemiluminescence detection method for detecting PARP enzyme, characterized in that, The electrochemiluminescence detection method for detecting PARP enzyme includes the following steps: (1) Human peripheral blood mononuclear cells were obtained by centrifugation after incubation with human whole blood, PARP inhibitor and PARG inhibitor. (2) Collect human peripheral blood mononuclear cell pellets for lysis, detect total protein concentration and add samples, add cell lysate to electrochemiluminescence plate for incubation, wash, add PARP enzyme detection antibody for incubation again, and wash. (3) Add the detection secondary antibody and incubate to form a capture antibody-PARP enzyme-detection antibody-secondary antibody complex, then add the reading solution for plate reading.

2. The method according to claim 1, characterized in that, The step (1) of obtaining human peripheral blood mononuclear cells by centrifugation further includes: mixing the obtained human peripheral blood mononuclear cells with a stimulant and incubating them, followed by centrifugation; Preferably, the stimulant comprises hydrogen peroxide.

3. The method according to claim 1 or 2, characterized in that, The volume ratio of whole blood, PARP inhibitor, and PARG inhibitor in step (1) is 1:(1×10⁻⁶). -2 ~1×10 6 ):(1×10 -2 ~1×10 6 ).

4. The method according to any one of claims 1-3, characterized in that, The centrifugation speed in step (1) is 500×g to 1000×g, and the centrifugation time is 20 to 60 min.

5. The method according to any one of claims 1-4, characterized in that, The incubation time in step (2) is 1 to 3 hours, and the re-incubation time is 1 to 3 hours; Preferably, the incubation time in step (3) is 0.5 to 1.5 hours.

6. The method according to any one of claims 1-5, characterized in that, The electrochemiluminescence plate described in step (2) is coated and sealed. Preferably, the plate coating includes: sealing the unused wells of the electrochemiluminescence plate with a sealing film, adding a diluted capture antibody solution to each remaining well, sealing the plate, shaking at 500-1000 rpm for 20-60 seconds at room temperature, and then transferring it to an environment of 2℃-8℃ for overnight incubation for 16-18 hours. Preferably, the plate sealing includes: adding 100-200 μL of sealing solution to each well, sealing the film, and incubating at 23°C-27°C and 500-1000 rpm for 0.5-2 hours.

7. The method according to any one of claims 1-6, characterized in that, The sample addition in step (2) includes adding 50-150 μL of standard, 50-150 μL of quality control and 50-150 μL of test sample to each well, sealing the membrane and incubating at 23℃-27℃ and 500-1000 rpm for 1-3 h. Preferably, the addition of PARP enzyme detection antibody in step (2) includes: adding 20-100 μL of diluted detection antibody solution to each well, sealing the membrane, and incubating at 23°C-27°C and 500-1000 rpm for 1-3 hours; Preferably, the addition of the detection secondary antibody in step (3) includes: adding 20-100 μL of diluted conjugated antibody solution to each well, sealing the membrane, and incubating at 23°C-27°C and 500-1000 rpm for 0.5-2 h; Preferably, the plate reading in step (3) includes: adding 100-120 μL of plate reading solution to each well and reading the plate immediately.

8. An electrochemiluminescence detection kit for detecting PARP enzyme, characterized in that, The kit includes: a high-affinity electrochemiluminescence plate, a capture antibody, a detection antibody, standards, quality control products, a secondary antibody labeled with an electrochemiluminescence marker, and a reading solution.

9. The electrochemiluminescence detection kit for detecting PARP enzyme according to claim 8, characterized in that, The PARP enzyme is derived from peripheral blood mononuclear cells; Preferably, the capture antibody comprises a mouse monoclonal antibody; Preferably, the detection antibody includes rabbit polyclonal antibodies; Preferably, the standard comprises an ADP polymer; Preferably, the secondary antibody comprises goat anti-rabbit antibody; Preferably, the electrochemiluminescence detection kit further includes an stimulant; Preferably, the stimulant comprises hydrogen peroxide.

10. The use of the electrochemiluminescence detection method for detecting PARP enzyme according to any one of claims 1-7 or the electrochemiluminescence detection kit for detecting PARP enzyme according to claim 8 or 9 in the preparation of products for detecting PARP enzyme content or evaluating the efficacy of PARP inhibitors and / or PARG inhibitors.

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