A method for preparing immunoglobulin A from normal human serum

Abstract

The application discloses a method for preparing immunoglobulin A from normal human serum and belongs to the technical field of biotechnology. The method comprises the following steps: taking normal human serum and performing centrifugal filtration treatment to collect supernatant to obtain pretreated serum; placing the pretreated serum into a dialysis bag to perform dialysis with purified water, centrifugally collecting supernatant to obtain dialysis supernatant; sequentially adding a phosphate buffer and ammonium sulfate into the dialysis supernatant to perform salting-out, centrifugally collecting precipitate, redissolving the precipitate with the phosphate buffer, performing dialysis, centrifugally filtering and obtaining crude immunoglobulin A solution; and loading the crude immunoglobulin A solution into a Sephacryl S-300 gel filtration chromatography column, eluting with the phosphate buffer, collecting a first elution peak and obtaining a purified immunoglobulin A solution. The immunoglobulin M is removed by dialysis with purified water, and the immunoglobulin A is obtained by combining salting-out and one-step gel filtration chromatography, so that the method is simple in process and controllable in cost, the purity of the obtained immunoglobulin A is more than 95%, and the method is suitable for preparation of raw materials of in-vitro diagnostic reagents.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing immunoglobulin A from normal human serum. Background Technology

[0002] Immunoglobulin A (IGA) is an important antibody in human serum, second only to IGA G in abundance, accounting for approximately 10%–20% of the total serum immunoglobulins. Serum IGA exists in the blood in monomeric form and has significant clinical diagnostic value. Detecting serum IGA levels can assess the body's humoral immune function status and aid in the diagnosis of immunoproliferation, immunodeficiency, infection, and autoimmune diseases. Particularly in the diagnosis and treatment of IGA nephropathy, purified IGA and its glycosylated variants, as core raw materials for diagnostic reagents, have become a hot research area in clinical practice. Antibody detection kits developed based on purified IGA antigen can achieve non-invasive diagnosis of IGA nephropathy; therefore, high-purity, high-activity purified human serum IGA products have become a crucial requirement in the in vitro diagnostics industry.

[0003] Currently, purification methods for immunoglobulin A mainly include salting out, ion exchange chromatography, affinity chromatography, and gel filtration chromatography, as well as various combinations thereof. Existing techniques have reported methods, such as purifying immunoglobulin A from normal human plasma through seven steps: ammonium sulfate precipitation of euglobulins, buffer dialysis, DEAE-Sephadex A50 batch processing, cold ethanol precipitation, ammonium sulfate precipitation, TEAE-cellulose chromatography, and bromoacetylcellulose immunosorbent assay. However, this method is extremely cumbersome, takes several days, and the introduction of organic solvents such as cold ethanol can easily lead to protein denaturation. In addition, purification methods for secretory immunoglobulin A have been reported, such as using affinity chromatography columns to separate and purify secretory immunoglobulin A from human oral secretions, or extracting secretory immunoglobulin A from colostrum through centrifugation and salting out. However, these methods target secretory immunoglobulin A derived from exocrine fluids, whose sample source, molecular morphology, and purification strategies differ fundamentally from those of serum immunoglobulin A. Furthermore, the media used in affinity chromatography are expensive and unsuitable for large-scale preparation of monomeric immunoglobulin A from serum samples.

[0004] Therefore, there is an urgent need in this field for a method for extracting immunoglobulin A from normal human serum that is extremely simple in process, low in cost, easy to operate, has good purification effect, and can maintain the natural immune activity of immunoglobulin A, so as to meet the preparation requirements of core raw materials for in vitro diagnostic reagents. Summary of the Invention

[0005] The purpose of this invention is to address the problems of existing methods for purifying immunoglobulin A from normal human serum, such as cumbersome steps, long time consumption, easy introduction of organic solvents leading to protein denaturation, and high cost of affinity media, and to propose a method for preparing immunoglobulin A from normal human serum.

[0006] The technical solution adopted by this invention to solve its technical problem is as follows: This invention proposes a method for preparing immunoglobulin A from normal human serum, comprising the following steps: S1. Take normal human serum and centrifuge and filter it to remove cell debris and particulate impurities. Collect the supernatant to obtain pretreated serum. S2. Place the pretreated serum into a dialysis bag and use purified water as the dialysate for dialysis. After dialysis, centrifuge to separate the serum, collect the supernatant, discard the precipitate, and obtain the dialysis supernatant. S3. Add phosphate buffer and ammonium sulfate to the dialysis supernatant in sequence for salting out. After centrifugation, collect the precipitate, reconstitute it with phosphate buffer, and then dialyze it in phosphate buffer. After dialysis, centrifuge and filter to obtain crude immunoglobulin A extract. S4. Load the crude immunoglobulin A extract onto a Sephacryl S-300 gel filtration chromatography column equilibrated with phosphate buffer. After adding phosphate buffer to form layers, elute with the same phosphate buffer and collect the first elution peak to obtain the purified immunoglobulin A solution.

[0007] Preferably, the normal human serum in step S1 is a mixed normal human serum that has been discarded and stored in a hospital laboratory at -20°C.

[0008] Preferably, in step S1, the centrifugation speed is 9000-11000 rpm and the centrifugation time is 9-11 min.

[0009] Preferably, the dialysis conditions in step S2 are: the ratio of serum volume to purified water volume is greater than 100 times during each dialysis, the number of dialysis sessions is 2 to 3, and the duration of each dialysis session is 12 hours.

[0010] Preferably, in step S2, the centrifugation speed is 9000-11000 rpm and the centrifugation time is 9-11 min.

[0011] Preferably, in step S3, the amount of ammonium sulfate added is such that the saturation of ammonium sulfate in the dialysis supernatant reaches 35% to 45%, and the phosphate buffer is a 10mM phosphate buffer with pH 7.4.

[0012] Preferably, in step S3, the centrifugation speed after the salting-out reaction is 7000-9000 rpm and the centrifugation time is 18-22 min; after dialysis, the centrifugation filtration speed is 9000-11000 rpm and the centrifugation time is 9-11 min.

[0013] Preferably, in step S4, the Sephacryl S-300 gel filtration chromatography column has a diameter of 3.5 cm, a column height of 85 cm, and an elution flow rate of 1.2 mL / min.

[0014] Preferably, the ratio of sample volume to gel volume in step S4 does not exceed 3%.

[0015] Preferably, in step S4, the absorbance of the eluent is monitored in real time at a wavelength of 280 nm, and the first elution peak that appears after the baseline stabilizes is collected based on the change in absorbance.

[0016] In summary, compared with the prior art, the method for preparing immunoglobulin A from normal human serum provided by the present invention has the following beneficial effects: (1) In the purified water dialysis step, the present invention utilizes the characteristic that immunoglobulin M has reduced solubility and preferential precipitation in purified water environment with low ionic strength, so that immunoglobulin M with the largest molecular weight is specifically precipitated and removed, while the target product immunoglobulin A and the main impurity immunoglobulin G are retained in the supernatant, thereby effectively reducing the separation burden of subsequent gel filtration chromatography, so that high-purity products can be obtained with only one gel filtration chromatography step.

[0017] (2) The present invention uses ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography in combination. It utilizes the dual separation principle of different proteins having different solubilities under the same ammonium sulfate saturation and molecular sieve effect to effectively remove immunoglobulin G and other impurity proteins. After purification, the purity of immunoglobulin A was identified by SDS-PAGE electrophoresis as reaching more than 95%.

[0018] (3) The present invention requires only four purification steps to obtain high-purity immunoglobulin A from normal human serum. The process is clear, easy to operate, and has a short total time, making it easy to standardize.

[0019] (4) The present invention uses conventional chromatography media, completely avoiding the use of ion exchange chromatography and expensive affinity ligands, effectively reducing purification costs and making it suitable for laboratory-scale and pilot-scale production.

[0020] (5) The present invention uses purified water dialysis combined with gel filtration chromatography, without the involvement of organic solvents and extreme pH conditions, which is conducive to maintaining the native conformation and immune activity of immunoglobulin A. The obtained product is qualitatively tested by colloidal gold double antibody sandwich method, and its activity is comparable to that of commercial serum immunoglobulin A. Attached Figure Description

[0021] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a flowchart of the method for preparing immunoglobulin A from normal human serum according to the present invention; Figure 2 This is a protein reduction electrophoresis image after dialysis with purified water in an embodiment of the present invention; Figure 3 This is the reduced SDS-PAGE pattern of immunoglobulin A in this embodiment of the invention; Figure 4 for Figure 3 Purity analysis chart of the band corresponding to lane 5 in the middle swimming lane; Figure 5 This is the non-reduced SDS-PAGE pattern of immunoglobulin A in this embodiment of the invention; Figure 6 for Figure 5 Purity analysis chart of the band corresponding to lane 7 in the middle swimming lane; Figure 7 This is a chromatogram of immunoglobulin A obtained by Sephacryl S-300 gel chromatography in an embodiment of the present invention. Detailed Implementation

[0022] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0024] Figure 1 A flowchart of the method for preparing immunoglobulin A from normal human serum according to the present invention is shown, as follows: Figure 1 As shown, the method includes the following steps: S1. Take normal human serum and centrifuge and filter it to remove cell debris and particulate impurities. Collect the supernatant to obtain pretreated serum.

[0025] Specifically, the starting material is a mixed serum of healthy individuals stored at -20°C in a hospital laboratory. The serum is centrifuged. In one specific embodiment, the centrifugation speed is 9000–11000 rpm, and the centrifugation time is 9–11 min. In this embodiment, the centrifugation speed is 10000 rpm, and the centrifugation time is 10 min. After centrifugation, the supernatant is collected to remove cell debris and insoluble particulate impurities. Then, it is filtered using a microfiltration membrane. In one specific embodiment, the pore size of the microfiltration membrane is 0.45 μm or 0.22 μm to further remove fine particulate impurities. The filtered supernatant is collected to obtain the pretreated serum.

[0026] S2. Place the pretreated serum into a dialysis bag and use purified water as the dialysate for dialysis. After dialysis, centrifuge to separate the serum, collect the supernatant, and discard the precipitate. The precipitate is normal human immunoglobulin M, and the dialysis supernatant is obtained.

[0027] Specifically, the pretreated serum obtained in step S1 is placed into a dialysis bag. In one specific embodiment, the molecular weight cutoff of the dialysis bag is 3 kDa. Purified water is used as the dialysis fluid. The dialysis operating conditions are: the ratio of normal human serum volume to purified water volume is greater than 100 times per dialysis session; the number of dialysis sessions is 2-3 times; the dialysis time is 12 hours per session; and the dialysis temperature is 4°C. During dialysis, immunoglobulin M has reduced solubility in the low ionic strength purified water environment and preferentially precipitates, forming a visible precipitate. As the number of dialysis sessions increases, the amount of precipitate gradually increases until it stops increasing, indicating that immunoglobulin M has been fully precipitated. After dialysis, centrifugation is performed. In one specific embodiment, the centrifugation speed is 9000-11000 rpm and the centrifugation time is 9-11 minutes. In this embodiment, the centrifugation speed is 10000 rpm and the centrifugation time is 10 minutes. After centrifugation, the supernatant is collected, the precipitate is discarded, and the dialysis supernatant is obtained.

[0028] Figure 2 The diagram shows a protein reduction electrophoresis image after dialysis with purified water in an embodiment of the present invention. Lane 4 is the molecular weight standard marker, lane 5 is BSA protein, lane 6 is normal human serum directly purified with ammonium sulfate after centrifugation, lane 7 is normal human serum dialysis treated with 10mM pH 7.4 phosphate buffer, lane 8 is normal human serum dialysis treated with purified water after centrifugation and filtration, lane 9 is commercially available serum immunoglobulin A, and lane 10 is normal human serum treated only by centrifugation and filtration. Figure 2 It can be seen that there were no obvious normal human immunoglobulin M bands of about 70kDa in lane 8, indicating that purified water dialysis can effectively remove immunoglobulin M.

[0029] S3. Add phosphate buffer and ammonium sulfate to the dialysis supernatant in sequence for salting out. After centrifugation, collect the precipitate, reconstitute it with phosphate buffer, and then dialyze it in phosphate buffer. After dialysis, centrifuge and filter to obtain crude immunoglobulin A extract.

[0030] Specifically, an equal volume of phosphate buffer is added sequentially to the dialysis supernatant obtained in step S2, followed by the addition of saturated ammonium sulfate to achieve a saturation level of 35%–45%. In one specific embodiment, saturated ammonium sulfate is added to achieve a saturation level of 40%. The phosphate buffer is a 10 mM pH 7.4 phosphate buffer, composed of Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, purified water, and ProClin 300. The phosphate buffer is added while stirring, and after addition, the mixture is thoroughly stirred for reaction. In one specific embodiment, the reaction is carried out at 4°C for 30–60 minutes to ensure complete precipitation of immunoglobulin A in the ammonium sulfate solution. After the reaction is complete, centrifugation is performed. In one specific embodiment, the centrifugation speed is 7000–9000 rpm for 18–22 minutes. In this embodiment, the centrifugation speed is 8000 rpm for 20 minutes. After centrifugation, the supernatant is discarded, and the precipitate is collected. The collected precipitate was reconstituted with 10 mM pH 7.4 phosphate buffer to ensure complete dissolution. The reconstituted solution was then dialyzed again in 10 mM pH 7.4 phosphate buffer under the following conditions: the volume ratio of solution to phosphate buffer was greater than 100 times during each dialyze, 2–3 dialyzes were performed, each dialyze lasted 6–12 hours, and the dialyze temperature was 4°C, to thoroughly remove residual ammonium sulfate and salt impurities. After dialysis, centrifugation was performed. In one specific embodiment, the centrifugation speed was 9000–11000 rpm, and the centrifugation time was 9–11 min. In this embodiment, the centrifugation speed was 10000 rpm, and the centrifugation time was 10 min. The filtrate was collected after centrifugation to obtain crude immunoglobulin A extract.

[0031] S4. Load the crude immunoglobulin A extract onto a Sephacryl S-300 gel filtration chromatography column equilibrated with phosphate buffer. After adding phosphate buffer to form layers, elute with the same phosphate buffer and collect the first elution peak to obtain the purified immunoglobulin A solution.

[0032] Specifically, a Sephacryl S-300 gel filtration chromatography column is prepared. In one specific embodiment, the column has a diameter of 3.5 cm and a height of 85 cm, with a gel volume of 817 mL. The UV detector and recorder are turned on, and the column is equilibrated using 10 mM pH 7.4 phosphate buffer as the equilibration buffer. During equilibration, 2-3 column volumes of 10 mM pH 7.4 phosphate buffer are used for washing. The crude immunoglobulin A extract obtained in step S3 is carefully loaded into the chromatography column, taking care not to damage the gel surface. The ratio of the sample volume to the gel volume should not exceed 3%. After all the sample has been added, 10 mM pH 7.4 phosphate buffer is carefully added. At this point, due to the concentration difference, a clear stratification occurs between the sample layer and the buffer. The chromatography column and peristaltic pump are connected, and isocratic elution is performed with 10 mM pH 7.4 phosphate buffer at a flow rate of 1.2 mL / min. The absorbance of the eluent was monitored in real time at a wavelength of 280 nm. Since immunoglobulin M had been removed by dialysis with purified water in step S2, the first elution peak appearing after the absorbance baseline stabilized during gel filtration chromatography was immunoglobulin A. This complete elution peak was collected to obtain a purified immunoglobulin A solution. In one specific embodiment, the time from protein loading to elution of the target protein was 291 min, and the elution volume was 108 mL.

[0033] The purified immunoglobulin A solution obtained in step S4 was identified by SDS-PAGE electrophoresis. Figure 3 The reduced SDS-PAGE pattern of immunoglobulin A in an embodiment of the present invention is shown, wherein lanes 2 and 4 are molecular weight standard markers, lanes 1 and 3 are two commercial serum types of immunoglobulin A, and lane 5 is the purified immunoglobulin A of the present invention. Figure 4 for Figure 3 The purity analysis diagram of the band corresponding to lane 5 in the middle swimming pool shows that the purity of the purified protein is 96.3%. Figure 5 The non-reduced SDS-PAGE pattern of immunoglobulin A in an embodiment of the present invention is shown, wherein lane 8 is commercial serum type immunoglobulin A and lane 7 is immunoglobulin A purified according to the present invention. Figure 6 for Figure 5 The purity analysis diagram of the band corresponding to lane 7 in the middle swimming pool shows that the purity of the purified protein is 96.7%. Figure 7 The chromatogram of immunoglobulin A obtained by Sephacryl S-300 gel chromatography in an embodiment of the present invention is shown, wherein the elution peak shown by UV280 monitoring is the collection peak of immunoglobulin A. The above results indicate that the purity of immunoglobulin A prepared by the method of the present invention reaches over 95%.

[0034] The purified immunoglobulin A solution obtained in step S4 was subjected to qualitative detection using a colloidal gold double-antibody sandwich method. A coated plate was prepared by coating with commercially available mouse anti-immunoglobulin A monoclonal antibody 1 from Hangzhou Longi, with a final coating concentration of 0.5 mg / mL. A binding pad was prepared using commercially available mouse anti-immunoglobulin A monoclonal antibody 2 from Hangzhou Longi, with 9 μL of K2CO3. Centrifugation was performed twice: 6000 rpm for 8 min and 10000 rpm for 10 min. The coated plate and binding pad were assembled into test strips, cut into strips 3.5 mm wide, to obtain the test strips. Commercially available serum-type immunoglobulin A and the purified immunoglobulin A obtained in step S4 were diluted to 1 mg / mL with 10 mM pH 7.4 phosphate buffer, and then further diluted to 10 mg / mL using a serial gradient. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 70 μL of each dilution solution was added to the test strip for testing. The color development was observed after 10 minutes, and the color intensity was scored and recorded. The higher the D value, the stronger the color development. Table 1 records the color development scores for each dilution gradient.

[0035] Table 1. Colorimetric results of colloidal gold double-antibody sandwich method for qualitative detection.

[0036] As shown in Table 1, the colorimetric scores of the purified product of this invention and commercial serum immunoglobulin A are basically consistent at each dilution gradient, indicating that the two have comparable immunoreactivity.

[0037] The immunoglobulin A eluent collected in step S4 is aliquoted and stored. In one specific embodiment, the collected immunoglobulin A solution is stored for a short period at 4°C, or for a long period at -20°C or -80°C, with a preservative added if necessary.

[0038] The recovery rate of immunoglobulin A obtained from normal human serum using the method of this invention was approximately 2.8 g / L, which is within the normal high range. This indicates that the method of this invention has good yield while ensuring purity, and is economically feasible for industrial application.

[0039] In summary, this invention provides a method for preparing immunoglobulin A from normal human serum. Utilizing the characteristic that immunoglobulin M preferentially precipitates due to reduced solubility in low ionic strength environments, a purified water dialysis step is used to specifically precipitate and remove immunoglobulin M. Combined with ammonium sulfate precipitation and one-step Sephacryl S-300 gel filtration chromatography for purification, only four steps are required to obtain immunoglobulin A with a purity of over 95% from normal human serum. The entire process is clear, simple to operate, and has a short total time. It completely avoids the use of ion exchange chromatography and expensive affinity ligands, effectively reducing purification costs and making it suitable for laboratory-scale and pilot-scale production. The entire process does not involve organic solvents or extreme pH conditions, which helps maintain the native conformation and immunomodulatory activity of immunoglobulin A. The activity of the obtained product is comparable to that of commercially available serum-type immunoglobulin A, fully meeting the needs of the in vitro diagnostic reagent industry for the preparation of high-purity, high-activity immunoglobulin A as a core raw material.

[0040] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A method for preparing immunoglobulin A from normal human serum, characterized in that, It consists of the following steps: S1. Take normal human serum and centrifuge and filter it to remove cell debris and particulate impurities. Collect the supernatant to obtain pretreated serum. S2. Place the pretreated serum into a dialysis bag, use purified water as the dialysate for dialysis, centrifuge after dialysis, collect the supernatant, discard the precipitate, and obtain the dialysis supernatant. S3. Add phosphate buffer and ammonium sulfate to the dialysis supernatant in sequence for salting out. After centrifugation, collect the precipitate, reconstitute it with phosphate buffer, and dialyze it in phosphate buffer. After dialysis, centrifuge and filter to obtain crude immunoglobulin A extract. S4. Load the crude immunoglobulin A extract onto a Sephacryl S-300 gel filtration chromatography column equilibrated with phosphate buffer, add phosphate buffer to form layers, elute with the same phosphate buffer, collect the first elution peak, and obtain the purified immunoglobulin A solution.

2. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, The normal human serum mentioned in step S1 is a mixed normal human serum that has been discarded and stored in a hospital laboratory at -20°C.

3. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, The centrifugation speed in step S1 is 9000-11000 rpm, and the centrifugation time is 9-11 min.

4. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, The dialysis conditions described in step S2 are as follows: the ratio of serum volume to purified water volume is greater than 100 times during each dialysis session, the number of dialysis sessions is 2 to 3, and the duration of each dialysis session is 12 hours.

5. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, In step S2, the centrifugation speed is 9000-11000 rpm and the centrifugation time is 9-11 min.

6. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, The amount of ammonium sulfate added in step S3 is such that the saturation of ammonium sulfate in the dialysis supernatant reaches 35% to 45%, and the phosphate buffer is a 10mM phosphate buffer with pH 7.

4.

7. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, In step S3, the centrifugation speed after the salting-out reaction is 7000-9000 rpm and the centrifugation time is 18-22 min; after the dialysis is completed, the centrifugation filtration speed is 9000-11000 rpm and the centrifugation time is 9-11 min.

8. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, The Sephacryl S-300 gel filtration chromatography column mentioned in step S4 has a diameter of 3.5 cm, a column height of 85 cm, and an elution flow rate of 1.2 mL / min.

9. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, The ratio of sample volume to gel volume in step S4 shall not exceed 3%.

10. The method for preparing immunoglobulin A from normal human serum according to claim 1, characterized in that, In step S4, the absorbance of the eluent is monitored in real time at a wavelength of 280 nm, and the first elution peak that appears after the baseline stabilizes is collected based on the change in absorbance.

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