Methods of making neutrophil gelatinase-associated lipocalin and antibodies

Abstract

This invention relates to the field of bioengineering technology, specifically to a method for preparing neutrophil gelatinase-associated lipid transport protein and its antibody. Specifically, using pET23a(+) as the expression vector, the NGAL gene with six His tags fused to its N-terminus under promoter control is introduced into *E. coli* for induced expression. After purification using a Ni Focurose 6FF column and a molecular sieve column, tag-free NGAL protein with a purity ≥95% is obtained. Subsequently, the NGAL protein is used to repeatedly immunize Japanese rabbits, and serum is collected after each immunization. Qualified serum is screened using a two-dimensional agar diffusion assay, and then purified using the aforementioned antigen to prepare an antigen affinity purification column, yielding a highly active NGAL polyclonal antibody. The high-purity recombinant NGAL of this invention not only possesses activity but is also low-cost, not limited by raw materials, and can be mass-produced, possessing potential and wide-ranging application value in clinical and in vitro diagnostics.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for preparing neutrophil gelatinase-associated lipid transport protein and antibody. Background Technology

[0002] Neutrophil gelatinase-associated lipocarboxin (NGAL) is a 25 kDa protein composed of 178 amino acid residues. It is a highly sensitive marker of acute kidney injury and has been found expressed in various human tissues, including the uterus, prostate, lung, trachea, stomach, colon, and kidney. NGAL is a sensitive marker of renal tubular injury. Normally, NGAL in the kidney is mainly secreted by the loop of Henle and the distal tubules, and is almost entirely reabsorbed in the proximal tubules. Therefore, only trace amounts of NGAL are detectable in healthy individuals.

[0003] NGAL (neutrophil gelatinase-associated lipotransferase) is an important biomarker in fields such as acute kidney injury (AKI). Its most crucial advantage lies in its extremely early warning capability. When the kidneys are damaged due to ischemia (e.g., after major surgery, shock), infection, or nephrotoxic drugs (e.g., certain antibiotics, chemotherapy drugs, contrast agents), renal tubular epithelial cells rapidly produce large amounts of NGAL. Its level rises significantly within 2 hours of injury, while serum creatinine (Scr) typically takes 24-72 hours to change. This allows doctors to gain a critical "golden window" for intervention, preventing irreversible damage to kidney function. NGAL is also suitable for detecting chronic kidney disease (CKD), diabetic nephropathy, lupus nephropathy, IgA tubulointerstitial nephropathy, and cardiorenal syndrome (CRS).

[0004] NGAL detection requires high-purity NGAL antigen and high-performance NGAL antibody. Currently, NGAL is mainly obtained through two routes: natural extraction and genetic engineering. However, direct extraction of natural NGAL from natural tissues presents significant challenges, including limited sources, complex composition, and difficulty in guaranteeing purity, making it unsuitable for large-scale or high-purity applications. Therefore, researchers prefer to synthesize active recombinant NGAL through genetic engineering, which not only avoids the limitations of raw material sources but also reduces costs and allows for large-scale production. High-quality NGAL immunogen is crucial for preparing high-quality NGAL antibodies. Currently, NGAL detection laboratories primarily use immunological methods, with commonly used methods including immunoturbidimetry (ITA), immunoturbidimetric assay (INA), fluorescence assays, and enzyme-linked immunosorbent assays (ELISA), all of which require NGAL antibodies with high specificity and sensitivity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing neutrophil gelatinase-associated lipid transport protein and antibody, producing inexpensive, active, high-purity NGAL and its high-performance NGAL polyclonal antibody, thus solving the problem of difficulty in obtaining NGAL antigen and high-performance NGAL antibody in the prior art.

[0006] To achieve the above objectives, this invention proposes a method for preparing neutrophil gelatinase-associated lipotransferase and its antibody, using the aforementioned neutrophil gelatinase-associated lipotransferase, comprising the following steps:

[0007] Step 1: Using pET23a(+) as the expression vector, the NGAL gene with 6 His tags fused to the N-terminus under the control of the promoter was introduced into E. coli for induced expression to obtain crude protein extract;

[0008] Step 2: Purify the solution using nickel ion affinity chromatography to obtain a concentrated solution;

[0009] Step 3: Purify using a molecular sieve column to obtain untagged NGAL protein with a purity ≥95%;

[0010] Step 4: Immunize Japanese rabbits multiple times with the unlabeled NGAL protein with a purity ≥95% obtained in Step 3. After each immunization, blood is collected, and qualified serum is screened by antiserum two-way agar diffusion experiment. The serum is then purified by antigen affinity purification column to obtain NGAL polyclonal antibody.

[0011] Optionally, step 1 includes a fermentation process using the recombinant NGAL expression strain, wherein the *E. coli* strain is *Ecoli BL21(DE3). The fermentation conditions are as follows: seed culture is inoculated at a 5% inoculum volume into 1000 ml of fermentation medium containing 100 ug / mL AMP; culture is carried out at 37 ℃ and 220 rpm for approximately 1-2 h; when OD... 600 When the concentration rises to 0.8, add IPTG to make the final concentration of IPTG in the fermentation broth 0.4 mM / L, and continue to incubate in a shaker at 18 ℃ and 220 rpm for 20-24 h.

[0012] Optionally, the fermentation medium contains 15 g / L tryptone, 25 g / L yeast extract, 0.6 g / L MgSO4, 23 g / L Na2HPO4, 5 g / L NaH2PO4, 15 g / L glucose, and has a pH of 7.2.

[0013] Optionally, the execution process of step 1 also includes the collection and lysis of bacterial cells. After fermentation, the bacterial solution is centrifuged to collect the bacterial cells, which are then further subjected to ultrasonic cell disruption and centrifugation to obtain crude protein extract.

[0014] Optionally, in step 2, Ni Focurose 6FF is used as the affinity chromatography medium. The eluent is placed in a dialysis bag with a capacity of 3500-14000 Da and concentrated with sucrose to obtain a concentrate.

[0015] Optionally, in step 3, molecular sieve purification is performed using a Superdex 75 packed column, and the column is equilibrated with NGAL Buffer B4.

[0016] Optionally, the antigen affinity purification column in step 4 is obtained by coupling a certain volume of NHS-activated Bestarose 4FF with unlabeled NGAL protein packing material with a purity ≥95%.

[0017] This invention provides a method for preparing neutrophil gelatinase-associated lipid transport protein and antibody. Specifically, using pET23a(+) as the expression vector, the NGAL gene with six His tags fused to its N-terminus under promoter control is introduced into *E. coli* for induced expression. After purification using a Ni Focurose 6FF column and a molecular sieve column, tag-free NGAL protein with a purity ≥95% is obtained. Subsequently, the NGAL protein is used to immunize Japanese rabbits multiple times, and the serum collected after each immunization is used to screen qualified serum through a two-way agar diffusion assay. Then, an antigen affinity purification column is prepared using the above-mentioned antigen, and the serum is purified to obtain a highly active NGAL polyclonal antibody. The high-purity recombinant NGAL of this invention is not only active but also inexpensive, not limited by raw materials, and can be mass-produced, showing potential and wide-ranging application value in clinical and in vitro diagnostics. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the purity test of NGAL antigen in an embodiment of the present invention.

[0020] Figure 2 This is a schematic diagram of the purity test of the NGAL polyclonal antibody in an embodiment of the present invention.

[0021] Figure 3 This is a schematic diagram comparing the linear relationship between absorbance and concentration between the reagent kit of the present invention and the control reagent kit in an embodiment of the present invention. Detailed Implementation

[0022] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0023] This invention proposes a method for preparing neutrophil gelatinase-associated lipid transport protein and antibody, comprising the following steps:

[0024] Step 1: Using pET23a(+) as the expression vector, the NGAL gene with 6 His tags fused to the N-terminus under the control of the promoter was introduced into E. coli for induced expression to obtain crude protein extract;

[0025] Step 2: Purify the solution using nickel ion affinity chromatography to obtain a concentrated solution;

[0026] Step 3: Purify using a molecular sieve column to obtain untagged NGAL protein with a purity ≥95%;

[0027] Step 4: Immunize Japanese rabbits multiple times with the unlabeled NGAL protein with a purity ≥95% obtained in Step 3. After each immunization, blood is collected, and qualified serum is screened by antiserum two-way agar diffusion experiment. The serum is then purified by antigen affinity purification column to obtain NGAL polyclonal antibody.

[0028] Specifically, the NGAL gene sequence with 6 His tags fused at the N-terminus is shown in SEQ ID NO.1: MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG.

[0029] Please see Figures 1 to 3 The following detailed explanation, in conjunction with specific embodiments and execution steps, further illustrates the process:

[0030] Step 1 includes the fermentation process of the recombinant NGAL expression strain and the collection and lysis of the bacterial cells.

[0031] Step 1.1: Fermentation of recombinant NGAL expression strain

[0032] The BL21 (pET23a-6×His-NGAL) recombinant strain, constructed by Sangon Biotech Co., Ltd., with good activity, was removed from a -20℃ freezer, thawed, and 50 μL of bacterial culture was added to 5 ml of LB medium containing 100 μg / mL AMP. After mixing, it was cultured at 37℃ and 220 rpm for 15-17 hours. The 5 mL of the cultured seed culture was then transferred to 50 ml of LB medium containing 100 μg / mL AMP and cultured at 37℃ and 220 rpm for approximately 2-4 hours. When the OD600 rose to approximately 0.8, the 50 mL of the cultured seed culture was transferred to 1000 ml of fermentation medium containing 100 μg / mL AMP and cultured at 37℃ and 220 rpm for approximately 1-2 hours. 600 When the concentration rises to 0.8, add IPTG to make the final concentration of IPTG in the fermentation broth 0.4 mM / L, and continue to incubate in a shaker at 18 ℃ and 220 rpm for 20-24 h.

[0033] Step 1.2: Collection and lysis of bacterial cells

[0034] After fermentation, the bacterial culture was centrifuged at 5000 rpm for 15 min at 4 ℃, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were washed with an equal volume of cold PBS buffer, and then centrifuged at 6000 rpm for 15 min at 4 ℃, and the bacterial cells were collected. The bacterial cells were resuspended in PBS with 1 / 20 of the fermentation broth volume and magnetically stirred in a refrigerator for 60 min. After the bacterial cells were thoroughly mixed, a certain volume of PMSF was added to bring the final concentration to 2 mM / L, and stirring was continued for 10 min to obtain a bacterial suspension. The bacterial suspension was placed in an ice-water mixture and ultrasonically disrupted. The ultrasonic instrument operating parameters were: ultrasonic power 75%, ultrasonic for 5 s, interval for 10 s, total time 35 min, alarm temperature 20 ℃. The bacterial lysate was centrifuged at 9000 rpm for 30 min at 4 ℃, and the supernatant was the crude protein extract.

[0035] The nickel ion affinity chromatography purification process in step 2 is as follows:

[0036] After packing the column, wash with 10 column volumes of deionized water, then equilibrate with 10 column volumes of Ni Focurose 6FF equilibration buffer. After equilibration, pass the crude protein extract through the column. Wash with 10 column volumes of Ni Focurose 6FF washing buffer. Finally, elute with 1.5-3 volumes of Ni Focurose 6FF eluent and collect the eluent with the peak (detected using a protein analyzer). Place the eluent in a dialysis bag (3500-14000 Da) and concentrate with sucrose to obtain 5-10 mL of concentrate for further molecular sieve purification.

[0037] Step 3: Molecular sieve column purification

[0038] Molecular sieve purification was performed using a Superdex 75 column (sample loading volume not exceeding 3% of the column volume, e.g., 30 ml sample on 1 L of packing material). The column was connected to a nucleic acid analyzer, and equilibrated with approximately 1.5 column volumes of NGAL Buffer B4 at a flow rate of 1.5-2 mL / min. After the column reached baseline equilibration, the concentrate was loaded, and the breakthrough buffer was collected. Elution of the protein continued with NGAL Buffer B4 at a flow rate of 1.5-2 mL / min. After peak formation, the eluent was collected; the eluent contained high-purity recombinant NGAL protein. The collected eluent was subjected to purity testing (SDS-PAGE). Breakthrough buffer with a purity ≥95% was considered high-purity recombinant NGAL protein. Purity testing is as follows: Figure 1 As shown.

[0039] Step 4 includes the preparation of the antigen affinity purification column and the purification process of the NGAL polyclonal antibody.

[0040] Step 4.1: Preparation of antigen affinity purification column

[0041] Take a certain volume of NHS-activated Bestarose 4FF packing material and wash it with pre-cooled solution A (1 mM HCl) for at least 30 min, using approximately 20 mL of solution A per 1 mL of packing material. After washing, dilute the packing material with solution A (approximately 0.5 mL of solution A per 1 mL of packing material). Redissolve the high-purity antigen in solution B (0.1 M NaHCO3 + 0.5 M NaCl, pH 8.3) to couple the packing material to a concentration of 5-10 mg / mL. Mix the diluted packing material with an equal volume of antigen solution, incubate at room temperature for 4 h, and then incubate overnight at 4°C. Pack the coupled packing material onto a column, elute the coupling supernatant, add blocking buffer (0.1 M Tris-HCl, pH 8.5), mix well, and block at room temperature for 4 h. Drain the blocking buffer and wash 3-6 times alternately with washing buffer 1 (0.1M Tris-HCl + 0.5M NaCl, pH 8-9) and washing buffer 2 (0.1M acetate buffer + 0.5M NaCl, pH 3-4), using 3 times the gel volume of solution each time. Finally, wash with 1xPBS and set aside for use.

[0042] Step 4.2: Purification of NGAL polyclonal antibody

[0043] The NGAL protein prepared above was used to immunize Japanese rabbits multiple times. Blood was collected after each immunization, and qualified serum was screened using a two-dimensional agar diffusion experiment. The NGAL polyclonal antibody was purified using an antigen affinity method. The collected antiserum was centrifuged at 9000 rpm for 20 min at 4 ℃, the precipitate was removed by filtration, and the antiserum was mixed with an equal volume of 1xPBS. The antigen affinity column was equilibrated with 5 column volumes of 1xPBS, and the antiserum was loaded at a ratio of packing material to original antiserum volume of 1:3, repeated twice. After column chromatography, the column was washed sequentially with 5 column volumes of 1xPBS followed by 5 column volumes of 1xPBS (pH=5.0). Finally, elution was performed with 0.1M glycine, and the washed buffer containing the peak was collected (detected using a protein analyzer). The pH of the eluent was adjusted to neutral using 0.2M Tris-HCl (pH 8-9). This eluent was the NGAL polyclonal antibody.

[0044] Add (NH4)2SO4 to the eluent at a ratio of 32 g (NH4)2SO4 per 100 mL of eluent while stirring continuously. Continue stirring for 0.5 h after the solid has completely dissolved, then let stand for 4 h. Centrifuge at 9000 rpm for 20 min at 4℃, remove the supernatant, and reconstitute the precipitate with 1xPBS, controlling the total protein concentration of the reconstituted solution to be 15-25 mg / mL. Place the reconstituted solution into a dialysis bag (molecular weight cutoff 7-14 kDa), clamp the dialysis clamp, and then immerse the dialysis bag in 5 L of 4℃ 1xPBS buffer for dialyzing. Change the buffer every 6-10 h until no significant change is observed in the protein solution when measured with Nessler's reagent. After centrifugation, filter the reconstituted solution through a 0.22 μm filter membrane to obtain high-purity NGAL polyclonal antibody. Purity testing is as follows. Figure 2 .

[0045] Furthermore, the present invention also validated the NGAL polyclonal antibody by preparing an NGAL immunoturbidimetric kit and testing the calibration value.

[0046] Reagent R1 includes buffer 1 (e.g., phosphate, citrate), surfactant (e.g., Triton X100), stabilizer (e.g., NaCl), chelating agent (e.g., EDTA), sensitizer (e.g., PEG6000), and preservative (e.g., NaN3).

[0047] Reagent R2: Dilute the above-mentioned rabbit anti-human NGAL polyclonal antibody and the commercially available rabbit anti-human NGAL polyclonal antibody purchased from Manufacturer A to the same concentration of 5-10 mg / mL using PBS buffer 1 (pH 7-8); dilute the latex microspheres (carboxylated polystyrene latex, purchased from Ninebot, particle size 160 nm) to 5% (solid content) with purified water, then add 5-10% (EDC / latex microsphere mass ratio) of EDC, stir and react at 25-30℃ for 20-30 min, then add the diluted antibody (1 mL latex microspheres correspond to 5-6 mg of antibody), stir and react at 25-30℃ for 90-150 min, add the stop reaction solution to terminate the reaction, centrifuge to remove the supernatant, and add a buffer (e.g., phosphate, citrate) and a surfactant (e.g., Triton) to terminate the reaction. The buffer solution 2, composed of X100, stabilizer (e.g., NaCl), chelating agent (e.g., EDTA), protectant (e.g., BSA), and preservative (e.g., NaN3), is sonicated for 120-180 seconds to obtain reagent R2.

[0048] Calibrators: A set of calibrators was prepared by adding different volumes of natural human NGAL protein to a buffer solution consisting of buffer 3 (e.g., phosphate, citrate), stabilizer (e.g., NaCl), chelating agent (e.g., EDTA), and preservative (e.g., NaN3) to obtain final concentrations of 5000.00, 2500.00, 1250.00, 625.00, 156.25, and 0 ng / mL (purified water as a control).

[0049] Reagent kit performance testing: Using the control kit, reagent 2 used a commercially available rabbit anti-human polyclonal antibody from manufacturer A. All other parameters were the same. Test parameters were: main wavelength 600nm, no secondary wavelength, R1:R2:S = 240:60:5. Testing instrument: Hitachi 3500.

[0050] (1) Linearity range verification: The reagent kit of this invention, the control kit, and one high-value test sample were used to determine the concentration of each calibrator and absorbance. A working curve was obtained with calibrator concentration as the independent variable and absorbance as the dependent variable. Figure 3 As shown in the figure. The results show that the detection range of the kit of the present invention has a linear correlation coefficient of 0.99 compared with the control kit in the range of 0-5000 ng / mL.

[0051] (2) Validation of antibody specificity (analytical sensitivity): The sample of (500.0±25) ng / mL was tested three times using the kit of the present invention and the control kit. The absorbance A generated by the reagent under the specified parameters was recorded and converted into the absorbance difference (ΔA) of 500.0 mg / L according to formula (1), as shown in Table 1. The data in the table show that the kit of the present invention has higher analytical sensitivity, that is, better specificity, when testing a sample of 500.0 mg / L.

[0052] (1)

[0053] (3) Detection limit verification: Blank samples were repeatedly measured 20 times using the kit of the present invention and the control kit. The mean (M) and standard deviation (SD) were calculated. The detection limit = M - 2SD, as shown in Table 2. The data in the table show that the kit of the present invention has a lower detection limit, that is, higher sensitivity.

[0054] Table 1. Comparison of absorbance differences between the kit of the present invention and the control kit.

[0055]

[0056] Table 2. Comparison of detection limits between the reagent kit of the present invention and the control reagent kit.

[0057]

[0058] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing neutrophil gelatinase-associated lipid transport protein and antibody, characterized in that, Includes the following steps: Step 1: Using pET23a(+) as the expression vector, the NGAL gene with 6 His tags fused to the N-terminus under the control of the promoter was introduced into E. coli for induced expression to obtain crude protein extract; Step 2: Purify the solution using nickel ion affinity chromatography to obtain a concentrated solution; Step 3: Purify using a molecular sieve column to obtain untagged NGAL protein with a purity ≥95%; Step 4: Immunize Japanese rabbits multiple times with the unlabeled NGAL protein with a purity ≥95% obtained in Step 3. After each immunization, blood is collected, and qualified serum is screened by antiserum two-way agar diffusion experiment. The serum is then purified by antigen affinity purification column to obtain NGAL polyclonal antibody.

2. The method for preparing neutrophil gelatinase-associated lipid transport protein and antibody as described in claim 1, characterized in that, Step 1 includes the fermentation process of the recombinant NGAL expression strain, in which the Escherichia coli is Ecoli BL21(DE3). The fermentation conditions are as follows: the seed culture is inoculated at a 5% inoculum into 1000 ml of fermentation medium containing 100 ug / mL AMP, and cultured at 37 ℃ and 220 rpm for about 1-2 h to scale up the culture. 600 When the concentration rises to 0.8, add IPTG to make the final concentration of IPTG in the fermentation broth 0.4 mM / L, and continue to incubate in a shaker at 18 ℃ and 220 rpm for 20-24 h.

3. The method for preparing neutrophil gelatinase-associated lipid transport protein and antibody as described in claim 2, characterized in that, The fermentation medium contains 15 g / L tryptone, 25 g / L yeast extract, 0.6 g / L MgSO4, 23 g / L Na2HPO4, 5 g / L NaH2PO4, 15 g / L glucose, and has a pH of 7.

2.

4. The method for preparing neutrophil gelatinase-associated lipid transport protein and antibody as described in claim 3, characterized in that, The execution process of step 1 also includes the collection and lysis of bacterial cells. After fermentation, the bacterial liquid is centrifuged to collect the bacterial cells, which are then further subjected to ultrasonic cell disruption and centrifugation to obtain crude protein extract.

5. The method for preparing neutrophil gelatinase-associated lipid transport protein and antibody as described in claim 4, characterized in that, In step 2, Ni Focurose 6FF was used as the affinity chromatography medium. The eluent was placed in a dialysis bag with a capacity of 3500-14000 Da and concentrated with sucrose to obtain a concentrate.

6. The method for preparing neutrophil gelatinase-associated lipid transport protein and antibody as described in claim 5, characterized in that, In step 3, molecular sieve purification was performed using a Superdex 75 packed column, and the column was equilibrated with NGAL Buffer B4 to elute the proteins.

7. The method for preparing neutrophil gelatinase-associated lipid transport protein and antibody as described in claim 6, characterized in that, The antigen affinity purification column in step 4 was obtained by coupling a certain volume of NHS-activated Bestarose 4FF with unlabeled NGAL protein packing material with a purity ≥95%.

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