SPINK1 specific ELISA detection kit for senile detection and preparation method thereof

Abstract

The present application relates to the field of biomedical detection, specifically to a SPINK1 specific ELISA detection kit for aging detection, the kit contains polyclonal antibodies specific to SPINK1 protein and a color developing reaction system for assisting detection; the kit is also configured with E. coli derived recombinant SPINK1 protein with a purity of more than 99% as a standard for concentration calibration; the specific reagent list supplied in the kit includes horseradish peroxidase labeled antibody, blocking solution, washing buffer, the color developing reaction system is composed of 3,3',5,5'-tetramethylbenzidine solution, and the termination solution for interrupting the color developing chemical reaction is independently packaged. Through prokaryotic expression technology and targeted optimization of core induction conditions, combined with protein affinity chromatography purification process based on imidazole elution mechanism, the present application successfully realizes high-efficiency soluble expression of SPINK1 protein in E. coli vector and obtains recombinant protein with a purity of more than 99%.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a SPINK1-specific ELISA detection kit for aging detection and its preparation method. Background Technology

[0002] Aging is a complex biological process accompanied by a comprehensive decline in cellular physiological functions and changes in the expression of various specific secretory phenotypic markers. SPINK1 protein, a classic serine protease inhibitor, has recently been shown to play a unique role in cell life cycle and the aging process. In senescent cells and tissues, SPINK1 expression levels are closely correlated with remaining cell lifespan; generally, higher expression levels are associated with shorter cell lifespan. This significant negative correlation makes this protein a potential high-value biomarker for assessing the degree of aging and cellular senescence.

[0003] Current immunological detection technologies for extracting and measuring trace proteins in complex biological microenvironments largely rely on traditional immunoblotting procedures or the use of conventional commercially available general-purpose ELISA kits. Conventional detection and evaluation systems often directly purchase commercially available non-specific or universal antibodies for target capture and identification. In current clinical testing and life science research practices, obtaining biological samples containing the target and directly using these conventional antibodies in combination with a universal secondary antibody system for colorimetric reading constitutes the mainstream operational standard for assessing the abundance of the target protein.

[0004] Investigating the specific role of SPINK1 in the aging process and accurately quantifying it inevitably faces many substantial difficulties and challenges. Endogenous SPINK1 expression levels are often relatively low, and in the extremely complex microenvironment of aging tissues, its actual content is easily and severely interfered with by various unknown matrices and contaminating proteins, making it difficult for conventional detection methods to accurately capture and quantify free SPINK1 protein. There is a severe lack of commercially available specialized antibodies with high specificity and affinity specifically targeting the native conformation of SPINK1. This industry situation severely limits the functional exploration, accurate quantification, and widespread application of SPINK1 in the assessment of aging indicators at the protein level. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a SPINK1-specific ELISA detection kit for aging detection and its preparation method, which solves the problems of poor specificity, low sensitivity, and difficulty in accurately quantifying aging status in existing technologies for SPINK1 protein detection.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a SPINK1-specific ELISA kit for aging detection, wherein the kit contains a polyclonal antibody that binds highly specifically to the SPINK1 protein and a colorimetric reaction system for auxiliary detection; the kit also contains recombinant SPINK1 protein from E. coli with a purity of over 99% as a standard for concentration calibration.

[0007] Preferably, the specific reagent list supplied in the kit includes horseradish peroxidase-labeled antibody, blocking solution, washing buffer, and the colorimetric reaction system consists of 3,3',5,5'-tetramethylbenzidine solution, and is separately packaged with a stop solution for interrupting the colorimetric chemical reaction.

[0008] Preferably, a method for preparing a SPINK1-specific ELISA detection kit for aging detection is provided, wherein the method begins with constructing an expression vector and completing the soluble induction expression and chromatographic purification of recombinant SPINK1 protein; using the obtained high-purity target protein, an in vivo animal immunization procedure is performed to prepare polyclonal antibodies; subsequent in-depth antibody purification steps are completed and the antibodies are physically assembled with conventional ELISA auxiliary biochemical reagents.

[0009] Preferably, the specific process steps for controlling the expression of recombinant SPINK1 protein are as follows:

[0010] The PET expression plasmid containing the SPINK1 gene was successfully transformed into E. coli competent cells to establish a stable engineered strain;

[0011] The fermentation broth was intervened with highly targeted induction conditions. Specifically, when the host bacterial culture grew and metabolized to the point where the OD value reached 0.6, isopropyl-β-D-thiogalactoside was added to the fermentation system at a final concentration of 1 mM as an external inducer.

[0012] The induction temperature was set at 37℃ throughout the entire process, and the culture was carried out at a constant temperature to promote the efficient and soluble expression of recombinant SPINK1 protein in E. coli.

[0013] After centrifuging to collect bacterial cells and performing disruption, the supernatant was obtained. Protein affinity chromatography was used to separate impurity proteins and obtain recombinant SPINK1 protein that meets the requirements for immunoassay grade.

[0014] Preferably, the preparation steps of the polyclonal antibody are as follows:

[0015] Injecting high-purity recombinant SPINK1 antigen stimulates the animal's immune system to produce a specific response. After immunization, live serum from the animals is collected, and the crude serum is purified by alternating ammonium sulfate precipitation and Protein A affinity chromatography. The purified antibody is then labeled by indirect ELISA, and its immunogenicity is controlled at 1:128,000.

[0016] Preferably, the preparation steps for the concentration calibration standard in the kit are as follows:

[0017] The prepared recombinant SPINK1 protein stock solution was serially diluted at an initial concentration of 300 ng / mL to construct a standard system with an absorbance linear range covering the OD value range of 0.137 to 3.436.

[0018] Preferably, an in vitro detection method for assessing aging process using a specific ELISA detection kit is characterized in that the procedure is directly based on quantitative determination data of the absolute content of SPINK1 protein in the test biological sample, and the types of biological samples to be tested include body serum obtained by centrifugation of whole blood and supernatant of senescent cells collected in an in vitro culture system.

[0019] Preferably, the specific steps for using this kit to complete the standardized sample determination are as follows:

[0020] In the sample pretreatment stage, the pre-extracted blood sample is placed in a centrifuge and centrifuged at 3000 r / min for 5 minutes to extract the serum to be tested.

[0021] Add 100 μL of standard or sample to be tested to the microplate according to the preset array, seal the wells and transfer the whole plate into a 37°C incubator for 1 hour.

[0022] After discarding the free liquid in the wells and rinsing thoroughly three times with washing buffer, pat dry and inject 100 μL of HRP-labeled detection antibody solution into each well. Incubate at 37°C for 1 hour.

[0023] Repeat the washing and tapping motions to remove unbound material, add 100 μL of TMB colorimetric solution, and initiate the enzymatic colorimetric reaction at room temperature in the dark. The reaction time should be controlled between 20 and 30 minutes.

[0024] 50 μL of stop solution was rapidly injected into each independent reaction system to destroy enzyme activity. The absorbance value of the corresponding microwell was read using a professional microplate reader at an excitation wavelength of 450 nm. The accurate expression concentration of SPINK1 in the sample was scientifically calculated using the standard curve equation.

[0025] This invention provides a SPINK1-specific ELISA detection kit for aging detection and its preparation method. It has the following beneficial effects:

[0026] 1. This invention, by relying on prokaryotic expression technology and specifically optimizing core induction conditions (1 mM IPTG concentration, 37°C isothermal temperature, and precise control of the induction start point at an OD value of 0.6) combined with a protein affinity chromatography purification process based on imidazole elution mechanism, successfully achieved efficient soluble expression of SPINK1 protein in E. coli vectors and obtained recombinant protein with a purity of over 99%. This establishes an extremely solid material foundation for the construction of the internal standard system of the detection kit and the subsequent acquisition of high-quality immunogenic antigens.

[0027] 2. This invention utilizes purified high-purity recombinant SPINK1 protein as an antigen to implement a standardized animal immunization program on experimental rabbits, including primary immunization and multiple rounds of booster immunization. Simultaneously, a dual deep purification mechanism is introduced, combining 33% ammonium sulfate precipitation with Protein A affinity chromatography, ultimately obtaining a high-affinity polyclonal antibody with an immunogenicity of 1:128,000. This purification strategy significantly improves the kit's sensitivity to antigen specificity and absolute accuracy in dealing with biological samples with complex compositions.

[0028] 3. This invention successfully constructs a precise ELISA detection system with a wide linear response range of 0.137-3.436 OD values ​​by coating self-made high-titer polyclonal antibodies onto ELISA plates and assembling kits with serially diluted recombinant SPINK1 protein standards at a maximum concentration of 300 ng / mL. This system can accurately and stably quantify the trace SPINK1 expression levels in the supernatant of senescent cells and complex body serum samples, effectively establishing a new biochemical indicator system and reliable judgment basis for the scientific assessment of aging in clinical practice. Attached Figure Description

[0029] Figure 1 This is an SDS-PAGE electrophoresis analysis of SPINK1 protein before and after induction of expression in this invention;

[0030] Figure 2 This is an SDS-PAGE electrophoresis analysis of the SPINK1 protein purified by affinity chromatography according to the present invention.

[0031] Figure 3 This is an SDS-PAGE electrophoresis analysis of the SPINK1 protein concentrate of this invention;

[0032] Figure 4 This is an SDS-PAGE electrophoresis analysis of the purified SPINK1 antibody after salt precipitation according to the present invention;

[0033] Figure 5 This is an SDS-PAGE electrophoresis analysis of the SPINK1 antibody of this invention purified by Protein A chromatography;

[0034] Figure 6 This is an SDS-PAGE electrophoresis analysis of the antibody concentrate of this invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1:

[0037] This invention provides a method for inducing expression and extracting and detecting SPINK1 protein, as detailed below:

[0038] Transformed single-clone colonies were inoculated into a container containing 10 mL of LB liquid medium (containing ampicillin, with a final concentration controlled at 100 μg / mL) and incubated on a shaker at 37°C for 16 hours for activation. 2.5 mL of the well-activated PET32a-SPINK1 engineered bacterial culture was then inoculated into 500 mL of fresh LB liquid medium (also maintaining an ampicillin final concentration of 100 μg / mL) for further expansion. Micro-samples of the bacterial culture were taken at set time intervals to measure their OD values. When the OD value of the fermentation broth reached the range of 0.6 to 0.8, 100 μL of 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) solution was precisely added to initiate the induction program. A portion of homologous bacterial culture without IPTG was reserved as a negative control for protein expression. SDS-PAGE electrophoresis analysis results before and after SPINK1 protein induction are shown below. Figure 1 As shown, 1 represents before SPINK1 induction, 2 represents after SPINK1 induction, and M represents the protein molecular weight standard. Compared with the bacterial culture without IPTG induction, a band with a particularly high expression level can be seen at around 20 KD, and the band size is consistent with the target protein.

[0039] After maintaining induced expression at 37℃ for 4 hours, 1 mL of bacterial culture before and after the induction point was transferred to centrifuge tubes. The centrifuge was run at 12000 rpm and 4℃ for 3 minutes to completely discard the supernatant and collect the precipitated bacterial cells. 1 mL of PBS buffer was added to the enriched bacterial cell tube, and the cells were repeatedly resuspended using a pipette. Then, 40 μL of the suspension was transferred to a clean EP tube, and 10 μL of loading buffer was added. The EP tube was then directly placed in a boiling water bath for 10 minutes to destroy the higher-order structure of the protein. The treated samples before and after induction were extracted, pumped into gel channels, and separated by SDS-PAGE electrophoresis. Coomassie brilliant blue staining was used to visually verify the expression level and preliminary purity of the target protein.

[0040] Example 2:

[0041] This invention provides an affinity chromatography purification and concentration method for SPINK1 protein, as detailed below:

[0042] 500 mL of fermented bacterial cells were extracted and transferred to a large-capacity centrifuge tube for centrifugation and concentration. After discarding the supernatant, 20 mL of specially prepared lysis buffer was added to the precipitate. An ultrasonic cell disruptor was then used to mechanically disrupt the resuspension. The instrument's operating parameters were strictly calibrated as follows: 3 seconds of ultrasonic excitation, followed by a 5-second pause, for a total processing time of 30 minutes. The disrupted liquid was centrifuged again, and the supernatant enriched with soluble target proteins was carefully aspirated for later use.

[0043] The protein purification gravity chromatography column (with built-in affinity packing material) was removed from the 4°C refrigerated environment and allowed to stand to return to room temperature. 30 mL of binding buffer was slowly injected, allowing it to flow naturally under gravity to equilibrate the column microenvironment. The previously collected SPINK1 protein-rich supernatant was robustly loaded into the column, and the runoff was collected using a collection container. The column bed was then repeatedly washed with a wash buffer containing 20 mM imidazole to remove non-specifically adsorbed impurities, and the wash buffer was collected. Next, a precise elution program was performed using an elution buffer containing 260 mM imidazole, collecting the eluent containing high-purity target protein into the corresponding test tubes. The resin was then deeply regenerated and eluted using 10 mL of 500 mM imidazole regeneration buffer to remove residues, and the runoff was collected. All collected solutions were subjected to SDS-PAGE gel electrophoresis. Coomassie brilliant blue staining was used to identify the target SPINK1 protein elution fraction with a purity of over 99%. The SDS-PAGE electrophoresis analysis results of the SPINK1 protein purified by affinity chromatography are as follows: Figure 2As shown, 1 represents SPINK1 flow-through buffer, 2 represents 20 mM imidazole washing buffer, 3 and 4 represent 260 mM imidazole elution buffer, 5 represents 500 mM imidazole regeneration buffer, and M represents the protein molecular weight standard. Lane 1 indicates successful elution of non-bound impurities; lane 2 indicates successful removal of non-specifically bound contaminating proteins; lanes 3 and 4 show a single, clear band approximately 20 kDa in size with almost no other bands, indicating that the target protein was obtained with high purity. Therefore, the expected purification effect was achieved, and the results prove that the protein purification was successful.

[0044] In the concentration process, the enriched high-purity protein solution was transferred to an ultrafiltration tube with a matched molecular weight cutoff, and centrifuged at 3000 rpm for 1 hour. After the initial ultrafiltration, TGE solution was added to the concentration tube until the 15 mL mark was reached, and the tube was centrifuged again at 2500 rpm for 1 hour to replace the system. This centrifugation cycle was repeated. PBS buffer was then added to the 15 mL mark, and the tube was centrifuged at 2500 rpm for 1 hour to wash and replace the system, and this process was repeated. After multiple replacements and concentrations, the high-concentration SPINK1 protein solution at the bottom of the tube was carefully aspirated, and a small amount of the concentrated product was extracted for final SDS-PAGE electrophoresis purity verification. The SDS-PAGE electrophoresis results of the SPINK1 protein concentrate are shown below. Figure 3 As shown, 1 represents protein concentrate and M represents protein molecular weight standard. A clear protein band appears in lane 1. Compared with the marker on the right, the molecular weight range corresponding to this band position is between 17-25 kDa, which is consistent with the expected molecular weight of SPINK1 protein, indicating that the target protein was successfully concentrated.

[0045] Example 3:

[0046] This invention provides a method for the preparation, purification, and titer identification of SPINK1 polyclonal antibodies, as detailed below:

[0047] High-quality purified SPINK1 protein was selected as the specific antigen to initiate the in vivo animal immunization protocol. Healthy rabbits were selected as experimental subjects. During the first primary immunization, a quantitative amount of purified SPINK1 protein antigen was vigorously emulsified and mixed with Freund's complete adjuvant in a glass container, ensuring that the aqueous antigen was perfectly encapsulated within the core of the microscopic oil droplets formed by the adjuvant. The emulsion was injected into the rabbits via deep intramuscular injection to establish a basic immune response. After a one-week rest period following the primary immunization, a second booster immunization was initiated. Subsequent third and fourth consolidation immunizations were administered continuously at one-week intervals. On the 7th day after the end of each single immunization cycle, a suitable amount of blood was slowly drawn from the marginal ear vein of the rabbit using a syringe for later use.

[0048] The collected blood samples were centrifuged to separate the blood layers, and the clear aqueous phase containing antiserum was carefully aspirated from the supernatant. Deep purification of the antibody was achieved through a two-step tandem process of ammonium sulfate precipitation and Protein A column chromatography. Under the protection of an ice-water bath and continuous vortexing with a magnetic stirrer, a 33% saturated ammonium sulfate solution was added dropwise to the SPINK1 antibody-rich serum stock solution. The mixture was kept in an ice-water bath with stirring for up to 1 hour to force the antibody molecules to salt out and precipitate completely. Subsequently, the mixture was centrifuged at 14000 rpm for 30 minutes at 4°C, and the supernatant was discarded, collecting the protein precipitate containing the antibody. The precipitate was reconstituted using an appropriate amount of buffer, and the above salting-out precipitation step was repeated once. The initial serum, the supernatant from each precipitation separation, and the final purified precipitate solution were collected for SDS-PAGE electrophoresis for comparison. Protein A affinity purification was initiated, and the affinity chromatography column was pre-equilibrated with 30 mL of binding buffer. The crude antibody solution, after salting out and reconstitution, was slowly loaded onto the packing bed, with the flow-through waste collected simultaneously. The column was flushed with a dedicated wash buffer, and the corresponding wash buffer was collected. The bound antibody was then dissociated using acidic elution buffer, carefully collected, and immediately neutralized. The column matrix was flushed with regeneration buffer for later use. The collected chromatographic solutions were also verified for antibody integrity and purity by SDS-PAGE electrophoresis with Coomassie brilliant blue staining.

[0049] The SDS-PAGE electrophoresis analysis results of the purified SPINK1 antibody after salt precipitation are as follows: Figure 4 As shown, 1 represents SPINK1 serum, 2 represents the supernatant after the first precipitation with 33% ammonium sulfate, 3 represents the supernatant after the second precipitation with 33% ammonium sulfate, 4 represents the crude purified antibody, and M represents the protein molecular weight standard. Lane 2 still shows obvious impurity bands, indicating that the supernatant after the first precipitation contains a large amount of contaminating proteins. Lane 3 shows no obvious protein bands, indicating that contaminating proteins have been removed after two ammonium sulfate precipitations. Lane 4 shows obvious protein bands with molecular weights meeting expectations, indicating successful purification of SPINK1 antibody, and a significant reduction in impurity bands, indicating good purification results.

[0050] The results of SDS-PAGE electrophoresis analysis of SPINK1 antibody purified by Protein A chromatography are as follows: Figure 5As shown, 1 represents flow-through buffer, 2 represents washing buffer, 3-4 represent elution buffer, 5 represents regeneration buffer, and M represents protein molecular weight standard. Lane 1 shows no obvious specific band, indicating that impurities not bound to the column have been removed; lane 2 shows no obvious band, indicating no non-specifically adsorbed proteins; the collected elution buffer from lanes 3-4 shows a single, clear band with the expected molecular weight, indicating successful purification of SPINK1 antibody; lane 5 shows a clear antibody band, indicating that the regeneration buffer successfully removed residual SPINK1 antibody from the column.

[0051] In the subsequent concentration stage, the purified SPINK1 antibody dilute solution was first centrifuged at 3000 rpm for 1 hour at 4°C to remove impurities. The pretreated, clear antibody solution was then transferred to a high-retention ultrafiltration centrifuge tube and centrifuged at 10000 rpm for 30 minutes in a precision centrifuge to force dehydration. After centrifugation, a sample of the high-concentration polyclonal antibody solution retained at the bottom of the tube was taken for SDS-PAGE electrophoresis to verify its final concentrated quality.

[0052] The results of SDS-PAGE electrophoresis analysis of the antibody concentrate are as follows: Figure 6 As shown, 1 represents protein concentrate and M represents protein molecular weight standard. Lane 1 shows a clear and single band, and its position matches the theoretical molecular weight of the SPINK1 polyclonal antibody, indicating that the SPINK1 polyclonal antibody was successfully concentrated.

[0053] The immunogenicity of extracted antibodies was assessed using an indirect ELISA method. Antigen coating: The diluted SPINK1 antigen solution was evenly distributed into the microplate wells, ensuring a volume of 200 μL per well. The plate was then smoothly transferred to a 37°C incubator for 1 hour to promote antigen adsorption. Blocking: The plate was inverted to remove any residual liquid. Washing buffer was continuously injected, followed by agitation and washing three times. After washing, the plate was firmly patted dry on clean absorbent paper. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for another hour. Three standardized washes were then performed, followed by patting dry. Binding: The obtained SPINK1 antibody was pre-diluted with a strict gradient, covering a range from 1:4000 to 1:128000. These different dilutions of antibody solution were injected into the corresponding wells of the ELISA plate, 100 μL per well. To ensure the scientific rigor of the data, a blank control group containing only PBS solution and a negative control group containing only antibody buffer were added concurrently. The plate with samples added was incubated at 37°C for 1 hour to allow sufficient time for antigen-antibody targeting. After the incubation period, three washes with washing buffer were performed to thoroughly remove any free antibodies that failed to form specific binding. For the addition of secondary antibody: HRP (horseradish peroxidase)-labeled secondary antibody solution diluted with blocking buffer at a specific ratio was injected, with a uniform volume of 200 μL per well. The plate was incubated again at 37°C for 1 hour, followed by three standardized washes with washing buffer to remove redundant secondary antibody. For color development and reading: TMB (3,3',5,5'-tetramethylbenzidine) was used as the substrate chromogenic agent, with 100 μL of substrate solution added to each well. The plate was placed in a dark chamber at room temperature for 30 minutes to stimulate color development. Immediately after color development, an acidic stop solution is added to halt the enzymatic reaction. The operator uses a professional microplate reader to select a 450nm detection wavelength and sequentially reads the precise absorbance (OD) values ​​in each well. By calculating the ratio (P / N) of the absorbance data of the positive test sample (P) to the negative control sample (N), the obtained polyclonal antibody is finally determined and verified to possess an excellent immunogenicity of up to 1:128,000.

[0054] Example 4:

[0055] This invention provides a method for the large-scale preparation and sample detection application of an ELISA detection kit. The kit preparation involves pre-coating a microplate. A validated high-affinity polyclonal antibody is diluted to an appropriate working concentration using coating buffer and quantitatively added to the detection wells of the matching microplate. The plate is then placed horizontally in a 37°C incubator for 1 hour to complete physical adsorption. Next, the wells are washed three times with washing buffer and patted dry on paper. Sufficient blocking solution is immediately added to fill any blank sites on the bottom of the plate, and the plate is incubated at 37°C for 1 hour.

[0056] When adding samples for evaluation, wash the plate three times with washing buffer and blot dry. Prepare a series of recombinant SPINK1 protein standard solutions with gradient concentrations, setting the starting concentration of the standard curve at 300 ng / mL and extending the dilution proportionally towards the lower concentration end. Transfer the above standards into the corresponding wells, ensuring an injection volume of 100 μL per well. Incubate at 37°C for 1 hour, then wash three more times with washing buffer and blot dry. Introduce the chromogenic tracking molecule: precisely add 100 μL of HRP-labeled targeted detection antibody solution to each well. Place the plate in a 37°C incubator for the final 1 hour of incubation.

[0057] After the incubation process is complete, wash the plate three times with washing buffer to remove background impurities. Colorimetric reaction stage: Add 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) colorimetric reaction solution to each well to be read. Allow the loaded microplate to react at room temperature in the dark for 30 minutes to promote blue color development. Reaction termination and data entry stage: Quickly squeeze 50 μL of dedicated chemical stop solution into each well; the original blue product instantly turns yellow, indicating that the reaction has completely frozen. Use an optical microplate reader to read the absorbance (OD) of each individual well at a fixed absorption wavelength of 450 nm. A linear range with a stable OD value between 0.137 and 3.436 was established based on various concentrations of standards, with a correlation coefficient R... 2 The perfect standard curve that reaches the extreme value 1.

[0058] In the actual verification of aging-related SPINK1 content detection, the extracted blood sample was first placed in a centrifuge and centrifuged at 3000 rpm for 5 minutes. The supernatant serum was carefully aspirated after separation. 100 μL of the processed serum sample or the supernatant of senescent cells cultured in vitro was then inoculated into the test wells of the kit. The ELISA plate was then incubated at 37°C for 1 hour. After removal, it was washed three times with washing buffer and then agitated to dry. 100 μL of activated HRP-labeled antibody reagent was added to the wells, and the plate was incubated at 37°C for another hour.

[0059] After incubation and a standard three-wash, blot-drying process, 100 μL of TMB chromogenic solution was promptly added to initiate the final chemical color development, and the reaction was carried out for 20 minutes in a dark environment at room temperature. Immediately after the time was up, 50 μL of stop solution was sprayed in to fix the color intensity. The accurate absorbance (OD) values ​​of the test wells were obtained at a wavelength of 450 nm using the measuring instrument. The tester simply substitutes the read parameters into a pre-plotted and calibrated precision standard curve equation to accurately calculate and deduce the absolute presence and content of natural SPINK1 protein in the test sample, thereby completing an objective quantitative assessment of the aging state of the corresponding organism.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A SPINK1-specific ELISA assay kit for aging detection, characterized in that, The kit contains a polyclonal antibody that binds to the SPINK1 protein with high specificity and a colorimetric reaction system to assist in detection; the kit also contains recombinant SPINK1 protein from E. coli with a purity of over 99% as a standard for concentration calibration.

2. The SPINK1-specific ELISA detection kit for aging detection according to claim 1, characterized in that, The kit includes a list of specific reagents, such as horseradish peroxidase-labeled antibody, blocking solution, and washing buffer. The colorimetric reaction system consists of a 3,3',5,5'-tetramethylbenzidine solution and is also separately packaged with a stop solution for interrupting the colorimetric chemical reaction.

3. A method for preparing a SPINK1-specific ELISA detection kit for aging detection according to claim 1 or 2, characterized in that, The method begins with constructing an expression vector and completing the soluble induction expression and chromatographic purification of recombinant SPINK1 protein; based on the obtained high-purity target protein, an in vivo animal immunization procedure is carried out to prepare polyclonal antibodies, and subsequent in-depth antibody purification processes are completed and physically assembled with conventional ELISA-assisted biochemical reagents.

4. The method for preparing a SPINK1-specific ELISA detection kit for aging detection according to claim 3, characterized in that, The specific process steps for controlling recombinant SPINK1 protein expression are as follows: The PET expression plasmid containing the SPINK1 gene was successfully transformed into E. coli competent cells to establish a stable engineered strain; The fermentation broth was intervened with highly targeted induction conditions. Specifically, when the host bacterial culture grew and metabolized to the point where the OD value reached 0.6, isopropyl-β-D-thiogalactoside was added to the fermentation system at a final concentration of 1 mM as an external inducer. The induction temperature was set at 37℃ throughout the entire process, and the culture was carried out at a constant temperature to promote the efficient and soluble expression of recombinant SPINK1 protein in E. coli. After centrifuging to collect bacterial cells and performing disruption, the supernatant was obtained. Protein affinity chromatography was used to separate impurity proteins and obtain recombinant SPINK1 protein that meets the requirements for immunoassay grade.

5. The method for preparing a SPINK1-specific ELISA detection kit for aging detection according to claim 3, characterized in that, The specific steps for preparing the polyclonal antibody are as follows: Injecting high-purity recombinant SPINK1 antigen stimulates the animal's immune system to produce a specific response. After immunization, live serum from the animals is collected, and the crude serum is purified by alternating ammonium sulfate precipitation and Protein A affinity chromatography. The purified antibody is then labeled by indirect ELISA, and its immunogenicity is controlled at 1:128,000.

6. The method for preparing a SPINK1-specific ELISA detection kit for aging detection according to claim 3, characterized in that, The preparation steps for the concentration calibration standards in the kit are as follows: The prepared recombinant SPINK1 protein stock solution was serially diluted at an initial concentration of 300 ng / mL to construct a standard system with an absorbance linear range covering the OD value range of 0.137 to 3.

436.

7. An in vitro detection method for assessing aging process using the specific ELISA detection kit of claim 1, characterized in that, This procedure is based directly on the quantitative determination data of the absolute content of SPINK1 protein in the test biological samples. The types of biological samples required for the test include body serum obtained by centrifugation of whole blood and supernatant of senescent cells collected in an in vitro culture system.

8. The in vitro detection method according to claim 7, characterized in that, The specific steps for using this kit to complete the standardized sample assay are as follows: In the sample pretreatment stage, the pre-extracted blood sample is placed in a centrifuge and centrifuged at 3000 r / min for 5 minutes to extract the serum to be tested. Add 100 μL of standard or sample to be tested to the microplate according to the preset array, seal the wells and transfer the whole plate into a 37°C incubator for 1 hour. After discarding the free liquid in the wells and rinsing thoroughly three times with washing buffer, pat dry and inject 100 μL of HRP-labeled detection antibody solution into each well. Incubate at 37°C for 1 hour. Repeat the washing and tapping motions to remove unbound material, add 100 μL of TMB colorimetric solution, and initiate the enzymatic colorimetric reaction at room temperature in the dark. The reaction time should be controlled between 20 and 30 minutes. 50 μL of stop solution was rapidly injected into each independent reaction system to destroy enzyme activity. The absorbance value of the corresponding microwell was read using a professional microplate reader at an excitation wavelength of 450 nm. The accurate expression concentration of SPINK1 in the sample was scientifically calculated using the standard curve equation.

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