A purification method for high-titer prealbumin antibodies and its application

Abstract

This invention relates to a high-titer prealbumin antibody purification method and its application, belonging to the field of biotechnology. The method utilizes Protein A affinity chromatography to capture total IgG from antiserum, followed by purification using an affinity chromatography column prepared by targeted conjugation of a specific sequence peptide. The antibody purified by this method achieves a titer of 1:256,000 in ELISA detection, with a half-maximal effective concentration (EC50). 50 The antibody concentration was 3.2 ng / mL, exhibiting extremely high affinity and reactivity. When used in a double-antibody sandwich chemiluminescent immunoassay, it achieved a detection limit of 0.08 µg / mL, a wide dynamic range (0.08–400 µg / mL), and a titer retention rate as high as 88% after accelerated storage at 37°C for 14 days, demonstrating significantly better stability than antibodies prepared by traditional methods. This invention provides a highly active and stable core antibody raw material for the preparation of high-performance immunodiagnostic reagents.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for purifying high-titer prealbumin antibodies and their applications. Background Technology

[0002] Prealbumin (PA), also known as transthyretin, is a sensitive biomarker reflecting nutritional status, liver function, and acute inflammatory responses. It is widely used in in vitro diagnostic reagents such as immunoturbidimetry, chemiluminescence, and enzyme-linked immunosorbent assay (ELISA). In these detection systems, the quality of the antibodies used, especially their titer, affinity, and specificity, directly determines the detection limit, sensitivity, accuracy, and interference resistance of the reagent.

[0003] Polyclonal antibodies remain a core ingredient in many immunoassay reagents due to their advantages, including the ability to recognize multiple epitopes of antigens, high affinity, and relatively low preparation cost. Among these, high-affinity antibodies targeting key functional epitopes of prealbumin are crucial for accurate detection of low-concentration samples. However, efficiently isolating and enriching such high-value antibodies from complex antiserum compositions presents significant technical challenges.

[0004] Currently, the mainstream methods for purifying polyclonal antibodies mainly include Protein A / G affinity chromatography and immunoaffinity chromatography based on intact antigens. Protein A / G chromatography can efficiently capture total IgG, but it cannot distinguish between specific and non-specific antibodies against the target antigen, resulting in limited product purity and specificity. While immunoaffinity chromatography based on intact recombinant prealbumin can improve specificity, its elution process usually relies on strong acid or strong alkaline conditions. This vigorous elution can disrupt the native conformation of the antibody-antigen binding site (paratope), leading to irreversible denaturation, aggregation, or loss of activity in some high-affinity antibodies. Although immediate neutralization after elution can mitigate the damage to some extent, the resulting conformational perturbation still significantly affects the final functional titer and long-term storage stability of the antibody, manifesting as decreased sensitivity of calibration curves in immunoassays.

[0005] To obtain antibodies with higher specificity, some studies have attempted to use antigen fragments or peptides as affinity ligands. However, traditional peptide affinity purification still largely employs acid elution strategies similar to those used for whole antigen purification, failing to fundamentally address the damage to antibody activity caused by the elution process. Furthermore, how to directionally isolate antibody subsets targeting a specific dominant epitope (especially linear epitopes directly related to detection performance) from polyclonal antiserum, and ensure that these antibody subsets retain their native, highly active binding conformation after detachment from the solid support, remains a key problem that has not yet been well resolved in current technologies. This not only limits further improvements in the performance of high-sensitivity diagnostic reagents but also makes batch-to-batch consistency of antibody raw materials difficult to control.

[0006] Therefore, developing a purification method that can efficiently and directionally separate high-affinity components targeting specific key epitopes from anti-prealbumin polyclonal antibodies under near-physiological mild conditions, while maximizing the preservation of their native conformation and long-term stability, is of urgent industrial demand and significant technological value. Summary of the Invention

[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide a method for purifying high-titer prealbumin antibodies and its application. This process can achieve high-purity target antibody separation while perfectly maintaining its biological activity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for purifying a high-titer prealbumin antibody, comprising the following steps: S1. After centrifugation and filtration pretreatment, the prealbumin antiserum was loaded onto a Protein A affinity chromatography column. After rinsing with neutral buffer and gentle elution with low pH buffer, neutralization buffer was added to the elution buffer to adjust to neutral to obtain an IgG solution. The neutralized IgG solution was then replaced with a peptide affinity chromatography binding buffer to obtain a total IgG sample. S2. Synthesize the polypeptide shown in the amino acid sequence of human prealbumin from position 10 to 25, add a cysteine ​​residue at the N-terminus, and directionally couple the polypeptide to a pre-activated chromatography medium to obtain an affinity chromatography column with the synthetic polypeptide as a ligand. S3. Load the total IgG sample from S1 onto an affinity chromatography column with the synthetic peptide as a ligand, wash with binding buffer, and then elute with PBS buffer containing the peptide shown in the amino acid sequence of free human prealbumin from amino acid position 10 to 25. S4. Collect the eluent from step S3, remove the free polypeptides, and obtain purified prealbumin antibody.

[0009] Preferably, the centrifugation in S1 is 10000g for 8-10 minutes, and the filtration is performed using a 0.45μm filter membrane.

[0010] Preferably, the neutral buffer in S1 is PBS buffer with pH 7.2, and the low pH buffer is 0.1M sodium citrate buffer with pH 3.8.

[0011] Preferably, the neutralization buffer in S1 is a 1M Tris-HCl buffer with pH=9.0, and the volume ratio of the neutralization buffer to the elution buffer is 1:(8-10).

[0012] Preferably, the buffer replacement of the IgG solution in S1 is performed by desalting column or dialysis, and the chromatography medium of the Protein A affinity chromatography column is Protein A agarose gel.

[0013] The amino acid sequence of human prealbumin from position 10 to 25 in S2 is shown in SEQ ID NO.1, and is as follows: CPLMVKVLDAVRGSPA.

[0014] Preferably, the pre-activated chromatography medium in S2 is an NHS-activated Sepharose 4FF medium.

[0015] In a second aspect, the present invention provides the application of the above-described purification method in the preparation of antibodies for in vitro detection of human prealbumin.

[0016] Preferably, the prepared antibody for in vitro detection of human prealbumin has at least one of the following properties: (a) In ELISA assays coated with human prealbumin, the titer is ≥1:128,000; (b) Half-maximal effective concentration (EC) 50 ≤5.0 ng / mL; (c) When used to construct a double-antibody sandwich chemiluminescent immunoassay, the detection limit for prealbumin is ≤0.15µg / mL; (d) After accelerated storage at 37°C for 14 days, its potency retention rate is ≥80%.

[0017] In a third aspect, the present invention provides a prealbumin immunoassay reagent, wherein the prealbumin immunoassay reagent uses an antibody purified by the above method as its active ingredient.

[0018] The beneficial effects of this invention are: 1. This invention employs a mild elution strategy based on free peptide competition, eluting prealbumin antibodies under completely mild physiological conditions (almost close to physiological pH=7.4), completely avoiding the destruction of the antibody complementarity-determining region (CDR) conformation by traditional strong acid (such as pH=2.5-3.0) or strong alkaline elution conditions, thus fully preserving the antibody's high affinity binding activity.

[0019] 2. This invention uses synthetic peptides designed to correspond to key functional epitopes of prealbumin as ligands. The peptide affinity column captures only antibodies specific to the designed epitopes, and the purified products have extremely low cross-reactivity, making them suitable for high-specificity detection and significantly improving the specificity and targeting of antibodies.

[0020] 3. The antibodies purified by the method of this invention, while maintaining their native conformation, exhibit significantly higher functional titers and affinity than those obtained using traditional acid elution methods or commercially available similar products. This provides a core raw material for high-sensitivity detection and enhances the overall performance of the reagents. Furthermore, the antibodies obtained by this invention demonstrate extremely high stability during long-term storage. Accelerated stability testing shows that after storage at 37°C for 14 days, their titer retention rate remains above 88%, far superior to the control sample. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0023] Example 1: Purification Process Operation 1. Pre-enrichment: Take 10 mL of rabbit anti-human prealbumin antiserum, centrifuge at low speed (10000g, 10 min), remove the precipitate, filter through a 0.45 μm filter membrane, and load the filtered sample onto a 5 mL Protein A agarose gel column. Wash with PBS (pH 7.2), elute with 0.1 M sodium citrate (pH 3.8), and immediately neutralize with 1 / 10 volume of 1 M Tris-HCl (pH 9.0). Pass the neutralized sample through a PD-10 desalting column and replace it with peptide affinity column binding buffer.

[0024] 2. Preparation of peptide affinity column: A peptide containing the amino acid sequence of human prealbumin from positions 10 to 25 was synthesized, and a cysteine ​​residue was added to the N-terminus. The peptide was then directionally coupled to an NHS-activated Sepharose 4FF medium and packed into a column.

[0025] 3. Competitive affinity purification: The pre-enriched and buffer-replaced IgG sample was loaded onto a peptide affinity column. After rinsing with binding buffer, it was eluted with PBS buffer containing 5 mg / mL of free homologous peptide, and the elution peak was collected.

[0026] 4. Removal of free peptides: Load the eluent onto a strong cation exchange column (which is negatively charged in the pH range of 1-14). Under pH 4.0 conditions, the antibody binds, and the free peptides (negatively charged) flow through. Then, elute the antibody with high-salt buffer (1M NaCl + 20mM sodium acetate) to obtain the final pure product.

[0027] Example 2: Performance verification of the purified product 1. Sample: Sample A (of the present invention): Prealbumin antibody prepared in Example 1.

[0028] Sample B (conventional control): The same peptide affinity column as in Example 1 was used, but the elution conditions were changed to 0.1 M glycine-HCl, pH 2.7. The neutralization and subsequent steps were the same.

[0029] Sample C (commercial control): Commercially available rabbit anti-human prealbumin antibody 2. Analytical Methods and Results: (1) Purity and concentration analysis Reduced SDS-PAGE: Under reduction conditions, all three samples showed clear ~50 kDa (heavy chain) and ~25 kDa (light chain) bands, with a purity of >95%.

[0030] SEC-HPLC: A TSKgel G3000SWxl column was used, with PBS as the mobile phase. The monomer content of sample A was 99.3%, sample B was 97.8%, and sample C was 96.5%. Sample A had the lowest polymer (>150 kDa) content (<0.5%).

[0031] (2) Valence determination Methods: Human prealbumin (2 μg / mL) was coated onto an ELISA plate. Three antibodies were serially diluted 2-fold (from 1:500 to 1:512,000) starting from the same initial concentration (1 mg / mL). The diluted antibodies were added and incubated; subsequent secondary antibody and colorimetric steps were performed as usual. A positive result was determined by using 2.1 times the OD value of the negative control well (i.e., the cut-off value).

[0032] Results (effectiveness value): Sample A (of this invention): Potency 1:256,000 Sample B (conventional acid elution): titer 1:64,000 Sample C (commercial antibody): titer 1:32,000 Conclusion: Under the same initial protein content, the highest dilution at which the antibody of this invention can produce a positive signal is 4 times that of traditional acid-eluting antibodies and 8 times that of commercial antibodies, directly proving its extremely high titer.

[0033] (3) Comparison of functional affinity Methods: Antibodies were coated with the same antigen concentration. Serial dilutions were performed, and dose-response curves were plotted. The antibody concentration (EC50) required to reach half of the maximum binding signal (plateau OD value) was calculated. 50 EC 50 The lower the value, the higher the functional affinity.

[0034] result: Sample A: EC 50 = 3.2 ng / mL Sample B: EC 50 = 8.1 ng / mL Sample C: EC 50 = 12.5 ng / mL Conclusion: The EC5 of the antibody of this invention 50 At its lowest concentration, the concentration required to achieve half-maximal binding is only 40% of that required by conventional methods, indicating significantly higher functional affinity, which is closely related to its high valence.

[0035] (4) Performance in actual testing systems Methods: A double-antibody sandwich CLIA was constructed. Each test antibody was used as a capture antibody and immobilized on magnetic beads. A biotinylated mouse anti-human prealbumin monoclonal antibody was used as the detection antibody, coupled with streptavidin-ALP. A series of prealbumin standards (0-500 μg / mL) were detected.

[0036] Key performance indicator results: Limit of detection (LoD): Sample A: 0.08 μg / mL; Sample B: 0.21 μg / mL; Sample C: 0.35 μg / mL.

[0037] Dynamic range: Sample A exhibits excellent linearity (R0) in the range of 0.08–400 μg / mL. 2 Sample B showed good linearity in the range of 0.21-200 μg / mL; Sample C showed acceptable linearity in the range of 0.35-150 μg / mL.

[0038] Hook effect: When a very high concentration of antigen (800 μg / mL) was added, only sample A did not show a decrease in signal, while samples B and C showed varying degrees of hook effect precursors (signal growth slowed down significantly).

[0039] Conclusion: The antibody of this invention, due to its high titer and high affinity, endows the detection system with a lower detection limit, a wider dynamic range, and a stronger ability to resist hook effects, which is a direct manifestation of its role as a core raw material for high-performance diagnostic reagents.

[0040] (5) Accelerated stability test (potency retention rate) Methods: The three antibodies (1 mg / mL) were incubated at 37°C for accelerated degradation. Samples were taken on days 0, 1, 3, 7, and 14. The efficacy at each time point was re-determined using the ELISA endpoint dilution method described above.

[0041] Results (valence retention rate %): Table 1: Valence Retention Rate Detection Conclusion: After accelerated elution at 37°C for 14 days, the antibody of this invention maintained a titer retention rate as high as 88%, while the control antibody showed a significant decrease. This demonstrates that the mild competitive elution process not only yielded a high-titer antibody, but also that this highly active state remained exceptionally stable during storage.

[0042] In summary, the purification process of this invention, through the core technology of targeted peptide affinity and physiological competitive elution, not only achieves gentle treatment of antibodies but also selectively screens and completely preserves antibodies with the highest binding capacity (high titer) and high affinity targeting key epitopes. Example 2 demonstrates this through endpoint dilution titer determination and EC50 assay. 50 Analysis, CLIA performance testing, and stability tracking data from multiple dimensions fully validated the superior performance of this invention in achieving the core objective of "high efficiency and high valence," which is unattainable by traditional acid / base elution methods. This process exhibits excellent reproducibility, and the product quality far surpasses existing technologies, providing a reliable raw material guarantee for the development of high-performance immunodiagnostic reagents.

[0043] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. A method for purifying a high-titer prealbumin antibody, characterized in that, Includes the following steps: S1. After centrifugation and filtration pretreatment, the prealbumin antiserum was loaded onto a Protein A affinity chromatography column. After rinsing with neutral buffer and gentle elution with low pH buffer, neutralization buffer was added to the elution buffer to adjust to neutral to obtain an IgG solution. The neutralized IgG solution was then replaced with a peptide affinity chromatography binding buffer to obtain a total IgG sample. S2. Synthesize the polypeptide shown in the amino acid sequence of human prealbumin from position 10 to 25, add a cysteine ​​residue at the N-terminus, and directionally couple the polypeptide to a pre-activated chromatography medium to obtain an affinity chromatography column with the synthetic polypeptide as a ligand. S3. Load the total IgG sample from S1 onto an affinity chromatography column with the synthetic peptide as a ligand, wash with binding buffer, and then elute with PBS buffer containing the peptide shown in the amino acid sequence of free human prealbumin from amino acid position 10 to 25. S4. Collect the eluent from step S3, remove the free polypeptides, and obtain purified prealbumin antibody.

2. The purification method according to claim 1, characterized in that, The centrifugation described in S1 is 10000g for 8-10 minutes, and the filtration is performed using a 0.45μm filter membrane.

3. The purification method according to claim 1, characterized in that, The neutral buffers mentioned in S1 are all PBS buffers with a pH of 7.2, and the low pH buffers are 0.1M sodium citrate buffers with a pH of 3.

8.

4. The purification method according to claim 1, characterized in that, The neutralization buffer described in S1 is a 1M Tris-HCl buffer with pH=9.0, and the volume ratio of the neutralization buffer to the elution buffer is 1:(8-10).

5. The purification method according to claim 1, characterized in that, The buffer replacement of the IgG solution in S1 is performed by desalting column or dialysis, and the chromatography medium of the Protein A affinity chromatography column is Protein A agarose gel.

6. The purification method according to claim 1, characterized in that, The amino acid sequence of human prealbumin from position 10 to 25 in S2 is shown in SEQ ID NO.

1.

7. The purification method according to claim 1, characterized in that, The pre-activated chromatography medium mentioned in S2 is an NHS-activated Sepharose 4FF medium.

8. Use of the purification method according to any one of claims 1-7 in the preparation of antibodies for in vitro detection of human prealbumin.

9. The application according to claim 8, characterized in that, The prepared antibody for in vitro detection of human prealbumin has at least one of the following properties: (a) In ELISA assays coated with human prealbumin, the titer is ≥1:128,000; (b) Half-maximal effective concentration (EC) 50 ≤5.0 ng / mL; (c) When used to construct a double-antibody sandwich chemiluminescent immunoassay, the detection limit for prealbumin is ≤0.15 µg / mL; (d) After accelerated storage at 37°C for 14 days, its potency retention rate is ≥80%.

10. A prealbumin immunoassay reagent, characterized in that, The prealbumin immunoassay reagent uses antibodies purified by the method described in any one of claims 1-7 as its active ingredient.

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